U.S. patent application number 11/090563 was filed with the patent office on 2006-06-01 for assays for identifying ubiquitin agents and for identifying agents that modify the activity of ubiquitin agents.
This patent application is currently assigned to Rigel Pharmaceuticals, Inc.. Invention is credited to Jianing Huang, Sarkiz D. Issakani, Todd R. Pray, Julie Sheung.
Application Number | 20060115864 11/090563 |
Document ID | / |
Family ID | 27808881 |
Filed Date | 2006-06-01 |
United States Patent
Application |
20060115864 |
Kind Code |
A1 |
Issakani; Sarkiz D. ; et
al. |
June 1, 2006 |
Assays for identifying ubiquitin agents and for identifying agents
that modify the activity of ubiquitin agents
Abstract
Provided are methods and compositions for assaying for ubiquitin
agents that are enzymatic components of ubiquitin-mediated
proteolysis and, more particularly, methods and compositions for
assaying for agents that modulate the activity of such ubiquitin
agents.
Inventors: |
Issakani; Sarkiz D.; (San
Jose, CA) ; Huang; Jianing; (Foster City, CA)
; Sheung; Julie; (San Francisco, CA) ; Pray; Todd
R.; (San Francisco, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Rigel Pharmaceuticals, Inc.
South San Francisco
CA
|
Family ID: |
27808881 |
Appl. No.: |
11/090563 |
Filed: |
March 25, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10152156 |
May 20, 2002 |
6979551 |
|
|
11090563 |
Mar 25, 2005 |
|
|
|
10108767 |
Mar 26, 2002 |
6919184 |
|
|
10152156 |
May 20, 2002 |
|
|
|
10109460 |
Mar 26, 2002 |
|
|
|
10108767 |
Mar 26, 2002 |
|
|
|
10091139 |
Mar 4, 2002 |
|
|
|
10109460 |
Mar 26, 2002 |
|
|
|
10091174 |
Mar 4, 2002 |
|
|
|
10091139 |
Mar 4, 2002 |
|
|
|
09826312 |
Apr 3, 2001 |
6737244 |
|
|
10091174 |
Mar 4, 2002 |
|
|
|
09542497 |
Apr 3, 2000 |
6740495 |
|
|
09826312 |
Apr 3, 2001 |
|
|
|
Current U.S.
Class: |
435/7.92 |
Current CPC
Class: |
C12Q 1/25 20130101; G01N
33/542 20130101; G01N 2500/02 20130101; Y10S 436/805 20130101; G01N
33/573 20130101; G01N 33/5008 20130101; Y10T 436/255 20150115; G01N
2500/00 20130101; G01N 33/6842 20130101; C12Q 1/37 20130101; G01N
2500/04 20130101; G01N 33/68 20130101; G01N 2333/9015 20130101 |
Class at
Publication: |
435/007.92 |
International
Class: |
G01N 33/537 20060101
G01N033/537; G01N 33/53 20060101 G01N033/53; G01N 33/543 20060101
G01N033/543 |
Claims
1. A method of assaying for an agent that modulates the attachment
of a ubiquitin moiety to at least one ubiquitin agent, said method
comprising: a) combining: i) a first ubiquitin agent; ii) a
candidate agent; and iii) a ubiquitin moiety; and b) assaying for
the attachment of said ubiquitin moiety to said first agent.
2. The method according to claim 1, wherein said first ubiquitin
agent is an ubiquitin activating agent.
3. The method according to claim 2, wherein said ubiquitin
activating agent is an E1.
4. The method according to claim 1, further comprising including a
second ubiquitin agent in said combining step.
5. The method according to claim 4, wherein said first agent is a
ubiquitin conjugating agent and said second agent is a ubiquitin
activating agent.
6. The method according to claim 5, wherein said ubiquitin
conjugating agent is an E2 and said ubiquitin activating agent is
an E1.
7. The method according to claim 4, wherein said first agent is a
ubiquitin ligating agent and said second agent is a ubiquitin
conjugating agent comprising said ubiquitin moiety.
8. The method according to claim 7, wherein said ubiquitin ligating
agent is an E3 and said ubiquitin conjugating agent is an E2
comprising said ubiquitin moiety.
9. The method according to claim 5, wherein said ubiquitin
conjugating agent is an E2 and said ubiquitin activating agent is
an E1 comprising said ubiquitin moiety.
10. The method according to claim 9 further comprising including a
third ubiquitin agent in said combining step.
11. The method according to claim 10, wherein said third agent is a
ubiquitin ligating agent.
12. The method according to claim 11, wherein said ubiquitin
ligating agent is an E3.
13. The method according to claim 1, wherein said first ubiquitin
agent is attached to a solid support.
14. The method according to claims 4, wherein said second ubiquitin
agent is attached to a solid support.
15. The method according to claim 7, wherein said first ubiquitin
agent is attached to a solid support.
16. The method according to any of claims 13, 14, and 15, wherein
said solid support is a microtiter plate.
17. The method according to any of claims 13, 14, and 15, wherein
said solid support is a bead.
18. The method according to claims 1 or 7, wherein said first agent
comprises a tag.
19. The method according to claim 4, wherein said second agent
comprises a tag.
20. The method according to any of claims 1, 4, and 7, wherein said
ubiquitin moiety comprises a tag.
21. The method according to claims 1 or 7, wherein said first agent
comprises an attachment tag.
22. The method according to claim 4, wherein said second agent
comprises an attachment tag.
23. The method according to claims 1 or 7, wherein said first agent
comprises a label
24. The method according to claim 4, wherein said second agent
comprises a label.
25. The method according to claims 1 or 7, wherein said first agent
comprises an epitope tag.
26. The method according to claim 4, wherein said second agent
comprises an epitope tag.
27. The method according to any of claims 1, 4, and 7, wherein at
least a first and a second ubiquitin moiety is used, wherein said
first and second ubiquitin moiety moieties comprise different
fluorescent labels, and wherein said labels form a fluorescence
resonance energy transfer (FRET) pair.
28. A method of assaying for an agent that modulates the attachment
of a ubiquitin moiety to at least one ubiquitin agent, said method
comprising: a) combining: i) a first ubiquitin agent comprising a
ubiquitin ligating agent; ii) a second ubiquitin agent; iii) a
candidate agent; iv) a ubiquitin moiety; and v) a target protein;
and assaying for the attachment of said ubiquitin moiety to said
first agent.
29. The method according to claim 28, wherein said second agent is
a ubiquitin conjugating agent comprising said ubiquitin moiety.
30. The method according to claim 28 further comprising including a
third ubiquitin agent in said combining step, wherein said third
agent is a ubiquitin activating agent; wherein said substrate and
said ubiquitin moiety comprise different fluorescent labels, and
wherein said labels form a fluorescence resonance energy transfer
(FRET) pair.
31. The method according to claim 1 or 28, wherein said ubiquitin
moiety is a mammalian ubiquitin moiety.
32. The method according to claim 28, wherein said ubiquitin moiety
comprises a label.
33. The method according to claim 28, wherein said label comprises
an epitope tag.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/152,156, filed May 20, 2002; which is a continuation-in-part
of U.S. application Ser. Nos. 10/108,767, filed Mar. 26, 2002; and
is a continuation-in-part of 10/109,460, filed Mar. 26, 2002, now
abandoned; and is a continuation-in-part of 10/091,139, filed Mar.
4, 2002, now abandoned; and is a continuation-in-part of
10/091,174, filed Mar. 4, 2002, now abandoned; and is a
continuation-in-part of 09/826,312, filed Apr. 3, 2001, now U.S.
Pat. No. 6,737,244; and is a continuation-in-part of 09/542,497,
filed Apr. 3, 2000, now U.S. Pat. No. 6,740,495; and claims the
benefit of 60/291,836, filed May 18, 2001; all of which are
incorporated in their entirety. In particular, Page 83, Table 1 and
FIG. 18 of U.S. application Ser. No. 60/291,836 are incorporated by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to the field of ubiquitin-mediated
proteolysis. In particular, the invention relates to methods and
compositions for assaying for ubiquitin agents that are enzymatic
components of ubiquitin-mediated proteolysis and, more
particularly, to methods and compositions for assaying for agents
that modulate the activity of such ubiquitin agents.
BACKGROUND OF THE INVENTION
[0003] Ubiquitin is a highly conserved 76 amino acid protein
expressed in all eukaryotic cells. The levels of many intracellular
proteins are regulated by a ubiquitin-mediated proteolytic process.
This process involves the covalent ligation of ubiquitin to a
target protein, resulting in a poly-ubiquitinated target protein
which is rapidly detected and degraded by the 26S proteasome.
[0004] The ubiquitination of these target proteins is known to be
mediated by the enzymatic activity of three ubiquitin agents.
Ubiquitin is first activated in an ATP-dependent manner by a
ubiquitin activating agent, for example, an E1. The C-terminus of a
ubiquitin forms a high energy thiolester bond with the ubiquitin
activating agent. The ubiquitin is then transferred to a ubiquitin
conjugating agent, for example, an E2 (also called ubiquitin moiety
carrier protein), also linked to this second ubiquitin agent via a
thiolester bond. The ubiquitin is finally linked to its target
protein to form a terminal isopeptide bond under the guidance of a
ubiquitin ligating agent, for example, an E3. In this process,
monomers or oligomers of ubiquitin are attached to the target
protein. On the target protein, each ubiquitin is covalently
ligated to the next ubiquitin through the activity of a ubiquitin
ligating agent.
[0005] The enzymatic components of the ubiquitination pathway have
received considerable attention (for a review, see Weissman, Nature
Reviews 2:169-178 (2001)). The members of the E1 ubiquitin
activating agents and E2 ubiquitin conjugating agents are
structurally related and well characterized enzymes. There are
numerous species of E2 ubiquitin conjugating agents, some of which
act in preferred pairs with specific E3 ubiquitin ligating agents
to confer specificity for different target proteins. While the
nomenclature for the E2 ubiquitin conjugating agents is not
standardized across species, investigators in the field have
addressed this issue and the skilled artisan can readily identify
various E2 ubiquitin conjugating agents, as well as species
homologues (See Haas and Siepmann, FASEB J. 11:1257-1268
(1997)).
[0006] Generally, ubiquitin ligating agents contain two separate
activities: a ubiquitin ligase activity to attach, via an
isopeptide bond, monomers or oligomers of ubiquitin to a target
protein, and a targeting activity to physically bring the ligase
and substrate together. The substrate specificity of different
ubiquitin ligating agents is a major determinant in the selectivity
of the ubiquitin-mediated protein degradation process.
[0007] In eukaryotes, some ubiquitin ligating agents contain
multiple subunits that form a complex called the SCF having
ubiquitin ligating activity. SCFs play an important role in
regulating G1 progression, and consists of at least three subunits,
SKP 1, Cullins (having at least seven family members) and an F-box
protein (of which hundreds of species are known) which bind
directly to and recruit the substrate to the complex. The
combinatorial interactions between the SCF's and a recently
discovered family of RING finger proteins, the ROC/APC11 proteins,
have been shown to be the key elements conferring ligase activity
to ubiquitin ligating agents. Particular ROC/Cullin combinations
can regulate specific cellular pathways, as exemplified by the
function of APC11-APC2, involved in the proteolytic control of
sister chromatid separation and exit from telophase into G1 in
mitosis (see King et al., supra; Koepp et al., Cell 97:431-34
(1999)), and ROC1-Cullin 1, involved in the proteolytic degradation
of I.sub..kappa.B.sub.'' in NF-.sub..kappa.B/I.sub..kappa.B
mediated transcription regulation (Tan et al., Mol. Cell
3(4):527-533 (1999); Laney et al., Cell 97:427-30 (1999)).
[0008] The best characterized ubiquitin ligating agent is the APC
(anaphase promoting complex), which is multi-component complex that
is required for both entry into anaphase as well as exit from
mitosis (see King et al., Science 274:1652-59 (1996) for review).
The APC plays a crucial role in regulating the passage of cells
through anaphase by promoting ubiquitin-mediated proteolysis of
many proteins. In addition to degrading the mitotic B-type cyclin
for inactivation of CDC2 kinase activity, the APC is also required
for degradation of other proteins for sister chromatid separation
and spindle disassembly. Most proteins known to be degraded by the
APC contain a conserved nine amino acid motif known as the
"destruction box" that targets them for ubiquitin ubiquitination
and subsequent degradation. However, proteins that are degraded
during G1, including G1 cyclins, CDK inhibitors, transcription
factors and signaling intermediates, do not contain this conserved
amino acid motif. Instead, substrate phosphorylation appears to
play an important role in targeting their interaction with a
ubiquitin ligating agent for ubiquitin ubiquitination (see Hershko
et al., Ann. Rev. Biochem. 67:429-75 (1998)).
[0009] Two major classes of E3 ubiquitin ligating agents are known:
the HECT (homologous to E6-AP carboxy terminus) domain E3 ligating
agents; and the RING finger domain E3 ligating agents. E6AP is the
prototype for the HECT domain subclass of E3 ligating agents and is
a multi-subunit complex that functions as a ubiquitin ligating
agent for the tumor suppressor p53 which is activated by
papillomavirus in cervical cancer (Huang et al. (1999) Science
286:1321-1326). Members of this class are homologous to the
carboxyl terminus of E6AP and utilize a Cys active site to form a
thiolester bond with ubiquitin, analogous to the E1 activating
agents and E2 conjugating agents. However, in contrast, the members
of the RING finger domain class of E3 ligating agents are thought
to interact with an ubiquitin-conjugated-E2 intermediate to
activate the complex for the transfer of ubiquitin to an acceptor.
Examples of the RING domain class of E3 ligating agents are TRAF6,
involved in IKK activation; Cbl, which targets insulin and EGF;
Sina/Siah, which targets DCC; Itchy, which is involved in
haematopoesis (B, T and mast cells); IAP, involved with inhibitors
of apoptosis; and Mdm2 which is involved in the regulation of
p53.
[0010] The RING finger domain subclass of E3 ligating agents can be
further grouped into two subclasses. In one subclass, the RING
finger domain and the substrate recognition domain are contained on
different subunits of a complex forming the ubiquitin ligating
agent (e.g., the RBx1 and the F-box subunit of the SCF complex). In
the second subclass of ubiquitin ligating agents, the ligating
agents have the RING finger domain and substrate recognition domain
on a single subunit. (e.g., Mdm2 and cbl) (Tyers et al. (1999)
Science 284:601, 603-604; Joazeiro et al. (2000) 102:549-552). A
further class of ligating agents are those having a "PHD" domain
and are homologs of the RING finger domain ligating agents (Coscoy
et al. (2001) J. Cell Biol. 155(7):1265-1273), e.g., MEKK1. The PHD
domain ligating agents are a novel class of membrane-bound E3
ligating agents.
[0011] Mdm2 belongs to the second subclass of single subunit E3
ligating agents and is involved in regulating the function and
stability of p53, an important tumor suppressor. In cells, p53
functions as a DNA-binding transcription factor which induces the
expression of genes involved in DNA repair, apoptosis, and the
arrest of cell growth. In approximately 50% of all human cancer p53
is inactivate by deletion or mutation. The level of p53 in the cell
is maintained at low steady-state levels, and is induced and
activated post-translationally by various signal pathways
responsive to cellular stress (Lakin et al. (1999) Oncogene
18:7644-7655; Oren, M. (1999) J. Biol. Chem 274:36031-36,034).
Stimuli that trigger the stress response and activate p53 include
oxygen stress, inappropriate activation of oncogenes and agents
that cause damage to DNA (e.g., ionizing radiation, chemicals, and
ultra violet light).
[0012] The carboxyl terminus of Mdm2 contains a variant of the RING
finger domain (Saurin et al. (1996) Trends Biochem. Sci.
21:208-214) that is critical for the activity of this E3 ligating
agent. Recent studies have shown that Mdm2 mediates the
ubiquitination of itself resulting in the formation of
poly-ubiquitin chains on the protein (Zhihong et al. (2001) J.B.C.
276:31,357-31,367; Honda et al. (2000) Oncogene 19:1473-1476;
Shengyun et al. (2000) 275:8945-8951). Further, the ubiquitin
ligating activity of Mdm2 is dependent on its RING finger
domain.
[0013] Typically, the ubiquitination of target proteins by E3 in
cells results in the formation of poly-ubiquitin chains. An
isopeptide bond is formed between the carboxyl terminus of the
ubiquitin and the .epsilon.-amino group of Lys in the target
protein. The extension or formation of ubiquitin chains results
from the formation of additional isopeptide bonds with the
Lys.sup.48 (and sometimes Lys.sup.63) of a previously conjugated
ubiquitin and the carboxyl-terminal Gly of an additional ubiquitin.
The efficient recognition of a ubiquitinated target protein by a
proteosome requires at least four ubiquitins linked in this
configuration. However, in the case of Mdm2-mediated ubiquitination
of p53, neither Lys.sup.48 or Lys.sup.63 is involved in the
formation of poly-ubiquitin chains. Recent studies show that human
Mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism
requiring enzyme isomerization (Zhihong et al. (2001) J. Biol.
Chem. 276:31,357-31,367). Further, in vitro, the transfer of
ubiquitin to p53 can occur independent of E1 when using an E2
pre-conjugated with ubiquitin. These results suggest that the
pre-conjugated E2 can bind to Mdm2 and thereafter transfer the
ubiquitin to the Mdm2 in the absence of an E1.
[0014] Thus, ubiquitin agents, such as the ubiquitin activating
agents, ubiquitin conjugating agents, and ubiquitin ligating
agents, are key determinants of the ubiquitin-mediated proteolytic
pathway that results in the degradation of targeted proteins and
regulation of cellular processes. Consequently, agents that
modulate the activity of such ubiquitin agents may be used to
upregulate or downregulate specific molecules involved in cellular
signal transduction. Disease processes can be treated by such up-
or down regulation of signal transducers to enhance or dampen
specific cellular responses. This principle has been used in the
design of a number of therapeutics, including phosphodiesterase
inhibitors for airway disease and vascular insufficiency, kinase
inhibitors for malignant transformation and Proteasome inhibitors
for inflammatory conditions such as arthritis.
[0015] Due to the importance of ubiquitin-mediated proteolysis in
cellular process, for example cell cycle regulation, there is a
need for a fast and simple means for identifying ubiquitin agents
that are catalytic components of this enzymatic pathway, and for
identifying agents that modulate the activity of these catalytic
components. Thus, an object of the present invention is to provide
methods of assaying for ubiquitin agents that are catalytic
components of ubiquitin-mediated proteolysis and, more
particularly, methods of assaying for agents that modulate the
activity of the ubiquitin agents.
SUMMARY OF THE INVENTION
[0016] In accordance with the above objects, the present invention
provides methods and compositions for assaying for ubiquitin agents
that are enzymatic components of ubiquitin-mediated proteolysis.
More particularly, the present invention provides methods and
compositions for assaying for an agent that modulates the activity
of a ubiquitin agent that is an enzymatic component of
ubiquitin-mediated proteolysis. Specifically, the methods of the
present invention are directed to identifying ubiquitin agents such
as ubiquitin activating agents, ubiquitin conjugating agents, and
ubiquitin ligating agents; and to identifying agents that modulate
the activity of these ubiquitin agents. In one aspect, the
invention provides assaying methods that do not require a ubiquitin
target protein. In the methods of the present invention the
ubiquitin agents are combined in different combinations with a
ubiquitin moiety to assay for the attachment of the ubiquitin
moiety, or the modulation of this attachment, to at least one of
the following substrate molecules: a ubiquitin agent, a target
protein, or a mono- or poly-ubiquitin moiety which is preferably
attached to a ubiquitin agent or target protein.
[0017] In aspects of the methods and compositions of the present
invention, the ubiquitin activating agent is an E1; the ubiquitin
conjugating agent is an E2; and/or the ubiquitin ligating agent is
an E3. In other aspects, the target protein is a mammalian target
protein, and in further aspects, the target protein is a human
target protein. In other aspects, the ubiquitin moiety is a
mammalian ubiquitin, and in further aspects, the ubiquitin moiety
is a human ubiquitin. In another aspect, the ubiquitin moiety is a
ubiquitin derivative. In some aspects, the candidate agent a small
molecule, and in further aspects, the candidate agent is a peptide.
In some aspects, the ubiquitin moiety comprises a label, and in
further aspects, the label comprises an epitope tag. In other
aspects, at least a first and a second ubiquitin moiety is used,
wherein the first and second ubiquitin moieties comprise different
fluorescent labels, and wherein the labels form a fluorescence
resonance energy transfer (FRET) pair.
[0018] In one aspect, the invention provides a method of assaying
for an agent that modulates the attachment of a ubiquitin moiety to
at least one ubiquitin agent involving the steps of: a) combining a
first ubiquitin agent, a candidate agent, and a ubiquitin moiety;
and b) assaying for the attachment of the ubiquitin moiety to the
first agent. In an additional aspect, the first ubiquitin agent is
an ubiquitin activating agent. In a further aspect, the ubiquitin
activating agent is an E1.
[0019] In another aspect, the method further comprises including a
second ubiquitin agent in the combining step. In a further aspect,
the first agent is a ubiquitin conjugating agent and the second
agent is a ubiquitin activating agent. In a further aspect, the
ubiquitin conjugating agent is an E2 and the ubiquitin activating
agent is an E1. Also in a further aspect, the ubiquitin conjugating
agent is an E2 and the ubiquitin activating agent is an E1
comprising the ubiquitin moiety.
[0020] In another aspect, the first agent is a ubiquitin ligating
agent and the second agent is a ubiquitin conjugating agent
comprises the ubiquitin moiety. In a further aspect, the ubiquitin
ligating agent is an E3 and the ubiquitin conjugating agent is an
E2 comprising the ubiquitin moiety.
[0021] In another aspect, the method further comprises a third
ubiquitin agent in the combining step. In a further aspect, the
third agent is a ubiquitin ligating agent. Also in a further
aspect, the ubiquitin ligating agent is an E3.
[0022] In the methods where the assaying concerns the attachment of
the ubiquitin moiety to the first ubiquitin agent, the following
additional aspects are provided. In one aspect, the first ubiquitin
agent comprises a tag. In a further aspect, the first ubiquitin
agent comprises an epitope tag. In another aspect, first ubiquitin
agent comprises a label. Also in a further aspect, the first
ubiquitin agent comprises an attachment tag. In another aspect, the
first ubiquitin agent is attached to a solid support; and in a
further aspect, the solid support is a microtiter plate or a
bead.
[0023] In the methods where the assaying concerns the attachment of
the ubiquitin moiety to a second ubiquitin agent, the following
additional aspects are provided. In one aspect, the second
ubiquitin agent comprises a tag. In a further aspect, the second
ubiquitin agent comprises an epitope tag. In another aspect, second
ubiquitin agent comprises a label. Also in a further aspect, the
second ubiquitin agent comprises an attachment tag. In another
aspect, the second ubiquitin agent is attached to a solid support,
and in a further aspect, the solid support is a microtiter plate or
a bead.
[0024] In another aspect, the invention provides a method of
assaying for an agent that modulates the attachment of a ubiquitin
moiety to at least one ubiquitin agent involving the steps of: a)
combining a first ubiquitin agent comprising a ubiquitin ligating
agent; a second ubiquitin agent, a candidate agent, a ubiquitin
moiety, and a substrate; and b) assaying for the attachment of the
ubiquitin moiety to the first agent. In an additional aspect, the
second agent is a ubiquitin conjugating agent comprising the
ubiquitin moiety.
[0025] In an additional aspect, the method further comprises a
third ubiquitin agent in the combining step, wherein the third
agent is a ubiquitin activating agent; wherein the substrate and
the ubiquitin moiety comprise different fluorescent labels, and
wherein the labels form a fluorescence resonance energy transfer
(FRET) pair.
[0026] In the following aspects of the present invention, the
ubiquitin ligating agent is preferably an Mdm2 protein and the
target protein is preferably p53. In a preferred embodiment, the
ubiquitin ligating agent is an Mdm2 fusion protein, and more
preferably an Mdm2-GST fusion protein.
[0027] In one aspect of the present methods, the Mdm2 protein
comprises a first FRET label and the ubiquitin moiety comprises a
second FRET label. In another aspect, the Mdm2 protein comprises an
attachment tag. In another aspect, the Mdm2 protein is provided on
a solid support; and in a further aspect, the solid support
comprises a microtiter plate or a bead. In another aspect, the Mdm2
protein is a mammalian Mdm2, and in a further aspect, the Mdm2 is a
human Mdm2.
[0028] In another aspect, the p53 protein comprises a first FRET
label and the ubiquitin moiety comprises a second FRET label. In
another aspect, the p53 protein comprises an attachment tag. In
another aspect, the p53 protein is provided on a solid support; and
in a further aspect, the solid support comprises a microtiter plate
or a bead.
[0029] In one aspect, the invention provides a method of assaying
for a candidate agent that modulates the attachment of a ubiquitin
moiety to an Mdm2 protein involving the steps of: a) combining a
first ubiquitin agent comprising at least one ubiquitin moiety, an
Mdm2 protein, and a candidate agent; and b) assaying for the
attachment of the ubiquitin moiety to the Mdm2 protein. In an
additional aspect, the first ubiquitin agent is a ubiquitin
conjugating agent.
[0030] In an additional aspect, the method further comprises
combining a ubiquitin activating agent comprising the ubiquitin
moiety, thereby forming the ubiquitin conjugating agent comprising
the ubiquitin moiety, in step a).
[0031] In an additional aspect, the method further comprises
combining a ubiquitin activating agent and the ubiquitin moiety,
thereby forming the ubiquitin conjugating agent comprising the
ubiquitin moiety.
[0032] In another aspect, the invention provides a method of
assaying for a candidate agent that modulates the attachment of a
ubiquitin moiety to a p53 protein involving the steps of: a)
combining a conjugating agent comprising at least one ubiquitin
moiety, an Mdm2 protein, a p53 protein, and a candidate agent; and
b) assaying for the attachment of the ubiquitin moiety to the p53
protein.
[0033] In an additional aspect, the method further comprises
combining a ubiquitin conjugating agent and the ubiquitin moiety,
thereby forming the ubiquitin conjugating agent comprising the
ubiquitin moiety.
[0034] In an additional aspect, the method further comprises
combining a ubiquitin activating agent comprising the ubiquitin
moiety, thereby forming the ubiquitin conjugating agent comprising
the ubiquitin moiety, in step a).
[0035] In an additional aspect, the method further comprises
combining a ubiquitin activating agent and the ubiquitin moiety,
thereby forming the ubiquitin conjugating agent comprising the
ubiquitin moiety.
[0036] In another aspect, the invention provides a method of
assaying for a candidate agent that modulates the attachment of a
ubiquitin moiety to an Mdm2 protein involving the steps of: a)
combining a ubiquitin activating agent, a ubiquitin conjugating
agent, an Mdm2 protein, a candidate agent, and a ubiquitin moiety;
and b) assaying for the attachment of the ubiquitin moiety to the
Mdm2 protein.
[0037] In another aspect, the invention provides a method of
assaying for a candidate agent that modulates the attachment of a
ubiquitin moiety to a p53 protein involving the steps of: a)
combining a ubiquitin activating agent, a ubiquitin conjugating
agent, an Mdm2 protein, a p53 protein, a candidate agent, and a
ubiquitin moiety; and b) assaying for the attachment of the
ubiquitin moiety to the p53 protein.
[0038] In another aspect, the invention provides a method of
assaying for a candidate agent that modulates the attachment of a
second ubiquitin moiety to a p53 protein involving the steps of: a)
combining a ubiquitin activating agent, a ubiquitin conjugating
agent, an Mdm2 protein, a p53 protein comprising a first ubiquitin
moiety, wherein the first ubiquitin moiety is labeled with a first
FRET label, a candidate agent, and a second ubiquitin moiety
labeled with a second FRET label; and b) assaying for the
attachment of the second ubiquitin moiety to the p53 protein by
detecting a FRET reaction.
[0039] In another aspect, the invention provides a method of
assaying for a candidate agent that modulates the attachment of a
first ubiquitin moiety to a p53 protein involving the steps of: a)
combining a ubiquitin conjugating agent comprising a first
ubiquitin moiety labeled with a first FRET, an Mdm2 protein, a p53
protein comprising a second ubiquitin moiety, wherein the first
ubiquitin moiety is labeled with a second FRET label, and a
candidate agent; and b) assaying for the attachment of the first
ubiquitin moiety to the p53 protein by detecting a FRET
reaction.
[0040] In another aspect, the invention provides a method of
assaying for a candidate agent that modulates the attachment of a
first ubiquitin moiety to a p53 protein involving the steps of: a)
combining a ubiquitin activating agent comprising a first ubiquitin
moiety labeled with a first FRET, a ubiquitin conjugating agent, an
Mdm2 protein, a p53 protein comprising a second ubiquitin moiety,
wherein the first ubiquitin moiety is labeled with a second FRET
label, and a candidate agent; and b) assaying for the attachment of
the first ubiquitin moiety to the p53 protein by detecting a FRET
reaction.
[0041] Other aspects of the invention will become apparent to the
skilled artisan from the following description of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 shows the relative amounts of attachment of
fluorescently labeled ubiquitin moiety to an E2 resulting from
combining a ubiquitin activating agent, ubiquitin conjugating
agent, and ubiquitin moiety. In these experiments, E2 is
His-Ubch5c.
[0043] FIG. 2 shows the relative amounts of attachment of ubiquitin
moiety to an E3 resulting from various combinations of ubiquitin
agents and ubiquitin moiety. In these experiments, E3 comprises the
RING finger protein ROC1 and the Cullin Cul1.
[0044] FIG. 3 shows relative amounts of attachment of ubiquitin
moiety to an E3 resulting from combining E1, E2, E3, and ubiquitin
moiety. FIG. 3A shows relative amounts of attachment of ubiquitin
moiety to an E3 using varying amounts of E1 in the presence and
absence of DMSO. FIG. 3B shows relative amounts of attachment of
ubiquitin moiety to an E3 using varying amounts of ubiquitin moiety
and E3.
[0045] FIG. 4 shows the signal to noise ratio of fluorescent label
indicative of the relative amounts of attachment of ubiquitin
moiety to an E3, in an assay combining Flag-ubiquitin moiety and an
anti-Flag/anti-mouse antibody conjugated to HRP and Luminol
fluorescent HRP substrate. The signal was measured from a reaction
composition combining ubiquitin moiety, E1, E2, and E3, where the
E3 specifically bound the reaction receptacle surface substrate.
The background was measured as the amount of fluorescence present
after performing the assay in the absence of E3.
[0046] FIG. 5 shows the concentration-dependent effect of two
candidate agents that modulate the attachment of ubiquitin moiety
to an using two different E3 ubiquitin ligating agents. FIG. 5A
shows a concentration-dependent reduction in the attachment of
ubiquitin moiety to an E3, in assays comprising either ROC1/Cul1 or
ROC2/Cul5 as the components of the E3 ubiquitin ligating agent.
FIG. 5B shows a slightly different pattern of
concentration-dependent reduction of attachment of ubiquitin moiety
to an E3, by another candidate agent.
[0047] FIG. 6 shows the proportions of attachment of ubiquitin
moiety to an E3 and attachment of ubiquitin moiety to an E2, in the
presence and absence of two candidate agents that modulate the
attachment of ubiquitin moiety to an E3 by combining ubiquitin
moiety, and E1, E2, and E3 ubiquitin agents and by combining
ubiquitin moiety and E1 and E2 ubiquitin agents. FIG. 6A shows a
candidate agent that only modulates the attachment of ubiquitin
moiety to an E3. FIG. 6B shows candidate agent that modulates the
attachment of ubiquitin moiety to ubiquitin agents other than
E3.
[0048] FIG. 7 shows the concentration-dependent effects of two
candidate agents that modulate the attachment of ubiquitin moiety
to an E3 and the attachment of ubiquitin moiety to an E2. FIG. 7A
shows the results of a candidate agent having a
concentration-dependent effect on the attachment of ubiquitin
moiety to an E3 (by combining ubiquitin moiety, E1, E2, and E3),
but does not have an effect on the attachment of ubiquitin moiety
to an E2 (by combining ubiquitin moiety, E1, and E2), thus
affecting only the attachment of ubiquitin moiety to an E3. FIG. 7B
shows the results for a candidate modulator having a
concentration-dependent effect on both the attachment of ubiquitin
moiety to an E2 and the attachment of ubiquitin moiety to an E3,
thus affecting a component other than the E3.
[0049] FIGS. 8A and 8B show the nucleic acid sequence encoding
rabbit E1 ubiquitin activating agent and the amino acid sequence of
rabbit E1 (SEQ ID NOS:1 and 2), respectively.
[0050] FIGS. 9A and 9B show the nucleic acid sequence encoding the
E2 Ubch5c and the amino acid sequence of the E2 Ubch5c (SEQ ID
NOS:3 and 4), respectively.
[0051] FIG. 10 shows the amino acid sequence of the RING finger
protein APC11 (SEQ ID NO:5).
[0052] FIG. 11 shows the amino acid sequence of the RING finger
protein ROC1 (SEQ ID NO:6).
[0053] FIGS. 12A and 12B show the nucleic acid sequence encoding
the RING finger protein ROC2 and the amino acid sequence of ROC2
(SEQ ID NOS:7 and 8), respectively.
[0054] FIGS. 13A and 13B show the nucleic acid sequence encoding
the Cullin CUL5 and the amino acid sequence of CUL5 (SEQ ID NOS:9
and 10), respectively.
[0055] FIGS. 14A and 14B show the nucleic acid sequence encoding
the Cullin APC2 and the amino acid sequence of APC2 (SEQ ID NOS:11
and 12), respectively.
[0056] FIGS. 15A, 15B and 15C show the amino acid sequences of
human ubiquitin moiety, Flag-ubiquitin moiety and
Flag-Cys-ubiquitin moiety (SEQ ID NOS:13-15), respectively. The
Flag and Flag-Cys portions of the sequence are shown in bold.
[0057] FIGS. 16A and 16B show the E3 -dependent incorporation of
Flag-Ala-Cys-ubiquitin moiety labeled with FRET fluorophores into
E3-ubiquitin moiety complex. Isolation by HPLC shows emissions from
free ubiquitin moiety and ubiquitin moiety attached to the E3
ubiquitin ligating agent. The traces show fluorescent emission at
the wavelength described below, under excitation at 336 nm, the
optimal excitation wavelength for IAEDANS. FIG. 16A shows the
fluorescence signals of IAEDANS (490 nm; larger peak) and
fluorescein (515 nm; smaller peak) labeled ubiquitin moiety
following combination with E1 and E2 only. The free ubiquitin
moiety was isolation using high performance liquid chromatography
(HPLC). FIG. 16B shows the fluorescence signals of IAEDANS (490 nm;
larger peak at each elution volume) and fluorescein (515 nm;
smaller peak at each elution volume) labeled ubiquitin moiety
following combination with E1 and E2 and E3 (Roc1/Cul1). The dashed
line shows optical density of the protein solution (scale on
right), revealing the high sensitivity of the fluorophores despite
a very low concentration of protein.
[0058] FIG. 17 shows the fluorescence emission spectra of free
ubiquitin moiety labeled with the FRET donor/acceptor pair EDANS
and fluorescein under excitation at 336 nm. The dashed line shows
the emission spectra of free labeled ubiquitin moiety (reactants),
while the solid line shows the emission spectra of labeled
ubiquitin moiety bound to E3 (products). The greatly increased
515:490 nm emission ratio of the E3-bound ubiquitin moiety as
compared with the free ubiquitin moiety shows the energy transfer
from the EDANS donor to the fluorescein acceptor of this FRET
donor/acceptor pair.
[0059] FIG. 18 shows a schematic representation of GST-Mdm2 and
His-p53.
[0060] FIG. 19 shows a Western blot analysis of the attachment of
ubiquitin moiety to p53 by Mdm2, in vitro.
[0061] FIG. 20 shows a schematic of a nickel plate based assay for
the attachment of ubiquitin moiety to p53 by Mdm2.
[0062] FIG. 21 shows the results of measuring the luminescence
indicative of the amount of attachment of ubiquitin moiety to p53
by Mdm2 in the nickel plate based assay.
[0063] FIG. 22 depicts the key for the ubiquitin activating agent
(UAA ), ubiquitin conjugating agent (UCA), ubiquitin ligating agent
(ULA), ubiquitin moiety (U), and candidate agent (CA) used in the
schematics in Figures
[0064] FIG. 23 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent (UA-1) where the assay comprises: [0065] 1)
combining a UA-1+CA+U; and [0066] 2) assaying for the attachment of
the ubiquitin moiety to UA-1. In another preferred embodiment UA-1
is a UAA. In another preferred embodiment, UAA is an E1. In yet
another preferred embodiment, UA-1 comprises a label. In another
preferred embodiment, the ubiquitin moiety comprises a label.
[0067] FIG. 24 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a second
ubiquitin agent (UA-2) where the assay comprises: [0068] 1)
combining a first ubiquitin agent that is UAA.sub.1+UA-2+CA+U; and
[0069] 2) assaying for the attachment of the ubiquitin moiety to
UA-2. In another preferred embodiment, UA-2 comprises a label. In
yet another preferred embodiment, UA-2 comprises a label.
[0070] FIG. 25 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin conjugating agent UCA, where
the assay comprises: [0071] 1) combining a second ubiquitin agent
that is UAA.sub.2+UCA.sub.1+CA+U; and [0072] 2) assaying for the
attachment of the ubiquitin moiety to UCA.sub.1.
[0073] FIG. 26 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to ubiquitin
conjugating agent that is an E2 where the assay comprises: [0074]
1) combining a ubiquitin activating agent that is an E1+E2+CA+U;
and [0075] 2) assaying for the attachment of the ubiquitin moiety
to E2.
[0076] FIG. 27 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.1)
where the assay comprises: [0077] 1) combining a second ubiquitin
agent that is a ubiquitin conjugating agent and comprising a
ubiquitin moiety UCA.sub.2-U+ULA.sub.1+CA; and [0078] 2) assaying
for the attachment of the ubiquitin moiety to ULA.sub.1. In another
preferred embodiment, the ubiquitin moiety comprises a label. In
yet another preferred embodiment, ULA.sub.1 comprises a label. In a
preferred embodiment the ubiquitin ligating agent comprises an Mdm2
protein.
[0079] FIG. 28 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a a ubiquitin
ligating agent that is an E3 where the assay comprises: [0080] 1)
combining a ubiquitin conjugating agent that is an E2 and
comprising a ubiquitin moiety+E3+CA; and [0081] 2) assaying for the
attachment of the ubiquitin moiety to E3. In a preferred
embodiment, the E3 is an Mdm2 protein.
[0082] FIG. 29 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a ubiquitin
conjugating agent that is an E2 where the assay comprises: [0083]
1) combining a ubiquitin activating agent that is an E1 and
comprising a ubiquitin moiety+E2+CA; and [0084] 2) assaying for the
attachment of the ubiquitin moiety to E2.
[0085] FIG. 30 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a third
ubiquitin agent (UA-3) where the assay comprises: [0086] 1)
combining a ubiquitin activating agent that is an E1 and comprising
a ubiquitin moiety+a ubiquitin conjugating agent that is an
E2+UA-3+CA; and [0087] 2) assaying for the attachment of the
ubiquitin moiety to UA-3. In a preferred embodiment, UA-3 comprises
an Mdm2 protein.
[0088] FIG. 31 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a third
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.3)
where the assay comprises: [0089] 1) combining a ubiquitin
activating agent that is an E1 and comprising a ubiquitin moiety+a
ubiquitin conjugating agent that is an E2+ULA.sub.3+CA; and [0090]
2) assaying for the attachment of the ubiquitin moiety to
ULA.sub.3. In a preferred embodiment the ubiquitin ligating agent
comprises an Mdm2 protein.
[0091] FIG. 32 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a ubiquitin
ligating agent that is an E3 where the assay comprises: [0092] 1)
combining an E1 comprising a ubiquitin moiety+an E2+an E3+CA; and
[0093] 2) assaying for the attachment of the ubiquitin moiety to
E3. In a preferred embodiment, the E3 is Mdm2.
[0094] FIG. 33 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent (UA-1) that is attached to a solid support where
the assay comprises: [0095] 1) combining a UA-1 (that is attached
to a solid support)+CA+U; and [0096] 2) assaying for the attachment
of the ubiquitin moiety to UA-1. In another preferred embodiment
UA-1 is a UAA. In another preferred embodiment, UAA is an E1. In
another preferred embodiment, the solid support is a microtiter
plate. In another preferred embodiment, the solid support is a
bead.
[0097] FIG. 34 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a second
ubiquitin agent (UA-2) that is attached to a solid suport) where
the assay comprises: [0098] 1) combining a first ubiquitin agent
that is UAA.sub.1+UA-2 (attached to a solid support)+CA+U; and
[0099] 2) assaying for the attachment of the ubiquitin moiety to
UA-2. In another preferred embodiment, the solid support is a
microtiter plate. In another preferred embodiment, the solid
support is a bead. In a preferred embodiment, UA-2 comprises an
Mdm2 protein.
[0100] FIG. 35 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.1) that
is attached to a solid support where the assay comprises: [0101] 1)
combining a second ubiquitin agent that is a ubiquitin conjugating
agent UCA.sub.2 comprising a ubiquitin moiety+ULA.sub.1 (attached
to a solid support)+CA; and [0102] 2) assaying for the attachment
of the ubiquitin moiety to ULA.sub.1. In a preferred embodiment the
ubiquitin ligating agent comprises an Mdm2.
[0103] FIG. 36 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent (UA-1) that comprises a label where the assay
comprises: [0104] 1) combining a UA-1 (plus label)+CA+U; and [0105]
2) assaying for the attachment of the ubiquitin moiety to UA-1.
[0106] FIG. 37 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.1) that
comprises a label where the assay comprises: [0107] 1) combining a
second ubiquitin agent that is a ubiquitin conjugating agent
UCA.sub.2 comprising a ubiquitin moiety+ULA.sub.1 (plus label)+CA;
and [0108] 2) assaying for the attachment of the ubiquitin moiety
to ULA.sub.1. In a preferred embodiment the ubiquitin ligating
agent comprises an Mdm2 protein.
[0109] FIG. 38 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety that comprises a
label, to a first ubiquitin agent (UA-1) where the assay comprises:
[0110] 1) combining a UA-1+CA+U (plus label); and [0111] 2)
assaying for the attachment of the ubiquitin moiety to UA-1.
[0112] FIG. 39 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety that comprises a
label, to a second ubiquitin agent (UA-2) where the assay
comprises: [0113] 1) combining a first ubiquitin agent that is
UAA.sub.1+UA-2+CA+U (plus label); and [0114] 2) assaying for the
attachment of the ubiquitin moiety to UA-2.
[0115] FIG. 40 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety that comprises a
label, to a first ubiquitin agent that is a ubiquitin ligating
agent (ULA.sub.1) where the assay comprises: [0116] 1) combining a
second ubiquitin agent that is a ubiquitin conjugating agent
UCA.sub.2 comprising a ubiquitin moiety (plus label)+ULA.sub.1+CA;
and [0117] 2) assaying for the attachment of the ubiquitin moiety
to ULA.sub.1. In a preferred embodiment the ubiquitin ligating
agent comprises an Mdm2 protein.
[0118] FIG. 41 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.1)
which comprises a label where the assay comprises: [0119] 1)
combining a second ubiquitin agent that is a ubiquitin conjugating
agent UCA.sub.2 comprising a ubiquitin moiety+ULA.sub.1 (plus
label)+CA; and [0120] 2) assaying for the attachment of the
ubiquitin moiety to ULA.sub.1. In a preferred embodiment the
ubiquitin ligating agent comprises an Mdm2 protein.
[0121] FIG. 42 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a second
ubiquitin agent (UA-2) that comprises a label where the assay
comprises: [0122] 1) combining a first ubiquitin agent that is
UAA.sub.1+UA-2 (plus label)+CA+U; and [0123] 2) assaying for the
attachment of the ubiquitin moiety to UA-2. In a preferred
embodiment, UA-2 comprises an Mdm2 protein.
[0124] FIG. 43 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent (UA-1) that comprises an attachment tag (or
attachment moiety) where the assay comprises: [0125] 1) combining a
UA-1 (plus attachment tag)+CA+U; and [0126] 2) assaying for the
attachment of the ubiquitin moiety to UA-1.
[0127] FIG. 44 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.1) that
comprises an attachment tag (or attachment moiety) where the assay
comprises: [0128] 1) combining a second ubiquitin agent that is a
ubiquitin conjugating agent UCA.sub.2 comprising a ubiquitin
moiety+ULA.sub.1 (plus attachment tag)+CA; and [0129] 2) assaying
for the attachment of the ubiquitin moiety to ULA.sub.1. In a
preferred embodiment the ubiquitin ligating agent comprises an Mdm2
protein.
[0130] FIG. 45 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a second
ubiquitin agent (UA-2) that comprises an attachment tag (or
attachment moiety) where the assay comprises: [0131] 1) combining a
first ubiquitin agent that is UAA.sub.1+UA-2 (plus attachment
tag)+CA+U; and [0132] 2) assaying for the attachment of the
ubiquitin moiety to UA-2.
[0133] FIG. 46 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent (UA-1) that comprises an epitope tag (or epitope
label) where the assay comprises: [0134] 1) combining a UA-1 (plus
epitope tag)+CA+U; and [0135] 2) assaying for the attachment of the
ubiquitin moiety to UA-1.
[0136] FIG. 47 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a second
ubiquitin agent (UA-2) that comprises an epitope tag (or epitope
label) where the assay comprises: [0137] 1) combining a first
ubiquitin agent that is UAA.sub.1+UA-2 (plus epitope tag)+CA+U; and
[0138] 2) assaying for the attachment of the ubiquitin moiety to
UA-2.
[0139] FIG. 48 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a substrate
molecule(s) where the assay comprises: [0140] 1) combining a first
ubiquitin agent that is a ubiquitin ligating agent ULA.sub.1+a
second ubiquitin agent+substrate molecule+CA+U; and [0141] 2)
assaying for the attachment of the ubiquitin moiety to the
substrate molecule. In a preferred embodiment the ubiquitin
ligating agent comprises an Mdm2 protein and the substrate molecule
comprises p53.
[0142] FIG. 49 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a substrate
molecule (s) where the assay comprises: [0143] 1) combining a first
ubiquitin agent that is a ubiquitin ligating agent ULA.sub.1+a
second ubiquitin agent that is a ubiquitin conjugating agent and
comprising a ubiquitin moiety +substrate molecule+CA; and [0144] 2)
assaying for the attachment of the ubiquitin moiety to the
substrate molecule. In a preferred embodiment the ubiquitin
ligating agent comprises an Mdm2 protein and the substrate molecule
comprises p53.
[0145] FIG. 50 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a substrate
molecule(s) where the assay comprises: [0146] 1) combining a first
ubiquitin agent that is a ubiquitin ligating agent ULA.sub.1+a
second ubiquitin agent+a third ubiquitin agent that is a ubiquitin
activating agent+a ubiquitin moiety comprising a first FRET
tag+substrate molecule comprising a second FRET tag+CA; and [0147]
2) assaying for the attachment of the ubiquitin moiety to the
substrate molecule. In a preferred embodiment the ubiquitin
ligating agent comprises an Mdm2 protein and the substrate molecule
comprises p53.
[0148] FIG. 51 depicts the amino acid sequence (FIG. 51A; SEQ ID
NO:16) and the nucleic acid sequence (FIG. 51B; SEQ ID NO:17) of an
E2 in a preferred embodiment.
[0149] FIG. 52 depicts the amino acid sequence (FIG. 52A; SEQ ID
NO:18) and the nucleic acid sequence (FIG. 52B1 and FIG. 52B2; SEQ
ID NO:19) of an E2 in a preferred embodiment.
[0150] FIG. 53 depicts the amino acid sequence (FIG. 53A) and the
nucleic acid sequence (FIG. 53B1 and FIG. 53B2; SEQ ID NO:21) of an
E2 in a preferred embodiment.
[0151] FIG. 54 depicts the amino acid sequence (FIG. 54A; SEQ ID
NO:22) and the nucleic acid sequence (FIG. 54B; SEQ ID NO:23) of an
E2 in a preferred embodiment.
[0152] FIG. 55 depicts the amino acid sequence (FIG. 55A; SEQ ID
NO:24) and the nucleic acid sequence (FIG. 55B; SEQ ID NO:25) of an
E2 in a preferred embodiment.
DETAILED DESCRIPTION OF THE INVENTION
[0153] The present invention provides methods and compositions for
assaying for ubiquitin agents that are enzymatic components of
ubiquitin-mediated proteolysis. More particularly, the present
invention provides methods and compositions for assaying for an
agent that modulates the activity of a ubiquitin agent that is an
enzymatic component of ubiquitin-mediated proteolysis.
Specifically, the methods of the present invention are directed to
identifying ubiquitin agents such as ubiquitin activating agents,
ubiquitin conjugating agents, and ubiquitin ligating agents; and to
identifying agents that modulate the activity of these ubiquitin
agents.
[0154] The advantages of the present invention include providing
methods for assaying for the activity of ubiquitin agents in one
reaction vessel thus obviating the need for subsequent steps, for
example, for separating and purifying the products of the reaction.
Consequently, this approach allows multi-well array analysis and
high throughput screening techniques for agents that modulate the
activity of ubiquitin agents. In addition, the present invention
provides methods that allow the analysis of many different
combinations of ubiquitin agents, without requiring prior
identification of specific target proteins. In particular, the
present invention provides methods that allow the analysis of
different combinations of ubiquitin agents in the absence of a
target protein. Alternatively, the present invention provides
methods that allow the analysis of combinations of ubiquitin agents
in the presence of a target protein.
[0155] In the methods of the present invention the ubiquitin agents
are combined in different combinations with a ubiquitin moiety to
assay for the attachment of the ubiquitin moiety, or the modulation
of this attachment, to at least one of the following substrate
molecules: a ubiquitin agent, a target protein, or a mono- or
poly-ubiquitin moiety which is preferably attached to a ubiquitin
agent or target protein. For example, the invention provides the
following combination of ubiquitin agents, plus or minus a target
protein, for use in methods of:
[0156] 1) assaying for the attachment of a ubiquitin moiety to a
ubiquitin activating agent by combining a ubiquitin activating
agent and a ubiquitin moiety; or
[0157] 2) assaying for the attachment of a ubiquitin moiety to a
ubiquitin conjugating agent by combining a ubiquitin activating
agent, ubiquitin conjugating agent, and ubiquitin moiety; or
[0158] 3) assaying for the attachment of a ubiquitin moiety to a
ubiquitin conjugating agent by combining a ubiquitin activating
agent comprising a ubiquitin moiety and a ubiquitin conjugating
agent; or
[0159] 4) assaying for the attachment of a ubiquitin moiety to a
ubiquitin ligating agent by combining a ubiquitin conjugating agent
comprising a ubiquitin moiety and a ubiquitin ligating agent;
or
[0160] 5) assaying for the attachment of a ubiquitin moiety to a
ubiquitin ligating agent by combining a ubiquitin activating agent,
ubiquitin conjugating agent, ubiquitin ligating agent, and
ubiquitin moiety; or
[0161] 6) assaying for the attachment of a ubiquitin moiety to a
ubiquitin ligating agent by combining a ubiquitin activating agent
comprising a ubiquitin moiety, a ubiquitin conjugating agent, and
ubiquitin ligating agent; or
[0162] 7) assaying for the attachment of a ubiquitin moiety to a
target protein by combining a ubiquitin activating agent, a
ubiquitin conjugating agent, a ubiquitin ligating agent, a
ubiquitin moiety, and a target protein; or
[0163] 8) assaying for the attachment of a ubiquitin moiety to a
target protein by combining a ubiquitin activating agent comprising
a ubiquitin moiety, a ubiquitin conjugating agent, ubiquitin
ligating agent, and target protein; or
[0164] 9) assaying for the attachment of a ubiquitin moiety to a
target molecule by combining a ubiquitin activating agent
comprising a ubiquitin moiety, a ubiquitin conjugating agent, and a
target protein.
[0165] In particular, in the methods of the present invention, to
assay for a candidate agent that modulates the attachment of a
ubiquitin moiety to a substrate molecule of interest, a candidate
agent is included in the above examples of combinations.
[0166] The invention provides a variety of approaches using above
the combinations of ubiquitin agents to assay for the attachment of
a ubiquitin moiety to a substrate molecule of interest, or to assay
for an agent that modulates the attachment of a ubiquitin moiety to
a substrate molecule of interest. Examples of the approaches are as
follows:
[0167] 1) the components of the assay are combined in solution
phase, and then assayed for the attachment of ubiquitin moiety to
the substrate molecule of interest; or
[0168] 2) the components of the assay are combined in solid phase
by providing the substrate molecule of interest on a solid support,
and then assayed for the attachment of ubiquitin moiety to the
substrate molecule of interest; or
[0169] 3) the components of the assay are combined in solution
phase, then the substrate molecule of interest is attached to a
solid substrate, and then assayed for the attachment of ubiquitin
moiety to the substrate molecule of interest; or
[0170] 4) the components of the assay are combined in solution,
then the substrate molecule of interest that is attached to
ubiquitin moiety is purified, the purified product is then attached
to a solid substrate, and assayed for the attachment of ubiquitin
moiety to the substrate molecule.
[0171] Examples of ubiquitin agents are ubiquitin activating
agents, ubiquitin conjugating agents, and ubiquitin ligating
agents. In preferred embodiments, the ubiquitin activating agent is
preferably an E1 or a variant thereof; the ubiquitin conjugating
agent is preferably an E2 or a variant thereof; and the ubiquitin
ligating agent is preferably an E3 or variant thereof. In a
preferred embodiment, the E3 is Mdm2. In another preferred
embodiment, the Mdm2 is a fusion protein, and more preferably an
Mdm2-GST fusion protein. Thus, the present invention provides
methods of assaying for agents that modulate ubiquitin activating
activity, ubiquitin conjugating activity, and ubiquitin ligating
activity. More particularly, the present invention provides methods
of assaying for agents that modulate the attachment of a ubiquitin
moiety to a ubiquitin agent, target protein, or mono- or
poly-ubiquitin moiety preferably attached to a ubiquitin agent or
target protein.
[0172] In general, the methods involve combining a ubiquitin moiety
and one or more ubiquitin agents in the presence of or in the
absence of a target protein and measuring the amount of ubiquitin
moiety attached to at least one of the following substrate
molecules: a ubiquitin agent; a target protein; or a mono- or
poly-ubiquitin moiety which is preferably attached to a ubiquitin
agent or target protein. As used herein, "substrate molecule" or
"target substrate" and grammatical equivalents thereof means a
molecule, preferably a protein, to which a ubiquitin moiety is
bound or attached through the activity of a ubiquitin agent or by
the process of ubiquitination. As used herein, "the substrate
molecule of interest" is the ubiquitin agent, target protein, or
ubiquitin moiety to which the attachment of a ubiquitin moiety is
being assayed for in the methods of the present invention. As used
herein with reference to the activity of ubiquitin agents,
"attachment" refers to the transfer, binding, ligation, and/or
ubiquitination of a mono- or poly-ubiquitin ubiquitin moiety to a
substrate molecule. Thus, "ubiquitination" and grammatical
equivalents thereof means the attachment, or transfer, binding,
and/or ligation of ubiquitin moiety to a substrate molecule; and
"ubiquitination reaction" and grammatical equivlents thereof refer
to the combining of components under conditions that permit
ubiquitination (i.e., the attachment or transfer, binding, and/or
ligation of ubiquitin moiety to a substrate molecule).
[0173] In some preferred embodiments, the ubiquitin agent comprises
a ubiquitin moiety. As used herein with reference to a ubiquitin
agent, the phrase "comprising a ubiquitin moiety" or grammatical
equivalents thereof refers to the pre-loading, pre-conjugation, or
pre-attachment of a ubiquitin moiety to a ubiquitin agent (forming
a "pre-conjugated ubiquitin agent" or "pre-loaded ubiquitin agent")
such that the attachment of a ubiquitin moiety to a substrate
molecule of interest does not require combining all three ubiquitin
agents (i.e., an ubiquitin activating agent, ubiquitin conjugating
agent, and ubiquitin ligating agent) and/or combining ubiquitin
moiety that is not pre-conjugated. For example in the case of a
ubiquitin activating agent comprising a ubiquitin moiety, the
attachment of ubiquitin moiety to a ubiquitin conjugating agent can
be performed in the absence of ubiquitin moiety that is not
pre-conjugated. For example, in the case of a ubiquitin conjugating
agent comprising a ubiquitin moiety, the attachment of ubiquitin
moiety to a ubiquitin ligating agent can be performed in the
absence of a ubiquitin activating agent and ubiquitin moiety that
is not pre-conjugated. Also, for example, in the case of a
ubiquitin ligating agent comprising a ubiquitin moiety, the
attachment of ubiquitin moiety to a target molecule can be
performed in the absence of a ubiquitin activating agent, ubiquitin
conjugating agent, and ubiquitin moiety that is not pre-conjugated.
A pre-conjugated ubiquitin agent suitable for use in the methods
and compositions of the present invention can be prepared using
methods known in the art. In a preferred embodiment, pre-conjugated
ubiquitin agents are prepared according to Zhihong et al. (2001) J.
Biol. Chem. 276:31,357-31,367.
[0174] By "target protein" herein is meant a protein other than a
ubiquitin moiety to which a ubiquitin moiety is bound or attached
through the activity of a ubiquitin agent or by the process of
ubiquitination. In preferred embodiments, the target protein is a
mammalian target protein, and more preferably a human target
protein. In a preferred embodiment, the target protein is p53.
[0175] In the following preferred embodiments at least one
ubiquitin agent is combined with a ubiquitin moiety in the absence
of a target protein. [0176] In a preferred embodiment, the
invention provides a method of assaying for an agent that modulates
the attachment of a ubiquitin moiety to at least one ubiquitin
agent involving the steps of: a) combining a first ubiquitin agent,
a candidate agent, and a ubiquitin moiety; and b) assaying for the
attachment of the ubiquitin moiety to the first agent. In one
preferred embodiment the first ubiquitin agent is an ubiquitin
activating agent, and preferably an E1. In another embodiment, the
method further comprises including a second ubiquitin agent in the
combining step, where the first agent is preferably a ubiquitin
conjugating agent and more preferably and E2; and the second agent
is preferably a ubiquitin activating agent and more preferably an
E1. In another embodiment, the ubiquitin conjugating agent is
preferably an E2 and the ubiquitin activating agent is preferably
an E1 comprising the ubiquitin moiety. [0177] In another
embodiment, the first agent is a preferably a ubiquitin ligating
agent and more preferably an E3; and the second agent is preferably
a ubiquitin conjugating agent comprising the ubiquitin moiety and
more preferably an E2 comprising a ubiquitin moiety. [0178] In
another embodiment, the method further comprises a third ubiquitin
agent in the combining step. In one embodiment, the third agent is
preferably a ubiquitin ligating agent and more preferably an E2.
[0179] In the methods where the assaying concerns the attachment of
the ubiquitin moiety to the first ubiquitin agent, the following
preferred embodiments are provided. In one embodiment, the first
ubiquitin agent preferably comprises a tag and more preferably an
epitope tag or a label. In another embodiment, the first ubiquitin
agent preferably comprises an attachment tag. In another
embodiment, the first ubiquitin agent is preferably attached to a
solid support and more preferably is attached to a microtiter plate
or a bead. [0180] In the methods where the assaying concerns the
attachment of the ubiquitin moiety to the second ubiquitin agent,
the following preferred embodiments are provided. In one
embodiment, the second ubiquitin agent preferably comprises a tag
and more preferably an epitope tag or a label. In another
embodiment, the second ubiquitin agent preferably comprises an
attachment tag. In another embodiment, the second ubiquitin agent
is preferably attached to a solid support and more preferably is
attached to a microtiter plate or a bead. [0181] In a preferred
embodiment, the invention provides a method of assaying for a
candidate agent that modulates the attachment of a ubiquitin moiety
to an MdM2 protein involving the steps of: a) combining a first
ubiquitin agent comprising at least one ubiquitin moiety, an MdM2
protein, and a candidate agent; and b) assaying for the attachment
of the ubiquitin moiety to the MdM2 protein. In an additional
embodiment, the first ubiquitin agent is preferably a ubiquitin
conjugating agent. [0182] In another preferred embodiment, the
method further comprises combining a ubiquitin activating agent
comprising the ubiquitin moiety, thereby forming the ubiquitin
conjugating agent comprising the ubiquitin moiety, in step a).
[0183] In another preferred embodiment, the method further
comprises combining a ubiquitin activating agent and the ubiquitin
moiety, thereby forming the ubiquitin conjugating agent comprising
the ubiquitin moiety. [0184] In another preferred embodiment, the
invention provides a method of assaying for a candidate agent that
modulates the attachment of a ubiquitin moiety to an MdM2 protein
involving the steps of: a) combining a ubiquitin activating agent,
a ubiquitin conjugating agent, an MdM2 protein, a candidate agent,
and a ubiquitin moiety; and b) assaying for the attachment of the
ubiquitin moiety to the MdM2 protein.
[0185] Alternatively, the invention provides assays including a
target protein. In the following preferred embodiments a target
protein is combined with ubiquitin moiety and at least one
ubiquitin agent. [0186] In another preferred embodiment, the
invention provides a method of assaying for an agent that modulates
the attachment of a ubiquitin moiety to at least one ubiquitin
agent involving the steps of: a) combining a first ubiquitin agent
comprising a ubiquitin ligating agent; a second ubiquitin agent, a
candidate agent, a ubiquitin moiety, and a target protein; and b)
assaying for the attachment of the ubiquitin moiety to the first
agent. In an additional embodiment, the second agent is a ubiquitin
conjugating agent comprising the ubiquitin moiety. [0187] In
another preferred embodiment, the method further comprises a third
ubiquitin agent in the combining step, wherein the third agent is a
ubiquitin activating agent; wherein the substrate and the ubiquitin
moiety comprise different fluorescent labels, and wherein the
labels form a fluorescence resonance energy transfer (FRET) pair.
[0188] In another preferred embodiment, the invention provides a
method of assaying for a candidate agent that modulates the
attachment of a ubiquitin moiety to a p53 protein involving the
steps of: a) combining a conjugating agent comprising at least one
ubiquitin moiety, an Mdm2 protein, a p53 protein, and a candidate
agent; and b) assaying for the attachment of the ubiquitin moiety
to the p53 protein. [0189] In an additional preferred embodiment,
the method further comprises combining a ubiquitin conjugating
agent and the ubiquitin moiety, thereby forming the ubiquitin
conjugating agent comprising the ubiquitin moiety. [0190] In an
additional preferred embodiment, the method further comprises
combining a ubiquitin activating agent comprising the ubiquitin
moiety, thereby forming the ubiquitin conjugating agent comprising
the ubiquitin moiety, in step a). [0191] In an additional preferred
embodiment, the method further comprises combining a ubiquitin
activating agent and the ubiquitin moiety, thereby forming the
ubiquitin conjugating agent comprising the ubiquitin moiety. [0192]
In another preferred embodiment, the invention provides a method of
assaying for a candidate agent that modulates the attachment of a
ubiquitin moiety to a p53 protein involving the steps of: a)
combining a ubiquitin activating agent, a ubiquitin conjugating
agent, an MdM2 protein, a p53 protein, a candidate agent, and a
ubiquitin moiety; and b) assaying for the attachment of the
ubiquitin moiety to the p53 protein. [0193] In another preferred
embodiment, the invention provides a method of assaying for a
candidate agent that modulates the attachment of a second ubiquitin
moiety to a p53 protein involving the steps of: a) combining a
ubiquitin activating agent, a ubiquitin conjugating agent, an MdM2
protein, a p53 protein comprising a first ubiquitin moiety, wherein
the first ubiquitin moiety is labeled with a first FRET label, a
candidate agent, and a second ubiquitin moiety labeled with a
second FRET label; and b) assaying for the attachment of the second
ubiquitin moiety to the p53 protein by detecting a FRET reaction.
[0194] In another preferred embodiment, the invention provides a
method of assaying for a candidate agent that modulates the
attachment of a first ubiquitin moiety to a p53 protein involving
the steps of: a) combining a ubiquitin conjugating agent comprising
a first ubiquitin moiety labeled with a first FRET, an MdM2
protein, a p53 protein comprising a second ubiquitin moiety,
wherein the first ubiquitin moiety is labeled with a second FRET
label, and a candidate agent; and b) assaying for the attachment of
the first ubiquitin moiety to the p53 protein by detecting a FRET
reaction. [0195] In another preferred embodiment, the invention
provides a method of assaying for a candidate agent that modulates
the attachment of a first ubiquitin moiety to a p53 protein
involving the steps of: a) combining a ubiquitin activating agent
comprising a first ubiquitin moiety labeled with a first FRET, a
ubiquitin conjugating agent, an MdM2 protein, a p53 protein
comprising a second ubiquitin moiety, wherein the first ubiquitin
moiety is labeled with a second FRET label, and a candidate agent;
and b) assaying for the attachment of the first ubiquitin moiety to
the p53 protein by detecting a FRET reaction.
[0196] In a preferred embodiment, the substrate molecule of
interest is attached to the surface of a reaction vessel, such as
the well of a multi-well plate. This embodiment facilitates the
separation of the ubiquitin moiety that is attached to the
substrate molecule of interest from the unattached or free
ubiquitin moiety. Means for attaching ubiquitin agents or target
proteins to the surface of a reaction vessel are described below.
The present methods permits the entire assay to occur in one
vessel, making the assay useful for high-throughput screening
applications.
[0197] In a preferred embodiment, the ubiquitin moiety is labeled,
either directly or indirectly, as further described below, and the
amount of label is measured and indicative of the amount of
attachment of ubiquitin moiety to a substrate molecule of interest.
Thus, the invention provides methods that permit for easy and rapid
detection and measurement of the activity of ubiquitin agents,
making the assay useful for high-throughput screening applications.
In one preferred embodiment, the signal of the label varies with
the extent of the attachment of ubiquitin moiety to the substrate
molecule of interest, such as in the FRET system described below.
One of ordinary skill in the art will recognize the applicability
of the present invention to screening for agents which modulate
ubiquitin ubiquitination.
[0198] As used herein, "ubiquitin moiety" refers to a polypeptide
which is transferred or attached to another polypeptide by a
ubiquitin agent. The ubiquitin moiety can comprise a ubiquitin from
any species of organism, preferably a eukaryotic species. In
preferred embodiments the ubiquitin moiety comprises is a mammlian
ubiquitin, and more preferably a human ubiquitin. In a preferred
embodiment, the ubiquitin moiety comprises a 76 amino acid human
ubiquitin. In a preferred embodiment, the ubiquitin moiety
comprises the amino acid sequence depicted in FIG. 15A. Other
embodiments utilize variants of ubiquitin, as further described
below.
[0199] As used herein, "poly-ubiquitin moiety" refers to a chain of
ubiquitin moieties comprising more than one ubiquitin moiety. As
used herein, "mono-ubiquitin moiety" refers to a single ubiquitin
moiety. In the methods of the present invention, a mono- or
poly-ubiquitin moiety can serve as a substrate molecule for the
transfer or attachment of ubiquitin moiety (which can itself be a
mono- or poly-ubiquitin moiety).
[0200] In a preferred embodiment, when ubiquitin moiety is attached
to a target protein, that protein is targeted for degradation by
the 26S proteasome.
[0201] As used herein, "ubiquitin moiety" encompasses naturally
occurring alleles and man-made variants of such a 76 amino acid
polypeptide. In a preferred embodiment, the ubiquitin moiety
comprises an amino acid sequence or nucleic acid sequence
corresponding to a sequence of GENBANK accession number P02248,
incorporated herein by reference. In other preferred embodiments,
the ubiquitin moiety comprises an amino acid sequence or nucleic
acid sequence of a sequence corresponding to one of the following
GENBANK accession numbers: NM.sub.--006156 (NEDD8); NM.sub.--003352
(SUMO-1, aka, UBL1); XM.sub.--048691 (SUMO-1, aka, UBL1);
NM.sub.--006936 (smt3a); XM.sub.--009805 (smt3a); XM.sub.--095400
(smt3b); NM.sub.--006937 (smt3b); XM.sub.--041583 (smt3b);
NM.sub.--015783 (ISG15); or NM.sub.--005101 (ISG15), each
incorporated herein by reference.
[0202] GENBANK accession numbers and their corresponding amino acid
sequences or nucleic acid sequences are found in the Genbank data
base. Sequences corresponding to GenBank accession numbers cited
herein are incorporated herein by reference. GenBank is known in
the art, see, e.g., Benson, D A, et al., Nucleic Acids Research
26:1-7 (1998) and http://www.ncbi.nlm.nih.gov/. Preferably, the
ubiquitin moiety has the amino acid sequence depicted in FIG. 15A.
In a preferred embodiment, variants of ubiquitin moiety have an
overall amino acid sequence identity of preferably greater than
about 75%, more preferably greater than about 80%, even more
preferably greater than about 85% and most preferably greater than
90% of the amino acid sequence depicted in FIG. 15A. In some
embodiments the sequence identity will be as high as about 93 to 95
or 98%.
[0203] In another preferred embodiment, a ubiquitin moiety protein
has an overall sequence similarity with the amino acid sequence
depicted in FIG. 15A of greater than about 80%, more preferably
greater than about 85%, even more preferably greater than about 90%
and most preferably greater than 93%. In some embodiments the
sequence identity will be as high as about 95 to 98 or 99%.
[0204] As is known in the art, a number of different programs can
be used to identify whether a protein (or nucleic acid as discussed
below) has sequence identity or similarity to a known sequence.
Sequence identity and/or similarity is determined using standard
techniques known in the art, including, but not limited to, the
local sequence identity algorithm of Smith & Waterman, Adv.
Appl. Math. 2:482 (1981), by the sequence identity alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, PNAS
USA 85:2444 (1988), by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Drive, Madison, Wis.), the Best Fit sequence program described by
Devereux et al., Nucl. Acid Res. 12:387-395 (1984), preferably
using the default settings, or by inspection. Preferably, percent
identity is calculated by FastDB based upon the following
parameters: mismatch penalty of 1; gap penalty of 1; gap size
penalty of 0.33; and joining penalty of 30, "Current Methods in
Sequence Comparison and Analysis," Macromolecule Sequencing and
Synthesis, Selected Methods and Applications, pp 127-149 (1988),
Alan R. Liss, Inc.
[0205] An example of a useful algorithm is PILEUP. PILEUP creates a
multiple sequence alignment from a group of related sequences using
progressive, pairwise alignments. It can also plot a tree showing
the clustering relationships used to create the alignment. PILEUP
uses a simplification of the progressive alignment method of Feng
& Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is
similar to that described by Higgins & Sharp CABIOS 5:151-153
(1989). Useful PILEUP parameters including a default gap weight of
3.00, a default gap length weight of 0.10, and weighted end
gaps.
[0206] Another example of a useful algorithm is the BLAST
algorithm, described in Altschul et al., J. Mol. Biol. 215,
403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993). A
particularly useful BLAST program is the WU-BLAST-2 program which
was obtained from Altschul et al., Methods in Enzymology, 266:
460-480 (1996); http://blast.wustl/edu/blast/README.html].
WU-BLAST-2 uses several search parameters, most of which are set to
the default values. The adjustable parameters are set with the
following values: overlap span=1, overlap fraction=0.125, word
threshold (T)=11. The HSP S and HSP S2 parameters are dynamic
values and are established by the program itself depending upon the
composition of the particular sequence and composition of the
particular database against which the sequence of interest is being
searched; however, the values may be adjusted to increase
sensitivity.
[0207] An additional useful algorithm is gapped BLAST as reported
by Altschul et al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST
uses BLOSUM-62 substitution scores; threshold Tparameter set to 9;
the two-hit method to trigger ungapped extensions; charges gap
lengths of k a cost of 10+k; X.sub.u set to 16, and X.sub.g set to
40 for database search stage and to 67 for the output stage of the
algorithms. Gapped alignments are triggered by a score
corresponding to .about.22 bits.
[0208] A percent amino acid sequence identity value is determined
by the number of matching identical residues divided by the total
number of residues of the "longer" sequence in the aligned region.
The "longer" sequence is the one having the most actual residues in
the aligned region (gaps introduced by WU-Blast-2 to maximize the
alignment score are ignored).
[0209] The alignment may include the introduction of gaps in the
sequences to be aligned. In addition, for sequences which contain
either more or fewer amino acids than the amino acid sequence
depictd in FIG. 15A, it is understood that in one embodiment, the
percentage of sequence identity will be determined based on the
number of identical amino acids in relation to the total number of
amino acids. Thus, for example, sequence identity of sequences
shorter than that of the sequence depicted in FIG. 15A, as
discussed below, will be determined using the number of amino acids
in the shorter sequence, in one embodiment. In percent identity
calculations relative weight is not assigned to various
manifestations of sequence variation, such as, insertions,
deletions, substitutions, etc.
[0210] In one embodiment, only identities are scored positively
(+1) and all forms of sequence variation including gaps are
assigned a value of "0", which obviates the need for a weighted
scale or parameters as described below for sequence similarity
calculations. Percent sequence identity can be calculated, for
example, by dividing the number of matching identical residues by
the total number of residues of the "shorter" sequence in the
aligned region and multiplying by 100. The "longer" sequence is the
one having the most actual residues in the aligned region.
[0211] Ubiquitin moieties of the present invention are polypeptides
that may be shorter or longer than the amino acid sequence depicted
in FIG. 15A. Thus, in a preferred embodiment, included within the
definition of ubiquitin moiety are portions or fragments of the
amino acid sequence depicted in FIG. 15A. In one embodiment herein,
fragments of ubiquitin moiety are considered ubiquitin moieties if
they are attached to another polypeptide by a ubiquitin agent.
[0212] In addition, as is more fully outlined below, ubiquitin
moieties of the present invention are polypeptides that can be made
longer than the amino acid sequence depicted in FIG. 15A; for
example, by the addition of tags, the addition of other fusion
sequences, or the elucidation of additional coding and non-coding
sequences. As described below, the fusion of a ubiquitin moiety to
a fluorescent peptide, such as Green Fluorescent Peptide (GFP), is
particularly preferred.
[0213] The ubiquitin moiety, as well as other proteins of the
present invention, are preferably recombinant proteins. A
"recombinant protein" is a protein made using recombinant
techniques, i.e. through the expression of a recombinant nucleic
acid as described below. In a preferred embodiment, the ubiquitin
moiety of the invention is made through the expression of a nucleic
acid sequence corresponding to GENBANK accession number M26880 or
AB003730, or a fragment thereof. In a most preferred embodiment,
the nucleic acid encodes the amino acid sequence depicted in FIG.
15A. A recombinant protein is distinguished from naturally
occurring protein by at least one or more characteristics. For
example, the protein may be isolated or purified away from some or
all of the proteins and compounds with which it is normally
associated in its wild type host, and thus may be substantially
pure. For example, an isolated protein is unaccompanied by at least
some of the material with which it is normally associated in its
natural state, preferably constituting at least about 0.5%, more
preferably at least about 5% by weight of the total protein in a
given sample. A substantially pure protein comprises at least about
75% by weight of the total protein, with at least about 80% being
preferred, and at least about 90% being particularly preferred. The
definition includes the production of a protein from one organism
in a different organism or host cell. Alternatively, the protein
may be made at a significantly higher concentration than is
normally seen, through the use of an inducible promoter or high
expression promoter, such that the protein is made at increased
concentration levels. Alternatively, the protein may be in a form
not normally found in nature, as in the addition of an epitope tag
or amino acid substitutions, insertions and deletions, as discussed
below.
[0214] As used herein and further defined below, "nucleic acid" may
refer to either DNA or RNA, or molecules which contain both deoxy-
and ribonucleotides. The nucleic acids include genomic DNA, cDNA
and oligonucleotides including sense and anti-sense nucleic acids.
Such nucleic acids may also contain modifications in the
ribose-phosphate backbone to increase stability and half life of
such molecules in physiological environments.
[0215] The nucleic acid may be double stranded, single stranded, or
contain portions of both double stranded or single stranded
sequence. As will be appreciated by those in the art, the depiction
of a single strand ("Watson") also defines the sequence of the
other strand ("Crick"); thus the sequences depicted in FIGS. 1 and
3 also include the complement of the sequence. By the term
"recombinant nucleic acid" herein is meant nucleic acid, originally
formed in vitro, in general, by the manipulation of nucleic acid by
endonucleases, in a form not normally found in nature. Thus an
isolated nucleic acid, in a linear form, or an expression vector
formed in vitro by ligating DNA molecules that are not normally
joined, are both considered recombinant for the purposes of this
invention. It is understood that once a recombinant nucleic acid is
made and reintroduced into a host cell or organism, it will
replicate non-recombinantly, i.e. using the in vivo cellular
machinery of the host cell rather than in vitro manipulations;
however, such nucleic acids, once produced recombinantly, although
subsequently replicated non-recombinantly, are still considered
recombinant for the purposes of the invention.
[0216] The terms "polypeptide" and "protein" may be used
interchangeably throughout this application and mean at least two
covalently attached amino acids, which includes proteins,
polypeptides, oligopeptides and peptides. The protein may be made
up of naturally occurring amino acids and peptide bonds, or
synthetic peptidomimetic structures. Thus "amino acid", or "peptide
residue", as used herein means both naturally occurring and
synthetic amino acids. For example, homo-phenylalanine, citrulline
and noreleucine are considered amino acids for the purposes of the
invention. "Amino acid" also includes imino acid residues such as
proline and hydroxyproline. The side chains may be in either the
(R) or the (S) configuration. In the preferred embodiment, the
amino acids are in the (S) or L-configuration. If non-naturally
occurring side chains are used, non-amino acid substituents may be
used, for example to prevent or retard in vivo degradation.
[0217] In one embodiment, the present invention provides
compositions containing protein variants, for example ubiquitin
moiety, E1, E2 and/or E3 variants. These variants fall into one or
more of three classes: substitutional, insertional or deletional
variants. These variants ordinarily are prepared by site specific
mutagenesis of nucleotides in the DNA encoding a protein of the
present compositions, using cassette or PCR mutagenesis or other
techniques well known in the art, to produce DNA encoding the
variant, and thereafter expressing the DNA in recombinant cell
culture as outlined above. However, variant protein fragments
having up to about 100-150 residues may be prepared by in vitro
synthesis using established techniques. Amino acid sequence
variants are characterized by the predetermined nature of the
variation, a feature that sets them apart from naturally occurring
allelic or interspecies variation of the protein amino acid
sequence. The variants typically exhibit the same qualitative
biological activity as the naturally occurring analogue, although
variants can also be selected which have modified characteristics
as will be more fully outlined below.
[0218] While the site or region for introducing an amino acid
sequence variation is predetermined, the mutation per se need not
be predetermined. For example, in order to optimize the performance
of a mutation at a given site, random mutagenesis may be conducted
at the target codon or region and the expressed variants screened
for the optimal desired activity. Techniques for making
substitution mutations at predetermined sites in DNA having a known
sequence are well known, for example, M13 primer mutagenesis and
PCR mutagenesis. Rapid production of many variants may be done
using techniques such as the method of gene shuffling, whereby
fragments of similar variants of a nucleotide sequence are allowed
to recombine to produce new variant combinations. Examples of such
techniques are found in U.S. Pat. Nos. 5,605,703; 5,811,238;
5,873,458; 5,830,696; 5,939,250; 5,763,239; 5,965,408; and
5,945,325, each of which is incorporated by reference herein in its
entirety. Screening of the mutants is performed using the activity
assays of the present invention.
[0219] Amino acid substitutions are typically of single residues;
insertions usually will be on the order of from about 1 to 20 amino
acids, although considerably larger insertions may be tolerated.
Deletions range from about 1 to about 20 residues, although in some
cases deletions may be much larger.
[0220] Substitutions, deletions, insertions or any combination
thereof may be used to arrive at a final derivative. Generally
these changes are done on a few amino acids to minimize the
alteration of the molecule. However, larger changes may be
tolerated in certain circumstances. When small alterations in the
characteristics of the protein are desired, substitutions of an
original residue are generally made in accordance with exemplary
substitutions listed below. TABLE-US-00001 Original Exemplary
Residue Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser,
Ala Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val
Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser
Trp Tyr Tyr Trp, Phe Val Ile, Leu
[0221] Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative than
those shown in the above list. For example, substitutions may be
made which more significantly affect: the structure of the
polypeptide backbone in the area of the alteration, for example the
alpha-helical or beta-sheet structure; the charge or hydrophobicity
of the molecule at the target site; or the bulk of the side chain.
The substitutions which in general are expected to produce the
greatest changes in the polypeptide's properties are those in which
(a) a hydrophilic residue, e.g. seryl or threonyl, is substituted
for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, e.g. lysyl, arginyl, or histidyl, is
substituted for (or by) an electronegative residue, e.g. glutamyl
or aspartyl; or (d) a residue having a bulky side chain, e.g.
phenylalanine, is substituted for (or by) one not having a side
chain, e.g. glycine.
[0222] The variants typically exhibit the same qualitative
biological activity and will elicit the same immune response as the
naturally-occurring analogue, although variants also are selected
to modify the characteristics of the proteins as needed.
Alternatively, the variant may be designed such that the biological
activity of the protein is altered. For example, glycosylation
sites may be altered or removed.
[0223] Covalent modifications of polypeptides are included within
the scope of this invention. One type of covalent modification
includes reacting targeted amino acid residues of a polypeptide
with an organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues of a
polypeptide. Derivatization with bifunctional agents is useful, for
instance, for crosslinking a protein to a water-insoluble support
matrix or surface for use in the method for screening assays, as is
more fully described below. Commonly used crosslinking agents
include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0224] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the "-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0225] Another type of covalent modification of a polypeptide
included within the scope of this invention comprises altering the
native glycosylation pattern of the polypeptide. "Altering the
native glycosylation pattern" is intended for purposes herein to
mean deleting one or more carbohydrate moieties found in native
sequence polypeptide, and/or adding one or more glycosylation sites
that are not present in the native sequence polypeptide.
[0226] Addition of glycosylation sites to polypeptides may be
accomplished by altering the amino acid sequence thereof. The
alteration may be made, for example, by the addition of, or
substitution by, one or more serine or threonine residues to the
native sequence polypeptide (for O-linked glycosylation sites). The
amino acid sequence may optionally be altered through changes at
the DNA level, particularly by mutating the DNA encoding the
polypeptide at preselected bases such that codons are generated
that will translate into the desired amino acids.
[0227] Another means of increasing the number of carbohydrate
moieties on a polypeptide is by chemical or enzymatic coupling of
glycosides to the polypeptide. Such methods are described in the
art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and
Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
[0228] Removal of carbohydrate moieties present on the polypeptide
may be accomplished chemically or enzymatically or by mutational
substitution of codons encoding for amino acid residues that serve
as targets for glycosylation. Chemical deglycosylation techniques
are known in the art and described, for instance, by Hakimuddin, et
al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al.,
Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate
moieties on polypeptides can be achieved by the use of a variety of
endo-and exo-glycosidases as described by Thotakura et al., Meth.
Enzymol., 138:350 (1987).
[0229] Another type of covalent modification of a protein comprises
linking the polypeptide to one of a variety of nonproteinaceous
polymers, e.g., polyethylene glycol, polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0230] Polypeptides of the present invention may also be modified
in a way to form chimeric molecules comprising a first polypeptide
fused to another, heterologous polypeptide or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of a
substrate molecule (e.g., a ubiquitin moiety, ubiquitin agent, or
target protein) with a tag polypeptide which provides an epitope to
which an anti-tag antibody can selectively bind. The epitope tag is
generally placed at the amino-or carboxyl-terminus of the
polypeptide. The presence of such epitope-tagged forms of a
polypeptide can be detected using an antibody against the tag
polypeptide. Also, providing an epitope tag enables the polypeptide
to be readily purified by affinity purification using an anti-tag
antibody or another type of affinity matrix that binds to the
epitope tag. In an alternative embodiment, the chimeric molecule
may comprise a fusion of a polypeptide disclosed herein with an
immunoglobulin or a particular region of an immunoglobulin. For a
bivalent form of the chimeric molecule, such a fusion could be to
the Fc region of an IgG molecule. Tags for components of the
invention are defined and described in detail below.
[0231] The present invention provides methods for assaying for the
attachment of ubiquitin moiety to a substrate molecule of interest.
Preferred embodiments of the invention involve combining ubiquitin
moiety and ubiquitin agents, plus or minus target protein; and
further in the presence or absence of a candidate agent; under
conditions where ubiquitin moiety can attach to a substrate
molecule of interest; and assaying for the attachment of the
ubiquitin moiety to the substrate molecule of interest, for
example, by measuring the amount of ubiquitin moiety (mono- or
poly-ubiquitin moiety) attached to the substrate molecule. In these
assays, the activity resulting from the combination of different
ubiquitin agents and combination of different subunits of
individual ubiquitin agents; plus or minus target protein; and
further, in the presence or absence of a candidate ubiquitin agent;
can be observed and measured.
[0232] In a preferred embodiment, the invention is additionally
directed to a method of assaying for ubiquitin activating activity.
By "ubiquitin activating activity`, "ubiquitin moiety activation"
and grammatical equivalents thereof is meant the binding or
attachment of ubiquitin moiety to a substrate molecule that is
preferably a ubiquitin activating agent. In a preferred embodiment,
the ubiquitin activating agent is an E1. Preferably, the E1 forms a
high energy thiolester bond with the ubiquitin moiety.
[0233] In a preferred embodiment, the invention is also directed to
a method of assaying for ubiquitin conjugating activity. By
"ubiquitin conjugating activity", "ubiquitin moiety conjugation"
and grammatical equivalents thereof is meant the binding or
attachment of an activated ubiquitin moiety to a ubiquitin
conjugating agent. As will be appreciated by those in the art, due
to the presence of the high energy thiolester bond in the conjugate
of the ubiquitin moiety-ubiquitin conjugating agent, the attached
ubiquitin moiety may be joined to other ubiquitin moiety at a low
rate in the absence of the catalytic activity of a ubiquitin
ligating agent (e.g., E3). Therefore, some of the ubiquitin moiety
will be attached in the form of poly-ubiquitin moiety.
[0234] In a preferred embodiment, the invention is directed to a
method of assaying ubiquitin ligating activity. By "ubiquitin
ligating activity", "ubiquitin moiety ligation" and grammatical
equivalents thereof is meant the transfer or attachment of
ubiquitin moiety to a substrate molecule that is preferably a
target protein or mono- or poly-ubiquitin moiety preferably
attached to a target protein. Preferably, each ubiquitin moiety is
covalently attached by the ubiquitin ligating agent such that a
subsequent ubiquitin moiety may be attached to it, to form chains
(poly-ubiquitin moieties) comprising a plurality of ubiquitin
moiety molecules.
[0235] The present invention provides methods and compositions
comprising combining ubiquitin moiety with other components. By
"combining" is meant the addition of the various components into a
reaction vessel under conditions in which attachment of ubiquitin
moiety to a substrate molecule interest can occur. In a preferred
embodiment, the reaction vessel is a well of a 96 well plate or
other commercially available multiwell plate. In an alternate
preferred embodiment, the reaction vessel is in a FACS machine.
Other reaction vessels useful in the present invention include, but
are not limited to 384 well plates and 1536 well plates. Still
other reaction vessels useful in the present invention will be
apparent to the skilled artisan.
[0236] The addition of the components may be sequential or in a
predetermined order or grouping, as long as the conditions amenable
to the attachment of ubiquitin to a substrate molecule of interest
are obtained. Such conditions are well known in the art, and
further guidance is provided below.
[0237] In a preferred embodiment, one or more components of the
present invention comprise a tag. By "tag" is meant an attached
molecule or molecules useful for the identification or isolation of
the attached molecule(s), which are preferably substrate molecules.
For example, a tag can be an attachment tag or a label tag.
Components having a tag are referred to as "tag-X", wherein X is
the component. For example, a ubiquitin moiety comprising a tag is
referred to herein as "tag-ubiquitin moiety". Preferably, the tag
is covalently bound to the attached component. When more than one
component of a combination has a tag, the tags will be numbered for
identification, for example "tag1-ubiquitin moiety". Components may
comprise more than one tag, in which case each tag will be
numbered, for example "tag 1,2-ubiquitin moiety". Preferred tags
include, but are not limited to, a label, a partner of a binding
pair, and a surface substrate binding molecule (or attachment tag).
As will be evident to the skilled artisan, many molecules may find
use as more than one type of tag, depending upon how the tag is
used.
[0238] By "label" is meant a molecule that can be directly (i.e., a
primary label) or indirectly (i.e., a secondary label) detected;
for example a label can be visualized and/or measured or otherwise
identified so that its presence or absence can be known. As will be
appreciated by those in the art, the manner in which this is
performed will depend on the label. Preferred labels include, but
are not limited to, fluorescent labels, label enzymes and
radioisotopes.
[0239] By "fluorescent label" is meant any molecule that may be
detected via its inherent fluorescent properties. Suitable
fluorescent labels include, but are not limited to, fluorescein,
rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,
methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,
Cascade Blue.TM., Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640,
Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes
are described in the 1996 Molecular Probes Handbook by Richard P.
Haugland, hereby expressly incorporated by reference. Suitable
fluorescent labels also include, but are not limited to, green
fluorescent protein (GFP; Chalfie, et al., Science
263(5148):802-805 (Feb. 11, 1994); and EGFP; Clontech--Genbank
Accession Number U55762 ), blue fluorescent protein (BFP; 1.
Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th
Floor, Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H.
Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y.
Curr. Biol. 6:178-182 (1996)), enhanced yellow fluorescent protein
(EYFP; 1. Clontech Laboratories, Inc., 1020 East Meadow Circle,
Palo Alto, Calif. 94303), luciferase (Ichiki, et al., J. Immunol.
150(12):5408-5417 (1993)), $-galactosidase (Nolan, et al., Proc
Natl Acad Sci USA 85(8):2603-2607 (Apr 1988)) and Renilla WO
92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; U.S.
Pat. No. 5,292,658; U.S. Pat. No. 5,418,155; U.S. Pat. No.
5,683,888; U.S. Pat. No. 5,741,668; U.S. Pat. No. 5,777,079; U.S.
Pat. No. 5,804,387; U.S. Pat. No. 5,874,304; U.S. Pat. No.
5,876,995; and U.S. Pat. No. 5,925,558) All of the above-cited
references are expressly incorporated herein by reference.
[0240] In some instances, multiple fluorescent labels are employed.
In a preferred embodiment, at least two fluorescent labels are used
which are members of a fluorescence resonance energy transfer
(FRET) pair. FRET is phenomenon known in the art wherein excitation
of one fluorescent dye is transferred to another without emission
of a photon. A FRET pair consists of a donor fluorophore and an
acceptor fluorophore. The fluorescence emission spectrum of the
donor and the fluorescence absorption spectrum of the acceptor must
overlap, and the two molecules must be in close proximity. The
distance between donor and acceptor at which 50% of donors are
deactivated (transfer energy to the acceptor) is defined by the
Forster radius (R.sub.o), which is typically 10-100 .ANG.. Changes
in the fluorescence emission spectrum comprising FRET pairs can be
detected, indicating changes in the number of that are in close
proximity (i.e., within 100 .ANG. of each other). This will
typically result from the binding or dissociation of two molecules,
one of which is labeled with a FRET donor and the other of which is
labeled with a FRET acceptor, wherein such binding brings the FRET
pair in close proximity. Binding of such molecules will result in
an increased fluorescence emission of the acceptor and/or quenching
of the fluorescence emission of the donor.
[0241] FRET pairs (donor/acceptor) useful in the invention include,
but are not limited to, EDANS/fluorescien, IAEDANS/fluorescein,
fluorescein/tetramethylrhodamine, fluorescein/LC Red 640,
fluorescein/Cy 5, fluorescein/Cy 5.5 and fluorescein/LC Red
705.
[0242] In another aspect of FRET, a fluorescent donor molecule and
a nonfluorescent acceptor molecule ("quencher") may be employed. In
this application, fluorescent emission of the donor will increase
when quencher is displaced from close proximity to the donor and
fluorescent emission will decrease when the quencher is brought
into close proximity to the donor. Useful quenchers include, but
are not limited to, DABCYL, QSY 7 and QSY 33. Useful fluorescent
donor/quencher pairs include, but are not limited to EDANS/DABCYL,
Texas Red/DABCYL, BODIPY/DABCYL, Lucifer yellow/DABCYL,
coumarin/DABCYL and fluorescein/QSY 7 dye.
[0243] The skilled artisan will appreciate that FRET and
fluorescence quenching allow for monitoring of binding of labeled
molecules over time, providing continuous information regarding the
time course of binding reactions.
[0244] It is important to remember that ubiquitin moiety is ligated
to a substrate molecule by its terminal carboxyl group to a lysine
residue, including lysine residues on other ubiquitin moiety.
Therefore, attachment of labels or other tags should not interfere
with either of these active groups on the ubiquitin moiety. Amino
acids may be added to the sequence of protein, through means well
known in the art and described herein, for the express purpose of
providing a point of attachment for a label. In a preferred
embodiment, one or more amino acids are added to the sequence of a
component for attaching a tag thereto, preferably a fluorescent
label. In a preferred embodiment, the amino acid to which a
fluorescent label is attached is Cysteine.
[0245] By "label enzyme" is meant an enzyme which may be reacted in
the presence of a label enzyme substrate which produces a
detectable product. Suitable label enzymes for use in the present
invention include but are not limited to, horseradish peroxidase,
alkaline phosphatase and glucose oxidase. Methods for the use of
such substrates are well known in the art. The presence of the
label enzyme is generally revealed through the enzyme's catalysis
of a reaction with a label enzyme substrate, producing an
identifiable product. Such products may be opaque, such as the
reaction of horseradish peroxidase with tetramethyl benzedine, and
may have a variety of colors. Other label enzyme substrates, such
as Luminol (available from Pierce Chemical Co.), have been
developed that produce fluorescent reaction products. Methods for
identifying label enzymes with label enzyme substrates are well
known in the art and many commercial kits are available. Examples
and methods for the use of various label enzymes are described in
Savage et al., Previews 247:6-9 (1998), Young, J. Virol. Methods
24:227-236 (1989), which are each hereby incorporated by reference
in their entirety.
[0246] By "radioisotope" is meant any radioactive molecule.
Suitable radioisotopes for use in the invention include, but are
not limited to .sup.14C, .sup.3H, .sup.32P, .sup.33P, .sup.35S,
.sup.125I, and .sup.131I. The use of radioisotopes as labels is
well known in the art.
[0247] In addition, labels may be indirectly detected, that is, the
tag is a partner of a binding pair. By "partner of a binding pair"
is meant one of a first and a second moiety, wherein said first and
said second moiety have a specific binding affinity for each other.
Suitable binding pairs for use in the invention include, but are
not limited to, antigens/antibodies (for example,
digoxigenin/anti-digoxigenin, dinitrophenyl (DNP)/anti-DNP,
dansyl-X-anti-dansyl, Fluorescein/anti-fluorescein, lucifer
yellow/anti-lucifer yellow, and rhodamine/anti-rhodamine),
biotin/avidin (or biotin/streptavidin) and calmodulin binding
protein (CBP)/calmodulin. Other suitable binding pairs include
polypeptides such as the FLAG-peptide [Hopp et al., BioTechnology,
6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al.,
Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner et
al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10
protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci.
USA, 87:6393-6397 (1990)] and the antibodies each thereto.
Generally, in a preferred embodiment, the smaller of the binding
pair partners serves as the tag, as steric considerations in
ubiquitin moiety ligation may be important. As will be appreciated
by those in the art, binding pair partners may be used in
applications other than for labeling, as is further described
below.
[0248] As will be appreciated by those in the art, a partner of one
binding pair may also be a partner of another binding pair. For
example, an antigen (first moiety) may bind to a first antibody
(second moiety) which may, in turn, be an antigen for a second
antibody (third moiety). It will be further appreciated that such a
circumstance allows indirect binding of a first moiety and a third
moiety via an intermediary second moiety that is a binding pair
partner to each.
[0249] As will be appreciated by those in the art, a partner of a
binding pair may comprise a label, as described above. It will
further be appreciated that this allows for a tag to be indirectly
labeled upon the binding of a binding partner comprising a label.
Attaching a label to a tag which is a partner of a binding pair, as
just described, is referred to herein as "indirect labeling".
[0250] By "surface substrate binding molecule" or "attachment tag"
and grammatical equivalents thereof is meant a molecule have
binding affinity for a specific surface substrate, which substrate
is generally a member of a binding pair applied, incorporated or
otherwise attached to a surface. Suitable surface substrate binding
molecules and their surface substrates include, but are not limited
to poly-histidine (poly-his) or poly-histidine-glycine
(poly-his-gly) tags and Nickel substrate; the Glutathione-S
Transferase tag and its antibody substrate (available from Pierce
Chemical); the flu HA tag polypeptide and its antibody 12CA5
substrate [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the
c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibody
substrates thereto [Evan et al., Molecular and Cellular Biology,
5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D
(gD) tag and its antibody substrate [Paborsky et al., Protein
Engineering, 3(6):547-553 (1990)]. In general, surface binding
substrate molecules useful in the present invention include, but
are not limited to, polyhistidine structures (His-tags) that bind
nickel substrates, antigens that bind to surface substrates
comprising antibody, haptens that bind to avidin substrate (e.g.,
biotin) and CBP that binds to surface substrate comprising
calmodulin.
[0251] Production of antibody-embedded substrates is well known;
see Slinkin et al., Bioconj. Chem. 2:342-348 (1991); Torchilin et
al., supra; Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992);
King et al., Cancer Res. 54:6176-6185 (1994); and Wilbur et al.,
Bioconjugate Chem. 5:220-235 (1994) (all of which are hereby
expressly incorporated by reference), and attachment of or
production of proteins with antigens is described above.
[0252] Calmodulin-embedded substrates are commercially available,
and production of proteins with CBP is described in Simcox et al.,
Strategies 8:40-43 (1995), which is hereby incorporated by
reference in its entirety.
[0253] As will be appreciated by those in the art, tag-components
of the invention can be made in various ways, depending largely
upon the form of the tag. Components of the invention and tags are
preferably attached by a covalent bond.
[0254] The production of tag-polypeptides by recombinant means when
the tag is also a polypeptide is described below. Production of
FLAG-labeled proteins is well known in the art and kits for such
production are commercially available (for example, from Kodak and
Sigma). Methods for the production and use of FLAG-labeled proteins
are found, for example, in Winston et al., Genes and Devel.
13:270-283 (1999), incorporated herein in its entirety, as well as
product handbooks provided with the above-mentioned kits.
[0255] Biotinylation of target molecules and substrates is well
known, for example, a large number of biotinylation agents are
known, including amine-reactive and thiol-reactive agents, for the
biotinylation of proteins, nucleic acids, carbohydrates, carboxylic
acids; see chapter 4, Molecular Probes Catalog, Haugland, 6th Ed.
1996, hereby incorporated by reference. A biotinylated substrate
can be attached to a biotinylated component via avidin or
streptavidin. Similarly, a large number of haptenylation reagents
are also known (Id.).
[0256] Methods for labeling of proteins with radioisotopes are
known in the art. For example, such methods are found in Ohta et
al., Molec. Cell 3:535-541 (1999), which is hereby incorporated by
reference in its entirety.
[0257] Production of proteins having His-tags by recombinant means
is well known, and kits for producing such proteins are
commercially available. Such a kit and its use is described in the
QIAexpress Handbook from Qiagen by Joanne Crowe et al., hereby
expressly incorporated by reference.
[0258] The functionalization of labels with chemically reactive
groups such as thiols, amines, carboxyls, etc. is generally known
in the art. In a preferred embodiment, the tag is functionalized to
facilitate covalent attachment.
[0259] The covalent attachment of the tag may be either direct or
via a linker. In one embodiment, the linker is a relatively short
coupling moiety, that is used to attach the molecules. A coupling
moiety may be synthesized directly onto a component of the
invention, ubiquitin moiety for example, and contains at least one
functional group to facilitate attachment of the tag.
Alternatively, the coupling moiety may have at least two functional
groups, which are used to attach a functionalized component to a
functionalized tag, for example. In an additional embodiment, the
linker is a polymer. In this embodiment, covalent attachment is
accomplished either directly, or through the use of coupling
moieties from the component or tag to the polymer. In a preferred
embodiment, the covalent attachment is direct, that is, no linker
is used. In this embodiment, the component preferably contains a
functional group such as a carboxylic acid which is used for direct
attachment to the functionalized tag. It should be understood that
the component and tag may be attached in a variety of ways,
including those listed above. What is important is that manner of
attachment does not significantly alter the functionality of the
component. For example, in tag-ubiquitin moiety, the tag should be
attached in such a manner as to allow the ubiquitin moiety to be
covalently attached to another ubiquitin moiety to form
polyubiquitin moiety chains. As will be appreciated by those in the
art, the above description of covalent attachment of a label and
ubiquitin moiety applies equally to the attachment of virtually any
two molecules of the present disclosure.
[0260] In a preferred embodiment, the tag is functionalized to
facilitate covalent attachment, as is generally outlined above.
Thus, a wide variety of tags are commercially available which
contain functional groups, including, but not limited to,
isothiocyanate groups, amino groups, haloacetyl groups, maleimides,
succinimidyl esters, and sulfonyl halides, all of which may be used
to covalently attach the tag to a second molecule, as is described
herein. The choice of the functional group of the tag will depend
on the site of attachment to either a linker, as outlined above or
a component of the invention. Thus, for example, for direct linkage
to a carboxylic acid group of a ubiquitin moiety, amino modified or
hydrazine modified tags will be used for coupling via carbodiimide
chemistry, for example using
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) as is known
in the art (see Set 9 and Set 11 of the Molecular Probes Catalog,
supra; see also the Pierce 1994 Catalog and Handbook, pages T-155
to T-200, both of which are hereby incorporated by reference). In
one embodiment, the carbodiimide is first attached to the tag, such
as is commercially available for many of the tags described
herein.
[0261] In a preferred embodiment, ubiquitin moiety is in the form
of tag-ubiquitin moiety, wherein, tag is a partner of a binding
pair. Preferably in this embodiment the tag is FLAG and the binding
partner is anti-FLAG. Preferably in this embodiment, a label is
attached to the FLAG by indirect labeling. Preferably, the label is
a label enzyme. Most preferably, the label enzyme is horseradish
peroxidase, which is reacted with a fluorescent label enzyme
substrate. Preferably, the label enzyme substrate is Luminol.
Alternatively, the label is a fluorescent label.
[0262] In another preferred embodiment, ubiquitin moiety is in the
form of tag-ubiquitin moiety, wherein the tag is a fluorescent
label. In a particularly preferred embodiment, ubiquitin moiety is
in the form of tag1-ubiquitin moiety and tag2-ubiquitin moiety,
wherein tag1 and tag2 are the members of a FRET pair. In an
alternate preferred embodiment, ubiquitin moiety is in the form of
tag1-ubiquitin moiety and tag2-ubiquitin moiety, wherein tag1 is a
fluorescent label and tag2 is a quencher of the fluorescent label.
In either of these preferred embodiments, when tag1-ubiquitin
moiety and tag2-ubiquitin moiety are attached to a substrate
molecule of interest through the activity of a ubiquitin agent,
preferably tag1 and tag2 are within 100 .ANG. of each other, more
preferable within 70 .ANG., still more preferably within 50 .ANG.,
even more preferably within 40 .ANG., and in some cases, preferably
within 30 .ANG. or less.
[0263] In yet another preferred embodiment, ubiquitin moiety is in
the form of tag1,2-ubiquitin moiety and tag1,3-ubiquitin moiety,
wherein tag1 is a member of a binding pair, preferably FLAG, tag2
is a fluorescent label and tag3 is either a fluorescent label such
that tag2 and tag3 are members of a FRET pair or tag3 is a quencher
of tag2.
[0264] In a preferred embodiment, one or more amino acids are added
to the ubiquitin moiety sequence, using recombinant techniques as
described herein, to provide an attachment point for a tag,
preferably a fluorescent label or a quencher. In a preferred
embodiment, the one or more amino acids are Cys or Ala-Cys.
Preferably, the one or more amino acids are attached to the
N-terminal of the ubiquitin moiety. In a preferred embodiment, the
one or more amino acids intervenes the sequence of a FLAG tag and
the ubiquitin moiety. In a preferred embodiment, the tag,
preferably a fluorescent label or a quencher, is attached to the
added Cysteine.
[0265] In some embodiments, the methods of the present invention
comprise the use of a ubiquitin activating agent. As used herein
"ubiquitin activating agent" refers to a ubiquitin agent,
preferably a protein, capable of transferring or attaching a
ubiquitin moiety to a ubiquitin conjugating agent. In a preferred
embodiment, the ubiquitin activating agent forms a high energy
thiolester bond with ubiquitin moiety, thereby "activating" the
ubiquitin moiety. In another preferred embodiment, the ubiquitin
activating agent binds or attaches ubiquitin moiety. In another
preferred embodiment, the ubiquitin activating agent is capable of
transferring or attaching ubiquitin moiety to a substrate molecule
that is a mono- or poly-ubiquitin moiety. In a preferred
embodiment, the ubiquitin activating agent is capable of
transferring or attaching ubiquitin moiety to a mono- or
poly-ubiquitinated ubiquitin conjugating agent.
[0266] In a preferred embodiment the ubiquitin activating agent is
an E1. In a preferred embodiment, the E1 is capable of transferring
or attaching ubiquitin moiety to an E2, defined below.
[0267] In the methods and compositions of the present invention,
the ubiquitin activating agent comprises an amino acid sequence or
a nucleic acid corresponding to a sequence of an Genbank data base
accession number listed in Table 1 below and incorporated herein by
reference. TABLE-US-00002 TABLE 1 ACCESSION ORG SYMBOL DESCRIPTION
NO. Hs APPBPI amyloid beta precursor protein binding protein 1, 59
kD NM_003905 Hs FLJ23251 hypothetical protein FLJ23251 NM_024818 Hs
GSA7 ubiquitin activating enzyme E1-like protein NM_006395 Hs
similar to ubiquitin-activating enzyme E1 (A1S9T and XM_088743 BN75
temperature sensitivity complementing) (H. sapiens) Hs similar to
SUMO-1 activating enzyme subunit 1; SUMO-1 XM_090110 activating
enzyme E1 N subunit; sentrin/SUMO-activating protein AOS1;
ubiquitin-like protein SUMO-1 activating enzyme Hs SAE1 SUMO-1
activating enzyme subunit 1 NM_005500 and XM_009036 Dm Uba1
Ubiquitin activating enzyme 1 NG_000652 and NM_057962 Dm Uba2 Smt3
activating enzyme 2 NM_080017 Hs UBA2 SUMO-1 activating enzyme
subunit 2 NM_005499 Hs UBE1 ubiquitin-activating enzyme E1 (A1S9T
and BN75 NM_003334 temperature sensitivity complementing) and
XM_033895 Hs UBE1C ubiquitin-activating enzyme E1C (UBA3 homolog,
yeast) NM_003968 Rn Ube1c Ubiquitin-activating enzyme E1C NM_057205
Mm Ube1l Ubiquitin-activating enzyme E1-like Hs UBE1L
Ubiquitin-activating enzyme E1-like NM_003335 Mm Ube1x
ubiquitin-activating enzyme E1, Chr X NM_009457 Mm Ube1y1
ubiquitin-activating enzyme E1, Chr Y 1 NM_011667 Mm Ube1y1-
ubiquitin-activating enzyme E1, Chr Y, pseudogene 1 M88481 and ps1
U09053 Mm Ube1y1- ubiquitin-activating enzyme E1, Chr Y-1,
pseudogene 2 U09054 ps2
[0268] Sequences encoding a ubiquitin activating agent may also be
used to make variants thereof that are suitable for use in the
methods and compositions of the present invention. The ubiquitin
activating agents and variants suitable for use in the methods and
compositions of the present invention may be made as described
herein.
[0269] In a preferred embodiment, E1 proteins useful in the
invention include the polypeptides encoded by the amino acid
sequence corresponding to GENBANK accession numbers A38564, S23770,
AAA61246, P22314, CAA40296 and BAA33144, incorporated herein by
reference. In a preferred embodiment, E1 has the amino acid
sequence shown in FIG. 8B or is encoded by a nucleic acid
comprising the sequence shown in FIG. 8A. Preferably E1 is human
E1. E1 is commercially available from Affiniti Research Products
(Exeter, U.K.).
[0270] In a preferred embodiment, nucleic acids which may be used
for producing E1 proteins for the invention include, but are not
limited to, those disclosed by GENBANK accession numbers M58028,
X56976 and AB012190, incorporated herein by reference. In a
preferred embodiment, E1 is encoded by a nucleic acid having a
sequence consisting essentially of the sequence shown in FIG. 8A.
Variants of the cited E1 proteins, also included in the term "E1",
can be made as described herein.
[0271] In some embodiments, the methods of the present invention
comprise the use of a ubiquitin conjugating agent. As used herein
"ubiquitin conjugating agent" refers to a ubiquitin agent,
preferably a protein, capable of transferring or attaching
ubiquitin moiety to a ubiquitin ligating agent. In some cases, the
ubiquitin conjugating agent is capable of directly transferring or
attaching ubiquitin moiety to lysine residues in a target protein
(Hershko et al. (1983) J. Biol. Chem. 258:8206-8214). In a
preferred embodiment, the ubiquitin conjugating agent is capable of
transferring or attaching ubiquitin moiety to a mono- or
poly-ubiquitin moiety preferably attached to a ubiquitin agent or
target protein. In a preferred embodiment, the ubiquitin
conjugating agent is capable of transferring ubiquitin moiety to a
mono- or poly-ubiquitinated ubiquitin ligating agent.
[0272] In a preferred embodiment the ubiquitin conjugating agent is
an E2. In a preferred embodiment, ubiquitin moiety is transferred
from E1 to E2. In a preferred embodiment, the transfer results in a
thiolester bond formed between E2 and ubiquitin moiety. In a
preferred embodiment, E2 is capable of transferring or attaching
ubiquitin moiety to an E3, defined below.
[0273] In the methods and compositions of the present invention,
the ubiquitin activating agent comprises an amino acid sequence or
a nucleic acid sequence corresponding to a sequence of an Genbank
data base accession number listed in Table 2 below and incorporated
herein by reference. TABLE-US-00003 TABLE 2 Accession No. Accession
No. (nucleic acid (amino acid Name ALIAS sequences) sequences)
UBE2D1 Hs UBC4/5 UBE2D1, UBCH5A, UBC4/5 NM_003338.1 NP_003329.1
homolog homolog UBC9 Gallus gallus UBC9, SUMO-conjugating enzyme
AB069964.1 BAB68210.1 UBC9 Mus musculus mUB69 U76416.1 AAB18790.1
UBC9/UBE21 Hs ?? UBE21 U45328.1 AAA86662.1 UBC9 MGC: 3994, IMAGE:
2819732, BC004437.1 AAH04437.1 isoform/MGC: 3994 Hs UBC9 isoform
NM_003345.1 NP_003336.1 UBC9 Hs UBC9, UBE21 FTS homolog Hs+ 1aa
fused toes homolog, FLJ13258 NM_022476.1 NP_071921.1 FLJ13988 Hs
FLJ13988, clone Y79AA1002027, AK024050.1 BAB14800.1 MGC: 13396 Hs
sim to E2-18 BC010900.1 AAH10900.1 UBE2V2 Hs MGC: 13396, IMAGE:
4081461 NM_003350.2 NP_003341.1 MGC: 10481 Hs UBE2V2, EDAF-1, MMS2,
UEV2, BC004862.1 AAH04862.1 XM_054332.1 Hs DDVIT1, ED XM_054332.1
XP_054332.1 FLJ13855 Hs MGC: 10481, IMAGE: 3838157 XM_030444.3
XP_030444.1 E2-230K homolog Hs FLJ13855 NM_022066.1 NP_071349.1
UBE2V2 Hs E2-230K ortholog, FLJ12878, NM_003339.1 NO_003330.1
UBE2D3 Hs 1 SNP KIAA1734 NM_003340.1 NP_003331.1 Non-canon Ub-conj
UBE2D2, UBCH5B, UBC4, NM_016336.2 NP_057420.2 Enz (NCUBE1) UBC4/5
homolog NM_014176.1 NP_054895.1 HSPC150 Hs UBE2D3, UBCH5C, UBC4/5
NM_016252.1 NP_057336.1 Brain 1AP repeat homolog contain 6 (BIRC6)
NCUBE1, HSU93243, HSPC153, CGI-76 BIRC6, KIAA1289, apollon UBC8 Mus
E2-20K, UBE2H NM_009459.1 NP_033485.1 UBC8 Hs UBE2H, UBCH, UBCH2,
UBC8 NM_003344.1 NP_003335.1 UBC8 Hs 6SNP homolog NM-003344.1
NP-003335.1 UBC8 Hs no 5' UBE2H, UBCH, UBCH2, UBC8 homolog RAD6
homolog Hs UBE2B, RAD6B, HHR6B, UBC2, NM_003337.1 NP_003328.1 RAD6
homolog UBE2V1 var 3 Hs UBE2V1, CIR1, UEV1, UEV1A, NM_022442.2
NP_071887.1 UBE2V1 var 1 Hs early CROC-1, CRO NM_021988.2
NP_068823.1 stop, 56aa UBE2V1, CIR1, UEV1, UEV1A, NM_003349.3
NP_003340.1 UBE2V1 var 2 Hs CROC-1, CRO UBE2V1, CIR1, UEV1, UEV1A,
CROC-1, CRO UBE2L6 Hs UBE2L6, UBCH8, RIG-B NM_004223.1 NP_004214.1
UBE2L3 Hs 2 SNP UBE2L3, UBCH7 NM_003347.1 NP_003338.1 UBE2E1 Hs
UBE2E1, UBCH6, UBC4/5 NM_003341.1 NP_003332.1 RAD6/UBE2A Hs homolog
NM_003336.1 NP_003327.1 UBE2E3 Hs UBE2A, RAD6A, HHR6A, UBC2,
NM_006357.1 NP_006348.1 UBC12/UBE2M Hs RAD6 homolog NM_003969.1
NP_003960.1 UBC7/UBE2G1 Hs UBE2E3, UBCH9, UBC4/5 NM_003342.1
NP_003333.1 homolog UBE2M, HUBC12, UBC12 homolog UBE2G1, UBC7
homolog Huntingtin interact prot HIP2, LIG, E2-25K NM_005339.2
NP_005330.1 2 (HIP2) Hs LIG, HIP2 alternative splicing form
ABO22436.1 BAA78556.1 LIG/HIP2 variant Hs UBC6p Hs UBC6p, UBC6
NM_058167.1 NP_477515.1 UBC6 Hs UBC6 AF296658.1 AAK52609.1
HBUCE1/UBE2D2 var HBUCE1, LOC51619 NM_015983.1 NP_057067.1 Hs
UBE2G2, UBC7 homolog XM_036087.1 XP_036087.1 UBE2G2/UBC7 NCE2
NM_080678.1 NP_542409.1 homolog Hs CDC34, E2-CDC34, E2-32
NM_004359.1 NP_004350.1 NEDD8-conj enzyme 2 complementing
BC000848.1 AAH00848.1 (NCE2) Hs IMAGE: 3458173 CDC34 Hs IMAGE:
3458173/NICE- 5 var UBE2C Hs UBE2C, UBCH10 NM_007019.1 NP_008950.1
UBE2C possible short UBE2C, UBCH10 NM_007019.1 NP_008950.1 form Hs
UBC3/UBE2N Hs UBE2N, UBCH-BEN, UBC13 NM_003348.1 NP_003339.1
FLJ25157 Hs hom., sim to bend AK057886.1 BAB71605.1 TSG101 Hs 1 SNP
FLJ25157, highly similar to E2-23 NM_006292.1 NP_006283.1 MGC:
21212/NICE-5 Tumor susceptibility gene 101 BC017708.1 AAH17708.1
var Hs MCG: 21212, IMAGE: 3907760, sim to NICE-5
[0274] Sequences encoding a ubiquitin conjugating agent may also be
used to make variants thereof that are suitable for use in the
methods and compositions of the present invention. The ubiquitin
conjugatin agents and variants suitable for use in the methods and
compositions of the present invention may be made as described
herein.
[0275] In a preferred embodiment, the E2 used in the methods and
compositions of the present invention comprises an amino acid
sequence or nucleic acid sequence of a sequence corresponding to an
Genbank data base accession number in the following list: AC37534,
P49427, CAA82525, AAA58466, AAC41750, P51669, AAA91460, AAA91461,
CAA63538, AAC50633, P27924, AAB36017, Q16763, AAB86433, AAC26141,
CAA04156, BAAI 1675, Q16781, NP.sub.--003333, BAB18652, AAH00468,
CAC16955, CAB76865, CAB76864, NP.sub.--05536, 000762, XP 009804,
XP.sub.--009488, XP.sub.--006823, XP.sub.--006343, XP.sub.--005934,
XP.sub.--002869, XP.sub.--003400XP.sub.--009365, XP.sub.--010361,
XP.sub.--004699, XP.sub.--004019, 014933, P27924, P50550, P52485,
P51668, P51669, P49459, P37286, P23567, P56554, and CAB45853, each
of which is incorporated herein by reference. Particularly
preferred are sequences corresponding to Genbank data base
accession numbers NP00333 1, NP003330, NP003329, P49427, AAB53362,
NP008950, XP009488and AAC41750, also incorporated by reference. The
skilled artisan will appreciate that many different E2 proteins and
isozymes are known in the filed and may be used in the present
invention, provided that the E2 has ubiquitin conjugating activity.
Also specifically included within the term "E2" are variants of E2,
which can be made as described herein.
[0276] In a preferred embodiment, E2 is one of Ubc5 (Ubch5,
preferably Ubch5c), Ubc3 (Ubch3), Ubc4 (Ubch4) and UbcX (Ubc10,
Ubch10). In a preferred embodiment, E2 is Ubch5c. In a preferred
embodiment, E2 has the amino acid sequence shown in FIG. 9B or is
encoded by a nucleic acid consisting essentially of the sequence
shown in FIG. 9A.
[0277] The E2 used in the methods and compositions of the present
invention, comprises a nucleic acid sequence of a sequence
corresponding to Genbank data base accession number L2205, Z29328,
M92670, L40146, U39317, U39318, X92962, U58522, S81003, AF031141,
AF075599, AJ000519, XM009488, NM007019, U73379, L40146, or D83004,
each of which is incorporated herein by reference. As described
above, variants of these and other E2 encoding nucleic acids may
also be used to make variant E2 proteins.
[0278] In a preferred embodiment, the nucleic acid used to make E2
comprises the sequence shown in FIG. 9A.
[0279] In a preferred embodiment, E2 has a tag, as defined above,
with the complex being referred to herein as "tag-E2". Preferred E2
tags include, but are not limited to, labels, partners of binding
pairs and substrate binding elements. In a most preferred
embodiment, the tag is a His-tag or GST-tag.
[0280] In some embodiments, the methods of the present invention
comprise the use of a ubiquitin ligating agent. As used herein
"ubiquitin ligating agent" refers to a ubiquitin agent, preferably
a protein, capable of transferring or attaching a ubiquitin moiety
to a target molecule. In some cases, the ubiquitin agent is capable
of transferring or attaching ubiquitin moiety to itself or another
ubiquitin ligating agent. In a preferred embodiment, the ubiquitin
ligating agent is an E3.
[0281] As used herein "E3" refers to a ubiquitin ligating agent
comprising one or more subunits, preferably polypeptides,
associated with the activity of E3 as a ubiquitin ligating agent
(i.e., associated with the ligation or attachment of ubiquitin
moiety to a target protein, and in some cases, to itself or another
E3). In a preferred embodiment, E3 is a member of the HECT domain
E3 ligating agents. In another preferred embodiment, E3 is a member
of the RING finger domain E3 ligating agents. In a preferred
embodiment, E3 comprises a ring finger subunit and a Cullin
subunit. Examples of RING finger polypeptides suitable for use in
the methods and compositions of the present invention include, but
are not limited to, ROC1, ROC2 and APC11. Examples of Cullin
polypeptides suitable for use in the methods and compositions of
the present invention include, but are not limited to, CUL1, CUL2,
CUL3, CUL4A, CUL4B, CUL5 and APC2. In another preferred embodiment,
the E3 is mdm2.
[0282] In the methods and compositions of the present invention,
the ubiquitin ligating agent comprises an amino acid sequence or a
nucleic acid sequence of a sequence corresponding to an accession
number in the Genbank data base, European Molecular Biology
Laboratories (EMBL) data base, or ENSEMBL data base (a joint
project of the European Molecular Biology Laboratories and the
Sanger Institute) listed in Table 3 below and incorporated herein
by reference. The accession numbers from the Genbank data base can
be found as stated above. The accession numbers from the EMBL data
base are found at www.embl-heidelberg.de. The accession numbers
from the ENSEMBL data base are found at www.ensembl.or.
TABLE-US-00004 TABLE 3 Accession Accession Accession Accession
Accession Accession Accession Accession Accession No. No. No. No.
No. No. No. No. No. AAD15547 AAH22038 O75485 Q96BD4 Q96K03 Q96T88
Q9BYV6 Q9H073 Q9H920 AAF42995 AAH22403 O75592 Q96BD Q96K19 Q99496
Q9BZX6 Q9H083 Q9H9B0 AAF91315 AAH22510 O75598 5Q96BE6 Q96K21 Q99579
Q9BZX7 Q9H0A6 Q9H9B5 AAF97687 AAL30771 O75615 Q96BH1 Q96KD9 Q99675
Q9BZX8 Q9H0M8 Q9H9P5 AAG50176 AAL31641 O75866 Q96BL1 Q96KL0 Q99942
Q9BZX9 Q9H0V6 Q9H9T2 AAG50180 AAL36460 O76050 Q96BM5 Q96KM9 Q9BPW2
Q9BZY0 Q9H0X6 Q9H9V4 AAG53500 AAL40179 O76064 Q96BQ3 Q96LD4 Q9BQ47
Q9BZY1 Q9H270 Q9H9Y7 AAG53509 AAL40180 O94896 Q96BS3 Q96M70 Q9BQV0
Q9BZY2 Q9H2A8 Q9HA51 AAH00832 AAL76101 O94941 Q96BX2 Q96MJ7 Q9BRZ2
Q9BZY3 Q9H2S3 Q9HAC1 AAH02922 CAC81706 O94972 Q96C24 Q96MT1 Q9BS04
Q9BZY4 Q9H2S4 Q9HAM2 AAH04978 CAC85986 O95159 Q96CA5 Q96MX5 Q9BSE9
Q9BZY5 Q9H2S5 Q9HAP7 AAH05375 CAD19102 O95247 Q96CC2 Q96MZ7 Q9BSL8
Q9BZY6 Q9H348 Q9HBD2 AAH13580 O00237 O95277 Q96D24 Q96NI4 Q9BSM1
Q9BZY8 Q9H463 Q9HCL8 AAH15738 O00463 O95604 Q96D38 Q96NS4 Q9BSV9
Q9BZY9 Q9H4C2 Q9HCR0 AAH16174 O00635 O95627 Q96D59 Q96NT2 KIAA066
Q9C017 Q9H4C3 Q9HCR1 AAH16924 O14616 O95628 Q96DB4 Q96P09 Q9BTC5
Q9C018 Q9H4C4 Q9HCR2 AAH17370 O14686 O96028 Q96DV2 Q96PF7 Q9BTD9
Q9C019 Q9H4C5 Q9HCS6 AAH17585 O15057 Q14527 Q96DV3 Q96PH3 Q9BU73
Q9C021 Q9H4J2 Q9NPN4 AAH17592 O15262 Q14536 Q96DX4 Q96PK3 Q9BUW4
Q9C025 Q9H5E4 Q9NPP8 AAH17707 O15344 Q14848 Q96DY5 Q96PM5 Q9BUZ4
Q9C026 Q9H5F1 Q9NPQ1 AAH18104 O43164 Q15156 Q96EL5 Q96PR5 Q9BV68
Q9C027 Q9H5K0 Q9NQ86 AAH18107 O43255 Q15290 Q96EP1 Q96PU4 Q9BVG3
Q9C029 Q9H5L8 Q9NQP8 AAH18198 O43269 Q15521 Q96EP8 Q96PX1 Q9BW41
Q9C030 Q9H5P2 Q9NR13 AAH18337 O43270 Q15959 Q96EQ8 Q96QB5 Q9BW90
Q9C031 Q9H5S6 Q9NRL2 AAH18647 O43567 Q16030 Q96F06 Q96QB6 Q9BWF2
Q9C032 Q9H647 Q9NRT4 AAH19283 O60272 Q92550 Q96F37 Q96QY9 Q9BWL5
Q9C033 Q9H6D9 Q9NRT6 AAH19355 O60291 Q92897 Q96F67 Q96RF3 Q9BWP7
Q9C034 Q9H6S6 Q9NS55 AAH20556 O60372 Q969K3 Q96GF1 Q96RF8 Q9BX37
Q9C035 Q9H6W8 Q9NS56 AAH20964 O60630 Q969Q1 Q96GT5 Q96RW5 Q9BXI1
Q9C036 Q9H6Y7 Q9NS56 AAH20984 O75150 Q969V5 Q96H69 Q96SH4 Q9BY78
Q9C037 Q9H748 Q9NS91 AAH20994 KIAA0661 Q96A37 Q96IB6 Q96SJ1 Q9BYE7
Q9C038 Q9H874 Q9NSR1 AAH21258 O75162 Q96A61 Q96ID9 Q96SL3 Q9BYV2
Q9C039 Q9H890 Q9NSX7 AAH21570 O75188 Q96AK4 Q96J90 Q96SR5 Q9BYV3
Q9C040 Q9H8K2 Q9NTX6 AAH21571 O75341 Q96AX9 Q96JD3 Q96T06 Q9BYV4
Q9C0B0 Q9H8V9 Q9NTX7 AAH21925 O75382 Q96BD3 Q96JL5 Q96T18 Q9BYV5
Q9C0G7 Q9H8W5 Q9NU68 Accession Accession Accession Accession
Accession Accession Accession Accession No. No. No. No. No. No. No.
No. Q9NUH2 Q9NZS9 Q9UIG0 9UQPQ7 O15151 Q9BXT8 O94822 Q13263 Q9NUR4
Q9NZT8 Q9UIG1 Q9UPR2 O15541 Q9BYM8 O95376 Q13489 Q9NUW5 Q9P0J9
Q9UJ97 Q9UQ11 O60858 Q9BZR9 P15918 Q13490 Q9NVD5 Q9P0P0 Q9UJJ8
Q9Y225 O75678 Q9H000 P19474 Q13702 Q9NVP6 Q9P115 Q9UJL3 Q9Y254
P14373 Q9NS80 P22681 Q14839 Q9NW38 Q9P1Y6 Q9UJR9 Q9Y2E6 P28328
Q9NV58 P29590 Q15326 Q9NWD2 Q9P200 Q9UJV3 Q9Y2N1 P35226 Q9UDY6
P35227 Q92785 Q9NWX1 Q9P2G1 Q9UKI6 Q9Y3C5 P46100 Q9UHC7 P36406
Q99728 Q9NX39 Q9P2L3 Q9UKV5 Q9Y3V1 P51948 Q9ULX5 P38398 Q9HCM9
Q9NXC0 Q9P2M3 Q9ULK6 Q9Y3V3 Q12899 Q9UMT8 P49754 Q9NVW2 Q9NXD0
Q9UBF6 Q9ULT6 Q9Y4I0 Q12933 Q9Y4X5 P50876 Q9NYG5 Q9NXI6 Q9UDN7
Q9ULW4 Q9Y4K3 Q12986 Q9Y508 P53804 Q9ULV8 Q9NZI5 Q9UEK4 Q9UMH1
Q9Y4L5 Q13049 O00623 P98170 Q9UPN9 Q9NZB4 Q9UF32 Q9UMQ2 Q9Y577
Q13064 O15164 Q06587 Q9Y252 Q9NZE3 Q9UHE7 Q9UNR9 Q9Y5M7 Q13114
O60683 Q12873 Q9NZE9 Q9UHW2 Q9UPQ2 Q9Y6E4 Q13434 O75677 Q13191
Q9NZN6 Q9UID0 Q9UPQ4 Q9Y6U1 Q14258 O75679 Q13233 Ringfinger domain
Hect domain proteins proteins (Embl data base) (GenBank data base)
AAH19105 AAF50078 AAH19345 AAH21525 AAH21144 AAH02582 O00307
NP_055486 O00308 BAB13352 O14996 NP_492389 O15029 XP_048020 O15033
BAB28637 O15036 O43165 BAA20780 O43584 T39585 O94970 NP_060239
O95071 T39007 O95714 BAA92539 Q15386 CAC42101 Q15751 XP_083009
Q96BP4 AAF79338 Q96CZ2 NP_060382 Q96DE7 AAH00621 Q96F34 AAH09271
Q96F66 AAC62434 Q96GR7 AAF51314 Q96J02 T21546 Q96PU5 NP_188346
Q9BUI0 AAF49328 Q9BUI6 XP_082286 Q9BVR2 NP_035020 Q9BXZ4 NP_501120
Q9BY75 NP_055636 Q9H0M0 NP_003913 Q9H2G0 BAB02722 Q9H2W4 NP_497697
Q9H451 NP_490865 Q9H783 T14761 Q9H9E9 AAC83345 Q9HCC7 S70642 Q9HCH9
AAG53076 Q9NPL3 CAA03915 Q9NPS9 XP_085770 Q9NT88 CAC09387 Q9NWS4
NP_055421 Q9NXC0 NP_523779 Q9NZS4 XP_038999 Q9P0A9 AAD51453 Q9P2L3
AAB49301 Q9P2M6 T49799 Q9P2P5 AAG16783 Q9UDU3 NP_195572 Q9UFZ7
AAH21470 Q9UII4 NP_078878 Q9ULT8 Q9Y4D8 NP_073576 Q9HAU4 XP_028151
Q9HCE7 P46934 P46934 BAB28001 Q05086 NP_004658 Q14669 P46935 Q15034
NP_524296 T14346 NP_008944 S66562 NP_008945 NP_032421 AAK33088
AAL39551 NP_175982 AAF68076 AAF68077 AAH11571 XP_052430 AAF68079
AAH04712 T38951 BAA23711 BAB13451 AAF46512 NP_000453 AAL29143
AAL27259 AAF36539 BAA84697 NP_499392 AAF68080 I83196 NP_057407
AAF28950 XP_052223 AAF68082 AAF68083 T41750 AAH11658 NP_114087
Q05086 T49744 AAC51324 BAA92571 BAB03733 NP_500283 AAK28419
NP_446441 BAA86445 NP_190877 Q9HCE7 AAF50332 AAH09527 NP_490750
XP_003492 T37736 AAF47474 AAD34642 BAB23311 T40821 NP_192994
AAF57824 NP_080106 T37964 NP_035798 BAB14280 XP_084941 AAH15380
XP_080159 AAF08298 BAA19217 T01491 CAB92704 CAB09785 NP_177189
XP_030186 AAF61856 XP_057408 Q9PUN2 CAB99103 NP_195908 AAH11391
NP_012570 AAF52899 AAF88143 AAF68614 BAA20771 BAB13419 NP_011051
AAH13645 Q9CUN6 XP_046129 A38920 AAB47756 Q92462 NP_113671 CAA57291
XP_087357 AAC41731 BAB69424 T37900 T14317 P51593 AAH04085 BAA21482
NP_012915 AAF48495 XP_045232 AAF50913 T00390 NP_476753 T46412
XP_045095 NP_113584 NP_495842 AAC04845 XP_030175 1C4Z AAL13848
XP_004990 BAB29387
BAA92558 AAG45422 AAF36454 AAF36455 AAK14420 BAA74919 BAB24805
BAB30794 NP_004229 O08759 AAH19345 NP_011374 NP_056092 AAH21144
NP_056986 B38919 T38617 AAH06848 NP_490834 NP_010745 CAB95249
Ringfinger domain proteins (Ensembl data base) ENSP00000259945
ENSP00000254436 ENSP00000066988 ENSP00000275736 ENSP00000275735
ENSP00000203439 ENSP00000013772 ENSP00000225283 ENSP00000246907
ENSP00000225285 ENSP00000225286 ENSP00000230239 ENSP00000286909
ENSP00000286910 ENSP00000280609 ENSP00000263651 ENSP00000261395
ENSP00000277584 ENSP00000224833 ENSP00000254604 ENSP00000240395
ENSP00000240318 ENSP00000286945 ENSP00000281874 ENSP00000240802
ENSP00000267825 ENSP00000254586 ENSP00000293123 ENSP00000285805
ENSP00000257633 ENSP00000266119 ENSP00000233630 ENSP00000264033
ENSP00000275619 ENSP00000275637 ENSP00000280063 ENSP00000276333
ENSP00000263651 ENSP00000278302 ENSP00000264122 ENSP00000284559
ENSP00000266252 ENSP00000278350 ENSP00000259847 ENSP00000274855
ENSP00000259930 ENSP00000217214 ENSP00000283330 ENSP00000263535
ENSP00000291416 ENSP00000291414 ENSP00000253769 ENSP00000274786
ENSP00000289896 ENSP00000289898 ENSP00000265771 ENSP00000229866
ENSP00000286475 ENSP00000256257 ENSP00000253554 ENSP00000259654
ENSP00000280266 ENSP00000259941 ENSP00000259940 ENSP00000270086
ENSP00000289140 ENSP00000225507 ENSP00000261593 ENSP00000257847
ENSP00000262881 ENSP00000222033 ENSP00000290048 ENSP00000274327
ENSP00000282135 ENSP00000280460 ENSP00000280461 ENSP00000217740
ENSP00000227588 ENSP00000259944 ENSP00000279757 ENSP00000274773
ENSP00000276311 ENSP00000166144 ENSP00000292363 ENSP00000264616
ENSP00000272390 ENSP00000272396 ENSP00000264767 ENSP00000255499
ENSP00000264614 ENSP00000262482 ENSP00000261481 ENSP00000261658
ENSP00000288774 ENSP00000261675 ENSP00000266880 ENSP00000243674
ENSP00000284638 ENSP00000247668 ENSP00000285317 ENSP00000278480
ENSP00000240159 ENSP00000294256 ENSP00000279766 ENSP00000288204
ENSP00000269439 ENSP00000268061 ENSP00000268058 ENSP00000268059
ENSP00000268060 ENSP00000261825 ENSP00000288587 ENSP00000275693
ENSP00000244061 ENSP00000272598 ENSP00000289818 ENSP00000238349
ENSP00000280266 ENSP00000242855 ENSP00000276688 ENSP00000280268
ENSP00000274811 ENSP00000268363 ENSP00000274828 ENSP00000235150
ENSP00000211960 ENSP00000262843 ENSP00000266952 ENSP00000288300
ENSP00000291134 ENSP00000261947 ENSP00000288715 ENSP00000222704
ENSP00000293938 ENSP00000266030 ENSP00000287335 ENSP00000256649
ENSP00000249240 ENSP00000253953 ENSP00000267073 ENSP00000271813
ENSP00000248492 ENSP00000265981 ENSP00000270280 ENSP00000270279
ENSP00000254959 ENSP00000255977 ENSP00000283460 ENSP00000262370
ENSP00000253024 ENSP00000282369 ENSP00000253571 ENSP00000288913
ENSP00000288918 ENSP00000276573 ENSP00000237308 ENSP00000238203
ENSP00000227451 ENSP00000244360 ENSP00000244359 ENSP00000281105
ENSP00000268907 ENSP00000292962 ENSP00000280804 ENSP00000287546
ENSP00000248980 ENSP00000287559 ENSP00000264926 ENSP00000261737
ENSP00000170447 ENSP00000270944 ENSP00000289726 ENSP00000230099
ENSP00000237455 ENSP00000263550 ENSP00000264198 ENSP00000263464
ENSP00000259604 ENSP00000265673 ENSP00000248983 ENSP00000269391
ENSP00000249007 ENSP00000242719 ENSP00000217169 ENSP00000253642
ENSP00000227758 ENSP00000291190 ENSP00000261537 ENSP00000291733
ENSP00000274782 ENSP00000271287 ENSP00000261445 ENSP00000245836
ENSP00000267291 ENSP00000292195 ENSP00000216420 ENSP00000261464
ENSP00000260076 ENSP00000284244 ENSP00000292545 ENSP00000242669
ENSP00000288848 ENSP00000261809 ENSP00000262952 ENSP00000245937
ENSP00000275970 ENSP00000238647 ENSP00000268850 ENSP00000291963
ENSP00000286349 ENSP00000257600 ENSP00000281843 ENSP00000261245
ENSP00000245888 ENSP00000222704 ENSP00000245419 ENSP00000272023
ENSP00000274068 ENSP00000275233 ENSP00000265742 ENSP00000269475
ENSP00000265290 ENSP00000222597 ENSP00000292307
ENSP00000265267 ENSP00000263220 ENSP00000216225 ENSP00000293538
ENSP00000229766 ENSP00000242239 ENSP00000274616 ENSP00000286773
ENSP00000273480 ENSP00000217173 ENSP00000290337 ENSP00000281930
ENSP00000257575 ENSP00000287212 ENSP00000290788 ENSP00000282455
ENSP00000254247 ENSP00000290649 ENSP00000274542 ENSP00000224944
ENSP00000281418 ENSP00000289883 ENSP00000255325 ENSP00000255326
ENSP00000292543 ENSP00000277534 ENSP00000260947 ENSP00000278455
ENSP00000278454 ENSP00000274694 ENSP00000217740 ENSP00000262952
ENSP00000268154 ENSP00000265756 ENSP00000277490 ENSP00000266625
ENSP00000266624 ENSP00000258147 ENSP00000258148 ENSP00000258149
ENSP00000264512 ENSP00000261212 ENSP00000262642 ENSP00000264359
ENSP00000217537 ENSP00000264777 ENSP00000287880 ENSP00000272674
ENSP00000272662 ENSP00000293245 ENSP00000283875 ENSP00000262642
ENSP00000259865 ENSP00000217908 ENSP00000255004 ENSP00000275184
ENSP00000275183 ENSP00000200457 ENSP00000261537 ENSP00000257100
ENSP00000286349 ENSP00000252445 ENSP00000294213 ENSP00000259939
ENSP00000236892 ENSP00000238001 ENSP00000274657 ENSP00000274799
[0283] Sequences encoding a ubiquitin activating agent may also be
used to make variants thereof that are suitable for use in the
methods and compositions of the present invention. The ubiquitin
ligating agents and variants suitable for use in the methods and
compositions of the present invention may be made as described
herein.
[0284] In a preferred embodiment, RING finger subunits include, but
are not limited to, polypeptides having an amino acid sequence
corresponding to Genbank accession numbers AAD30147, AAD30146, or
6320196, incorporated herein by reference. In a more preferred
embodiment, the ring finger protein has a sequence selected from
the group consisting of that shown in FIG. 10, FIG. 11 and FIG.
12B.
[0285] In a preferred embodiment, Cullins include, but are not
limited to, polypeptides having an amino acid sequence
corresponding to Genbank accession number 4503161, AAC50544,
AAC36681, 4503163, AAC51190, AAD23581, 4503165, AAC36304, AAC36682,
AAD45191, AAC50548, Q13620, 4503167, or AAF05751, each of which is
incorporated herein by reference. In addition, in the context of
the invention, each of the RING finger proteins and Cullins
encompass variants of the known or listed sequences, as described
herein.
[0286] In a preferred embodiment, the Cullin has a sequence as
shown in FIG. 13B or 14B.
[0287] These E3 ligating agents and variants may be made as
described herein. In a preferred embodiment, nucleic acids used to
make the RING finger proteins include, but are not limited to,
those having the nucleic acid sequences disclosed in Genbank
accession numbers AF142059, AF142060 and nucleic acids 433493 to
433990 of NC 001136. In a preferred embodiment, Cullins are made
from nucleic acids including, but not limited to, those having
nucleic acid sequences disclosed in Genbank accession numbers NM
003592, U58087, AF062536, AF126404, NM 003591, U83410, NM 003590,
AB014517, AF062537, AF064087, AF077188, U58091, NM 003478, X81882
and AF191337, each of which is incorporated herein by reference. As
described above, variants of these sequences are also encompassed
by the invention.
[0288] In a preferred embodiment, nucleic acid used to produce ROC2
comprises the sequence depicted in FIG. 12A. In a preferred
embodiment, nucleic acid used to produce CUL5 comprises the
sequence depicted in FIG. 13A. In a preferred embodiment, nucleic
acid used to produce APC2 comprises the sequence depicted in FIG.
14A.
[0289] In a preferred embodiment, E3 comprises the RING finger
protein/Cullin combination APC11/APC2. In another preferred
embodiment, E3 comprises the RING finger protein/Cullin combination
ROC1/CUL1. In yet preferred embodiment, E3 comprises the RING
finger protein/Cullin combination ROC1/CUL2. In still another
preferred embodiment, E3 comprises the RING finger protein/Cullin
combination ROC2/CUL5. However, the skilled artisan will appreciate
that any combination of E3 components may be produced and used in
the invention described herein.
[0290] In an alternate embodiment, E3 comprises the ligase
E3-alpha, E3A (E6-AP), HERC2, SMURF1, TRAF6, Mdm2, Cbl, Sina/Siah,
Itchy, IAP or NEDD-4. In this embodiment, the ligase has the amino
acid sequence of that disclosed in Genbank accession number
AAC39845, Q05086, CAA66655, CAA66654, CAA66656, AAD08657,
NP.sub.--002383, XP.sub.--006284, AAC51970, XP.sub.--013050,
BAB39389, Q00987, AAF08298 or P46934, each of which is incorporated
herein by reference. As above, variants are also encompassed by the
invention. Nucleic acids for making E3 for this embodiment include,
but are not limited to, those having the sequences disclosed in
Genbank accession numbers AF061556, XM006284, U76247, XM013050,
X898032, X98031, X98033, AF071172, Z12020, AB056663, AF199364 and
D42055 and variants thereof.
[0291] E3 may also comprise other components, such as SKP 1 and
F-box proteins. The amino acid and nucleic acid sequences for SKP1
correspond to GENBANK accession numbers AAC50241 and U33760,
respectively. Many F-box proteins are known in the art and their
amino acid and nucleic acid sequences are readily obtained by the
skilled artisan from various published sources.
[0292] In a preferred embodiment, the E3 components are produced
recombinantly, as described herein. In a preferred embodiment, the
E3 components are co-expressed in the same host cell. Co-expression
may be achieved by transforming the cell with a vector comprising
nucleic acids encoding two or more of the E3 components, or by
transforming the host cell with separate vectors, each comprising a
single component of the desired E3 protein complex. In a preferred
embodiment, the RING finger protein and Cullin are expressed in a
single host transfected with two vectors, each comprising nucleic
acid encoding one or the other polypeptide, as described in further
detail in the Examples.
[0293] In a preferred embodiment, E3 has a tag, and this complex is
referred to herein as "tag-E3". Preferably, the tag is attached to
only one component of the E3. Preferred E3 tags include, but are
not limited to, labels, partners of binding pairs and substrate
binding elements. More preferably, the tag is a surface substrate
binding molecule. Most preferably, the tag is a His-tag or
GST-tag.
[0294] Ubiquitin moieties, ubiquitin agents, and target molecules
suitable for use in the methods and compositions of the present
invention can be cloned and expressed as described below. Thus,
probe or degenerate polymerase chain reaction (PCR) primer
sequences may be used to find other related or variant ubiquitin
moieties, ubiquitin agents, and target proteins from humans or
other organisms. As will be appreciated by those in the art,
particularly useful probe and/or PCR primer sequences include the
unique areas of a nucleic acid sequence. As is generally known in
the art, preferred PCR primers are from about 15 to about 35
nucleotides in length, with from about 20 to about 30 being
preferred, and may contain inosine as needed. The conditions for
the PCR reaction are well known in the art. It is therefore also
understood that provided along with the sequences in the sequences
cited herein are portions of those sequences, wherein unique
portions of 15 nucleotides or more are particularly preferred. The
skilled artisan can routinely synthesize or cut a nucleotide
sequence to the desired length.
[0295] Once isolated from its natural source, e.g., contained
within a plasmid or other vector or excised therefrom as a linear
nucleic acid segment, the recombinant nucleic acid can be
further-used as a probe to identify and isolate other nucleic
acids. It can also be used as a "precursor" nucleic acid to make
modified or variant nucleic acids and proteins.
[0296] Using the nucleic acids of the present invention which
encode a protein, a variety of expression vectors are made. The
expression vectors may be either self-replicating extrachromosomal
vectors or vectors which integrate into a host genome. Generally,
these expression vectors include transcriptional and translational
regulatory nucleic acid operably linked to the nucleic acid
encoding the protein. The term "control sequences" refers to DNA
sequences necessary for the expression of an operably linked coding
sequence in a particular host organism. The control sequences that
are suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0297] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. As another example, operably linked refers
to DNA sequences linked so as to be contiguous, and, in the case of
a secretory leader, contiguous and in reading fram. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adapters or linkers are used
in accordance with conventional practice. The transcriptional and
translational regulatory nucleic acid will generally be appropriate
to the host cell used to express the protein; for example,
transcriptional and translational regulatory nucleic acid sequences
from Bacillus are preferably used to express the protein in
Bacillus. Numerous types of appropriate expression vectors, and
suitable regulatory sequences are known in the art for a variety of
host cells.
[0298] In general, the transcriptional and translational regulatory
sequences may include, but are not limited to, promoter sequences,
ribosomal binding sites, transcriptional start and stop sequences,
translational start and stop sequences, and enhancer or activator
sequences. In a preferred embodiment, the regulatory sequences
include a promoter and transcriptional start and stop
sequences.
[0299] Promoter sequences encode either constitutive or inducible
promoters. The promoters may be either naturally occurring
promoters or hybrid promoters. Hybrid promoters, which combine
elements of more than one promoter, are also known in the art, and
are useful in the present invention.
[0300] In addition, the expression vector may comprise additional
elements. For example, the expression vector may have two
replication systems, thus allowing it to be maintained in two
organisms, for example in mammalian or insect cells for expression
and in a prokaryotic host for cloning and amplification.
Furthermore, for integrating expression vectors, the expression
vector contains at least one sequence homologous to the host cell
genome, and preferably two homologous sequences which flank the
expression construct. The integrating vector may be directed to a
specific locus in the host cell by selecting the appropriate
homologous sequence for inclusion in the vector. Constructs for
integrating vectors are well known in the art.
[0301] In addition, in a preferred embodiment, the expression
vector contains a selectable marker gene to allow the selection of
transformed host cells. Selection genes are well known in the art
and will vary with the host cell used.
[0302] A preferred expression vector system is a retroviral vector
system such as is generally described in PCT/US97/01019 and
PCT/US97/01048, both of which are hereby expressly incorporated by
reference.
[0303] Proteins of the present invention are produced by culturing
a host cell transformed with an expression vector containing
nucleic acid encoding the protein, under the appropriate conditions
to induce or cause expression of the protein. The conditions
appropriate for protein expression will vary with the choice of the
expression vector and the host cell, and will be easily ascertained
by one skilled in the art through routine experimentation. For
example, the use of constitutive promoters in the expression vector
will require optimizing the growth and proliferation of the host
cell, while the use of an inducible promoter requires the
appropriate growth conditions for induction. In addition, in some
embodiments, the timing of the harvest is important. For example,
the baculoviral systems used in insect cell expression are lytic
viruses, and thus harvest time selection can be crucial for product
yield.
[0304] Appropriate host cells include yeast, bacteria,
archaebacteria, fungi, and insect and animal cells, including
mammalian cells. Of particular interest are Drosophila melanogaster
cells, Pichia pastoris and P. methanolica, Saccharomyces cerevisiae
and other yeasts, E. coli, Bacillus subtilis, SF9 cells, SF21
cells, C129 cells, Saos-2 cells, Hi-5 cells, 293 cells, Neurospora,
BHK, CHO, COS, and HeLa cells. Of greatest interest are Pichia
pastoris and P. methanolica, E. coli, SF9 cells, SF21 cells and
Hi-5 cells.
[0305] In a preferred embodiment, the proteins are expressed in
mammalian cells. Mammalian expression systems are also known in the
art, and include retroviral systems. A mammalian promoter is any
DNA sequence capable of binding mammalian RNA polymerase and
initiating the downstream (3') transcription of a coding sequence
for a protein into mRNA. A promoter will have a transcription
initiating region, which is usually placed proximal to the 5' end
of the coding sequence, and a TATA box, using a located 25-30 base
pairs upstream of the transcription initiation site. The TATA box
is thought to direct RNA polymerase II to begin RNA synthesis at
the correct site. A mammalian promoter will also contain an
upstream promoter element (enhancer element), typically located
within 100 to 200 base pairs upstream of the TATA box. An upstream
promoter element determines the rate at which transcription is
initiated and can act in either orientation. Of particular use as
mammalian promoters are the promoters from mammalian viral genes,
since the viral genes are often highly expressed and have a broad
host range. Examples include the SV40 early promoter, mouse mammary
tumor virus LTR promoter, adenovirus major late promoter, herpes
simplex virus promoter, and the CMV promoter.
[0306] Typically, transcription termination and polyadenylation
sequences recognized by mammalian cells are regulatory regions
located 3' to the translation stop codon and thus, together with
the promoter elements, flank the coding sequence. The 3' terminus
of the mature mRNA is formed by site-specific post-translational
cleavage and polyadenylation. Examples of transcription terminator
and polyadenylation signals include those derived form SV40.
[0307] The methods of introducing exogenous nucleic acid into
mammalian hosts, as well as other hosts, is well known in the art,
and will vary with the host cell used. Techniques include
dextran-mediated transfection, calcium phosphate precipitation,
polybrene mediated transfection, protoplast fusion,
electroporation, viral infection, encapsulation of the
polynucleotide(s) in liposomes, and direct microinjection of the
DNA into nuclei.
[0308] In a preferred embodiment, proteins are expressed in
bacterial systems. Bacterial expression systems are well known in
the art.
[0309] A suitable bacterial promoter is any nucleic acid sequence
capable of binding bacterial RNA polymerase and initiating the
downstream (3') transcription of the coding sequence of a protein
into mRNA. A bacterial promoter has a transcription initiation
region which is usually placed proximal to the 5' end of the coding
sequence. This transcription initiation region typically includes
an RNA polymerase binding site and a transcription initiation site.
Sequences encoding metabolic pathway enzymes provide particularly
useful promoter sequences. Examples include promoter sequences
derived from sugar metabolizing enzymes, such as galactose, lactose
and maltose, and sequences derived from biosynthetic enzymes such
as tryptophan. Promoters from bacteriophage may also be used and
are known in the art. In addition, synthetic promoters and hybrid
promoters are also useful; for example, the tac promoter is a
hybrid of the trp and lac promoter sequences. Furthermore, a
bacterial promoter can include naturally occurring promoters of
non-bacterial origin that have the ability to bind bacterial RNA
polymerase and initiate transcription.
[0310] In addition to a functioning promoter sequence, an efficient
ribosome binding site is desirable. In E. coli, the ribosome
binding site is called the Shine-Delgarno (SD) sequence and
includes an initiation codon and a sequence 3-9 nucleotides in
length located 3-11 nucleotides upstream of the initiation
codon.
[0311] The expression vector may also include a signal peptide
sequence that provides for secretion of the protein in bacteria.
The signal sequence typically encodes a signal peptide comprised of
hydrophobic amino acids which direct the secretion of the protein
from the cell, as is well known in the art. The protein is either
secreted into the growth media (gram-positive bacteria) or into the
periplasmic space, located between the inner and outer membrane of
the cell (gram-negative bacteria).
[0312] The bacterial expression vector may also include a
selectable marker gene to allow for the selection of bacterial
strains that have been transformed. Suitable selection genes
include genes which render the bacteria resistant to drugs such as
ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and
tetracycline. Selectable markers also include biosynthetic genes,
such as those in the histidine, tryptophan and leucine biosynthetic
pathways.
[0313] These components are assembled into expression vectors.
Expression vectors for bacteria are well known in the art, and
include vectors for Bacillus subtilis, E. coli, Streptococcus
cremoris, and Streptococcus lividans, among others.
[0314] The bacterial expression vectors are transformed into
bacterial host cells using techniques well known in the art, such
as calcium chloride treatment, electroporation, and others.
[0315] In one embodiment, proteins are produced in insect cells.
Expression vectors for the transformation of insect cells, and in
particular, baculovirus-based expression vectors, are well known in
the art.
[0316] In a preferred embodiment, proteins are produced in yeast
cells. Yeast expression systems are well known in the art, and
include expression vectors for Saccharomyces cerevisiae, Candida
albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces
fragilis and K. lactis, Pichia guillerimondii P. methanolica and P.
pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
Preferred promoter sequences for expression in yeast include the
inducible GAL1,10 promoter, the promoters from alcohol
dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,
glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,
phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase,
and the acid phosphatase gene. Yeast selectable markers include
ADE2, HIS4, LEU2, TRP1, and ALG7, which confers resistance to
tunicamycin; the neomycin phosphotransferase gene, which confers
resistance to G418; and the CUP1 gene, which allows yeast to grow
in the presence of copper ions.
[0317] The protein may also be made as a fusion protein, using
techniques well known in the art. Thus, for example, the protein
may be made as a fusion protein to increase expression, or for
other reasons. For example, when the protein is a peptide, the
nucleic acid encoding the peptide may be linked to other nucleic
acid for expression purposes. Similarly, proteins of the invention
can be linked to protein labels, such as green fluorescent protein
(GFP), red fluorescent protein (RFP), blue fluorescent protein
(BFP), yellow fluorescent protein (YFP), etc.
[0318] In a preferred embodiment, the protein is purified or
isolated after expression. Proteins may be isolated or purified in
a variety of ways known to those skilled in the art depending on
what other components are present in the sample. Standard
purification methods include electrophoretic, molecular,
immunological and chromatographic techniques, including ion
exchange, hydrophobic, affinity, and reverse-phase HPLC
chromatography, and chromatofocusing. For example, the ubiquitin
moiety protein may be purified using a standard anti-ubiquitin
moiety antibody column. Ultrafiltration and diafiltration
techniques, in conjunction with protein concentration, are also
useful. For general guidance in suitable purification techniques,
see Scopes, R., Protein Purification, Springer-Verlag, N.Y. (1982).
The degree of purification necessary will vary depending on the use
of the protein. In some instances no purification will be
necessary.
[0319] Once made, the compositions find use in a number of
applications, including, but not limited to, assaying for agents
that modulate the activity of a ubiquitin agent. In particular, the
compositions can be used to assay for agents that modulate the
transfer or attachment of ubiquitin moiety to a substrate molecule.
The term "modulate" as used herein with reference to the activity
of a ubiquitin agent refers to the increase or decrease in an
activity of a ubiquitin agent, for example, activating activity,
conjugating activity, ligating activity, and more specifically the
attachment of ubiquitin moiety to a substrate molecule. The term
"attachment" as used herein with reference to the activity of a
ubiquitin agent refers to the binding, transfer, or attachment of a
ubiquitin moiety to a substrate molecule. The skilled artisan will
appreciate that agents that modulate the activity of ubiquitin
agents (or "modulators") may affect enzyme activity, enzyme
interaction with a substrate, interaction between ubiquitin moiety
and the substrate, or a combination of these.
[0320] By "candidate", "candidate agent", "candidate modulator",
"candidate ubiquitination modulator" or grammatical equivalents
herein is meant any candidate molecule, e.g. a protein (which
herein includes a protein, polypeptide, and peptide), small organic
or inorganic molecule, polysaccharide, or polynucleotide which are
to be tested for the ability to modulate the activity of a
ubiquitin agent, and more specifically, for the ability to modulate
the attachment of ubiquitin moiety to a substrate molecule.
Candidate agents encompass numerous chemical classes. In a
preferred embodiment, the candidate agents are small molecules. In
another preferred embodiment, the candidate agents are organic
molecules, particularly small organic molecules, comprising
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and typically include at
least an amine, carbonyl, hydroxyl or carboxyl group, preferably at
least two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more
chemical functional groups.
[0321] Candidate agents are obtained from a wide variety of
sources, as will be appreciated by those in the art, including
libraries of synthetic or natural compounds. As will be appreciated
by those in the art, the present invention provides a rapid and
easy method for screening any library of candidate modulators,
including the wide variety of known combinatorial chemistry-type
libraries.
[0322] In a preferred embodiment, candidate agents are synthetic
compounds. Any number of techniques are available for the random
and directed synthesis of a wide variety of organic compounds and
biomolecules, including expression of randomized oligonucleotides.
See for example WO 94/24314, hereby expressly incorporated by
reference, which discusses methods for generating new compounds,
including random chemistry methods as well as enzymatic methods. As
described in WO 94/24314, one of the advantages of the present
method is that it is not necessary to characterize the candidate
agent prior to the assay. Using the methods of the present
invention, any candidate agents can be screened for the ability to
increase or decease the activity of a ubiquitin agent, or more
specifically for the ability to increase or decrease the attachment
of ubiquitin moiety to a substrate. In addition, as is known in the
art, coding tags using split synthesis reactions may be used to
essentially identify the chemical moieties tested.
[0323] Alternatively, a preferred embodiment utilizes libraries of
natural compounds, as candidate agents, in the form of bacterial,
fungal, plant and animal extracts that are available or readily
produced.
[0324] Additionally, natural or synthetically produced libraries
and compounds are readily modified through conventional chemical,
physical and biochemical means. Known pharmacological agents may be
subjected to directed or random chemical modifications, including
enzymatic modifications, to produce structural analogs.
[0325] In a preferred embodiment, candidate agents include
proteins, nucleic acids, and chemical moieties.
[0326] In a preferred embodiment, the candidate agents are
proteins, as defined above. In a preferred embodiment, the
candidate agents are naturally occurring proteins or fragments of
naturally occurring proteins. Thus, for example, cellular extracts
containing proteins, or random or directed digests of proteinaceous
cellular extracts, may be tested, as is more fully described below.
In this way libraries of prokaryotic and eukaryotic proteins may be
made for screening against any number of candidate agents.
Particularly preferred in this embodiment are libraries of
bacterial, fungal, viral, and mammalian proteins, with the latter
being preferred, and human proteins being especially preferred.
[0327] In a preferred embodiment, the candidate agents are peptides
of from about 2 to about 50 amino acids, with from about 5 to about
30 amino acids being preferred, and from about 8 to about 20 being
particularly preferred. The peptides may be digests of naturally
occurring proteins as is outlined above, random peptides, or
"biased" random peptides. By "randomized" or grammatical
equivalents herein is meant that each nucleic acid and peptide
consists of essentially random nucleotides and amino acids,
respectively. Since generally these random peptides (or nucleic
acids, discussed below) are chemically synthesized, they may
incorporate any nucleotide or amino acid at any position. The
synthetic process can be designed to generate randomized proteins
or nucleic acids, to allow the formation of all or most of the
possible combinations over the length of the sequence, thus forming
a library of randomized candidate bioactive proteinaceous
agents.
[0328] The library should provide a sufficiently structurally
diverse population of randomized agents to effect a
probabilistically sufficient range of diversity to allow
interaction with a particular ubiquitin ligating agent enzyme.
Accordingly, an interaction library must be large enough so that at
least one of its members will have a structure that interacts with
a ubiquitin agents or other components of a ubiquitin reaction, for
example, ubiquitin moiety or target protein. Although it is
difficult to gauge the required absolute size of an interaction
library, nature provides a hint with the immune response: a
diversity of 10.sup.7-10.sup.8 different antibodies provides at
least one combination with sufficient affinity to interact with
most potential antigens faced by an organism. Published in vitro
selection techniques have also shown that a library size of
10.sup.7 to 10.sup.8 is sufficient to find structures with affinity
for a target. A library of all combinations of a peptide 7 to 20
amino acids in length, such as generally proposed herein, has the
potential to code for 20.sup.7 (10.sup.9l ) to 20.sup.20. Thus,
with libraries of 10.sup.7 to 10.sup.8 different molecules the
present methods allow a "working" subset of a theoretically
complete interaction library for 7 amino acids, and a subset of
shapes for the 20.sup.20 library. Thus, in a preferred embodiment,
at least 10.sup.6, preferably at least 10.sup.7, more preferably at
least 10.sup.8 and most preferably at least 10.sup.9 different
sequences are simultaneously analyzed in the subject methods.
Preferred methods maximize library size and diversity.
[0329] In one embodiment, the library is fully randomized, with no
sequence preferences or constants at any position. In a preferred
embodiment, the library is biased. That is, some positions within
the sequence are either held constant, or are selected from a
limited number of possibilities. For example, in a preferred
embodiment, the nucleotides or amino acid residues are randomized
within a defined class, for example, of hydrophobic amino acids,
hydrophilic residues, sterically biased (either small or large)
residues, towards the creation of cysteines, for cross-linking,
prolines for SH-3 domains, serines, threonines, tyrosines or
histidines for phosphorylation sites, etc., or to purines, etc.
[0330] In a preferred embodiment, the bias is towards peptides or
nucleic acids that interact with known classes of molecules. For
example, when the candidate agent is a peptide, it is known that
much of intracellular signaling is carried out via short regions of
polypeptides interacting with other polypeptides through small
peptide domains. For instance, a short region from the HIV-1
envelope cytoplasmic domain has been previously shown to block the
action of cellular calmodulin. Regions of the Fas cytoplasmic
domain, which shows homology to the mastoparan toxin from Wasps,
can be limited to a short peptide region with death-inducing
apoptotic or G protein inducing functions. Magainin, a natural
peptide derived from Xenopus, can have potent anti-tumor and
anti-microbial activity. Short peptide fragments of a protein
kinase C isozyme (.beta.PKC), have been shown to block nuclear
translocation of .beta.PKC in Xenopus oocytes following
stimulation. And, short SH-3 target peptides have been used as
psuedosubstrates for specific binding to SH-3 proteins. This is of
course a short list of available peptides with biological activity,
as the literature is dense in this area. Thus, there is much
precedent for the potential of small peptides to have activity on
intracellular signaling cascades. In addition, agonists and
antagonists of any number of molecules may be used as the basis of
biased randomization of candidate modulators as well.
[0331] Thus, a number of molecules or protein domains are suitable
as starting points for the generation of biased randomized
candidate modulators. A large number of small molecule domains are
known, that confer a common function, structure or affinity. In
addition, as is appreciated in the art, areas of weak amino acid
homology may have strong structural homology. A number of these
molecules, domains, and/or corresponding consensus sequences, are
known, including, but are not limited to, SH-2 domains, SH-3
domains, Pleckstrin, death domains, protease cleavage/recognition
sites, enzyme inhibitors, enzyme substrates, and Traf.
[0332] In a preferred embodiment, the candidate agents are nucleic
acids. With reference to candidate agents, by "nucleic acid" or
"oligonucleotide" or grammatical equivalents herein means at least
two nucleotides covalently linked together. A nucleic acid of the
present invention will generally contain phosphodiester bonds,
although in some cases, as outlined below, nucleic acid analogs are
included that may have alternate backbones, comprising, for
example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925
(1993) and references therein; Letsinger, J. Org. Chem. 35:3800
(1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et
al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805
(1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and
Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate
(Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No.
5,644,048), phosphorodithioate (Briu et al, J. Am. Chem. Soc.
111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein,
Oligonucleotides and Analogues: A Practical Approach, Oxford
University Press), and peptide nucleic acid backbones and linkages
(see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem.
Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);
Carlsson etal., Nature 380:207 (1996), all of which are
incorporated by reference). Other analog nucleic acids include
those with positive backbones (Denpcy et al., Proc. Natl. Acad.
Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos.
5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991);
Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et
al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3,
ASC Symposium Series 580, "Carbohydrate Modifications in Antisense
Research", Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al.,
Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al.,
J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996))
and non-ribose backbones, including those described in U.S. Pat.
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium
Series 580, "Carbohydrate Modifications in Antisense Research", Ed.
Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more
carbocyclic sugars are also included within the definition of
nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995)
ppl69-176). Several nucleic acid analogs are described in Rawls, C
& E News Jun. 2, 1997 page 35. All of these references are
hereby expressly incorporated by reference. These modifications of
the ribose-phosphate backbone may be done to facilitate the
addition of additional moieties such as labels, or to increase the
stability and half-life of such molecules in physiological
environments.
[0333] As will be appreciated by those in the art, all of these
nucleic acid analogs may find use in the present invention. In
addition, mixtures of naturally occurring nucleic acids and analogs
can be made. Alternatively, mixtures of different nucleic acid
analogs, and mixtures of naturally occurring nucleic acids and
analogs may be made. Particularly preferred are peptide nucleic
acids (PNA) which includes peptide nucleic acid analogs. These
backbones are substantially non-ionic under neutral conditions, in
contrast to the highly charged phosphodiester backbone of naturally
occurring nucleic acids.
[0334] The nucleic acids may be single stranded or double stranded,
as specified, or contain portions of both double stranded or single
stranded sequence. The nucleic acid may be DNA, both genomic and
cDNA, RNA or a hybrid, where the nucleic acid contains any
combination of deoxyribo- and ribo-nucleotides, and any combination
of bases, including uracil, adenine, thymine, cytosine, guanine,
inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc. As
used herein, the term "nucleoside" includes nucleotides and
nucleoside and nucleotide analogs, and modified nucleosides such as
amino modified nucleosides. In addition, "nucleoside" includes
non-naturally occurring analog structures. Thus for example the
individual units of a peptide nucleic acid, each containing a base,
are referred to herein as a nucleoside.
[0335] As described above generally for proteins, nucleic acid
candidate agent may be naturally occurring nucleic acids, random
nucleic acids, or "biased" random nucleic acids. For example,
digests of prokaryotic or eukaryotic genomes may be used as is
outlined above for proteins. Where the ultimate expression product
is a nucleic acid, at least 10, preferably at least 12, more
preferably at least 15, most preferably at least 21 nucleotide
positions need to be randomized, with more preferable if the
randomization is less than perfect. Similarly, at least 5,
preferably at least 6, more preferably at least 7 amino acid
positions need to be randomized; again, more are preferable if the
randomization is less than perfect.
[0336] In a preferred embodiment, the candidate agents are organic
moieties. In this embodiment, as is generally described in WO
94/24314, candidate agents are synthesized from a series of
substrates that can be chemically modified. "Chemically modified"
herein includes traditional chemical reactions as well as enzymatic
reactions. These substrates generally include, but are not limited
to, alkyl groups (including alkanes, alkenes, alkynes and
heteroalkyl), aryl groups (including arenes and heteroaryl),
alcohols, ethers, amines, aldehydes, ketones, acids, esters,
amides, cyclic compounds, heterocyclic compounds (including
purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines,
cephalosporins, and carbohydrates), steroids (including estrogens,
androgens, cortisone, ecodysone, etc.), alkaloids (including
ergots, vinca, curare, pyrollizdine, and mitomycines),
organometallic compounds, hetero-atom bearing compounds, amino
acids, and nucleosides. Chemical (including enzymatic) reactions
may be done on the moieties to form new substrates or candidate
agents which can then be tested using the present invention.
[0337] As will be appreciated by those in the art, it is possible
to screen more than one type of candidate agent at a time. Thus,
the library of candidate agents used may include only one type of
agent (i.e. peptides), or multiple types (peptides and organic
agents). The assay of several candidates at one time is further
discussed below.
[0338] The present invention provides methods and compositions
comprising combining different combinations of ubiquitin agents,
with ubiquitin moiety, in the presence or absence of a target
protein. In preferred embodiments, a candidate agent is included in
the combining to assay for an agent that modulates the attachment
of a ubiquitin moiety to a substrate molecule. In preferred
embodiments the ubiquitin moiety and/or the substrate molecule of
interest in the assay comprises a tag.
[0339] Preferably the tag is a label, a partner of a binding pair,
or a substrate binding molecule (or attachment tag). In a preferred
embodiment, the tag is an epitope tag. In another preferred
embodiment, the tag is a label. More preferably, the tag is a
fluorescent label or a binding pair partner. In a preferred
embodiment, the tag is a binding pair partner and the ubiquitin
moiety is labeled by indirect labeling. In the indirect labeling
embodiment, preferably the label is a fluorescent label or a label
enzyme. In an embodiment comprising a label enzyme, preferably the
substrate for that enzyme produces a fluorescent product. In a
preferred embodiment, the label enzyme substrate is luminol. In a
preferred embodiment, combining specifically excludes combining the
components with a target protein.
[0340] In another preferred embodiment, a preferred combination is
Tag1-ubiquitin moiety, tag2-ubiquitin moiety. Preferably, tag1 and
tag2 are labels, preferably fluorescent labels, most preferably
tag1 and tag2 constitute a FRET pair.
[0341] In a preferred embodiment, a preferred combination is
tag1-ubiquitin moiety and tag2-substrate molecule of interest.
Preferably, tag1 is a label, a partner of a binding pair, or a
substrate binding molecule and tag2 is a different label, partner
of a binding pair, or substrate binding molecule. More preferably,
tag1 is a fluorescent label or a member of a binding pair. When
tag1 is a member of a binding pair, preferably tagl is indirectly
labeled. Still more preferably, tag-1 is indirectly labeled with a
label enzyme. Preferably the label enzyme substrate used to reveal
the presence of the enzyme produces a fluorescent product, and more
preferably is luminol. In the presently described combination,
preferably tag2 is a surface substrate binding element, more
preferably a His-tag.
[0342] In a preferred embodiment, the methods of the invention do
not comprise a target protein. In a preferred embodiment, a mono-
or poly-ubiquitin moiety is a substrate molecule, as discussed
above. Because the different combinations of ubiquitin agents are
specific for particular target proteins, the present assays are
much more versatile then conventional assays which require a target
protein. However, the activity of these ubiquitin agents can be
assayed in the methods of the present invention because the methods
permit the use of any variation of such combinations without first
identifying the specific target protein to which the combination is
directed.
[0343] The components of the present assays may be combined in
varying amounts. In a preferred embodiment, ubiquitin moiety is
combined at a final concentration of from 20 to 200 ng per 100
.mu.l reaction solution, most preferable at about 100 ng per 100
.mu.l reaction solution.
[0344] In a preferred embodiment, the ubiquitin activating agent,
preferably an E1, is combined at a final concentration of from 1 to
50 ng per 100 .mu.l reaction solution, more preferably from 1 ng to
20 ng per 100 .mu.l reaction solution, most preferably from 5 ng to
10 ng per 100 .mu.l reaction solution.
[0345] In a preferred embodiment, the ubiquitin conjugating agent,
preferably an E2, is combined at a final concentration of 10 to 100
ng per 100 .mu.l reaction solution, more preferably 10-50 ng per
100 .mu.l reaction solution.
[0346] In a preferred embodiment, the ubiquitin ligating agent,
preferably an E3, is combined at a final concentration of from 1 ng
to 500 ng per 100 .mu.l reaction solution, more preferably from 50
to 400 ng per 100 .mu.l reaction solution, still more preferably
from 100 to 300 ng per 100 .mu.l reaction solution, and still more
preferably about 100 ng per 100 .mu.l reaction solution. In a
preferred embodiment, the ubiquitin ligating agent is combined at a
final concentration of from 50 to 100 ng per 100 .mu.l reaction
solution, still more preferably from 20 to 50 ng per 100 .mu.l
reaction solution, and still more preferably about 10 ng to 20 ng
per 100 .mu.l reaction solution.
[0347] The components of the present assays are combined under
reaction conditions that favor the activity of the ubiquitin agents
of the present invention, and more specifically favir the
attachment of ubiquitin moiety to a substrate molecule of interest
in the assay. Generally, this will be under physiological
conditions. Incubations may be performed at any temperature which
facilitates optimal activity, typically between 4 and 40.degree. C.
Incubation periods are selected for optimum activity, but may also
be optimized to facilitate rapid high through put screening.
Typically between 0.5 and 1.5 hours will be sufficient.
[0348] A variety of other reagents may be included in the
compositions. These include reagents like salts, solvents, buffers,
neutral proteins, e.g. albumin or detergents which may be used to
facilitate optimal activity of ubiquitin agents, and more
specifically facilitate the attachment of ubiquitin moiety to a
substrate molecule of interest in the assay; and/or reduce
non-specific or background interactions. Also reagents that
otherwise improve the efficiency of the assay, such as protease
inhibitors, nuclease inhibitors, anti-microbial agents, etc., may
be used. The compositions will also preferably include adenosine
tri-phosphate (ATP).
[0349] The mixture of components may be added in any order that
promotes the activity ubiquitin agents, and more specifically
promotes the attachment of ubiquitin moiety to a substrate molecule
of interest in the assay; or optimizes identification of the
modulating activity of a candidate agent. In a preferred
embodiment, ubiquitin moiety is provided in a reaction buffer
solution, followed by addition of the ubiquitin ubiquitination
enzymes. In an alternate preferred embodiment, ubiquitin moiety is
provided in a reaction buffer solution, a candidate agent is then
added, followed by the addition of ubiquitin agents.
[0350] Once combined, in a preferred embodiment, the amount of
ubiquitin moiety attached to a substrate molecule of interest in an
assay of the present invention, is measured. As will be understood
by one of ordinary skill in the art, the mode of measuring may
depend on the specific tag attached to the ubiquitin moiety. As
will also be apparent to the skilled artisan, the amount of
ubiquitin moiety attached to a substrate molecule will encompass
not only the particular ubiquitin moiety bound directly to the
substrate molecule, but also a mono- or poly-ubiquitin moiety
preferably attached to the substrate molecule.
[0351] In a preferred embodiment, the tag attached to the ubiquitin
moiety is a fluorescent label. In a preferred embodiment, the tag
attached to ubiquitin moiety is an enzyme label or a binding pair
member which is indirectly labeled with an enzyme label. In this
latter preferred embodiment, the enzyme label substrate produces a
fluorescent reaction product. In these preferred embodiments, the
amount of ubiquitin moiety bound is measured by luminescence.
[0352] In other preferred embodiments, at least a first and a
second ubiquitin moiety is used, wherein the first and second
ubiquitin moieties comprise different fluorescent labels, and
wherein the labels form a FRET pair.
[0353] As used herein, "luminescence" or "fluorescent emission"
means photon emission from a fluorescent label. In an embodiment
where FRET pairs are used, fluorescence measurements may be taken
continuously or at time-points during the ligation reaction.
Equipment for such measurement is commercially available and easily
used by one of ordinary skill in the art to make such a
measurement.
[0354] Other modes of measuring the attachment of ubiquitin moiety
to a substrate molecule of are well known in the art and easily
identified by the skilled artisan for each of the labels described
herein. For example, radioisotope labeling may be measured by
scintillation counting, or by densitometry after exposure to a
photographic emulsion, or by using a device such as a
Phosphorimager. Likewise, densitometry may be used to measure the
attachment of ubiquitin moiety following a reaction with an enzyme
label substrate that produces an opaque product when an enzyme
label is used.
[0355] In a preferred embodiment, the substrate molecule of
interest in the assays of the present invention is bound to a
surface substrate. This may be achieved as described above for the
binding of a label to ubiquitin moiety.
[0356] In another preferred embodiment, a ubiquitin activating
agent is bound to a surface substrate in the absence of a ubiquitin
conjugating agent and ubiquitin ligating agent. This may be
achieved, as described above for the binding of a label to
ubiquitin moiety. This may also be accomplished using tag-ubiquitin
activating agent, wherein the tag is a surface substrate binding
molecule.
[0357] In another preferred embodiment, a ubiquitin conjugating
agent is bound to a surface substrate in the absence of a ubiquitin
ligating agent. This may be achieved, as described above for the
binding of a label to ubiquitin moiety. This may also be
accomplished using tag-ubiquitin conjugating agent, wherein the tag
is a surface substrate binding molecule.
[0358] In another preferred embodiment, a ubiquitin ligating agent
is bound to a surface substrate in the absence of a target protein.
This may be achieved, as described above for the binding of a label
to ubiquitin moiety. This may also be accomplished using
tag-ubiquitin ligating agent, wherein the tag is a surface
substrate binding molecule.
[0359] In general, any substrate binding molecule can be used. In a
preferred embodiment, the tag is a His-tag and the surface
substrate is nickel. In a preferred embodiment, the nickel surface
substrate is present on the surface of the wells of a multi-well
plate, such as a 96 well plate. Such multi-well plates are
commercially available. The binding of the enzyme to a surface
substrate facilitates the separation of bound ubiquitin moiety from
unbound ubiquitin moiety. In the present embodiment, the unbound
ubiquitin moiety is easily washed from the receptacle following the
ligation reaction. As will be appreciated by those of skill in the
art, the use of any surface substrate binding element and
receptacle having the surface substrate to which it binds will be
effective for facilitating the separation of bound and unbound
ubiquitin moiety.
[0360] In an alternative embodiment, the substrate molecule of
interest in the assays of the present invention comprise a bead
that is attached to the substrate molecule directly or via a
substrate binding element. Following ligation, the beads may be
separated from the unbound ubiquitin moiety and the bound ubiquitin
moiety measured. In a preferred embodiment, the substrate molecule
of interest in the assay of the present invention comprises a bead
and the ubiquitin agents in the assay are combined with a
tag-ubiquitin moiety wherein the tag is a fluorescent label. In
this embodiment, the beads with bound ubiquitin moiety may be
separated using a fluorescence-activated cell sorting (FACS)
machine. Methods for such use are described in U.S. patent
application Ser. No. 09/047,119, which is hereby incorporated in
its entirety. The amount of bound ubiquitin moiety can then be
measured.
[0361] In another embodiment, none of the ubiquitin agents are
bound to a surface substrate. Preferably in this embodiment, the
assays comprise a tag1-ubiquitin moiety and tag2-ubiquitin moiety.
Preferably, tag1 and tag2 are labels, preferably fluorescent
labels, most preferably tag1 and tag2 constitute a FRET pair. In
this embodiment, the attachment of ubiquitin moiety to the
substrate molecule of interest is measured by measuring the
fluorescent emission spectrum. This measuring may be continuous or
at one or more times following the combination of the components.
Alteration in the fluorescent emission spectrum of the combination
as compared with unligated ubiquitin moiety indicates the amount of
ubiquitin ubiquitination. The skilled artisan will appreciate that
in this embodiment, alteration in the fluorescent emission spectrum
results from ubiquitin moiety bearing different members of the FRET
pair being brought into close proximity, either through the
formation of poly-ubiquitin moiety and/or by binding nearby
locations on a protein, preferably a target protein.
[0362] In one preferred embodiment of the present methods, the
ubiquitin ligating agent is an MdM2 protein and comprises a first
FRET label and the ubiquitin moiety comprises a second FRET label.
In another embodiment, the MdM2 protein comprises an attachment
tag. In another embodiment, the MdM2 protein is preferably provided
on a solid support, and more preferably the solid support comprises
a microtiter plate or a bead. In another embodiment, the mdm2
protein is preferably a mammalian mdm2 and more preferably a human
mdm2.
[0363] In another preferred embodiment, the target protein is p53
and comprises a first FRET label and the ubiquitin moiety comprises
a second FRET label.
[0364] In another embodiment, the p53 protein preferably comprises
an attachment tag. In another embodiment, the p53 protein is
preferably provided on a solid support, and more preferably the
solid support comprises a microtiter plate or a bead.
[0365] In a preferred embodiment, the compositions of the invention
are used to identify agents that modulate the attachment of
ubiquitin moiety to a substrate molecule. In this embodiment, the
composition includes a candidate agent. In a preferred embodiment,
the measured amount and/or rate of tag-ubiquitin moiety binding to
the substrate molecule is compared with that when the candidate
agent is absent from the composition, whereby the presence or
absence of the agent's effects on the attachment of ubiquitin
moiety to a substrate molecule is determined. In this embodiment,
whether the agent enhances or inhibits, or reduces or increases the
attachment of ubiquitin moiety to the substrate molecule is
determined.
[0366] In a preferred embodiment, the composition of the invention
containing a candidate agent lacks E3 and the amount and/or rate of
ubiquitin moiety attached to E2 is measured. This embodiment may
also comprise the step of comparing the amount and/or rate of
ubiquitin moiety attached to E2 in a composition lacking both E3
and a candidate agent, whereby the modulating activity of the
candidate agent is determined. In a preferred embodiment, the
percentage difference in the amount of ubiquitin moiety attached to
E2 in the presence and absence of the candidate agent is compared
with the percentage difference in the amount attached to E3 in the
presence and absence of candidate agent, whereby the point of
effect of the candidate agent in the cascade of enzymatic activity
and attachment of ubiquitin moiety to a substrate molecule is
determined. That is, it is determined whether the candidate agent
affects the attachment of ubiquitin moiety to E1, E2, and/or
E3.
[0367] In another preferred embodiment, the compositions of the
invention are used to identify agents that modulate the attachment
of ubiquitin moiety to a substrate molecule of interest in the
assay. In this embodiment, the present assays include a candidate
agent. In a preferred embodiment, where tag1 and tag2 constitute a
FRET pair, the measured amount and/or rate of tag1-ubiquitin moiety
and tag2-ubiquitin moiety binding to a substrate molecule (as a
poly-ubiquitin moiety and/or ubiquitin moiety attached to a
substrate molecule) is compared with the amount or rate of such
attachment in the absence of the candidate agent, whereby the
presence or absence of the candidate agent's effect on the
attachment of ubiquitin moiety to a substrate molecule is
determined. In this embodiment, whether the candidate agent
enhances or inhibits, or increases or decreases, the attachment of
ubiquitin moiety to the substrate molecule is also determined.
[0368] In a preferred embodiment, multiple assays are performed
simultaneously in a high throughput screening system. In this
embodiment, multiple assays may be performed in multiple
receptacles, such as the wells of a 96 well plate or other
multi-well plate. As will be appreciated by one of skill in the
art, such a system may be applied to the assay of multiple
candidate agents and/or multiple combinations of ubiquitin agents
with ubiquitin moiety. In a preferred embodiment, the present
invention is used in a high-throughput screening system for
determining the attachment of ubiquitin moiety to a substrate
molecule of interest, by combining different combinations of
ubiquitin agents, in the presence or absence of a target protein.
In an alternate preferred embodiment, the present invention is used
in a high throughput screening system for simultaneously testing
the effect of individual candidate agents by additionally
combinining a candidate agent.
[0369] In another aspect, the invention provides a method of
assaying for the attachment of a ubiquitin moiety to a substrate
molecule of in a mixture. Ubiquitin moiety is introduced into a
cell or mixture of protein, preferably a cell lysate, under
conditions in which the attachment of ubiquitin moiety to a
substrate molecule of interest can take place. In this embodiment,
the ubiquitin moiety is in the form of tag1-ubiquitin moiety and
tag2-ubiquitin moiety, wherein tag1 and tag2 constitute a FRET pair
or tag1 is a fluorescent label and tag2 is a quencher of tag1.
Fluorescent emission spectrum is measured as an indication of
whether ubiquitin ubiquitination activity is present in the mixture
or cell. In a preferred embodiment, the ubiquitin moiety also
comprises a member of a binding pair, such as FLAG. In this latter
embodiment, components involved in ubiquitin ubiquitination can be
isolated from the mixture using any one of a number of
affinity-based separation means such as fluorescent beads coated
with anti-FLAG antibody or amino precipitation using anti-FLAG
antibodies, or using anti-FLAG antibody attached to a solid
support. Other means of separating ubiquitin moiety attached
components of the cell or mixture will be readily apparent to the
skilled artisan. Ubiquitin moiety attached components so separated
in this method may include ubiquitin agents and target proteins.
The skilled artisan will appreciate that separation of these
components for individual identification or subsequent
investigation may be obtained by several means well known in the
art, such as by HPLC or electrophoresis.
[0370] It is understood by the skilled artisan that the steps of
the assays provided herein can vary in order. It is also
understood, however, that while various options (of compounds,
properties selected or order of steps) are provided herein, the
options are also each provided individually, and can each be
individually segregated from the other options provided herein.
Moreover, steps which are obvious and known in the art that will
increase the sensitivity of the assay are intended to be within the
scope of this invention. For example, there may be additionally
washing steps, blocking steps, etc.
[0371] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. All references cited herein
are expressly incorporated by reference in their entirety.
EXAMPLES
Example 1
Production of E2, E3, and Ubiquitin Moiety
E2 Production
[0372] The open reading frame of E2 (Ubch5c) was amplified by PCR
and cloned into the pGex-6p-1 E. Coli. expression vector (Amersham
Pharmacia) as BglII-EcoRI fragments, with N-terminus in frame fused
to the GST-tag.
Materials and Methods
[0373] Plasmid is transformed in BL21 DE3 competent E. coli
(Stratagene, cat # 230132). Cells are grown at 37.degree. C. in
TB+100 ug/ml ampicillin and 0.4% glucose to an OD600 of about 0.6,
induced with addition of 320 uM IPTG and allowed to grow for
another 3 h before harvest. The pellets are washed once with cold
PBS, then resuspended in about 6 volumes of lysis buffer (20 mM
Tris, 10% glycerol, 0.5 M Nacl, 2.5mM EDTA, 1 mM TCEP plus
Complete-EDTA Free Protease inhibitor tablets, 1 tablet/25 ml of
resuspended cells, pH 8.0). The suspension is homogenized and
sonicated 3.times.30 sec. NP40, then added to a final concentration
of 0.5% and the tubes are rocked for 30 min at 4.degree. C.
Following centrifugation at 11000 rpm for 25 to 30 min, the
supernatant is incubated with Glutathione Sepharose 4B (Amersham,
cat # 17-0756-01) at a ratio of 1 ml of beads per 100 ml of
original culture volume for 1 to 2 hours at 4.degree. C. with
gentle rocking. The beads are pelleted and washed once with 10 bed
volumes of the lysis buffer, then twice with 10 bed volumes of
Prescission Protease buffer (50 mM Tris-HCL, 150 mM NaCl, 1 mM
EDTA, 1 mM DTT, 0.1% NP-40, pH 7.0.). Prescission Protease
(Amersham, product # 27-0843) is added at a ratio of 80 ul (160
Units) per ml of GST resin, and allowed to incubate for 4 h at
4.degree. C. The supernatant containing the cleaved E2 protein is
collected, and the resin is washed twice with one bed volume of
Prescission buffer. All three fractions are analyzed by SDS-PAGE
and pooled when appropriate.
Ubiquitin Moiety Production
[0374] Ubiquitin moiety was cloned into the pFlag-Mac Expression
Vector (Sigma) as a HindIII-EcoRI fragment by PCR. This results in
expression of amino-terminal Flag fusion ubiquitin moiety in E.
Coli.
Materials and Methods
[0375] The induction of protein expression and cell lysis is
similar to the above GST-E2 preparation, except that the
supernatant is loaded over a FLAG-affinity resin (VWR, cat # IB
13020) at a ratio of 15 ml of beads per 1 L of original culture.
The resin is then washed with 10 bed volumes of lysis buffer. The
protein is eluted from the column with: 100 mM Acetic acid, 10%
glycerol, 200 mM NaCl, 2.5 mM EDTA, 0.1% NP-40, pH 3.5. The
elutions are collected as 1 bed volume fractions into tubes that
contain 1/10.sup.th volume of 2M Tris, 80 mM B-ME, pH 9.0 to
neutralize the pH. The elution fractions are analyzed by SDS-PAGE
and the appropriate fractions are pooled and dialyzed against 400
volumes of 20 mM Tris, 10% glycerol, 200 mM NaCl, 2.5 mMEDTA, pH
8.0.
Production of E3
[0376] Coding sequences for E3 complex were also amplified by PCR
and baculoviruses were generated using the Bac-to-Bac system
(GibcoBRL). E3 contains two subunits, which are expressed by
co-infection of the two baculovirus in the same Hi-5 insect cells.
One of the subunit is His-tagged, with the other associating
subunit untagged. The detail procedure was done following the Bac
to Bac Baculovirus Expression system by GibcoBRL. For example, ROC1
was cloned into the pFastBacHtb vector with a N-terminal His6-tag
(SEQ ID NO:26), while CUL1 was insert into the pFastBacl vector
without any fusing tag. After transposition and Bacmid DNA
transfection into SF-9 cells, Baculoviruses were harvested,
amplified, and used to co-infect Hi-5 cells for protein
expression.
Materials and Methods
[0377] Cells are harvested, washed once with cold PBS, and
resuspended in about 6 volumes of lysis buffer (20 mM Tris, 20%
glycerol, 0.5 M Nacl, 15 mM imidazole, 1 mM TCEP plus Complete-EDTA
Free Protease inhibitor tablets, 1 tablet/25 ml of resuspended
cells, pH 8.0.). The suspension is then sonicated 3.times.30 sec,
followed by addition of NP40 to a final concentration of 0.5% and
incubation for 30 min at 4.degree. C. The lysate is then
centrifuged and the supernatant is incubated with pre-equilibrated
(lysis buffer+NP40) Ni- NTA Agarose beads (Qiagen, cat # 1000632)
for 1 to 2 hrs. The pelleted beads are washed 2 times with lysis
buffer, resuspended in 1 to 2 volumes of lysis buffer and
transferred to a disposable column for elution. Elution is
accomplished using 5.times.1-bed volume aliquots of Lysis
buffer+250 mM imidazole. Elution fractions are analyzed by SDS-PAGE
and appropriate fractions are pooled. The elution pool is then
desalted using either a desalting column or a centrifugal
concentration device (more often used for large volumes.) When
using centrifugal devices, the eluted pool is diluted 1:1 with
lysis buffer that has no imidazole and spun at the appropriate
speed until the volume is reduced by half. At this point an equal
volume of fresh buffer is added and the device is respun. This is
done a total of four times resulting in a 32 fold exchange.
Example 2
E1+E2 Assay
[0378] The attachment of ubiquitin moiety to an E2, by combining
E1+E2 and ubiquitin moiety, was assayed using the following
protocol with Flag-ubiquitin, purified from E. coli, and the E2
Ubch5c, purified as His-Ubch5c from E. coli.
Materials and Methods
[0379] The following procedures were used for assays measuring the
attachment of ubiquitin moiety to E2. The wells of Nickel-substrate
96-well plates (Pierce Chemical) are blocked with 100 .mu.l of 1%
casein/phosphate buffered saline (PBS) for 1 hour at room
temperature, then washed with 200 .mu.l of PBST (0.1% Tween-20 in
PBS) 3 times. To each well is added the following Flag-ubiquitin
moiety (see above) reaction solution:
Final Concentration
[0380] 62.5 mM Tris pH 7.5 [0381] 6.25 mM MgCl2 [0382] 0.75 mM DTT
[0383] 2.5 mM ATP [0384] 2.5 mM NaF [0385] 12.5 nM Okadaic acid
[0386] 100 ng Flag-ubiquitin moiety (made as described above).
[0387] The buffer solution is brought to a final volume of 80 .mu.l
with milipore-filtered water, followed by the addition of 10 .mu.l
of DMSO.
[0388] To the above solution is then added 10 .mu.l of E1, His-E2
in 20 mM Tris buffer, pH 7.5, and 5% glycerol. His-E2 is made as
described above. E1 is obtained commercially (Affiniti Research
Products, Exeter, U.K.). The following amounts of each enzyme are
used for these assays: 5 ng/well of E1; 25 nl/well E2. The reaction
is then allowed to proceed at room temperature for 1 hour.
[0389] Following the ubiquitin reaction, the wells are washed with
200 .mu.l of PBST 3 times. For measurement of the E2-attached
ubiquitin moiety, 100 .mu.l of Mouse anti-Flag (1:10,000) and
ant-Mouse Ig-HRP (1:15,000) in PBST are added to each well and
allowed to incubate at room temperature for 1 hour. The wells are
then washed with 200 .mu.l of PBST 3 times, followed by the
addition of 100 .mu.l of luminol substrate (1/5 dilution).
Luminescence for each well is then measured using a
fluorimeter.
Results
Attachment of Ubiquitin Moiety to E1 and Attachment of Ubiquitin
Moiety to E2
[0390] FIG. 1A shows the luminescence measured for E1 alone and for
E1+his-E2, as described above.
Example 3
E1+E2+E3 Assay
[0391] The attachment of ubiquitin moiety to E3, by combining
E1+E2+E3 and ubiquitin moiety, was assayed using the following
protocol with Flag-ubiquitin, purified from E. coli, the E2 Ubch5c,
purified as GST-Ubch5c from E. coli with the GST tag removed, and
the E3 His-ROC1/Cul1 complex purified from Hi-5 cells by
Baculovirus co-infection. This assay was also used to show the
effects of candidate agents on the attachment of ubiquitin moiety
to E3.
Materials and Methods
[0392] The wells of Nickel-substrate 96-well plates (Pierce
Chemical) are blocked with 100 .mu.l of 1 casein/phosphate buffered
saline (PBS) for 1 hour at room temperature, then washed with 200
.mu.l of PBST (0.1% Tween-20 in PBS) 3 times. To each well is added
the following Flag-ubiquitin moiety (see above) reaction
solution:
Final Concentration
[0393] 62.5 mM Tris pH 7.5 [0394] 6.25 mM MgCl.sub.2 [0395] 0.75 mM
DTT [0396] 2.5 mM ATP [0397] 2.5 mM NaF [0398] 12.5 nM Okadaic acid
[0399] 100 ng Flag-ubiquitin moiety (made as described above).
[0400] The buffer solution is brought to a final volume of 80 .mu.l
with milipore-filtered water.
[0401] For assays directed to identifying agents that modulate the
attachment of ubiquitin moiety to E3, 10 .mu.l of a candidate agent
in DMSO is then added to the solution. If no candidate agent is
added, 10 .mu.l of DMSO is added to the solution.
[0402] To the above solution is then added 10 .mu.l containing the
ubiquitin agents in 20 mM Tris buffer, pH 7.5, and 5% glycerol.
E2-Ubch5c and E3-HisROC1/Cul1 are made as described above. E1 is
obtained commercially (Affiniti Research Products, Exeter, U.K.).
The following amounts of each enzyme are used for these assays: 5
ng/well of E1; 25 nl/well E2; and 100 ng/well His-E3. The reaction
is then allowed to proceed at room temperature for 1 hour.
[0403] Following the ubiquitin ubiquitination reaction, the wells
are washed with 200 .mu.l of PBST 3 times. For measurement of the
E3-attached ubiquitin moiety, 100 .mu.l of Mouse anti-Flag
(1:10,000) and ant-Mouse Ig-HRP (1:15,000) in PBST are added to
each well and allowed to incubate at room temperature for 1 hour.
The wells are then washed with 200 .mu.l of PBST 3 times, followed
by the addition of 100 .mu.l of luminol substrate (1/5 dilution).
Luminescence for each well is then measured using a
fluorimeter.
Results
Attachment of Ubiquitin Moiety to E3
[0404] FIG. 2 shows the luminescence measured for several different
combinations of ubiquitin agents. In these experiments, only E3 was
in the form His-E3. The luminescence measurements show that the
assay specifically measures the activity of the entire cascade of
activity or attachment of ubiquitin moiety by the ubiquitin agents,
which requires the presence of all three ubiquitin agents (i.e.,
E1+E2+E3) in this reaction.
Varying the Amounts of Ubiquitin Agents
[0405] FIG. 3A shows the relative effect of varying the amount of
E1 on the attachment of ubiquitin moiety to E3 in the above
procedure, in presence and absence of DMSO. The addition of about
10 ng per 100 .mu.l reaction solution provides maximum amounts of
attachment of ubiquitin moiety to E3 with the other components of
the reaction maintained as detailed above. The presence of DMSO
does not significantly affect the activity of the ubiquitin
agents.
[0406] The relative effect of varying E3 and ubiquitin moiety
concentration in the ubiquitin reaction is shown in FIG. 3B.
Generally speaking, maximum amounts of attachment of ubiquitin
moiety to E3 was obtained with 200 to 300 ng per 100 .mu.l of E3 at
each concentration of ubiquitin moiety, while increasing ubiquitin
moiety concentration generally increased the amount of attachment
of ubiquitin moiety to E3 at each concentration of E3.
[0407] It was also found that blocking of the wells with 1% casein
improved the signal to noise ratio over either no blocking or
blocking with 5% bovine serum albumen (BSA). Background was
determined after combining all of the components as above except
His-E3 and measuring the resulting fluorescence after pre-treating
the wells with 5% BSA, 1% casein or nothing. Results are shown in
FIG. 4.
Identification of Agents that Modulate the Attachment of Ubiquitin
Moiety to E3
[0408] To show that the assay is useful for identifying agents that
modulate the attachment of ubiquitin moiety to E3, several
candidate agents were combined with the ubiquitin agents (E1+E2+E3)
and ubiquitin moiety, at varying concentrations as described above.
FIG. 5 shows the results from two identified agents that modulate
the attachment of ubiquitin moiety to E3. The modulators decreased
the attachment of ubiquitin moiety to E3 in a dose-dependent
fashion corresponding to the concentration of the ubiquitin agents
present in the reaction which comprised either ROC1/Cul1 or
ROC2/Cul5 as the E3 component.
[0409] Comparison of the effect of the modulators on the attachment
of ubiquitin moiety to E3, as described above, either containing
E1, E2 and His-E3 or containing E1, His-E2 and lacking E3, shows
whether the modulator affects E3 or a ubiquitin agent other than
E3. In FIG. 6A, the identified modulator decreases the attachment
of ubiquitin to E3 in the presence of E3, but does not modulate the
attachment in the absence of E3, showing that the modulator has a
specific effect on the attachment of ubiquitin moiety to E3. In
contrast, results shown in FIG. 6B for another modulator reveals
that this agent reduces activity whether or not E3 is present,
showing that the affects of this agent effect the activity of
ubiquitin agents other than E3.
Example 4
FRET Anaylsis of Ubiquitin Moiety Attached to E3
[0410] Ubiquitin moiety was prepared, labeled with either EDANS or
fluorescein, and the fluorescence of each of these labels and their
interaction as a FRET pair was measured to show attachment of the
labeled ubiquitin moiety to E3 and FRET activity of the attached
ubiquitin moiety.
Materials and Methods
[0411] Ubiquitin moiety were produced incorporating Cys residues
into the FLAG-ubiquitin moiety sequence by site-directed
mutagenesis using either the primer TABLE-US-00005 (SEQ ID NO:27)
5'-CCCCCCAAGCTTTGCATGCAGATTTTCGTGAAGACCCTGACC-3'
[0412] to produce FLAG-Cys-ubiquitin moiety, or the primer
TABLE-US-00006 (SEQ ID NO:28)
5'-CCCCCCAAGCTTGCGTGCATGCAGATTTTCGTGAAGAC CCTGACC-3'
to produce FLAG-Ala-Cys-ubiquitin moiety. Protein was expressed and
purified as described above.
[0413] Either fluorescein 5-maleimide (peak emission at 515 nm) or
1,5-iodacetamide EDANS (IAEDANS; peak emission at 490 nm) was
reacted with the thiol group on the cysteine of the ubiquitin
moiety produced as above to form a thioether. The labeling was
performed in PBS with 1 mM TCEP. Labeled protein was separated from
free label by gel filtration.
[0414] The ubiquitin assay was performed substantially as described
above, with a few modifications. No nickel substrate was used in
the reaction wells, so all of the components were free in solution.
Equal amounts of fluorescein labeled ubiquitin moiety and IAEDANS
labeled ubiquitin moiety were used. The reaction was performed at
room temperature for 2 hours in a volume of 100-150 .mu.l, then
stopped with 50 .mu.l of 0.5M EDTA, pH 8.
[0415] Following the reaction, the products were separated in PBS
with 1 mM TCEP by HPLC on a Superdex-75 HR 10/30 size-exclusion
column using fluorescence emission detection. A larger molecular
weight cutoff gel-filtration column (e.g., Superdex 200 HR 10/30)
could be used to resolve individual ligation species.
Results
[0416] FIGS. 16A and 16B show the E3-dependent incorporation of
Flag-Ala-Cys-ubiquitin moiety labeled with FRET fluorophores into
E3-ubiquitin moiety complex. Isolation by HPLC shows emissions from
free ubiquitin moiety and ubiquitin moiety attached to the E3
ubiquitin ligating agent. The traces show fluorescent emission at
the wavelength described below, under excitation at 336 nm, the
optimal excitation wavelength for IAEDANS. FIG. 16A shows the
fluorescence signals of IAEDANS (490 nm; larger peak) and
fluorescein (515 nm; smaller peak) labeled ubiquitin moiety
following combination with E1 and E2 only. The free ubiquitin
moiety was isolation using high performance liquid chromatography
(HPLC). FIG. 16B shows the fluorescence signals of IAEDANS (490 nm;
larger peak at each elution volume) and fluorescein (515 nm;
smaller peak at each elution volume) labeled ubiquitin moiety
following combination with E1 and E2 and E3 (Roc1/Cul1). The dashed
line shows optical density of the protein solution (scale on
right), revealing the high sensitivity of the fluorophores despite
a very low concentration of protein.
[0417] Fluorescein labeled ubiquitin moiety and IAEDANS labeled
ubiquitin moiety was attached to E3 in approximately equal amounts.
A comparison of the spectral analysis of fluorescent emission from
the free (unligated) ubiquitin moiety labeled with both
fluorophores and the E3-attached ubiquitin moiety shows a distinct
increase in ratio of emission at 515 nm versus 490 nm (FIG. 17).
This shows that in the attached ubiquitin moiety, the fluorophores
on different ubiquitin moieties are sufficiently close for FRET to
be measured.
Example 5
E1+E2+MDM2+P53 Assay
[0418] The attachment of ubiquitin moiety to p53, by combining
E1+E2Ubch5c+Gst-Mdm2+His-p53, and Flag-ubiquitin moiety, was
assayed using the following protocol with: E1 obtained commercially
(Affiniti Research Products, Exeter, U.K.); Flag-ubiquitin moiety
purified from E. coli; E2 Ubch5c (also called Ubc-5) purified as
GST-Ubch5c from E. coli with the GST tag removed; GST-Mdm2
(schematically depicted in FIG. 18) purified from Hi-5 cells by
Baculovirus infection with the GST tag intact; and p53 purified
from Hi-5 cells by Baculovirus infection (schematically depicted in
FIG. 18). E2 Ubch5c was made as described above. Gst-Mdm2 and
His-p53 were made as described above for GST-Ubch5c and E3
His-ROC1/Cul1, respectively.
Materials and Methods
[0419] The following procedures were used for assays measuring the
attachment of ubiquitin moiety to p53 by Western blot analysis. The
following combinations of ubiquitin agents, ubiquitin moiety, and
p53 were combined in a reaction mixture: E1+E2 Ubch5c+p53 (as a
control); Mdm2+p53 (as a control); E1+E2Ubch5c (as a control); and
E1+E2 Ubch5c+Mdm2+p53 (as a control). To each reaction mixture is
added the following:
Final Concentration
[0420] 50 mM Tris pH 7.5 [0421] 5 mM MgCl.sub.2 [0422] 0.6 mM DTT
[0423] 2.0 mM ATP [0424] 100 ng Flag-ubiquitin moiety (made as
described above) [0425] 100 ng His-p53. [0426] The buffer solution
is brought to a final volume of 80 .mu.l with milipore-filtered
water, followed by the addition of 10 .mu.l of DMSO.
[0427] To the above solution is then added 10 .mu.l of E1+E2
Ubch5c+p53; Mdm2+p53; E1+E2 Ubch5c; or E1+E2 Ubch5c+Mdm2+p53, in 20
mM Tris buffer, pH 7.5, and 5% glycerol. The His-E2 and Mdm2 is
made as described above, and E1 is obtained commercially (as
described above). The following amounts of each enzyme are used for
these assays: 5 ng of E1; 15 ng E2 Ubch5c; and 50 ng Mdm2. The
reaction is then allowed to proceed at 37.degree. C. for 1
hour.
[0428] The products of the reaction were then resolved by SDS-PAGE;
analyzed by Western blot using Mouse anti-Flag and ant-Mouse
Ig-HRP.
Results
Attachment of Ubiquitin Moiety to p53
[0429] FIG. 19 shows the attachment of ubiquitin moiety measured
for E1+E2 Ubch5c+p53; Mdm2+p53; E1+E2 Ubch5c; and E1 +E2
Ubch5c+Mdm2+p53, as described above.
Example 6
E1+E2+MDM2+P53 Assay
[0430] The attachment of ubiquitin moiety to p53, by combining
E1+E2 Ubch5c+Gst-Mdm2+His-p53, and Flag-ubiquitin moiety, was
assayed using the following protocol with: E1 obtained commercially
(Affiniti Research Products, Exeter, U.K.); Flag-ubiquitin moiety
purified from E. coli; E2 Ubch5c (also called Ubc-5) purified as
GST-Ubch5c from E. coli with the GST tag removed; GST-Mdm2
(schematically depicted in FIG. 18) purified from Hi-5 cells by
Baculovirus infection with the GST tag intact; and p53 purified
from Hi-5 cells by Baculovirus infection (schematically depicted in
FIG. 18). E2 Ubch5c was made as described above. Gst-Mdm2 and
His-p53 were made as described above for GST-Ubch5c and E3
His-ROC1/Cul1, respectively.
Materials and Methods
[0431] The following procedures were used for assays measuring the
attachment of ubiquitin moiety to p53, and are illustrated
schematically in FIG. 20. The wells of Nickel-substrate 96-well
plates (Pierce Chemical) are blocked with 100 .mu.l of 1%
casein/phosphate buffered saline (PBS) for 1 hour at room
temperature, then washed with 200 .mu.l of PBS 3 times. To each
well is added the following Flag-ubiquitin moiety (see above)
reaction solution:
Final Concentration
[0432] 50 mM Tris pH 7.5 [0433] 5 mM MgCl.sub.2 [0434] 0.6 mM DTT
[0435] 2.0 mM ATP [0436] 100 ng Flag-ubiquitin moiety (made as
described above) [0437] 100 ng His-p53. [0438] The buffer solution
is brought to a final volume of 80 .mu.l with milipore-filtered
water, followed by the addition of 10 .mu.l of DMSO.
[0439] To the above solution is then added 10 .mu.l of E1, His-E2,
and Mdm2 in 20 mM Tris buffer, pH 7.5, and 5% glycerol. The
controls contained either Mdm2 alone or His-p53 alone. The His-E2
and Mdm2 is made as described above, and E1 is obtained
commercially (as described above). The following amounts of each
enzyme are used for these assays: 5 ng/well of E1; 15 ng/well E2
Ubch5c; and 50 ng/well Mdm2. The reaction is then allowed to
proceed at room temperature for 1 hour.
[0440] Following the ubiquitin reaction, the wells are washed with
200 .mu.l of PBS 3 times. For measurement of the p53-attached
ubiquitin moiety, 100 .mu.l of Mouse anti-Flag (1:10,000) and
ant-Mouse Ig-HRP (1:15,000) in PBS are added to each well and
allowed to incubate at room temperature for 1 hour. The wells are
then washed with 200 .mu.l of PBS 3 times, followed by the addition
of 100 .mu.l of luminol substrate (1/5 dilution). Luminescence for
each well is then measured using a fluorimeter.
Results
Attachment of Ubiquitin Moiety to p53
[0441] FIG. 21 shows the luminescence measured for His-p53 alone,
Mdm2 alone, and for Mdm2+His-p53 as described above.
[0442] Examples of preferred embodiments are depicted in the
following figures.
[0443] FIG. 22 depicts the key for the ubiquitin activating agent
(UAA), ubiquitin conjugating agent (UCA), ubiquitin ligating agent
(ULA), ubiquitin moiety (U), and candidate agent (CA) used in the
schematics in Figures
[0444] FIG. 23 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent (UA-1) where the assay comprises: [0445] 1)
combining a UA-1+CA+U; and [0446] 2) assaying for the attachment of
the ubiquitin moiety to UA-1. In another preferred embodiment UA-1
is a UAA. In another preferred embodiment, UAA is an E1. In yet
another preferred embodiment, UA-1 comprises a label. In another
preferred embodiment, the ubiquitin moiety comprises a label.
[0447] FIG. 24 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a second
ubiquitin agent (UA-2) where the assay comprises: [0448] 1)
combining a first ubiquitin agent that is UAA.sub.1+UA-2+CA+U; and
[0449] 2) assaying for the attachment of the ubiquitin moiety to
UA-2. In another preferred embodiment, UA-2 comprises a label. In
yet another preferred embodiment, UA-2 comprises a label.
[0450] FIG. 25 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin conjugating agent UCA, where
the assay comprises: [0451] 1) combining a second ubiquitin agent
that is UAA.sub.2+UCA.sub.1+CA+U; and [0452] 2) assaying for the
attachment of the ubiquitin moiety to UCA.sub.1.
[0453] FIG. 26 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to ubiquitin
conjugating agent that is an E2 where the assay comprises: [0454]
1) combining a ubiquitin activating agent that is an E1+E2+CA+U;
and [0455] 2) assaying for the attachment of the ubiquitin moiety
to E2.
[0456] FIG. 27 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.1)
where the assay comprises: [0457] 1) combining a second ubiquitin
agent that is a ubiquitin conjugating agent and comprising a
ubiquitin moiety UCA.sub.2-U+ULA.sub.1+CA; and [0458] 2) assaying
for the attachment of the ubiquitin moiety to ULA.sub.1. In another
preferred embodiment, the ubiquitin moiety comprises a label. In
yet another preferred embodiment, ULA.sub.1 comprises a label. In a
preferred embodiment the ubiquitin ligating agent comprises an Mdm2
protein.
[0459] FIG. 28 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a a ubiquitin
ligating agent that is an E3 where the assay comprises: [0460] 1)
combining a ubiquitin conjugating agent that is an E2 and
comprising a ubiquitin moiety+E3+CA; and [0461] 2) assaying for the
attachment of the ubiquitin moiety to E3. In a preferred
embodiment, the E3 is an Mdm2 protein.
[0462] FIG. 29 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a ubiquitin
conjugating agent that is an E2 where the assay comprises: [0463]
1) combining a ubiquitin activating agent that is an E1 and
comprising a ubiquitin moiety+E2+CA; and [0464] 2) assaying for the
attachment of the ubiquitin moiety to E2.
[0465] FIG. 30 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a third
ubiquitin agent (UA-3) where the assay comprises: [0466] 1)
combining a ubiquitin activating agent that is an E1 and comprising
a ubiquitin moiety+a ubiquitin conjugating agent that is an
E2+UA-3+CA; and [0467] 2) assaying for the attachment of the
ubiquitin moiety to UA-3. In a preferred embodiment, UA-3 comprises
an Mdm2 protein.
[0468] FIG. 31 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a third
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.3)
where the assay comprises: [0469] 1) combining a ubiquitin
activating agent that is an E1 and comprising a ubiquitin moiety+a
ubiquitin conjugating agent that is an E2+ULA.sub.3+CA; and [0470]
2) assaying for the attachment of the ubiquitin moiety to
ULA.sub.3. In a preferred embodiment the ubiquitin ligating agent
comprises an Mdm2 protein.
[0471] FIG. 32 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a ubiquitin
ligating agent that is an E3 where the assay comprises: [0472] 1)
combining an E1 comprising a ubiquitin moiety+an E2+an E3+CA; and
[0473] 2) assaying for the attachment of the ubiquitin moiety to
E3. In a preferred embodiment, the E3 is Mdm2.
[0474] FIG. 33 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent (UA-1) that is attached to a solid support where
the assay comprises: [0475] 1) combining a UA-1 (that is attached
to a solid support)+CA+U; and [0476] 2) assaying for the attachment
of the ubiquitin moiety to UA-1. In another preferred embodiment
UA-1 is a UAA. In another preferred embodiment, UAA is an E1. In
another preferred embodiment, the solid support is a microtiter
plate. In another preferred embodiment, the solid support is a
bead.
[0477] FIG. 34 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a second
ubiquitin agent (UA-2) that is attached to a solid suport) where
the assay comprises: [0478] 1) combining a first ubiquitin agent
that is UAA.sub.1+UA-2 (attached to a solid support)+CA+U; and
[0479] 2) assaying for the attachment of the ubiquitin moiety to
UA-2. In another preferred embodiment, the solid support is a
microtiter plate. In another preferred embodiment, the solid
support is a bead. In a preferred embodiment, UA-2 comprises an
Mdm2 protein.
[0480] FIG. 35 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.1) that
is attached to a solid support where the assay comprises: [0481] 1)
combining a second ubiquitin agent that is a ubiquitin conjugating
agent UCA.sub.2 comprising a ubiquitin moiety+ULA.sub.1 (attached
to a solid support)+CA; and [0482] 2) assaying for the attachment
of the ubiquitin moiety to ULA.sub.1. In a preferred embodiment the
ubiquitin ligating agent comprises an Mdm2.
[0483] FIG. 36 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent (UA-1) that comprises a label where the assay
comprises: [0484] 1) combining a UA-1 (plus label)+CA+U; and [0485]
2) assaying for the attachment of the ubiquitin moiety to UA-1.
[0486] FIG. 37 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.1) that
comprises a label where the assay comprises: [0487] 1) combining a
second ubiquitin agent that is a ubiquitin conjugating agent
UCA.sub.2 comprising a ubiquitin moiety+ULA.sub.1 (plus label)+CA;
and [0488] 2) assaying for the attachment of the ubiquitin moiety
to ULA.sub.1. In a preferred embodiment the ubiquitin ligating
agent comprises an Mdm2 protein.
[0489] FIG. 38 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety that comprises a
label, to a first ubiquitin agent (UA-1) where the assay comprises:
[0490] 1) combining a UA-1+CA+U (plus label); and [0491] 2)
assaying for the attachment of the ubiquitin moiety to UA-1.
[0492] FIG. 39 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety that comprises a
label, to a second ubiquitin agent (UA-2) where the assay
comprises: [0493] 1) combining a first ubiquitin agent that is
UAA.sub.1+UA-2+CA+U (plus label); and [0494] 2) assaying for the
attachment of the ubiquitin moiety to UA-2.
[0495] FIG. 40 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety that comprises a
label, to a first ubiquitin agent that is a ubiquitin ligating
agent (ULA.sub.1) where the assay comprises: [0496] 1) combining a
second ubiquitin agent that is a ubiquitin conjugating agent
UCA.sub.2 comprising a ubiquitin moiety (plus label)+ULA.sub.1+CA;
and [0497] 2) assaying for the attachment of the ubiquitin moiety
to ULA.sub.1. In a preferred embodiment the ubiquitin ligating
agent comprises an Mdm2 protein.
[0498] FIG. 41 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.1)
which comprises a label where the assay comprises: [0499] 1)
combining a second ubiquitin agent that is a ubiquitin conjugating
agent UCA.sub.2 comprising a ubiquitin moiety+ULA.sub.1 (plus
label)+CA; and [0500] 2) assaying for the attachment of the
ubiquitin moiety to ULA.sub.1. In a preferred embodiment the
ubiquitin ligating agent comprises an Mdm2 protein.
[0501] FIG. 42 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a second
ubiquitin agent (UA-2) that comprises a label where the assay
comprises: [0502] 1) combining a first ubiquitin agent that is
UAA.sub.1+UA-2 (plus label)+CA+U; and [0503] 2) assaying for the
attachment of the ubiquitin moiety to UA-2. In a preferred
embodiment, UA-2 comprises an Mdm2 protein.
[0504] FIG. 43 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent (UA-1) that comprises an attachment tag (or
attachment moiety) where the assay comprises: [0505] 1) combining a
UA-1(plus attachment tag)+CA+U; and [0506] 2) assaying for the
attachment of the ubiquitin moiety to UA-1.
[0507] FIG. 44 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent that is a ubiquitin ligating agent (ULA.sub.1) that
comprises an attachment tag (or attachment moiety) where the assay
comprises: [0508] 1) combining a second ubiquitin agent that is a
ubiquitin conjugating agent UCA.sub.2 comprising a ubiquitin
moiety+ULA.sub.1 (plus attachment tag)+CA; and [0509] 2) assaying
for the attachment of the ubiquitin moiety to ULA.sub.1. In a
preferred embodiment the ubiquitin ligating agent comprises an Mdm2
protein.
[0510] FIG. 45 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a second
ubiquitin agent (UA-2) that comprises an attachment tag (or
attachment moiety) where the assay comprises: [0511] 1) combining a
first ubiquitin agent that is UAA.sub.1+UA-2 (plus attachment
tag)+CA+U; and [0512] 2) assaying for the attachment of the
ubiquitin moiety to UA-2.
[0513] FIG. 46 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a first
ubiquitin agent (UA-1) that comprises an epitope tag (or epitope
label) where the assay comprises: [0514] 1) combining a UA-1 (plus
epitope tag)+CA+U; and [0515] 2) assaying for the attachment of the
ubiquitin moiety to UA-1.
[0516] FIG. 47 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a second
ubiquitin agent (UA-2) that comprises an epitope tag (or epitope
label) where the assay comprises: [0517] 1) combining a first
ubiquitin agent that is UAA.sub.1+UA-2 (plus epitope tag)+CA+U; and
[0518] 2) assaying for the attachment of the ubiquitin moiety to
UA-2.
[0519] FIG. 48 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a substrate
molecule (s) where the assay comprises: [0520] 1) combining a first
ubiquitin agent that is a ubiquitin ligating agent ULA.sub.1+a
second ubiquitin agent+substrate molecule+CA+U; and [0521] 2)
assaying for the attachment of the ubiquitin moiety to the
substrate molecule. In a preferred embodiment the ubiquitin
ligating agent comprises an Mdm2 protein and the substrate molecule
comprises p53.
[0522] FIG. 49 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a substrate
molecule (s) where the assay comprises: [0523] 1) combining a first
ubiquitin agent that is a ubiquitin ligating agent ULA.sub.1+a
second ubiquitin agent that is a ubiquitin conjugating agent and
comprising a ubiquitin moiety+substrate molecule+CA; and [0524] 2)
assaying for the attachment of the ubiquitin moiety to the
substrate molecule. In a preferred embodiment the ubiquitin
ligating agent comprises an Mdm2 protein and the substrate molecule
comprises p53.
[0525] FIG. 50 schematically depicts a preferred embodiment for
assaying for the attachment of ubiquitin moiety to a substrate
molecule (s) where the assay comprises: [0526] 1) combining a first
ubiquitin agent that is a ubiquitin ligating agent ULA.sub.1+a
second ubiquitin agent+a third ubiquitin agent that is a ubiquitin
activating agent+a ubiquitin moiety comprising a first FRET
tag+substrate molecule comprising a second FRET tag+CA; and [0527]
2) assaying for the attachment of the ubiquitin moiety to the
substrate molecule. In a preferred embodiment the ubiquitin
ligating agent comprises an Mdm2 protein and the substrate molecule
comprises p53.
[0528] As depicted in FIG. 51, in a preferred embodiment, the E2
has the amino acid sequence (FIG. 51A) and the nucleic acid
sequence (FIG. 51B).
[0529] As depicted in FIG. 52, in a preferred embodiment, the E2
has the amino acid sequence (FIG. 52A) and the nucleic acid
sequence (FIG. 52B1 and FIG. 52B2 ).
[0530] As depicted in FIG. 53, in a preferred embodiment, the E2
has the amino acid sequence (FIG. 53A) and the nucleic acid
sequence (FIG. 53B1 and FIG. 53B2).
[0531] As depicted in FIG. 54, in a preferred embodiment, the E2
has the amino acid sequence (FIG. 54A) and the nucleic acid
sequence (FIG. 54B).
[0532] As depicted in FIG. 55, in a preferred embodiment, the E2
has the amino acid sequence (FIG. 55A) and the nucleic acid
sequence (FIG. 55B).
Sequence CWU 1
1
28 1 3177 DNA Oryctolagus cuniculus rabbit E1 1 atgtccagct
cgccgctgtc caagaaacgt cgcgtgtccg ggcctgatcc aaagccgggt 60
tctaactgct cccctgccca gtccgtgttg ccccaagtgc cctcggcgcc aaccaacgga
120 atggcgaaga acggcagtga agcagacatc gatgagggcc tttactcccg
gcagctgtat 180 gtgttgggcc atgaggcgat gaagcggctc cagacatcca
gcgttctggt gtcaggcctg 240 cggggcctgg gggtagagat cgcgaagaac
atcatccttg gcggggtcaa ggccgtgacc 300 ctccatgacc agggcacggc
ccagtgggct gacctctcct cccagttcta cctgcgagag 360 gaggacatag
ggaaaaaccg cgctgaggtg tcacagcccc gccttgctga actcaatagc 420
tacgtgcctg tcaccgccta cactgggccg ctggttgagg acttcctcag tggcttccag
480 gtggtggtcc tcactaacag ccccctggag gaccagctgc gcgtgggcga
gttctgtcat 540 agccgtggca tcaagctggt agtggcagac acgagaggct
tgtttgggca actcttctgc 600 gactttggag aggaaatgat cctcacagat
tccaacgggg agcagcccct cagcaccatg 660 gtttctatgg tcaccaagga
caaccctggt gtggttacct gcctggatga ggcccgacat 720 gggtttgaga
gtggcgattt tgtttccttc tccgaagtac agggcatgac tgagctcaat 780
ggaaaccagc ccatagagat caaagtcctg ggtccttaca cctttagcat ctgtgacacc
840 tccaacttct ccgattacat ccgtggaggc attgtcagcc aggtcaaagt
acctaagaag 900 ataagcttta aatccttgtc agcctcgctg gcagagcctg
actttgtgat gacggacttc 960 gccaagtttt ctcgccccgc tcagcttcac
attggcttcc aggccttgca caagttctgt 1020 gcacagcaca gccggccacc
tagaccccgg aacgaggagg atgcagcaga gctggtgacc 1080 ctagcacgcg
ctgtgaactc taaagcctcg tcggcagtgc agcaagatag cctggatgag 1140
gacctcatcc ggaacctggc ctttgtggca gccggggacc tggcgcccat caatgccttc
1200 attgggggcc tggctgccca ggaagtcatg aaggcctgct ctgggaagtt
tatgcccatc 1260 atgcagtggc tgtactttga tgcccttgag tgtctcccgg
aggacaaaga atccctcaca 1320 gaggacaagt gcctcccgcg ccagaaccgt
tatgatgggc aggtggctgt gtttggctca 1380 gacctgcaag agaagctggg
caggcagaag tacttcctgg tgggtgcagg ggctattggc 1440 tgtgagctgc
tcaagaactt tgccatgatt gggctgggct gtggtgagaa cggagaaata 1500
attgtcacag acatggacac cattgagaaa tctaatctga accgacagtt tctattccgg
1560 ccctgggatg tcacgaagtt aaaatctgac acagctgctg cagctgtgca
ccagatgaat 1620 ccacatatcc gggtgacaag ccaccagaac cgtgtgggtc
ctgacactga acgtatctac 1680 gacgacgatt tcttccaaac tctggatggc
gtggccaacg ccttagacaa cgtggatgcc 1740 cgcatgtaca tggaccgccg
ctgcgtgtac taccggaagc cgctgctcga atcaggcacc 1800 ctgggcacca
agggcaacgt ccaggtggtg atccccttcc tgacagagtc ctacagctcc 1860
agccaagacc cacctgagaa gtccatcccc atctgtaccc tgaagaactt ccccaacgcc
1920 atcgaacaca ctcttcagtg ggctcgggat gaatttgaag gcctcttcaa
gcagccagcg 1980 gaaaatgtca accagtacct cacagaccct aagtttgtgg
agcggacatt gcggctggcg 2040 ggtacccagc cactggaggt gctggaggct
gtgcagcgca gcctggtgct gcagctaccg 2100 cagagctggg cagactgtgt
gacctgggcc tgccaccact ggcacaccca gtattctaac 2160 aatatccggc
agctgttgca caacttccct cccgaccagc tcacaagctc gggagctccc 2220
ttctggtctg ggcccaaacg ttgtcctcac ccactcacct ttgatgttag caaccctctg
2280 catctggact atgtgatggc tgctgccaac ctgtttgccc agacctacgg
gctggcaggc 2340 tctcaggacc gagctgctgt ggccacactc ctgcagtctg
tacaggtccc cgagtttacc 2400 cccaagtctg gcgtcaaaat ccacgtttct
gaccaggagc tgcagagcgc caatgcttct 2460 gttgacgaca gccgtttaga
ggagctcaag gctacgctgc ctagccccga caagctccct 2520 ggattcaaga
tgtaccccat tgactttgag aaggatgatg atagtaactt tcacatggac 2580
ttcattgtgg ccgcatccaa cctccgggcc gaaaactatg acattccccc tgcagaccgg
2640 cacaagagca agctgattgc agggaagatc atcccagcca ttgccacgac
cacagcagct 2700 gtcgttggcc ttgtgtgtct ggagctgtac aaggtagtgc
agggacaccg acacctcgac 2760 tcctacaaga atggtttcct caacctggcc
ctgccgtttt tcggtttctc tgaacctctg 2820 gctgcaccac gtcaccagta
ctataaccaa gagtggacat tgtgggatcg ctttgaggtt 2880 cagggactgc
agcccaacgg tgaggagatg accctcaaac aattcctcga ctactttaag 2940
acagagcaca aattggagat taccatgctg tcccagggtg tgtccatgct ctattccttc
3000 tttatgccag ctgcgaagct caaggaacgg ttggaccagc cgatgacaga
gattgtaagc 3060 cgtgtgtcga agcgaaagct gggccgccac gtgcgggcgc
tggtgcttga gctgtgctgc 3120 aacgacgaga gcggcgagga cgtcgaagtc
ccctacgtcc gatataccat ccgttaa 3177 2 1058 PRT Oryctolagus cuniculus
rabbit E1 2 Met Ser Ser Ser Pro Leu Ser Lys Lys Arg Arg Val Ser Gly
Pro Asp 1 5 10 15 Pro Lys Pro Gly Ser Asn Cys Ser Pro Ala Gln Ser
Val Leu Pro Gln 20 25 30 Val Pro Ser Ala Pro Thr Asn Gly Met Ala
Lys Asn Gly Ser Glu Ala 35 40 45 Asp Ile Asp Glu Gly Leu Tyr Ser
Arg Gln Leu Tyr Val Leu Gly His 50 55 60 Glu Ala Met Lys Arg Leu
Gln Thr Ser Ser Val Leu Val Ser Gly Leu 65 70 75 80 Arg Gly Leu Gly
Val Glu Ile Ala Lys Asn Ile Ile Leu Gly Gly Val 85 90 95 Lys Ala
Val Thr Leu His Asp Gln Gly Thr Ala Gln Trp Ala Asp Leu 100 105 110
Ser Ser Gln Phe Tyr Leu Arg Glu Glu Asp Ile Gly Lys Asn Arg Ala 115
120 125 Glu Val Ser Gln Pro Arg Leu Ala Glu Leu Asn Ser Tyr Val Pro
Val 130 135 140 Thr Ala Tyr Thr Gly Pro Leu Val Glu Asp Phe Leu Ser
Gly Phe Gln 145 150 155 160 Val Val Val Leu Thr Asn Ser Pro Leu Glu
Asp Gln Leu Arg Val Gly 165 170 175 Glu Phe Cys His Ser Arg Gly Ile
Lys Leu Val Val Ala Asp Thr Arg 180 185 190 Gly Leu Phe Gly Gln Leu
Phe Cys Asp Phe Gly Glu Glu Met Ile Leu 195 200 205 Thr Asp Ser Asn
Gly Glu Gln Pro Leu Ser Thr Met Val Ser Met Val 210 215 220 Thr Lys
Asp Asn Pro Gly Val Val Thr Cys Leu Asp Glu Ala Arg His 225 230 235
240 Gly Phe Glu Ser Gly Asp Phe Val Ser Phe Ser Glu Val Gln Gly Met
245 250 255 Thr Glu Leu Asn Gly Asn Gln Pro Ile Glu Ile Lys Val Leu
Gly Pro 260 265 270 Tyr Thr Phe Ser Ile Cys Asp Thr Ser Asn Phe Ser
Asp Tyr Ile Arg 275 280 285 Gly Gly Ile Val Ser Gln Val Lys Val Pro
Lys Lys Ile Ser Phe Lys 290 295 300 Ser Leu Ser Ala Ser Leu Ala Glu
Pro Asp Phe Val Met Thr Asp Phe 305 310 315 320 Ala Lys Phe Ser Arg
Pro Ala Gln Leu His Ile Gly Phe Gln Ala Leu 325 330 335 His Lys Phe
Cys Ala Gln His Ser Arg Pro Pro Arg Pro Arg Asn Glu 340 345 350 Glu
Asp Ala Ala Glu Leu Val Thr Leu Ala Arg Ala Val Asn Ser Lys 355 360
365 Ala Ser Ser Ala Val Gln Gln Asp Ser Leu Asp Glu Asp Leu Ile Arg
370 375 380 Asn Leu Ala Phe Val Ala Ala Gly Asp Leu Ala Pro Ile Asn
Ala Phe 385 390 395 400 Ile Gly Gly Leu Ala Ala Gln Glu Val Met Lys
Ala Cys Ser Gly Lys 405 410 415 Phe Met Pro Ile Met Gln Trp Leu Tyr
Phe Asp Ala Leu Glu Cys Leu 420 425 430 Pro Glu Asp Lys Glu Ser Leu
Thr Glu Asp Lys Cys Leu Pro Arg Gln 435 440 445 Asn Arg Tyr Asp Gly
Gln Val Ala Val Phe Gly Ser Asp Leu Gln Glu 450 455 460 Lys Leu Gly
Arg Gln Lys Tyr Phe Leu Val Gly Ala Gly Ala Ile Gly 465 470 475 480
Cys Glu Leu Leu Lys Asn Phe Ala Met Ile Gly Leu Gly Cys Gly Glu 485
490 495 Asn Gly Glu Ile Ile Val Thr Asp Met Asp Thr Ile Glu Lys Ser
Asn 500 505 510 Leu Asn Arg Gln Phe Leu Phe Arg Pro Trp Asp Val Thr
Lys Leu Lys 515 520 525 Ser Asp Thr Ala Ala Ala Ala Val His Gln Met
Asn Pro His Ile Arg 530 535 540 Val Thr Ser His Gln Asn Arg Val Gly
Pro Asp Thr Glu Arg Ile Tyr 545 550 555 560 Asp Asp Asp Phe Phe Gln
Thr Leu Asp Gly Val Ala Asn Ala Leu Asp 565 570 575 Asn Val Asp Ala
Arg Met Tyr Met Asp Arg Arg Cys Val Tyr Tyr Arg 580 585 590 Lys Pro
Leu Leu Glu Ser Gly Thr Leu Gly Thr Lys Gly Asn Val Gln 595 600 605
Val Val Ile Pro Phe Leu Thr Glu Ser Tyr Ser Ser Ser Gln Asp Pro 610
615 620 Pro Glu Lys Ser Ile Pro Ile Cys Thr Leu Lys Asn Phe Pro Asn
Ala 625 630 635 640 Ile Glu His Thr Leu Gln Trp Ala Arg Asp Glu Phe
Glu Gly Leu Phe 645 650 655 Lys Gln Pro Ala Glu Asn Val Asn Gln Tyr
Leu Thr Asp Pro Lys Phe 660 665 670 Val Glu Arg Thr Leu Arg Leu Ala
Gly Thr Gln Pro Leu Glu Val Leu 675 680 685 Glu Ala Val Gln Arg Ser
Leu Val Leu Gln Leu Pro Gln Ser Trp Ala 690 695 700 Asp Cys Val Thr
Trp Ala Cys His His Trp His Thr Gln Tyr Ser Asn 705 710 715 720 Asn
Ile Arg Gln Leu Leu His Asn Phe Pro Pro Asp Gln Leu Thr Ser 725 730
735 Ser Gly Ala Pro Phe Trp Ser Gly Pro Lys Arg Cys Pro His Pro Leu
740 745 750 Thr Phe Asp Val Ser Asn Pro Leu His Leu Asp Tyr Val Met
Ala Ala 755 760 765 Ala Asn Leu Phe Ala Gln Thr Tyr Gly Leu Ala Gly
Ser Gln Asp Arg 770 775 780 Ala Ala Val Ala Thr Leu Leu Gln Ser Val
Gln Val Pro Glu Phe Thr 785 790 795 800 Pro Lys Ser Gly Val Lys Ile
His Val Ser Asp Gln Glu Leu Gln Ser 805 810 815 Ala Asn Ala Ser Val
Asp Asp Ser Arg Leu Glu Glu Leu Lys Ala Thr 820 825 830 Leu Pro Ser
Pro Asp Lys Leu Pro Gly Phe Lys Met Tyr Pro Ile Asp 835 840 845 Phe
Glu Lys Asp Asp Asp Ser Asn Phe His Met Asp Phe Ile Val Ala 850 855
860 Ala Ser Asn Leu Arg Ala Glu Asn Tyr Asp Ile Pro Pro Ala Asp Arg
865 870 875 880 His Lys Ser Lys Leu Ile Ala Gly Lys Ile Ile Pro Ala
Ile Ala Thr 885 890 895 Thr Thr Ala Ala Val Val Gly Leu Val Cys Leu
Glu Leu Tyr Lys Val 900 905 910 Val Gln Gly His Arg His Leu Asp Ser
Tyr Lys Asn Gly Phe Leu Asn 915 920 925 Leu Ala Leu Pro Phe Phe Gly
Phe Ser Glu Pro Leu Ala Ala Pro Arg 930 935 940 His Gln Tyr Tyr Asn
Gln Glu Trp Thr Leu Trp Asp Arg Phe Glu Val 945 950 955 960 Gln Gly
Leu Gln Pro Asn Gly Glu Glu Met Thr Leu Lys Gln Phe Leu 965 970 975
Asp Tyr Phe Lys Thr Glu His Lys Leu Glu Ile Thr Met Leu Ser Gln 980
985 990 Gly Val Ser Met Leu Tyr Ser Phe Phe Met Pro Ala Ala Lys Leu
Lys 995 1000 1005 Glu Arg Leu Asp Gln Pro Met Thr Glu Ile Val Ser
Arg Val Ser Lys 1010 1015 1020 Arg Lys Leu Gly Arg His Val Arg Ala
Leu Val Leu Glu Leu Cys Cys 1025 1030 1035 1040 Asn Asp Glu Ser Gly
Glu Asp Val Glu Val Pro Tyr Val Arg Tyr Thr 1045 1050 1055 Ile Arg
3 444 DNA Homo sapiens E2 Ubc5c 3 atggcgctga aacggattaa taaggaactt
agtgatttgg cccgtgaccc tccagcacaa 60 tgttctgcag gtccagttgg
ggatgatatg tttcattggc aagccacaat tatgggacct 120 aatgacagcc
catatcaagg cggtgtattc tttttgacaa ttcattttcc tacagactac 180
cccttcaaac cacctaaggt tgcatttaca acaagaattt atcatccaaa tattaacagt
240 aatggcagca tttgtctcga tattctaaga tcacagtggt cgcctgcttt
aacaatttct 300 aaagttcttt tatccatttg ttcactgcta tgtgatccaa
acccagatga ccccctagtg 360 ccagagattg cacggatcta taaaacagac
agagataagt acaacagaat atctcgggaa 420 tggactcaga agtatgccat gtga 444
4 147 PRT Homo sapiens E2 Ubc5c 4 Met Ala Leu Lys Arg Ile Asn Lys
Glu Leu Ser Asp Leu Ala Arg Asp 1 5 10 15 Pro Pro Ala Gln Cys Ser
Ala Gly Pro Val Gly Asp Asp Met Phe His 20 25 30 Trp Gln Ala Thr
Ile Met Gly Pro Asn Asp Ser Pro Tyr Gln Gly Gly 35 40 45 Val Phe
Phe Leu Thr Ile His Phe Pro Thr Asp Tyr Pro Phe Lys Pro 50 55 60
Pro Lys Val Ala Phe Thr Thr Arg Ile Tyr His Pro Asn Ile Asn Ser 65
70 75 80 Asn Gly Ser Ile Cys Leu Asp Ile Leu Arg Ser Gln Trp Ser
Pro Ala 85 90 95 Leu Thr Ile Ser Lys Val Leu Leu Ser Ile Cys Ser
Leu Leu Cys Asp 100 105 110 Pro Asn Pro Asp Asp Pro Leu Val Pro Glu
Ile Ala Arg Ile Tyr Lys 115 120 125 Thr Asp Arg Asp Lys Tyr Asn Arg
Ile Ser Arg Glu Trp Thr Gln Lys 130 135 140 Tyr Ala Met 145 5 84
PRT Homo sapiens RING finger protein APC11 5 Met Lys Val Lys Ile
Lys Cys Trp Asn Gly Val Ala Thr Trp Leu Trp 1 5 10 15 Val Ala Asn
Asp Glu Asn Cys Gly Ile Cys Arg Met Ala Phe Asn Gly 20 25 30 Cys
Cys Pro Asp Cys Lys Val Pro Gly Asp Asp Cys Pro Leu Val Trp 35 40
45 Gly Gln Cys Ser His Cys Phe His Met His Cys Ile Leu Lys Trp Leu
50 55 60 His Ala Gln Gln Val Gln Gln His Cys Pro Met Cys Arg Gln
Thr Trp 65 70 75 80 Lys Phe Lys Glu 6 108 PRT Homo sapiens RING
finger protein ROC1 6 Met Ala Ala Ala Met Asp Val Asp Thr Pro Ser
Gly Thr Asn Ser Gly 1 5 10 15 Ala Gly Lys Lys Arg Phe Glu Val Lys
Lys Trp Asn Ala Val Ala Leu 20 25 30 Trp Ala Trp Asp Ile Val Val
Asp Asn Cys Ala Ile Cys Arg Asn His 35 40 45 Ile Met Asp Leu Cys
Ile Glu Cys Gln Ala Asn Gln Ala Ser Ala Thr 50 55 60 Ser Glu Glu
Cys Thr Val Ala Trp Gly Val Cys Asn His Ala Phe His 65 70 75 80 Phe
His Cys Ile Ser Arg Trp Leu Lys Thr Arg Gln Val Cys Pro Leu 85 90
95 Asp Asn Arg Glu Trp Glu Phe Gln Lys Tyr Gly His 100 105 7 342
DNA Homo sapiens RING finger protein ROC2 7 atggccgacg tggaagacgg
agaggaaacc tgcgccctgg cctctcactc cgggagctca 60 ggctcaacgt
cgggaggcga caagatgttc tccctcaaga agtggaaccc ggtggccatg 120
tggagctggg acgtggagtg cgatacgtgc gccatctgca gggtccaggt gatggatgcc
180 tgtcttagat gtcaagctga aaacaaacaa gaggactgtg ttgtggtctg
gggagaatgt 240 aatcattcct tccacaactg ctgcatgtcc ctgtgggtga
aacagaacaa tcgctgccct 300 ctctgccagc aggactgggt ggtccaaaga
atcggcaaat ga 342 8 113 PRT Homo sapiens RING finger protein ROC2 8
Met Ala Asp Val Glu Asp Gly Glu Glu Thr Cys Ala Leu Ala Ser His 1 5
10 15 Ser Gly Ser Ser Gly Ser Thr Ser Gly Gly Asp Lys Met Phe Ser
Leu 20 25 30 Lys Lys Trp Asn Pro Val Ala Met Trp Ser Trp Asp Val
Glu Cys Asp 35 40 45 Thr Cys Ala Ile Cys Arg Val Gln Val Met Asp
Ala Cys Leu Arg Cys 50 55 60 Gln Ala Glu Asn Lys Gln Glu Asp Cys
Val Val Val Trp Gly Glu Cys 65 70 75 80 Asn His Ser Phe His Asn Cys
Cys Met Ser Leu Trp Val Lys Gln Asn 85 90 95 Asn Arg Cys Pro Leu
Cys Gln Gln Asp Trp Val Val Gln Arg Ile Gly 100 105 110 Lys 9 2343
DNA Homo sapiens Cullin CUL5 9 atggcgacgt ctaatctgtt aaagaataaa
ggttctcttc agtttgaaga caaatgggat 60 tttatgcgcc cgattgtttt
gaagctttta cgccaggaat ctgttacaaa acagcagtgg 120 tttgatctgt
tttcggatgt gcatgcagtc tgtctttggg atgataaagg cccagcaaaa 180
attcatcagg ctttaaaaga agatattctt gagtttatta agcaggcaca ggcacgagta
240 ctgagccatc aagatgatac ggctttgcta aaagcatata ttgttgaatg
gcgaaagttc 300 tttacacaat gtgatatttt accaaaacct ttttgtcaac
tagagattac tttaatgggt 360 aaacagggca gcaataaaaa atcaaatgtg
gaagacagta ttgttcgaaa gcttatgctt 420 gatacatgga atgagtcaat
cttttcaaac ataaaaaaca gactccaaga tagtgcaatg 480 aagctggtac
atgctgagag attgggagaa gcttttgatt ctcagctggt tattggagta 540
agagaatcct atgttaacct ttgttctaat cctgaggata aacttcaaat ttatagggac
600 aattttgaga aggcatactt ggattcaaca gagagatttt atagaacaca
agcaccctcg 660 tatttacaac caaatggtgt acagaattat atgaaatatg
cagatgctaa attaaaagaa 720 gaagaaaaac gagcactacg ttatttagaa
acaagacgag aatgtaactc cgttgaagca 780 ctcatggaat gctgtgtaaa
tgccctggtg acatcattta aagagactat cttagctgag 840 tgccaaggca
tgatcaagag aaatgaaact gaaaaattac atttaatgtt ttcattgatg 900
gacaaagttc ctaatggtat agagccaatg ttgaaagact tggaggaaca tatcattagt
960 gctggcctgg cagatatggt agcagctgct gaaactatta ctactgactc
tgagaaatac 1020 gttgagcagt tacttacact atttaataga tttagtaaac
tcgtcaaaga agcttttcaa 1080 gatgatccac gatttcttac tgcaagagat
aaggcgtata aagcagttgt taatgatgct 1140 accatattta aacttgaatt
acctttgaag cagaaggggg tgggattaaa aactcagcct 1200 gaatcaaaat
gccctgagct gcttgccaat tactgtgaca tgttgctaag aaaaacacca 1260
ttaagcaaaa aactaacctc tgaagagatt gaagcaaagc ttaaagaagt gctcttggta
1320 cttaagtatg tacagaacaa agatgttttt atgaggtatc ataaagctca
tttgacacga 1380 cgtcttatat tagacatctc tgccgatagt
gaaattgaag aaaacatggt agagtggcta 1440 agagaagttg gtatgccagc
ggattatgta aacaagcttg ctagaatgtt tcaggacata 1500 aaagtatctg
aagatttgaa ccaagctttt aaggaaatgc acaaaaataa taaattggca 1560
ttaccagctg attcagttaa tataaaaatt ctgaatgctg gcgcctggtc aagaagttct
1620 gagaaagtct ttgtctcact tcctactgaa ctggaggact tgataccgga
agtagaagaa 1680 ttctacaaaa aaaatcatag tggtagaaaa ttacattggc
atcatctcat gtcaaatgga 1740 attataacat ttaagaatga agttggtcaa
tatgatttgg aggtaaccac gtttcagctc 1800 gctgtattgt ttgcatggaa
ccaaagaccc agagagaaaa tcagctttga aaatcttaag 1860 cttgcaactg
aactccctga tgctgaactt aggaggactt tatggtcttt agtagctttc 1920
ccaaaactca aacggcaagt ttttttgtat gaccctcaag tcaactcacc caaagacttt
1980 acagaaggta ccctcttctc agtgaaccag gagttcagtt taataaaaaa
tgcaaaggtt 2040 cagaaaaggg gtaaaatcaa cttgattgga cgtttgcagc
tcactacaga aaggatgaga 2100 gaagaagaga atgaaggaat agttcaacta
cgaatactaa gaacccagga agctatcata 2160 caaataatga aaatgagaaa
gaaaattagt aatgctcagc tgcagactga attagtagaa 2220 attttgaaaa
acatgttctt gccacaaaag aaaatgataa aagagcaaat agagtggcta 2280
atagagcaca aatacatcag aagagatgaa tctgatatca acactttcat atatatggca
2340 taa 2343 10 861 PRT Homo sapiens Cullin CUL5 10 Met Arg Ser
Phe Ala Trp Gly Ser Ser Gly Asp His Val Gly Asp Lys 1 5 10 15 Ser
Glu Glu Ala Pro Gly Ala Trp Asp Glu Val Ser Ala Val Gly Ala 20 25
30 Leu Leu Gln Arg Pro Pro His Pro Gly Ala Gly Pro Thr Gly Pro Gly
35 40 45 Pro Trp Trp Glu Leu Arg Pro Pro Val Lys Ala Trp Pro Gly
Arg Glu 50 55 60 Arg His Glu Phe Ser Arg Arg Leu Val Ser Arg Glu
Ser Lys Leu Lys 65 70 75 80 Asn Met Ala Thr Ser Asn Leu Leu Lys Asn
Lys Gly Ser Leu Gln Phe 85 90 95 Glu Asp Lys Trp Asp Phe Met Arg
Pro Ile Val Leu Lys Leu Leu Arg 100 105 110 Gln Glu Ser Val Thr Lys
Gln Gln Trp Phe Asp Leu Phe Ser Asp Val 115 120 125 His Ala Val Cys
Leu Trp Asp Asp Lys Gly Pro Ala Lys Ile His Gln 130 135 140 Ala Leu
Lys Glu Asp Ile Leu Glu Phe Ile Lys Gln Ala Gln Ala Arg 145 150 155
160 Val Leu Ser His Gln Asp Asp Thr Ala Leu Leu Lys Ala Tyr Ile Val
165 170 175 Glu Trp Arg Lys Phe Phe Thr Gln Cys Asp Ile Leu Pro Lys
Pro Phe 180 185 190 Cys Gln Leu Glu Ile Thr Leu Met Gly Lys Gln Gly
Ser Asn Lys Lys 195 200 205 Ser Asn Val Glu Asp Ser Ile Val Arg Lys
Leu Met Leu Asp Thr Trp 210 215 220 Asn Glu Ser Ile Phe Ser Asn Ile
Lys Asn Arg Leu Gln Asp Ser Ala 225 230 235 240 Met Lys Leu Val His
Ala Glu Arg Leu Gly Glu Ala Phe Asp Ser Gln 245 250 255 Leu Val Ile
Gly Val Arg Glu Ser Tyr Val Asn Leu Cys Ser Asn Pro 260 265 270 Glu
Asp Lys Leu Gln Ile Tyr Arg Asp Asn Phe Glu Lys Ala Tyr Leu 275 280
285 Asp Ser Thr Glu Arg Phe Tyr Arg Thr Gln Ala Pro Ser Tyr Leu Gln
290 295 300 Pro Asn Gly Val Gln Asn Tyr Met Lys Tyr Ala Asp Ala Lys
Leu Lys 305 310 315 320 Glu Glu Glu Lys Arg Ala Leu Arg Tyr Leu Glu
Thr Arg Arg Glu Cys 325 330 335 Asn Ser Val Glu Ala Leu Met Glu Cys
Cys Val Asn Ala Leu Val Thr 340 345 350 Ser Phe Lys Glu Thr Ile Leu
Ala Glu Cys Gln Gly Met Ile Lys Arg 355 360 365 Asn Glu Thr Glu Lys
Leu His Leu Met Phe Ser Leu Met Asp Lys Val 370 375 380 Pro Asn Gly
Ile Glu Pro Met Leu Lys Asp Leu Glu Glu His Ile Ile 385 390 395 400
Ser Ala Gly Leu Ala Asp Met Val Ala Ala Ala Glu Thr Ile Thr Thr 405
410 415 Asp Ser Glu Lys Tyr Val Glu Gln Leu Leu Thr Leu Phe Asn Arg
Phe 420 425 430 Ser Lys Leu Val Lys Glu Ala Phe Gln Asp Asp Pro Arg
Phe Leu Thr 435 440 445 Ala Arg Asp Lys Ala Tyr Lys Ala Val Val Asn
Asp Ala Thr Ile Phe 450 455 460 Lys Leu Glu Leu Pro Leu Lys Gln Lys
Gly Val Gly Leu Lys Thr Gln 465 470 475 480 Pro Glu Ser Lys Cys Pro
Glu Leu Leu Ala Asn Tyr Cys Asp Met Leu 485 490 495 Leu Arg Lys Thr
Pro Leu Ser Lys Lys Leu Thr Ser Glu Glu Ile Glu 500 505 510 Ala Lys
Leu Lys Glu Val Leu Leu Val Leu Lys Tyr Val Gln Asn Lys 515 520 525
Asp Val Phe Met Arg Tyr His Lys Ala His Leu Thr Arg Arg Leu Ile 530
535 540 Leu Asp Ile Ser Ala Asp Ser Glu Ile Glu Glu Asn Met Val Glu
Trp 545 550 555 560 Leu Arg Glu Val Gly Met Pro Ala Asp Tyr Val Asn
Lys Leu Ala Arg 565 570 575 Met Phe Gln Asp Ile Lys Val Ser Glu Asp
Leu Asn Gln Ala Phe Lys 580 585 590 Glu Met His Lys Asn Asn Lys Leu
Ala Leu Pro Ala Asp Ser Val Asn 595 600 605 Ile Lys Ile Leu Asn Ala
Gly Ala Trp Ser Arg Ser Ser Glu Lys Val 610 615 620 Phe Val Ser Leu
Pro Thr Glu Leu Glu Asp Leu Ile Pro Glu Val Glu 625 630 635 640 Glu
Phe Tyr Lys Lys Asn His Ser Gly Arg Lys Leu His Trp His His 645 650
655 Leu Met Ser Asn Gly Ile Ile Thr Phe Lys Asn Glu Val Gly Gln Tyr
660 665 670 Asp Leu Glu Val Thr Thr Phe Gln Leu Ala Val Leu Phe Ala
Trp Asn 675 680 685 Gln Arg Pro Arg Glu Lys Ile Ser Phe Glu Asn Leu
Lys Leu Ala Thr 690 695 700 Glu Leu Pro Asp Ala Glu Leu Arg Arg Thr
Leu Trp Ser Leu Val Ala 705 710 715 720 Phe Pro Lys Leu Lys Arg Gln
Val Phe Leu Tyr Asp Pro Gln Val Asn 725 730 735 Ser Pro Lys Asp Phe
Thr Glu Gly Thr Leu Phe Ser Val Asn Gln Glu 740 745 750 Phe Ser Leu
Ile Lys Asn Ala Lys Val Gln Lys Arg Gly Lys Ile Asn 755 760 765 Leu
Ile Gly Arg Leu Gln Leu Thr Thr Glu Arg Met Arg Glu Glu Glu 770 775
780 Asn Glu Gly Ile Val Gln Leu Arg Ile Leu Arg Thr Gln Glu Ala Ile
785 790 795 800 Ile Gln Ile Met Lys Met Arg Lys Lys Ile Ser Asn Ala
Gln Leu Gln 805 810 815 Thr Glu Leu Val Glu Ile Leu Lys Asn Met Phe
Leu Pro Gln Lys Lys 820 825 830 Met Ile Lys Glu Gln Ile Glu Trp Leu
Ile Glu His Lys Tyr Ile Arg 835 840 845 Arg Asp Glu Ser Asp Ile Asn
Thr Phe Ile Tyr Met Ala 850 855 860 11 2469 DNA Homo sapiens Cullin
APC2 11 atggcggcgg cagttgtggt ggcggagggg gacagcgact cccggcccgg
acaggagttg 60 ttagtggcct ggaacaccgt gagcaccggc ctggtgccgc
cggctgcgct ggggctggtg 120 tcttcccgga ccagcggtgc agtcccgcca
aaggaagagg agctccgggc ggcggtggag 180 gttctgaggg gccacgggct
acactcggtc ctggaggagt ggttcgtgga ggtgctgcag 240 aacgatctgc
aggccaacat ctcccctgag ttctggaatg ccatctccca atgcgagaac 300
tctgcggatg agccccagtg ccttttgcta ctccttgacg cttttggcct gctggagagc
360 cgcctggatc cctacctgcg tagcctagag ctgctggaga aatggactcg
cctgggcttg 420 ctgatgggca ctggtgctca ggggctgcga gaagaagtcc
acactatgtt gcgcggagtc 480 ttgttcttta gcacccccag aaccttccaa
gagatgatcc agcgtctgta tgggtgcttc 540 ttgagagtct atatgcagag
taagaggaag ggggaagggg gcacagaccc ggaactggaa 600 ggggagctgg
acagccggta tgcccgtcgc cggtactacc ggctcctgca gagcccgctg 660
tgtgcagggt gcagcagtga caagcaacag tgctggtgtc gccaggctct ggagcagttc
720 catcagctca gccaggtctt acacaggctc agtctgctgg agcgggtcag
tgccgaggct 780 gtgaccacca ccctgcacca ggtgacccgg gagaggatgg
aggaccgttg ccggggcgag 840 tacgagcgct ccttcctgcg tgagttccac
aagtggatcg agcgggtggt cggctggctc 900 ggcaaggtgt tcctgcagga
cggccccgcc aggcccgcat ctcccgaggc cggcaacacc 960 ctgcgccgct
ggcgctgcca cgtgcaaagg ttcttctacc gcatctacgc cagcctgcgc 1020
atcgaggagc tcttcagcat cgtccgagac ttcccagact cccggccagc catcgaggac
1080 ctcaagtact gcctggagag gacggaccag aggcagcagc tgctcgtgtc
cctcaaggct 1140 gccctggaga ctcggctcct gcatccaggc gtcaacacgt
gtgacatcat caccctctat 1200 atctctgcca tcaaggcgct gcgcgtgctg
gacccttcca tggtcatcct ggaggtggcc 1260 tgtgagccta tccgccgcta
cctgaggacg cgggaggaca cagtgcggca gattgtggct 1320 gggctgacgg
gggactcgga cgggacaggg gacctggctg ttgagctgtc caagaccgac 1380
ccggcgagcc tggagacagg ccaggacagt gaggatgact caggcgagcc agaggactgg
1440 gtcccggacc ctgtggatgc cgatccaggg aagtcgagct ccaagcggcg
ttcatcggac 1500 atcatcagcc tgctggtcag catctacggc agcaaggacc
tcttcatcaa tgagtaccgc 1560 tcgctgctgg ccgaccgcct gctgcaccag
ttcagcttca gccccgagcg ggagatccgc 1620 aacgtggagc tgctgaagct
gcgctttggc gaggccccaa tgcacttctg tgaagtcatg 1680 ctgaaggaca
tggcggactc ccgccgcatc aatgccaaca tccgggagga ggatgagaag 1740
cggccagcag aggagcagcc accgttcggg gtctacgctg tcatcctgtc cagtgagttc
1800 tggccgccct tcaaggacga gaagctggag gtccccgagg atatcagggc
agccctggag 1860 gcttactgca agaagtatga gcagctcaag gccatgcgga
ccctcagttg gaagcacacc 1920 ctgggcctgg tgaccatgga cgtggagctg
gccgaccgca cgctgtctgt ggcggtcacc 1980 ccagtacagg cggtgatctt
gctgtatttt caggaccaag ccagctggac cctggaggaa 2040 ctgagcaagg
cggtgaaaat gcccgtggcg ctgctgcggc ggcggatgtc cgtgtggctg 2100
cagcagggtg tgctgcgtga ggagcccccc ggcaccttct ctgtcattga ggaggagcgg
2160 cctcaggacc gggacaacat ggtgctcatt gacagtgacg acgagagcga
ctccggcatg 2220 gcctcccagg ccgaccagaa ggaggaggag ctgctgctct
tctggacgta catccaggcc 2280 atgctgacca acctggagag cctctcactg
gatcgtatct acaacatgct ccgcatgttt 2340 gtggtgactg ggcctgcact
ggccgagatt gacctgcagg agctgcaggg ctacctgcag 2400 aagaaggtgc
gggaccagca gctcgtctac tcggccggcg tctaccgcct gcccaagaac 2460
tgcagctga 2469 12 822 PRT Homo sapiens Cullin APC2 12 Met Ala Ala
Ala Val Val Val Ala Glu Gly Asp Ser Asp Ser Arg Pro 1 5 10 15 Gly
Gln Glu Leu Leu Val Ala Trp Asn Thr Val Ser Thr Gly Leu Val 20 25
30 Pro Pro Ala Ala Leu Gly Leu Val Ser Ser Arg Thr Ser Gly Ala Val
35 40 45 Pro Pro Lys Glu Glu Glu Leu Arg Ala Ala Val Glu Val Leu
Arg Gly 50 55 60 His Gly Leu His Ser Val Leu Glu Glu Trp Phe Val
Glu Val Leu Gln 65 70 75 80 Asn Asp Leu Gln Ala Asn Ile Ser Pro Glu
Phe Trp Asn Ala Ile Ser 85 90 95 Gln Cys Glu Asn Ser Ala Asp Glu
Pro Gln Cys Leu Leu Leu Leu Leu 100 105 110 Asp Ala Phe Gly Leu Leu
Glu Ser Arg Leu Asp Pro Tyr Leu Arg Ser 115 120 125 Leu Glu Leu Leu
Glu Lys Trp Thr Arg Leu Gly Leu Leu Met Gly Thr 130 135 140 Gly Ala
Gln Gly Leu Arg Glu Glu Val His Thr Met Leu Arg Gly Val 145 150 155
160 Leu Phe Phe Ser Thr Pro Arg Thr Phe Gln Glu Met Ile Gln Arg Leu
165 170 175 Tyr Gly Cys Phe Leu Arg Val Tyr Met Gln Ser Lys Arg Lys
Gly Glu 180 185 190 Gly Gly Thr Asp Pro Glu Leu Glu Gly Glu Leu Asp
Ser Arg Tyr Ala 195 200 205 Arg Arg Arg Tyr Tyr Arg Leu Leu Gln Ser
Pro Leu Cys Ala Gly Cys 210 215 220 Ser Ser Asp Lys Gln Gln Cys Trp
Cys Arg Gln Ala Leu Glu Gln Phe 225 230 235 240 His Gln Leu Ser Gln
Val Leu His Arg Leu Ser Leu Leu Glu Arg Val 245 250 255 Ser Ala Glu
Ala Val Thr Thr Thr Leu His Gln Val Thr Arg Glu Arg 260 265 270 Met
Glu Asp Arg Cys Arg Gly Glu Tyr Glu Arg Ser Phe Leu Arg Glu 275 280
285 Phe His Lys Trp Ile Glu Arg Val Val Gly Trp Leu Gly Lys Val Phe
290 295 300 Leu Gln Asp Gly Pro Ala Arg Pro Ala Ser Pro Glu Ala Gly
Asn Thr 305 310 315 320 Leu Arg Arg Trp Arg Cys His Val Gln Arg Phe
Phe Tyr Arg Ile Tyr 325 330 335 Ala Ser Leu Arg Ile Glu Glu Leu Phe
Ser Ile Val Arg Asp Phe Pro 340 345 350 Asp Ser Arg Pro Ala Ile Glu
Asp Leu Lys Tyr Cys Leu Glu Arg Thr 355 360 365 Asp Gln Arg Gln Gln
Leu Leu Val Ser Leu Lys Ala Ala Leu Glu Thr 370 375 380 Arg Leu Leu
His Pro Gly Val Asn Thr Cys Asp Ile Ile Thr Leu Tyr 385 390 395 400
Ile Ser Ala Ile Lys Ala Leu Arg Val Leu Asp Pro Ser Met Val Ile 405
410 415 Leu Glu Val Ala Cys Glu Pro Ile Arg Arg Tyr Leu Arg Thr Arg
Glu 420 425 430 Asp Thr Val Arg Gln Ile Val Ala Gly Leu Thr Gly Asp
Ser Asp Gly 435 440 445 Thr Gly Asp Leu Ala Val Glu Leu Ser Lys Thr
Asp Pro Ala Ser Leu 450 455 460 Glu Thr Gly Gln Asp Ser Glu Asp Asp
Ser Gly Glu Pro Glu Asp Trp 465 470 475 480 Val Pro Asp Pro Val Asp
Ala Asp Pro Gly Lys Ser Ser Ser Lys Arg 485 490 495 Arg Ser Ser Asp
Ile Ile Ser Leu Leu Val Ser Ile Tyr Gly Ser Lys 500 505 510 Asp Leu
Phe Ile Asn Glu Tyr Arg Ser Leu Leu Ala Asp Arg Leu Leu 515 520 525
His Gln Phe Ser Phe Ser Pro Glu Arg Glu Ile Arg Asn Val Glu Leu 530
535 540 Leu Lys Leu Arg Phe Gly Glu Ala Pro Met His Phe Cys Glu Val
Met 545 550 555 560 Leu Lys Asp Met Ala Asp Ser Arg Arg Ile Asn Ala
Asn Ile Arg Glu 565 570 575 Glu Asp Glu Lys Arg Pro Ala Glu Glu Gln
Pro Pro Phe Gly Val Tyr 580 585 590 Ala Val Ile Leu Ser Ser Glu Phe
Trp Pro Pro Phe Lys Asp Glu Lys 595 600 605 Leu Glu Val Pro Glu Asp
Ile Arg Ala Ala Leu Glu Ala Tyr Cys Lys 610 615 620 Lys Tyr Glu Gln
Leu Lys Ala Met Arg Thr Leu Ser Trp Lys His Thr 625 630 635 640 Leu
Gly Leu Val Thr Met Asp Val Glu Leu Ala Asp Arg Thr Leu Ser 645 650
655 Val Ala Val Thr Pro Val Gln Ala Val Ile Leu Leu Tyr Phe Gln Asp
660 665 670 Gln Ala Ser Trp Thr Leu Glu Glu Leu Ser Lys Ala Val Lys
Met Pro 675 680 685 Val Ala Leu Leu Arg Arg Arg Met Ser Val Trp Leu
Gln Gln Gly Val 690 695 700 Leu Arg Glu Glu Pro Pro Gly Thr Phe Ser
Val Ile Glu Glu Glu Arg 705 710 715 720 Pro Gln Asp Arg Asp Asn Met
Val Leu Ile Asp Ser Asp Asp Glu Ser 725 730 735 Asp Ser Gly Met Ala
Ser Gln Ala Asp Gln Lys Glu Glu Glu Leu Leu 740 745 750 Leu Phe Trp
Thr Tyr Ile Gln Ala Met Leu Thr Asn Leu Glu Ser Leu 755 760 765 Ser
Leu Asp Arg Ile Tyr Asn Met Leu Arg Met Phe Val Val Thr Gly 770 775
780 Pro Ala Leu Ala Glu Ile Asp Leu Gln Glu Leu Gln Gly Tyr Leu Gln
785 790 795 800 Lys Lys Val Arg Asp Gln Gln Leu Val Tyr Ser Ala Gly
Val Tyr Arg 805 810 815 Leu Pro Lys Asn Cys Ser 820 13 76 PRT Homo
sapiens human ubiquitin 13 Met Gln Ile Phe Val Lys Thr Leu Thr Gly
Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu
Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro
Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp
Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr
Leu His Leu Val Leu Arg Leu Arg Gly Gly 65 70 75 14 84 PRT
Artificial Sequence Description of Artificial Sequence
FLAG-ubiquitin 14 Met Asp Tyr Lys Asp Asp Asp Asp Lys Gln Ile Phe
Val Lys Thr Leu 1 5 10 15 Thr Gly Lys Thr Ile Thr Leu Glu Val Glu
Pro Ser Asp Thr Ile Glu 20 25 30 Asn Val Lys Ala Lys Ile Gln Asp
Lys Glu Gly Ile Pro Pro Asp Gln 35 40 45 Gln Arg Leu Ile Phe Ala
Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu 50 55 60 Ser Asp Tyr Asn
Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg 65 70 75 80 Leu Arg
Gly Gly 15 85 PRT Artificial
Sequence Description of Artificial SequenceFLAG-Cys-ubiquitin 15
Met Asp Tyr Lys Asp Asp Asp Asp Lys Cys Gln Ile Phe Val Lys Thr 1 5
10 15 Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr
Ile 20 25 30 Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile
Pro Pro Asp 35 40 45 Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu
Glu Asp Gly Arg Thr 50 55 60 Leu Ser Asp Tyr Asn Ile Gln Lys Glu
Ser Thr Leu His Leu Val Leu 65 70 75 80 Arg Leu Arg Gly Gly 85 16
170 PRT Homo sapiens E2 UBC7 16 Met Thr Glu Leu Gln Ser Ala Leu Leu
Leu Arg Arg Gln Leu Ala Glu 1 5 10 15 Leu Asn Lys Asn Pro Val Glu
Gly Phe Ser Ala Gly Leu Ile Asp Asp 20 25 30 Asn Asp Leu Tyr Arg
Trp Glu Val Leu Ile Ile Gly Pro Pro Asp Thr 35 40 45 Leu Tyr Glu
Gly Gly Val Phe Lys Ala His Leu Thr Phe Pro Lys Asp 50 55 60 Tyr
Pro Leu Arg Pro Pro Lys Met Lys Phe Ile Thr Glu Ile Trp His 65 70
75 80 Pro Asn Val Asp Lys Asn Gly Asp Val Cys Ile Ser Ile Leu His
Glu 85 90 95 Pro Gly Glu Asp Lys Tyr Gly Tyr Glu Lys Pro Glu Glu
Arg Trp Leu 100 105 110 Pro Ile His Thr Val Glu Thr Ile Met Ile Ser
Val Ile Ser Met Leu 115 120 125 Ala Asp Pro Asn Gly Asp Ser Pro Ala
Asn Val Asp Ala Ala Lys Glu 130 135 140 Trp Arg Glu Asp Arg Asn Gly
Glu Phe Lys Arg Lys Val Ala Arg Cys 145 150 155 160 Val Arg Lys Ser
Gln Glu Thr Ala Phe Glu 165 170 17 513 DNA Homo sapiens E2 UBC7 17
atgacggagc tgcagtcggc actgctactg cgaagacagc tggcagaact caacaaaaat
60 ccagtggaag gcttttctgc aggtttaata gatgacaatg atctctaccg
atgggaagtc 120 cttattattg gccctccaga tacactttat gaaggtggtg
tttttaaggc tcatcttact 180 ttcccaaaag attatcccct ccgacctcct
aaaatgaaat tcattacaga aatctggcac 240 ccaaatgttg ataaaaatgg
tgatgtgtgc atttctattc ttcatgagcc tggggaagat 300 aagtatggtt
atgaaaagcc agaggaacgc tggctcccta tccacactgt ggaaaccatc 360
atgattagtg tcatttctat gctggcagac cctaatggag actcacctgc taatgttgat
420 gctgcgaaag aatggaggga agatagaaat ggagaattta aaagaaaagt
tgcccgctgt 480 gtaagaaaaa gccaagagac tgcttttgag tga 513 18 165 PRT
Homo sapiens E2 UBC7 homolog 18 Met Ala Gly Thr Ala Leu Lys Arg Leu
Met Ala Glu Tyr Lys Gln Leu 1 5 10 15 Thr Leu Asn Pro Pro Glu Gly
Ile Val Ala Gly Pro Met Asn Glu Glu 20 25 30 Asn Phe Phe Glu Trp
Glu Ala Leu Ile Met Gly Pro Glu Asp Thr Cys 35 40 45 Phe Glu Phe
Gly Val Phe Pro Ala Ile Leu Ser Phe Pro Leu Asp Tyr 50 55 60 Pro
Leu Ser Pro Pro Lys Met Arg Phe Thr Cys Glu Met Phe His Pro 65 70
75 80 Asn Ile Tyr Pro Asp Gly Arg Val Cys Ile Ser Ile Leu His Ala
Pro 85 90 95 Gly Asp Asp Pro Met Gly Tyr Glu Ser Ser Ala Glu Arg
Trp Ser Pro 100 105 110 Val Gln Ser Val Glu Lys Ile Leu Leu Ser Val
Val Ser Met Leu Ala 115 120 125 Glu Pro Asn Asp Glu Ser Gly Ala Asn
Val Asp Ala Ser Lys Met Trp 130 135 140 Arg Asp Asp Arg Glu Gln Phe
Tyr Lys Ile Ala Lys Gln Ile Val Gln 145 150 155 160 Lys Ser Leu Gly
Leu 165 19 2878 DNA Homo sapiens E2 UBC7 homolog 19 cgcgcggctg
aggcgaggtc gctcggcgca gctgttgcgg ggccatggcg gggaccgcgc 60
tcaagaggct gatggccgag tacaaacaat taacactgaa tcctccggaa ggaattgtag
120 caggccccat gaatgaagag aacttttttg aatgggaggc attgatcatg
ggcccagaag 180 acacctgctt tgagtttggt gtttttcctg ccatcctgag
tttcccactt gattacccgt 240 taagtccccc aaagatgaga tttacctgtg
agatgtttca tcccaacatc taccctgatg 300 ggagagtctg catttccatc
ctccacgcgc caggcgatga ccccatgggc tacgagagca 360 gcgcggagcg
gtggagtcct gtgcagagtg tggagaagat cctgctgtcg gtggtgagca 420
tgctggcaga gcccaatgac gaaagtggag ctaacgtgga tgcgtccaaa atgtggcgcg
480 atgaccggga gcagttctat aagattgcca agcagatcgt ccagaagtct
ctgggactgt 540 gagacctggc ctcgcacagg cgcgcacaca ccgccaagca
gctcagcatt ctcccccggc 600 acacttagtg acagtgatgc tctgtgctgg
taccaaacaa ggcagacttg caagaaccat 660 ggcatctttt ttttttttca
aacctttcct acttcaaaca ggcttctctt ctgaaatgat 720 gacttaatgt
cgaatattga cagcttactg cagttttaca gtattcctca caaagggctt 780
caggtagatt atcagagctg tcagcactac ctctccccgc tgaaaccagc agttcatggc
840 ttcctgtgga ttccctccct ccctggagtg ttgagggggt tgtacctgcc
agacttccag 900 gggacgatgg aatacccaga acgctccttc tgaagaaatg
gggccctgta gctgcagcac 960 aggggaaggg cccggcaccc tttctgggtc
cttcctggtt ccctgtgggc cccatgagga 1020 gtccattact tcctttcttc
cttcatattt tacaggcaga tgcttttctt ataatctaat 1080 tacatctttt
catttgttat atattacaaa ccatcacact tagaaatact tccaggaaat 1140
gcttttttga agtgtgaatt aataagaaat ggggtaaata gaaaagaaat ttattgctga
1200 ttggccaggt gcggtggttc gtgcctgtaa tcccagctct ttgggaggcc
aaggcaggta 1260 gatcacaagg tcaggaaatt gagaccatcc tggctaatac
agtgaaaccc catgtctgct 1320 aaaattacaa aaaattagct gggcgtggtg
gtgcacgcct gtagtctcag ctactcagga 1380 ggctgaggca ggagaatcgc
ttgaacccgg gaggcagagg tagcagtgag ctgaagtccc 1440 gccactgcac
tccagcctgg gcaacagagc gagactcagt ctcaaaaaga aaaaagaaat 1500
ttattgctga tcacaaggac agacagtttt ttcccgacca tactcatcaa agatttacgt
1560 ttgtatatta gtaactagtg cattactaga gcaggtgcag gtgaggtctt
taaagtttca 1620 atgaaagttt cttctggatc tacagaaaaa attttttttt
ttcaatctaa aaactggaaa 1680 ttctagggtt tttgtacatt ttggatgcac
tgggaattta ttagcacaaa atcattcttt 1740 gcaactcaaa attcagaagg
gactctacca tatcttagct cagagcacag aggagtgcct 1800 tatccccaca
cttgactggg ctgtggaggt gggcatgtgg gcccctgggc ccaggctggg 1860
gacagagccc ttgttttgtg acttaggatt ttgatgtggt tcccatgttc tctaacaggg
1920 ccagctgagc agcacaggcc aggaggccac agtgtaagca ataacagatc
tgccacatgc 1980 agaagcaaat atcaggcctg tcgcacacgg gcggcattta
aataggaatt tctatttttg 2040 aaataaggga tggtctatga ggcatacagt
agatttgatg tgatcctttt ctccctccct 2100 tccataatgg atcgtggtct
gtgtgactga acccacacag agtgtcatgg gtgacagttt 2160 ctggttgaag
tagctccacg cctggcttct gtggacagca gattcttttc cttctcacaa 2220
ggggctcatt taaaatttgg aggctgggtg ctgtggctca cgcctgcaat cccagcactt
2280 tgggagactg aggcgggcgg atcatgaggt caggagatcg cgaccatcct
ggctaacagt 2340 gaaaccctgt ctccactaaa aatacaaaaa attagccggg
cgtggtggcg ggcgcctgta 2400 gtcccagcta ctctgaagac tgaggcagga
gaatggcgtg aacccaggag gcggagcttg 2460 cagtgagctg agatcacgcc
actgcactcc agcctgggca acagagtgag actctgtctc 2520 aaaaaaaaaa
aaaaaaaaaa tggaacgcag ggcaagaact cgtatttgga aggagatggg 2580
ggaaaggagc ggtattatac ctatgttgta tttgcaggca aatgagatgg agccctctct
2640 gtaaagaaga gtcatttgtg caagtagacg gggtctgtgg gtgcaggccc
tggaggggca 2700 cacaattgcc tggaggcttc tgtgagatcg ggagagggag
gagaggcagt ctcttgacaa 2760 aataaagtat ttttattcat ttgtatttat
taaatgaaaa aacaatccca tggtgtccct 2820 gttgtgtggt ggaacctaat
gactgttgaa ataaagttct gtgttttccc tgccctgc 2878 20 158 PRT Homo
sapiens E2 UBC9 20 Met Ser Gly Ile Ala Leu Ser Arg Leu Ala Gln Glu
Arg Lys Ala Trp 1 5 10 15 Arg Lys Asp His Pro Phe Gly Phe Val Ala
Val Pro Thr Lys Asn Pro 20 25 30 Asp Gly Thr Met Asn Leu Met Asn
Trp Glu Cys Ala Ile Pro Gly Lys 35 40 45 Lys Gly Thr Pro Trp Glu
Gly Gly Leu Phe Lys Leu Arg Met Leu Phe 50 55 60 Lys Asp Asp Tyr
Pro Ser Ser Pro Pro Lys Cys Lys Phe Glu Pro Pro 65 70 75 80 Leu Phe
His Pro Asn Val Tyr Pro Ser Gly Thr Val Cys Leu Ser Ile 85 90 95
Leu Glu Glu Asp Lys Asp Trp Arg Pro Ala Ile Thr Ile Lys Gln Ile 100
105 110 Leu Leu Gly Ile Gln Glu Leu Leu Asn Glu Pro Asn Ile Gln Asp
Pro 115 120 125 Ala Gln Ala Glu Ala Tyr Thr Ile Tyr Cys Gln Asn Arg
Val Glu Tyr 130 135 140 Glu Lys Arg Val Arg Ala Gln Ala Lys Lys Phe
Ala Pro Ser 145 150 155 21 1856 DNA Homo sapiens E2 UBC9 21
ggatgggaag cgagcatggt gagtcctcaa gtcgcagctg ggcctgccac gtgggagtgg
60 agggtggagg aacgtgtgga gtttcggagt ccagcccagt gcgagacagc
cttgaaaccg 120 tggttggcgg gcgctccact ccgctctggg ctcgaaccct
gcctgaccct agctgtgccc 180 cccactttct ccctgtctgg cccctgctcc
ccgccccctc acttagagga gggcacgggg 240 aagggcaaac ggtccagagg
gcgggcggct gcgggctcct ctgcatcatg tgaggagggc 300 gtggggaagg
acatcctggt ggggcccgat ctgggctgcc tccagcccgg gcctgtgtct 360
tggacttagt cgtggacctg gaggccagtg cccggctggc cctgtcaccc tctcgctgtg
420 acgccagcgc ctgctgactg gaggacccag gttccttcgc ctgctttttc
tcaggctgcc 480 ctgaggatct gtgtttggtg aaaaggagcc aaattcacct
gcagggcagg cggctctagc 540 agcttcagaa gcctggtgcc ctggcgacac
tggacctgcc ttggcttctt tgatcccaac 600 cccacccccg atttctgctc
tgctgactgg ggaagtcatc gtgccaccca gaacctgagt 660 gcgggcctct
cagagctcct tcgtccgtgg gtctgccggg gactgggcct tgtctccctg 720
gcgagtgcca ggtgaggctg cggcggctcc gacgcaggtg gagctgctga cctggcccct
780 ttctgcggct gcgagggact ttgaacatgt cggggatcgc cctcagcaga
ctcgcccagg 840 agaggaaagc atggaggaaa gaccacccat ttggtttcgt
ggctgtccca acaaaaaatc 900 ccgatggcac gatgaacctc atgaactggg
agtgcgccat tccaggaaag aaagggactc 960 cgtgggaagg aggcttgttt
aaactacgga tgcttttcaa agatgattat ccatcttcgc 1020 caccaaaatg
taaattcgaa ccaccattat ttcacccgaa tgtgtaccct tcggggacag 1080
tgtgcctgtc catcttagag gaggacaagg actggaggcc agccatcaca atcaaacaga
1140 tcctattagg aatacaggaa cttctaaatg aaccaaatat ccaagaccca
gctcaagcag 1200 aggcctacac gatttactgc caaaacagag tggagtacga
gaaaagggtc cgagcacaag 1260 ccaagaagtt tgcgccctca taagcagcga
ccttgtggca tcgtcagaag gaagggattg 1320 gtttggcaag aacttgttta
caacattttt gcaaatctaa agttgctcca tacaatgact 1380 agtcacctgg
gggggttggg cgggcgccat cttccattgc cgccgcgggt gtgcggtctc 1440
gattcgctga attgcccgtt tccatacagg gtctcttcct tcggtctttt gtatttttga
1500 ttgttatgta aaactcgctt ttattttaat attgatgtca gtatttcaac
tgctgtaaaa 1560 ttataaactt ttatacttgg gtaagtcccc caggcgagtt
cctcgctctg ggatgcaggc 1620 atgcttctca ccgtgcagag ctgcacttgg
cctcagctgg ctgtatggaa atgcaccctc 1680 cctcctgcgc tcctctctag
aacctgggct gtgctgcttt tgagcctcag accccagggc 1740 agcatctcgg
ttctgcgcca cttcctttgt gtttatatgg cgttttgtct gtgttgctgt 1800
ttaggtaaat aaactgttta tataaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 1856
22 179 PRT Homo sapiens E2 UBCH10 22 Met Ala Ser Gln Asn Arg Asp
Pro Ala Ala Thr Ser Val Ala Ala Ala 1 5 10 15 Arg Lys Gly Ala Glu
Pro Ser Gly Gly Ala Ala Arg Gly Pro Val Gly 20 25 30 Lys Arg Leu
Gln Gln Glu Leu Met Thr Leu Met Met Ser Gly Asp Lys 35 40 45 Gly
Ile Ser Ala Phe Pro Glu Ser Asp Asn Leu Phe Lys Trp Val Gly 50 55
60 Thr Ile His Gly Ala Ala Gly Thr Val Tyr Glu Asp Leu Arg Tyr Lys
65 70 75 80 Leu Ser Leu Glu Phe Pro Ser Gly Tyr Pro Tyr Asn Ala Pro
Thr Val 85 90 95 Lys Phe Leu Thr Pro Cys Tyr His Pro Asn Val Asp
Thr Gln Gly Asn 100 105 110 Ile Cys Leu Asp Ile Leu Lys Glu Lys Trp
Ser Ala Leu Tyr Asp Val 115 120 125 Arg Thr Ile Leu Leu Ser Ile Gln
Ser Leu Leu Gly Glu Pro Asn Ile 130 135 140 Asp Ser Pro Leu Asn Thr
His Ala Ala Glu Leu Trp Lys Asn Pro Thr 145 150 155 160 Ala Phe Lys
Lys Tyr Leu Gln Glu Thr Tyr Ser Lys Gln Val Thr Ser 165 170 175 Gln
Glu Pro 23 783 DNA Homo sapiens E2 UBCH10 23 ggcacgagcg agttcctgtc
tctctgccaa cgccgcccgg atggcttccc aaaaccgcga 60 cccagccgcc
actagcgtcg ccgccgcccg taaaggagct gagccgagcg ggggcgccgc 120
ccggggtccg gtgggcaaaa ggctacagca ggagctgatg accctcatga tgtctggcga
180 taaagggatt tctgccttcc ctgaatcaga caaccttttc aaatgggtag
ggaccatcca 240 tggagcagct ggaacagtat atgaagacct gaggtataag
ctctcgctag agttccccag 300 tggctaccct tacaatgcgc ccacagtgaa
gttcctcacg ccctgctatc accccaacgt 360 ggacacccag ggtaacatat
gcctggacat cctgaaggaa aagtggtctg ccctgtatga 420 tgtcaggacc
attctgctct ccatccagag ccttctagga gaacccaaca ttgatagtcc 480
cttgaacaca catgctgccg agctctggaa aaaccccaca gcttttaaga agtacctgca
540 agaaacctac tcaaagcagg tcaccagcca ggagccctga cccaggctgc
ccagcctgtc 600 cttgtgtcgt ctttttaatt tttccttaga tggtctgtcc
tttttgtgat ttctgtatag 660 gactctttat cttgagctgt ggtatttttg
ttttgttttt gtcttttaaa ttaagcctcg 720 gttgagccct tgtatattaa
ataaatgcat ttttgtcctt ttttaaaaaa aaaaaaaaaa 780 aaa 783 24 152 PRT
Homo sapiens E2 UBC13 24 Met Ala Gly Leu Pro Arg Arg Ile Ile Lys
Glu Thr Gln Arg Leu Leu 1 5 10 15 Ala Glu Pro Val Pro Gly Ile Lys
Ala Glu Pro Asp Glu Ser Asn Ala 20 25 30 Arg Tyr Phe His Val Val
Ile Ala Gly Pro Gln Asp Ser Pro Phe Glu 35 40 45 Gly Gly Thr Phe
Lys Leu Glu Leu Phe Leu Pro Glu Glu Tyr Pro Met 50 55 60 Ala Ala
Pro Lys Val Arg Phe Met Thr Lys Ile Tyr His Pro Asn Val 65 70 75 80
Asp Lys Leu Gly Arg Ile Cys Leu Asp Ile Leu Lys Asp Lys Trp Ser 85
90 95 Pro Ala Leu Gln Ile Arg Thr Val Leu Leu Ser Ile Gln Ala Leu
Leu 100 105 110 Ser Ala Pro Asn Pro Asp Asp Pro Leu Ala Asn Asp Val
Ala Glu Gln 115 120 125 Trp Lys Thr Asn Glu Ala Gln Ala Ile Glu Thr
Ala Arg Ala Trp Thr 130 135 140 Arg Leu Tyr Ala Met Asn Asn Ile 145
150 25 1203 DNA Homo sapiens E2 UBC13 25 actcgtgcgt gaggcgagag
gagccggaga cgagaccaga ggccgaactc gggttctgac 60 aagatggccg
ggctgccccg caggatcatc aaggaaaccc agcgtttgct ggcagaacca 120
gttcctggca tcaaagccga accagatgag agcaacgccc gttattttca tgtggtcatt
180 gctggccctc aggattcccc ctttgaggga gggactttta aacttgaact
attccttcca 240 gaagaatacc caatggcagc ccctaaagta cgtttcatga
ccaaaattta tcatcctaat 300 gtagacaagt tgggaagaat atgtttagat
attttgaaag ataagtggtc cccagcactg 360 cagatccgca cagttctgct
atcgatccag gccttgttaa gtgctcccaa tccagatgat 420 ccattagcaa
atgatgtagc ggagcagtgg aagaccaacg aagcccaagc catagaaaca 480
gctagagcat ggactaggct atatgccatg aataatattt aaattgatac gatcatcaag
540 tgtgcatcac ttctcctgtt ctgccaagac ttcctcctct ttgtttgcat
ttaatggaca 600 cagtcttaga aacattacag aataaaaaag cccagacatc
ttcagtcctt tggtgattaa 660 atgcacatta gcaaatctat gtcttgtcct
gattcactgt cataaagcat gagcagaggc 720 tagaagtatc atctggattg
ttgtgaaacg tttaaaagca gtggcccctc cctgctttta 780 ttcatttccc
ccatcctggt ttaagtataa agcactgtga atgaaggtag ttgtcaggtt 840
agctgcaggg gtgtgggtgt ttttatttta ttttatttta ttttattttt gaggggggag
900 gtagtttaat tttatgggct cctttccccc ttttttggtg atctaattgc
attggttaaa 960 agcagctaac caggtcttta gaatatgctc tagccaagtc
taactttatt tagacgctgt 1020 agatggacaa gcttgattgt tggaaccaaa
atgggaacat taaacaaaca tcacagccct 1080 cactaataac attgctgtca
agtgtagatt ccccccttca aaaaaagctt gtgaccattt 1140 tgtatggctt
gtctggaaac ttctgtaaat cttatgtttt agtaaaatat tttttgttat 1200 tct
1203 26 6 PRT Artificial Sequence Description of Artificial
SequenceN-terminal His6-tag 26 His His His His His His 1 5 27 42
DNA Artificial Sequence Description of Artificial
SequenceFLAG-Cys-ubiquitin site-directed mutagenesis primer 27
ccccccaagc tttgcatgca gattttcgtg aagaccctga cc 42 28 45 DNA
Artificial Sequence Description of Artificial
SequenceFLAG-Ala-Cys-ubiquitin site-directed mutagenesis primer 28
ccccccaagc ttgcgtgcat gcagattttc gtgaagaccc tgacc 45
* * * * *
References