U.S. patent application number 10/927588 was filed with the patent office on 2005-07-28 for assays for the detection of microtubule depolymerization inhibitors.
Invention is credited to Hartman, James J., Vale, Ronald D..
Application Number | 20050164230 10/927588 |
Document ID | / |
Family ID | 32852889 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050164230 |
Kind Code |
A1 |
Vale, Ronald D. ; et
al. |
July 28, 2005 |
Assays for the detection of microtubule depolymerization
inhibitors
Abstract
This invention provides methods for the screening and
identification of agents having potent effects on the progression
of the cell cycle. In one embodiment, the methods involve
contacting a polymerized microtubule with a microtubule severing
protein or a microtubule depolymerizing protein in the presence of
an ATP or a GTP and a test agent; and (ii) detecting the formation
of tubulin monomers, dimers or oligomers. The p60 subunit of
katanin provides a particularly preferred microtubule severing
protein possessing both ATPase and microtubule severing
activities.
Inventors: |
Vale, Ronald D.; (San
Francisco, CA) ; Hartman, James J.; (San Francisco,
CA) |
Correspondence
Address: |
MEDLEN & CARROLL, LLP
Suite 350
101 Howard Street
San Francisco
CA
94105
US
|
Family ID: |
32852889 |
Appl. No.: |
10/927588 |
Filed: |
August 25, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10927588 |
Aug 25, 2004 |
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09673222 |
Dec 4, 2000 |
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6872537 |
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09673222 |
Dec 4, 2000 |
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PCT/US99/08086 |
Apr 13, 1999 |
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60081734 |
Apr 14, 1998 |
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Current U.S.
Class: |
435/6.14 ;
435/7.23; 514/449 |
Current CPC
Class: |
G01N 33/68 20130101;
C12Q 1/37 20130101; C12Q 1/42 20130101; G01N 2333/4703 20130101;
C12N 9/14 20130101; G01N 21/6458 20130101; G01N 21/6428 20130101;
C12N 2799/026 20130101 |
Class at
Publication: |
435/006 ;
435/007.23; 514/449 |
International
Class: |
C12Q 001/68; G01N
033/574; A61K 031/337 |
Claims
1-25. (canceled)
26. An isolated polypeptide having microtubule severing activity,
said polypeptide comprising an amino acid sequence that is encoded
by a nucleic acid sequence that hybridizes under stringent
conditions with a nucleic acid sequence that encodes SEQ ID
NO:1.
27. The polypeptide of claim 26, wherein said polypeptide comprises
SEQ ID NO:1.
28. The polypeptide of claim 26, wherein said polypeptide: 1)
comprises at least 8 contiguous amino acids of SEQ ID NO: 1; 2)
elicits the production of an antibody that specifically binds to
SEQ ID NO:1; and 3) does not bind to antiserum that is raised
against SEQ ID NO:1, and that has been fully immunosorbed with SEQ
ID NO:1.
29. The polypeptide of claim 26, wherein said polypeptide is the
pelypeptide consists of SEQ ID NO:1.
30-33. (canceled)
34. A kit for screening for agents that modulate microtubule
depolymerization, said kit comprising one or more containers
containing one or more of an isolated microtubule severing protein
and a microtubule depolymerizing protein.
35. The kit of claim 34, further comprising a polymerized
microtubule labeled with 4'-6-diamidino-2-phenylindole (DAPI).
36. The kit of claim 34, wherein said microtubule is stabilized by
contact with an agent chosen from one or more of paclitaxel,
paclitaxel analogue, and non-hydrolyzable nucleotide GTP
analogue.
37. The kit of claim 36, wherein said microtubule is attached to a
solid surface.
38. The kit of claim 37, wherein said microtubule is attached to
said surface by binding with a motor protein.
39. The kit of claim 34, wherein said microtubule severing protein
or microtubule depolymerizing protein is selected from the group
consisting of katanin polypeptide, p60 subunit of katanin
polypeptide, Xenopus kinesin central motor 1 (XKCM1) polypeptide,
and a stathmin (OP18) polypeptide.
40-41. (canceled)
42. The kit of claim 34, wherein said one or more of microtubule
severing protein and microtubule depolymerizing protein is attached
to a solid surface.
43-59. (canceled)
60. The polypeptide of claim 26, wherein said nucleic acid sequence
that encodes SEQ ID NO:1 is listed in Genbank AF052191.
61. The polypeptide of claim 26, wherein said polypeptide comprises
SEQ ID NO:1 having conservative substitutions.
62. The polypeptide of claim 26, wherein said polypeptide consists
of SEQ ID NO:1 having conservative substitutions.
63. The polypeptide of claim 26, wherein said hybridization
conditions comprise hybridization at 42.degree. C. overnight in 50%
formamide.
64. The polypeptide of claim 26, wherein said isolated polypeptide
is recombinant.
65. An isolated polypeptide comprising SEQ ID NO:1 and having
microtubule severing activity.
66. An isolated polypeptide consisting of SEQ ID NO:1.
67. The kit of claim 34, further comprising tubulin that is labeled
with a label chosen from one or more of
4'-6-diamidino-2-phenylindole (DAPI), anilinonapthalene sulfonate
(ANS), bis-ANS (Bis-anilinonapthalene sulfonate),
N-phenyl-1-naphthylene (NPN), ruthernium red, cresol violet, and
4-(dicyanovinyl)julolidine (DCVJ).
68. The kit of claim 34, further comprising tubulin that is labeled
with 4'-6-diamidino-2-phenylindole (DAPI).
69. The kit of claim 34, wherein said microtubule depolymerizing
protein comprises Xenopus kinesin central motor 1 (XKCM1)
polypeptide.
70. The kit of claim 34, wherein said microtubule depolymerizing
protein comprises stathmin (OP18) polypeptide.
71. The kit of claim 34, wherein said microtubule depolymerizing
protein comprises katanin polypeptide.
72. The kit of claim 34, wherein said microtubule depolymerizing
protein comprises a p60 subunit of katanin polypeptide.
73. The kit of claim 37, wherein said microtubule is attached to
said surface by binding with a molecule chosen from one or more of
inactivated microtubule motor protein, avidin-biotin linkage,
anti-tubulin antibody, microtubule binding protein (MAP),
polyarginine, polyhistidine, and polylysine.
74. A kit comprising the polypeptide of claim 26.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. .sctn.
119(e) of provisional patent U.S. Ser. No. 60/081,734, filed on
Apr. 14, 1998, which is herein incorporated by reference in its
entirety for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] [ Not Applicable ]
FIELD OF THE INVENTION
[0003] This invention relates assay for agents that modulate (e.g.
upregulate, downregulate or completely inhibit) microtubule
depolymerizing or microtubule severing proteins. Such agents will
have profound effects on progression of the cell cycle and act as
potent anti-mitotic agents.
BACKGROUND OF THE INVENTION
[0004] The cytoskeleton constitutes a large family of proteins that
are involved in many critical processes of biology, such as
chromosome and cell division, cell motility and intracellular
transport (Vale and Kreis, (1993) Guidebook to the Cytoskeletal and
Motor Proteins New York: Oxford University Press; Alberts et al.,
(1994) Molecular Biology of the Cell, 788-858). Cytoskeletal
proteins are found in all cells and are involved in the
pathogenesis of a large range of clinical diseases. The
cytoskeleton includes a collection of polymer proteins,
microtubules, actin, intermediate filaments, and septins, as well
as a wide variety of proteins that bind to these polymers
(polymer-interacting proteins). Some of the polymer-interacting
proteins are molecular motors (myosins, kinesins, dyneins)
(Goldstein (1993) Ann. Rev. Genetics 27: 319-351; Mooseker and
Cheney (1995) Annu. Rev. Cell Biol. 11: 633-675) that are essential
for transporting material within cells (e.g., chromosomal movement
during metaphase), for muscle contraction, and for cell migration.
Other groups of proteins (e.g., vinculin, talin and alpha-actinin)
link different filaments, connect the cytoskeleton to the plasma
membrane, control the assembly and disassembly of the cytoskeletal
polymers, and moderate the organization of the polymers within
cells.
[0005] Given the central role of the cytoskeleton in cell division,
cell migration, inflammation, and fungal/parasitic life cycles, it
is a fertile system for drug discovery. Although much is known
about the molecular and structural properties of cytoskeletal
components, relatively little is known about how to efficiently
manipulate cytoskeletal structure and function. Such manipulation
requires the discovery and development of specific compounds that
can predictably and safely alter cytoskeletal structure and
function. However, at present, drug targets in the cytoskeleton
have been relatively untapped. Extensive work has been directed
towards drugs that interact with the cytoskeletal polymers
themselves (e.g., taxol and vincristine), and towards motility
assays (Turner et al. (1996) Anal. Biochem. 242 (1): 20-5; Gittes
et al. (1996) Biophys. J. 70 (1): 418-29; Shirakawa et al. (1995)
J. Exp. Biol. 198: 1809-15; Winkelmann et al. (1995) Biophys. J.
68: 2444-53; and Winkelmann et al. (1995) Biophys. J. 68: 72S).
Virtually no effort has been directed to finding drugs that target
the cytoskeletal proteins that bind to the different filaments,
which might be more specific targets with fewer unwanted side
effects.
SUMMARY OF THE INVENTION
[0006] This invention pertains to the discovery that proteins (e.g.
motor proteins) that either depolymerize or sever microtubules,
provide good targets for modulators of such activity. Without being
bound by a particular theory, it is believed that microtubule
depolymerizing or severing activity is critical for normal
formation and/or function of the mitotic spindle. Thus, agents that
modulate (e.g., upregulate, downregulate, or completely inhibit)
depolymerization or severing activity are expected to have a
significant activity on progression of the cell cycle (e.g. acting
as potent anti-mitotic agents).
[0007] This invention thus provides, in one embodiment, assays for
identifying an agent that modulates microtubule depolymerization.
The assays involve contacting a polymerized microtubule with a
microtubule severing protein or a microtubule depolymerizing
protein in the presence of an ATP or a GTP and the "test" agent;
and detecting the formation of tubulin monomers, dimers, or
oligomers. The microtubule can be labeled with any of a variety of
labels, however in a preferred embodiment, it is labeled with DAPI.
The formation of tubulin monomers, dimers, or oligomers can be
detected by any of a wide variety of methods including, but not
limited to changes in DAPI fluorescence, fluorescent resonance
energy transfer (FRET), centrifugation, and the like. The
microtubules are preferably microtubules that are either naturally
stable (e.g., axonemal microtubules) or microtubules that have been
stabilized (e.g., by contact with an agent such as paclitaxel, a
paclitaxel analogue, or a non-hydrolyzable nucleotide GTP analogue
such as guanylyl-(.alpha.,.beta.- )-methylene diphosphate
(GMPCPP)).
[0008] The assays can be run in solution or in solid phase (i.e.
where one or more assay components are attached to a solid surface.
In one embodiment, of solid-phase assays, the microtubule is
attached to the surface e.g., by direct binding or by binding with
an agent such as an inactivated microtubule motor protein, an
avidin-biotin linkage, an anti-tubulin antibody, a microtubule
binding protein (MAP), or a polylysine. The microtubule severing
protein or microtubule depolymerizing protein is preferably a
katanin, a p60 subunit of a katanin, an XKCM1, or an OP18
polypeptide. In a particularly preferred embodiment, the
microtubule severing protein is a katanin or a p60 subunit of a
katanin as described herein.
[0009] It was also a discovery of this invention that the katanin
p60 subunit exhibits both the ATPase and microtubule severing
activity observed in katanin. The p60 subunit thus provides a good
target for screening for potential therapeutic lead compounds.
Thus, in another embodiment, this invention provides methods for
screening and for identifying a therapeutic lead compound that
modulates depolymerization or severing of a microtubule system. The
methods involve providing an assay mixture comprising a katanin p60
subunit and a microtubule, contacting the assay mixture with a test
compound to be screened for the ability to inhibit or enhance the
microtubule-severing or ATPase activity of the p60 subunit; and
detecting specific binding of the test compound to said p60 subunit
or a change in the ATPase activity of the p60 subunit. The
detecting can be by any of a wide variety of methods including, but
not limited to detecting ATPase activity using malachite green as a
detection reagent. Binding activity can be easily detected in
binding assays in which the p60 subunit is labeled and said test
agent is attached to a solid support or conversely, the test agent
is labeled and the p60 subunit is attached to a solid support. In a
preferred embodiment, the ATPase assays are performed in the
presence of stabilized microtubules.
[0010] The assay methods of this invention are also amenable to
high throughput screening. Thus, in one embodiment, any of the
methods described herein is performed in an array where said array
comprises a multiplicity of reaction mixtures. each reaction
mixture comprising a distinct and distinguishable domain of said
array, and wherein the assay steps are performed in each reaction
mixture. The array can take a number of formats, however, in one
preferred format, the array comprises a microtitre plate,
preferably a microtitre plate comprising at least 48 and more
preferably at least 96 reaction mixtures. The test agent can be one
of a plurality of agents and each reaction mixture can comprise one
agent of the plurality of agents.
[0011] In addition, this invention provides for polypeptides having
microtubule severing activity. The polypeptides comprise an
isolated p60 subunit of a katanin, where the p60 subunit is encoded
by a nucleic acid that hybridizes under stringent conditions with a
nucleic acid that encodes the katanin p60 amino acid sequence (SEQ
ID NO:1). In a particularly preferred embodiment, the polypeptide
is the polypeptide of SEQ ID NO:1 or the polypeptide of SEQ ID NO:
1 having conservative substitutions. The polypeptide can comprise
at least 8 contiguous amino acids from a polypeptide sequence
encoded by a nucleic acid as set forth in SEQ ID NO:1, where the
polypeptide, when presented as an antigen, elicits the production
of an antibody that specifically binds to a polypeptide sequence
encoded by a nucleic acid as set forth in SEQ ID NO:1; and the
polypeptide does not bind to antisera raised against a polypeptide
encoded by a nucleic acid sequence as set forth in SEQ ID NO:1,
that has been fully immunosorbed with a polypeptide encoded by a
nucleic acid sequence as set forth in SEQ ID NO:1. In a most
preferred embodiment, the polypeptide is polypeptide of SEQ ID
NO:1.
[0012] This invention also provides an isolated nucleic acid that
encodes a katanin p60 subunit having microtubule severing activity.
The nucleic acid preferably comprises a nucleic acid that
specifically hybridizes with a nucleic acid that encodes the
polypeptide of SEQ ID NO:1 under stringent conditions. The nucleic
acid preferably encodes a polypeptide of SEQ ID NO:1 or
conservative substitutions thereof. The katanin p60 encoding
nucleic acid can be operably linked to a promoter (e.g. a
baculovirus promoter) and may be present in a vector.
[0013] In another embodiment, this invention provides methods of
screening for an agent that alters microtubule polymerization, or
depolymerization, or severing. The methods involve providing
labeled tubulin; contacting the labeled tubulin with the test agent
to produce contacted tubulin; and comparing the fluorescence
intensity or pattern of the contacted tubulin with the fluorescence
intensity or pattern of labeled tubulin that is not contacted with
the test agent where a difference in fluorescence pattern or
intensity between the contacted and the not contacted tubulin
indicates that the agent alters microtubule polymerization, or
depolymerization, or severing. In particularly preferred
embodiments, the labeled tubulin is in the form of tubulin
monomers, tubulin dimers, tubulin oligomers, or a microtubule. In
some embodiments, the microtubule is attached to a solid surface
(e.g., by binding with an agent selected from the group consisting
of an inactivated microtubule motor protein, an avidin-biotin
linkage, an anti-tubulin antibody, a microtubule binding protein
(MAP), a polyarginine, a polyhistidine, and a polylysine).
Preferred labels include DAPI, ANS, Bis-ANS, ruthenium red, cresol
violet, and DCVJ, with DAPI being most preferred. In some
embodiments, the "contacting" step can further comprise contacting
the tubulin with a microtubule depolymerizing protein or a
microtubule severing protein. Preferred microtubule severing or a
microtubule depolymerizing proteins include, but are not limited to
katanin, a p60 subunit of a katanin, an XKCM1, and a OP18
polypeptide. A preferred p60 subunit of a katanin is a polypeptide
of SEQ ID NO:1. The method can further involve listing the agents
that alter microtubule polymerization, depolymerization, or
severing into a database of therapeutic lead compounds that act on
the cytoskeletal system. This method can be performed in various
array embodiments as described herein.
[0014] This invention also provides kits for practice of any of the
methods described herein. In one embodiment, the kits comprise one
or more containers containing an isolated microtubule severing
protein or a microtubule depolymerizing protein. The kit can
further comprise a polymerized microtubule labeled with DAPI. The
microtubule can be stabilized by contact with paclitaxel or a
paclitaxel derivative. The microtubule can also optionally be
attached to a solid surface (e.g., by binding with an inactivated
motor protein). The microtubule severing protein or microtubule
depolymerizing protein is preferably selected from the group
consisting of a katanin, a p60 subunit of a katanin, an XKCM1, and
a OP18 polypeptide. In a particularly preferred embodiment, the
microtubule severing protein is a katanin or a p60 subunit of a
katanin.
DEFINITIONS
[0015] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides which have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g. degenerate codon substitutions) and
complementary sequences, as well as the sequence explicitly
indicated.
[0016] Specifically, degenerate codon substitutions may be achieved
by generating sequences in which the third position of one or more
selected (or all) codons is substituted with mixed-base and/or
deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:
5081; Ohtsuka et al. (1985) J. Biol Chem. 260: 2605-2608; Cassol et
al. (1992); and Rossolini et al, (1994) Mol Cell Probes 8: 91-98).
The term nucleic acid is used interchangeably with gene, cDNA, and
mRNA encoded by a gene.
[0017] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to designate a linear series of amino acid
residues connected one to the other by peptide bonds between the
alpha-amino and carboxy groups of adjacent residues. The amino acid
residues are preferably in the natural "L" isomeric form. However,
residues in the "D" isomeric form can be substituted for any
L-amino acid residue, as long as the desired functional property is
retained by the polypeptide. In addition, the amino acids, in
addition to the 20 "standard" amino acids, include modified and
unusual amino acids, which include, but are not limited to those
listed in 37 CFR .sctn.1.822(b)(4). Furthermore, it should be noted
that a dash at the beginning or end of an amino acid residue
sequence indicates either a peptide bond to a further sequence of
one or more amino acid residues or a covalent bond to a carboxyl or
hydroxyl end group.
[0018] The term "conservative substitution" is used in reference to
proteins or peptides to reflect amino acid substitutions that do
not substantially alter the activity (specificity or binding
affinity) of the molecule. Typically conservative amino acid
substitutions involve substitution one amino acid for another amino
acid with similar chemical properties (e.g. charge or
hydrophobicity). The following six groups each contain amino acids
that are typical conservative substitutions for one another:
[0019] 1) Alanine (A), Serine (S), Threonine (T);
[0020] 2) Aspartic acid (D), Glutamic acid (E);
[0021] 3) Asparagine (N), Glutamine (Q);
[0022] 4) Arginine (R), Lysine (K);
[0023] 5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V);
and
[0024] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0025] The terms "isolated" and "biologically pure" refer to
material which is substantially or essentially free from components
which normally accompany it as found in its native state. However,
the term "isolated" is not intended to refer to the components
present in an electrophoretic gel or other separation medium. An
isolated component is free from such separation media and in a form
ready for use in another application or already in use in the new
application/milieu.
[0026] The terms "identical," percent "identity," and percent
"homology" in the context of two or more nucleic acids or
polypeptide sequences, refer to two or more sequences or
subsequences that are the same or have a specified percentage of
amino acid residues or nucleotides that are the same, when compared
and aligned for maximum correspondence, as measured using one of
the following sequence comparison algorithms or by visual
inspection.
[0027] The phrase "substantially identical," in the context of two
nucleic acids or polypeptides, refers to two or more sequences or
subsequences that have at least 60%, preferably 80%, most
preferably 90-95% or even at least 98% amino acid residue identity
across a window of at least 30 nucleotides, preferably across a
window of at least 40 nucleotides, more preferably across a window
of at least 80 nucleotides, and most preferably across a window of
at least 100 nucleotides, 150 nucleotides, 200 nucleotides or
greater, when compared and aligned for maximum correspondence, as
measured using one of the following sequence comparison algorithms
or by visual inspection.
[0028] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0029] Optimal alignment of sequences for comparison can be
conducted, for example, by the local homology algorithm of Smith
& Waterman, Adv. Appl. Math. 2:482 (1981), by the homology
alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443
(1970), by the search for similarity method of Pearson &
Lipman, Proc. Natl. Acad Sci. 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 Dr., Madison, Wis.), or by visual
inspection (See generally, Ausubel et al., supra).
[0030] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle (1987) J. Mol. Evol. 3 5:3 51-360. The method
used is similar to the method described by Higgins & Sharp
(1989) CABIOS 5:151-153. The program can align up to 300 sequences,
each of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. For example, a reference sequence can be
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps.
[0031] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described in Altschul et al. (1990)
J. Mol Biol. 215:403-410. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length "W" in
the query sequence, which either match or satisfy some
positive-valued threshold score "T" when aligned with a word of the
same length in a database sequence. "T" is referred to as the
neighborhood word score threshold (Altschul et al, supra). These
initial neighborhood word hits act as seeds for initiating searches
to find longer HSPs containing them. The word hits are then
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Extension of the word
hits in each direction are halted when: the cumulative alignment
score falls off by the quantity "X" from its maximum achieved
value; the cumulative score goes to zero or below, due to the
accumulation of one or more negative-scoring residue alignments; or
the end of either sequence is reached. The BLAST algorithm
parameters "W," "T," and "X" determine the sensitivity and speed of
the alignment. The BLAST program uses as defaults a wordlength
("W") of 11, the BLOSUM62 scoring matrix (See Henikoff &
Henikoff (1989) Proc. Natl. Acad Sci USA 89: 10915) alignments (B)
of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both
strands.
[0032] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (See, e.g., Karlin & Altschul
(1993) Proc. Natl. Acad Sci. USA 90: 5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0033] A further indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the polypeptide encoded by the second nucleic acid, as
described below. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions. Another
indication that two nucleic acid sequences are substantially
identical is that the two molecules hybridize to each other under
stringent conditions, as described below.
[0034] The phrases "hybridizing specifically to," "specific
hybridization," and "selectively hybridize to," refer to the
binding, duplexing, or hybridizing of a nucleic acid molecule
preferentially to a particular nucleotide sequence under stringent
conditions when that sequence is present in a complex mixture
(e.g., total cellular) DNA or RNA.
[0035] The term "stringent conditions" refers to conditions under
which a probe will hybridize preferentially to its target
subsequence, and to a lesser extent to, or not at all to, other
sequences. Stringent hybridization and stringent hybridization wash
conditions in the context of nucleic acid hybridization experiments
such as Southern and Northern hybridizations are sequence
dependent, and are different under different environmental
parameters. An extensive guide to the hybridization of nucleic
acids is found in Tijssen (1993) Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes part I chapter 2 Overview of Principles of Hybridization and
the Strategy of Nucleic Acid Probe Assays, Elsevier, New York.
Generally, highly stringent hybridization and wash conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Very stringent conditions
are selected to be equal to the T.sub.m for a particular probe.
[0036] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or Northern
blot is 50% formamide with 1 mg of heparin at 42.degree. C., with
the hybridization being carried out overnight. An example of highly
stringent wash conditions is 0.15 M NaCl at 72.degree. C. for about
15 minutes. An example of stringent wash conditions is a
0.2.times.SSC wash at 65.degree. C. for 15 minutes (See, Sambrook
et al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol.
1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,
for a description of SSC buffer). Often, a high stringency wash is
preceded by a low stringency wash to remove background probe
signal. An example medium stringency wash for a duplex (e.g., of
more than 100 nucleotides), is 1.times.SSC at 45.degree. C. for 15
minutes. An example low stringency wash for a duplex (e.g., of more
than 100 nucleotides), is 4-6.times.SSC at 40.degree. C. for 15
minutes. In general, a signal to noise ratio of 2.times. (or
higher) than that observed for an unrelated probe in the particular
hybridization assay indicates detection of a specific
hybridization. Nucleic acids which do not hybridize to each other
under stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code.
[0037] The terms "katanin" or "katanin p60 subunit" refer to
katanin and the katanin p60 subunit as described herein, in the
references cited and in the sequence listings. The terms also
include proteins having substantial amino acid sequence identity
with katanin or the katanin p60 subunit sequences provided herein
that exhibit ATPase and microtubule severing activity.
[0038] The terms "taxol" and "taxol derivatives or analogues refer
to the drug taxol known generically as paclitaxel (NSC number:
125973). Paclitaxel (taxol) derivatives and analogues show similar
microtubule-stabilizing activity. Preferred derivatives include
taxotere and others.
[0039] Depolymerized microtubule components are defined so as to
include the products of microtubule depolymerization or severing,
and include tubulin monomers, dimers and oligomers.
[0040] The term "test agent" refers to an agent that is to be
screened in one or more of the assays described herein. The agent
can be virtually any chemical compound. It can exist as a single
isolated compound or can be a member of a chemical (e.g.,
combinatorial) library. In a particularly preferred embodiment, the
test agent will be a small organic molecule.
[0041] The term small organic molecules refers to molecules of a
size comparable to those organic molecules generally used in
pharmaceuticals. The term excludes biological macromolecules (e.g.,
proteins, nucleic acids, etc.). Preferred small organic molecules
range in size up to about 5000 Da, more preferably up to 2000 Da,
and most preferably up to about 1000 Da.
[0042] The terms "label" or "detectable label" are used herein to
refer to any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means. Such labels include biotin for staining with
labeled streptavidin conjugate, magnetic beads (e.g.,
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein, texas red,
rhodamine, green fluorescent protein, and the like), radiolabels
(e.g., .sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P),
enzymes (e.g., horse radish peroxidase, alkaline phosphatase and
others commonly used in an ELISA), and colorimetric labels such as
colloidal gold or colored glass or plastic (e.g., polystyrene,
polypropylene, latex, etc.) beads. Patents teaching the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241. Means of detecting
such labels are well known to those of skill in the art. Thus, for
example, radiolabels may be detected using photographic film or
scintillation counters, fluorescent markers may be detected using a
photodetector to detect emitted light. Enzymatic labels are
typically detected by providing the enzyme with a substrate and
detecting, the reaction product produced by the action of the
enzyme on the substrate, and colorimetric labels are detected by
simply visualizing the colored label.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIGS. 1A (SEQ ID NO:1) and 1B (SEQ ID NOS: 4-9) show the
sequence analysis of p60 katanin. FIG. 1A: Predicted protein
sequence of the S. purpuratus katanin p60 subunit (SEQ ID NO:1;
GENBANK AF052191). Sequences obtained by direct peptide
microsequencing are underlined. Differences between the predicted
peptide sequence and that obtained by direct sequencing are
indicated by doubled underlines (S95 was reported as F, H99 was
reported as P, and P138 was reported as T). The Walker A (P-loop)
motif is shaded. FIG. 1B: Amino acid sequence alignment of the p60
AAA domain (SEQ ID NO:4) with AAA members mei-1 (SEQ ID NO:5; C.
elegans, GenBank L25423), Suglp (SEQ ID NO:6; S. cerevisiae,
GenBank X66400), ftsH (SEQ ID NO:7; E. coli, GenBank M83138), Paslp
(SEQ ID NO:8; S. cerevisiae, GenBank M58676), and NSF (SEQ ID NO:9;
C. longicaudatus, GenBank X15652). Identical residues are shaded
black, residues conserved in >60% of the shown members are
shaded gray. Left hand numbering indicates the amino acid residue
in the corresponding sequence. Alignment was performed using PILEUP
(Genetics Computer Group) and the output was shaded using
MACBOXSHADE.
[0044] FIG. 2A (SEQ ID NO:2) and 2B (SEQ ID NOS: 10-13) show the
sequence analysis of p80 katanin. FIG. 2A: Predicted protein
sequence of the S. purpuratus katanin p80 subunit (SEQ ID NO:2;
GENBANK AF052433). Sequences obtained by direct peptide
microsequencing are underlined. Differences between the predicted
peptide sequence and that obtained by direct peptide sequencing, or
differences found between 2 different p80 cDNA clones are indicated
by double underlines. FIG. 2B: Amino acid sequence alignment of the
WD40 repeat region of p80 (SEQ ID NO:10) with a putative human
ortholog of p80 (SEQ ID NO:11; Homo sapiens p80, GenBank AF052432),
TFIID (SEQ ID NO:12; Homo sapiens, GenBank U80191), and putative
serine/threonine kinase PkwA (SEQ ID NO:13; Thermomonospora
curvata, GenBank P49695). Identical residues are shaded black,
residues found in at least 2 sequences are shaded in grey. Left
hand numbering indicates the amino acid residue in the
corresponding sequence. Alignment was performed using PILEUP
(Genetics Computer Group) and the output was shaded using
MACBOXSHADE.
[0045] FIG. 3 illustrates the results of expression and
purification of recombinant katanin subunits. Panel A shows
Coomassie-stained SDS-PAGE analysis of expressed katanin subunits.
6xHis-tagged (SEQ ID NO:14) katanin subunits were purified from
lysates of baculovirus-infected insect cells by binding to
Ni.sup.2+-NTA Superflow followed by elution with imidazole, as
described in the Experimental Procedures. Cells were infected with
either p60 virus alone, p80 virus alone, or coinfected with equal
amounts of p60 and p80 viruses. Panel B shows immunoprecipitation
performed on extracts of insect cells coinfected with p60- and
p80expressing baculoviruses using affinity-purified p60 antibody
crosslinked to protein A agarose. Proteins bound to the resin were
analyzed by SDS-PAGE followed by staining with Coomassie. This
immunoprecipitate shows that baculovirus-expressed p60 and p80 form
a complex with equal stoichiometry.
[0046] FIG. 4 shows the structure of katanin as visualized by
rotary-shadowing electron microscopy. Panel A shows 14-16 nm
diameter rings observed in preparations of recombinant p60. Panel B
shows single particles of recombinant p80; occasional aggregates
are seen (rightmost picture) but rings are never observed. Panels C
and D show different rings observed in recombinant p60/p80
preparations. Panel C shows a "splayed" complex, consisting of a
central p60-like ring surrounded by a halo particles that resemble
p80. In Panel D, intact 20 nm diameter rings are seen with bright
edges, suggesting they extend >10 nm above the mica surface. All
images are shown at 300,000.times.. The dimensions indicated above
include the platinum shadowing, which typically adds 2 nm of
material to the protein surface.
[0047] FIGS. 5A, 5B, and 5C illustrate the activities of
recombinant katanin subunits. FIG. 5A shows ATPase activities of
0.04 MM p60 katanin (squares) and co-expressed p60/p80 (circles)
determined at various microtubule concentrations as described in
the Experimental Procedures. Both p60 katanin and p60/p80 show
similar patterns of microtubule stimulation, with p60 katanin
having approximately one half of the maximally stimulated ATPase
activity of p60/p80. The insert in the upper right shows the
stimulation of ATPase activity at low (0-2 .mu.M) microtubule
concentration. FIG. 5B shows microtubule severing activity of
recombinant katanin subunits. Taxol-stabilized, rhodamine-labeled
microtubules were adsorbed onto the surface of a microscope
perfusion chamber, and then recombinant katanin subunits were
introduced. The time elapsed after perfusing p60/p80 (0.1 .mu.M),
p60 (0.1 .mu.M), or p80 (0.5 .mu.M) is shown. The recombinant
co-expressed p60/p80 and p60, but not p80, can sever and
disassemble microtubules. Scale bar, 10 .mu.m. FIG. 5C shows
quantitative measurement of microtubule disassembly using a DAPI
fluorescence assay. MT indicates microtubules (2 .mu.M) without
added protein, and tubulin indicates microtubules that had been
depolymerized by treatment with 10 mM CaCl.sub.2 on ice for 1.5 hr.
p60 katanin and p60/p80 were added at 0.2 .mu.M concentration, and
the fluorescence change as a function of time after protein
addition is shown. p80 did not cause a change in fluorescence that
was different from that shown for microtubules alone.
[0048] FIG. 6 shows that the WD40 repeats of p80 katanin are not
required for interaction with p60 katanin. Epitope-tagged
derivatives of p80 and p60 were synthesized in vitro in a combined
transcription-translation reaction. p60 and interacting proteins
were immunoprecipitated with a p60-specific antibody and the
resulting immunoprecipitates were resolved by SDS-PAGE and blotted
to nitrocellulose. In vitro translated proteins were detected by
chemiluminescence as described in Experimental Procedures. Lane 1:
molecular weight standards Mr: 100,000, 75,000, 50,000, 35,000 and
25,000; lane 2: p60 co-translated with full-length p80; lane 3: p60
co-translated with the .DELTA.560-690 derivative of p80; lane 4:
p60 co-translated with the .DELTA.1-302 derivative of p80; lane 5:
p60 co-translated with the .DELTA.303-690 derivative of p80. The
structure of each deletion derivative of p80 is shown at right. The
.DELTA.560-690 and .DELTA.303-690 translation products were
detected in the supernatants of the immunoprecipitations (not
shown).
[0049] FIG. 7 shows that human p80 katanin and a fusion protein of
the human p80 WD40 domain with GFP co-localize with .gamma.-tubulin
at centrosomes of MSU1.1 human fibroblasts. Panels A and B:
Co-localization of immunofluorescence staining by a human p80
katanin-specific antibody (Panel A) and a .gamma.-tubulin specific
antibody (Panel B). Panels C-F show co-localization of GFP
fluorescence (Panels C and E) with staining by a
.gamma.-tubulin-specific antibody (Panels D and F). Co-localization
to two centrosomes seen in Panel C and Panel D while
co-localization to a single centrosome is seen in Panels E and F.
The apparently higher background of cytoplasmic green fluorescence
in Panel E relative to Panel C is a display artifact. The
fluorescence intensity of the centrosomes in Panel C is at least 5
fold greater than that of the centrosome in Panel E. The p80
antibody was detected with an Oregon Green 488 second antibody and
the .gamma.-tubulin antibody was detected with a Texas Red-X second
antibody. Fluorescence signals were separated with fluorescein and
Texas Red filter sets (Chroma Technologies). Bar =14 .mu.m.
DETAILED DESCRIPTION
[0050] I. Introduction.
[0051] This invention provides assays for the identification of
agents that modulate the activity of microtubule depolymerizing or
severing proteins. The assays generally involve contacting a
polymerized microtubule with a microtubule severing or
depolymerizing protein (e.g., XKCM1, OP18, katanin, etc.) in the
presence of a test agent and a chemical energy source (e.g., ATP or
GTP). The effect of the agent on the depolymerization or severing
of the microtubules is then detected typically by detecting the
formation of microtubule degradation components (e.g., tubulin
monomers, tubulin dimers, or tubulin oligomers). Test agents that
alter the amount and or rate of depolymerization or severing of
microtubules as compared to one or more control assays are
identified as modulators of microtubule depolymerizing or severing
activity.
[0052] It was a discovery of this invention that certain proteins
that either depolymerize or sever microtubules, provide good
targets for modulators of normal mitotic spindle formation. Without
being bound by a particular theory, it is believed that microtubule
depolymerizing or severing activity is critical for normal mitotic
spindle formation and/or function. Agents that modulate (e.g.,
upregulate, downregulate, or completely inhibit) depolymerization
or severing activity are expected to have a significant activity on
progression of the cell cycle. Thus, for example, inhibitors of
microtubule depolymerization or severing will act as potent
antimitotic agents.
[0053] Anti-mitotic agents are useful in a wide variety of
contexts. As powerful anti-mitotics or anti-meiotics, the
inhibitors of microtubule depolymerizing or severing activity
identified by the screening (assay) methods described herein, will
have a wide variety of uses, particularly in the treatment (e.g.,
amelioration) of pathological conditions characterized by abnormal
cell proliferation. Such conditions include, but are not limited
to: fungal infections, abnormal stimulation of endothelial cells
(e.g., atherosclerosis), solid tumors and tumor metastasis, benign
tumors (for example, hemangiomas, acoustic neuromas, neurofibromas,
trachomas, and pyogenic granulomas), vascular malfunctions (e.g.,
arterio-venous malformations), abnormal wound healing, inflammatory
and immune disorders, Bechet's disease, gout or gouty arthritis,
abnormal angiogenesis accompanying: rheumatoid arthritis,
psoriasis, diabetic retinopathy, and other ocular angiogenic
diseases such as retinopathy of prematurity (retrolental
fibroplasic), macular degeneration, corneal overgrowth, corneal
graft rejection, neuroscular glaucoma, Oster Webber syndrome, and
the like.
[0054] The inhibitors of microtubule depolymerization or severing
will also have a variety of in vitro uses as well. For example,
they can be used to freeze cells in a particular stage of the cell
cycle for a variety of purposes (e.g., in the preparation of
samples for histological examination), in the isolation of nucleic
acids from a particular stage of the cell cycle, and so forth.
[0055] The modulators identified by the assays of this invention
are preferably characterized by specificity to the target
microtubule depolymerizing or severing proteins or the pathways
characteristic of the activity of these proteins. They therefor
provide novel lead compounds for the development of highly specific
inhibitors for depolymerizing and/or microtubule severing protein
families and subfamilies, thus allowing for precise chemical
intervention.
[0056] IL Assays for the Detection of Microtubule Depolymerization
Modulators.
[0057] A) Depolymerization Assay
[0058] In one embodiment, this invention provides assays for the
detection/identification of agents that have activity in modulating
the depolymerization or severing of microtubules. The assays
generally involve contacting a polymerized microtubule with a
microtubule severing protein or a microtubule depolymerizing
protein in the presence of a chemical energy source (e.g., ATP or
GTP) and said agent; and detecting and/or quantifying the formation
of microtubule degradation products (e.g., tubulin monomers).
Agents that inhibit the activity of the microtubule depolymerizing
or severing proteins will inhibit the breakdown of the polymerized
microtubules thereby delaying the formation of or reducing the
quantity of tubulin monomers or oligomers. Thus a decrease in the
rate of formation or amount of tubulin monomer or an increase in
the ratio of tubulin polymer (microtubule) to tubulin monomer
indicates an inhibitory modulating effect of the agent. Conversely,
an increase in the rate of formation or amount of tubulin monomer
or an increase in the ratio of tubulin polymer (microtubule) to
tubulin monomer indicates a microtubule stabilizing modulating
effect of the agent.
[0059] The increase or decrease is determined by reference to one
or more controls. A control is essentially an identical assay that
either lacks the test agent or contains a "reference" agent that
has a known activity. Assays lacking any test agent whatsoever act
as negative controls, while assays utilizing an agent that has
known modulating activity act as positive controls.
[0060] In a preferred embodiment, the assay is scored as positive
(i.e., the agent has activity modulating a microtubule
depolymerizing or severing protein) when there is a significant
difference between the negative control and test assay and/or when
there is no significant difference between the positive control and
test assay. The significant difference is preferably a
statistically significant difference, more preferably at least
about a 10% difference, and most preferably at least about a 20%,
30%, 50% or 100% difference.
[0061] The assays can be performed in solution or in solid phase
(i.e., with one or more components of the assay attached to a solid
surface) as described below. One particularly preferred embodiment
is described herein in Example I. The various components of the
assay are described below.
[0062] B) Binding Assays.
[0063] In another embodiment, this invention provides binding
assays to identify agents that inhibit binding of depolymerizing or
severing proteins to microtubules or for agents that specifically
bind to the microtubule depolymerizing or severing polypeptide or
polypeptide subunit.
[0064] In preferred binding assays, the ability of the test agent
to specifically bind to the depolymerizing or severing protein is
assayed. In a particularly preferred embodiment, the ability of the
test agent to specifically bind to a katanin p60 domain is
assayed.
[0065] There are a wide variety of formats for binding assays. In
one embodiment, the depolymerizing or severing protein or protein
subunit is immobilized on a surface and contacted with the test
agent or conversely test agent(s) are immobilized on a surface and
specific binding of the protein or protein subunit is assayed.
Binding is most easily detected where the moiety in solution (test
agent or depolymerizing or severing protein) is labeled and after
the contacting and washing off of unbound agents, identification of
the labeled moiety associated with the support suggests
binding.
[0066] Solution phase binding assays are also known to those of
skill in the art. For example, in one embodiment, the binding assay
is a cosedimentation assay. In this (pelleting) assay, when the
test agent binds to the microtubule severing or depolymerizing
protein or protein subunits, the bound agent and protein will
cosediment when centrifuged. Unbound polypeptide and test agent
will either sediment at a different rate or remain fully in
solution.
[0067] Methods of performing various binding assays can be found in
copending application U.S. Ser. No. 60/057,895 filed on Sep. 4,
1997. For a general description of different formats for protein
binding assays, including competitive binding assays and direct
binding assays (See, Stites and A. Terr (1991) Basic and Clinical
Immunology, 7th Edition; Maggio (1980) Enzyme Immunoassay, CRC
Press, Boca Raton, Fla.; and Tijssen (1985) Practice and Theory of
Enzyme Immunoassays, in Laboratory Techniques in Biochemisty and
Molecular Biology, Elsevier Science Publishers, B.V.
Amsterdam).
[0068] C) ATPase Assay.
[0069] It was a discovery of this invention that the katanin p60
subunit is a new member of the AAA family of ATPases and that
expressed p60 has microtubule-stimulated ATPase and
microtubule-severing activities in the absence of the p80 subunit.
Thus, in another embodiment, this invention provides assays for
agents that modulate the ATPase activity of a katanin p60
subunit.
[0070] ATPase assays are well known to those of skill in the art.
In one preferred embodiment, the assay can be performed according
to the methods described by Kodama et al. (1986) J Biochem. 99:
1465-1472. This assay, described in detail in Example 1, is
performed with the test agent present and the results are compared
to negative and/or positive control assays to determine the ability
of the test agent to alter (modulate) p60 ATPase activity.
[0071] D) Solid Phase Assays.
[0072] In one embodiment, the assays of this invention can be
performed in solid phase where one or more components of the assay
is attached to a solid surface. In solid phase assays, one or more
components of the assay is attached to a solid surface. Virtually
any solid surface is suitable, as long as the surface material is
compatible with the assay reagents and it is possible to attach the
component to the surface without unduly altering the reactivity of
the assay components. It is recognized that some components show
reduced activity in solid phase, but this is generally acceptable
so long as the activity is sufficient to detect and/or quantify
depolymerization or severing activity of the subject protein.
[0073] Solid supports include, essentially any solid surface, such
as a glass bead, planar glass, controlled pore glass, plastic,
porous plastic metal, or resin to which the molecule may be
adhered. One of skill will appreciate that the solid supports may
be derivatized with functional groups (e.g., hydroxyls, amines,
carboxyls, esters, and sulfhydryls) to provide reactive sites for
the attachment of linkers or the direct attachment of the
component(s).
[0074] Adhesion of the assay component (e.g., microtubule(s)) to
the solid support can be direct (i.e., the microtubule directly
contacts the solid support) or indirect (i.e., a particular
compound or compounds are bound to the support, and the assay
component binds to this compound or compounds rather than to the
solid support). The component can be immobilized either covalently
(e.g., utilizing single reactive thiol groups of cysteine for
anchoring protein components (Colliuod et al. (1993) Bioconjugate
Chem. 4, 528-536)), or non-covalently but specifically (e.g., via
immobilized antibodies or other specific binding proteins
(Schuhmann et al. (1991), Adv. Mater. 3: 388-391; and Lu et al.
(1995), Anal. Chem. 67: 83-87), the biotin/streptavidin system
(Iwane et al. (1997) Biophys. Biochem. Res. Comm. 230: 76-80), or
metal-chelating Langmuir-Blodgett films (Ng et al. (1995) Langmuir
11: 4048-4055; Schmitt et al. (1996) Angew. Chem. Int. Ed. Engl.
35: 317-320; Frey et al. (1996) Proc. Nat. Acad. Sci. USA
93:4937-4941; and Kubalek et al. (1994) J Struct. Biol.
113:117-1231) and metal-chelating self-assembled monolayers (Sigal
et al. (1996) Analytical Chem., 68: 490-497) for binding of
polyhistidine fusion proteins.
[0075] In a preferred embodiment, the microtubule(s) are
immobilized by attachment to an inactivated microtubule motor
protein, by an avidin biotin linkage (preferably with the biotin on
the microtubule and the avidin on the surface), by an anti-tubulin
antibody, by a microtubule binding protein (MAP), by an amino
silane, a polylysine, or through interaction with a polycationic
surface.
[0076] By manipulating the solid support and the mode of attachment
of the assay component to the support, it is possible to control
the orientation of the assay component(s). For example, copending
application U.S. Ser. No. 60/057,929, filed on Sep. 4, 1997,
describes the use of an arginine tail to attach cytoskeletal
proteins to a mica film.
[0077] In one preferred embodiment, the microtubules are
immobilized by coating the surface (e.g., a flow cell) with either
n-ethylmaleimide (NEM)-treated Xenopus egg extract (6 mg/ml protein
treated with 10 mM NEM for 10 minutes followed by addition of 100
mM dithiothreitol, a treatment that inactivates severing activity)
or Escherichia coli-expressed KAR3 protein (which binds
microtubules in a nucleotide-independent manner). After washing out
unbound protein, the stabilized microtubules (e.g., 100 .mu.g/ml in
BR80, 20 .mu.M taxol) are perfused onto the surface and allowed to
bind. After washing out unbound microtubules samples to be tested
can be contacted to the surface (e.g., perfused into a flow
cell).
[0078] E) High-throughput Screening of Candidate Agents that
Modulate Microtubule Depolymerizing or Severing Proteins.
[0079] Conventionally, new chemical entities with useful properties
are generated by identifying a chemical compound (called a "lead
compound") with some desirable property or activity, creating
variants of the lead compound, and evaluating the property and
activity of those variant compounds. However, the current trend is
to shorten the time scale for all aspects of drug discovery.
Because of the ability to test large numbers quickly and
efficiently, high throughput screening (HTS) methods are replacing
conventional lead compound identification methods.
[0080] In one preferred embodiment, high throughput screening
methods involve providing a library containing a large number of
potential therapeutic compounds (candidate compounds). Such
"combinatorial chemical libraries" are then screened in one or more
assays, as described herein, to identify those library members
(particular chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
[0081] i) Combinatorial Chemical Libraries
[0082] Recently, attention has focused on the use of combinatorial
chemical libraries to assist in the generation of new chemical
compound leads. A combinatorial chemical library is a collection of
diverse chemical compounds generated by either chemical synthesis
or biological synthesis by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks called amino acids in
every possible way for a given compound length (i.e., the number of
amino acids in a polypeptide compound). Millions of chemical
compounds can be synthesized through such combinatorial mixing of
chemical building blocks. For example, one commentator has observed
at the systematic, combinatorial mixing of 100 interchangeable
chemical building blocks results in the theoretical synthesis of
100 million tetrameric compounds or 10 billion pentameric compounds
(Gallop et al. (1994) 37(9): 1233-1250).
[0083] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (See, e.g., U.S. Pat. No. 5,010,175, Furka (1991)
Int. J Pept. Prot. Res., 37: 487-493; and Houghton et al. (1991)
Nature, 354: 84-88). Peptide synthesis is by no means the only
approach envisioned and intended for use with the present
invention. Other chemistries for generating chemical diversity
libraries can also be used. Such chemistries include, but are not
limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec.
1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct.
1993), random bio-oligomers (PCT Publication WO 92/00091, 9 Jan.
1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such
as hydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993)
Proc. Nat. Acad Sci. USA 90: 6909-6913), vinylogous polypeptides
(Hagihara et al. (1992) J Amer. Chem. Soc. 114: 6568), nonpeptidal
peptidomimetics with a Beta- D- Glucose scaffolding (Hirschmann et
al., (1992) J. Amer. Chem. Soc. 114: 9217-9218), analogous organic
syntheses of small compound libraries (Chen et al. (1994) J Amer.
Chem. Soc. 116: 2661), oligocarbamates (Cho, et al., (1993) Science
261: 1303), and/or peptidyl phosphonates (Campbell et al., (1994)
J. Org. Chem. 59: 658); See, generally, Gordon et al., (1994) J.
Med Chem. 37: 1385), nucleic acid libraries (See, e.g., Strategene,
Corp.), peptide nucleic acid libraries (See, e.g., U.S. Pat. No.
5,539,083) antibody libraries (See, e.g., Vaughn et al. (1996)
Nature Biotechnology, 14(3): 309-314), and PCT/US96/10287),
carbohydrate libraries (See, e.g., Liang et al. (1996) Science,
274: 1520-1522, and U.S. Pat. No. 5,593,853), and small organic
molecule libraries (See, e.g., benzodiazepines, Baum (1993)
C&EN, Jan 18, page 33; isoprenoids U.S. Pat. No. 5,569,588;
thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;
pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino
compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.
5,288,514; and the like).
[0084] Devices for the preparation of combinatorial libraries are
commercially available (See, e.g., 357 NIPS, 390 NTS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif, 9050 Plus, Millipore, Bedford,
Mass.).
[0085] A number of well known robotic systems have also been
developed for solution phase chemistries. These systems include
automated workstations like the automated synthesis apparatus
developed by Takeda Chemical Industries, LTD. Osaka, Japan) and
many robotic systems utilizing robotic arms (Zymate II, Zymark
Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto,
Calif.) which mimic the manual synthetic operations performed by a
chemist. Any of the above devices are suitable for use with the
present invention. The nature and implementation of modifications
to these devices (if any) so that they can operate as discussed
herein will be apparent to persons skilled in the relevant art. In
addition, numerous combinatorial libraries are themselves
commercially available (See, e.g., ComGenex, Princeton, N.J.,
Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd,
Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences,
Columbia, Md., etc.).
[0086] ii) High Throughput Assays of Chemical Libraries
[0087] Any of the assays for compounds modulating the activity of
microtubule depolymerizing or severing proteins (or other agents)
described herein are amenable to high throughput screening. As
described above, in a preferred embodiment, the assays screen for
agents that enhance or inhibit the activity of katanin, XKCM1, or
OP18. Preferred assays detect the rate or amount of
depolymerization of microtubules into tubulin monomers, tubulin
dimers, or tubulin oligomers.
[0088] High throughput implementation of the assays described
herein can be implemented with, at most, routine modification of
the assays format (e.g.,. for compatibility with robotic
manipulators, large plate readers, and the like). Various high
throughput screening systems (e.g., for protein binding, nucleic
acid binding, etc.) are described in U.S. Pat. Nos. 5,559,410,
5,585,639, 5,576,220, and 5,541,061.
[0089] In addition, high throughput screening systems are
commercially available (See, e.g., Zymark Corp., Hopkinton, Mass.;
Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc.
Fullerton, Calif.; Precision Systems, Inc., Natick, Mass., etc.).
These systems typically automate entire procedures including all
sample and reagent pipetting, liquid dispensing, timed incubations,
and final readings of the microplate in detector(s) appropriate for
the assay. These configurable systems provide high throughput and
rapid start up as well as a high degree of flexibility and
customization. The manufacturers of such systems provide detailed
protocols the various high throughput. Thus, for example, Zymark
Corp. provides technical bulletins describing screening systems for
detecting the modulation of gene transcription, ligand binding, and
the like.
[0090] III) Assay Components.
[0091] A) Polymerized Microtubules
[0092] As indicated above, the assays of this invention utilize
polymerized microtubules, which, in the presence of a
depolymerizing protein or severing protein are depolymerized or
cleaved to produce tubulin monomers or oligomers. When the
depolymerizing or severing proteins are inhibited, the formation of
tubulin monomers or oligomers is inhibited.
[0093] Virtually any microtubules can be used for the assays of
this invention. Means of obtaining such microtubules are well known
to those of skill in the art. Tubulin is available commercially or
can be isolated from a wide variety of sources (e.g., plants,
animal tissues, oocytes, etc.) For example, tubulin can be isolated
from Arabidopsis cells in stationary phase (day 10 to II) cultured
cells (200 to 500 gm fresh weight) by DEAE-Sephadex A50
chromatography as described by Morejohn et al. (1985). Cell Biol.
Int. Rep. 9(9): 849-857 with modifications described in Bokros et
al. (1993), Biochemistry 32(13): 3437-3447. Briefly, Arabidopsis
cells are homogenized in an isolation buffer (IB) consisting of 50
mM PIPES-KOH, pH 6.9, 1 mM EGTA, 0.5 mM MgS0.sub.4, 1 mM DTT and
0.1 mM GTP, supplemented with 50 mg/mL Na-p-tosyl-L-arginine methyl
ester (TAME), and 5 mg/mL each of pepstatin A, leupeptin
hemisulfate, and aprotinin. The cell homogenate is subjected to
DEAE-Sephadex A50 chromatography for tubulin isolation. Ammonium
sulfate precipitates of DEAE-isolated tubulin can be aliquoted and
stored at -80.degree. C. until use.
[0094] Microtubules can be purified to homogeneity by a single
taxol-induced microtubule polymerization step in IB supplemented
with 1 mM GTP as described previously (Bokros et al. (1993),
Biochemistry 32(13): 3437-47). Briefly, samples of DEAE-isolated
tubulin are thawed and resuspended in IB supplemented with 1 mM DTT
and 1 mM GTP, and clarified by centrifugation for 1 hr at
100,000.times. g (2.degree. C.) in a Beckman TL-100 ultracentrifuge
(TLA-100 rotor). Clarified tubulin is polymerized with a twofold
molar excess of taxol in a microtubule assembly buffer composed of
IB, 1 mM DTT, 1 mM GTP and 1% DMSO. Assembly of microtubules was
performed by gradual temperature ramping from 2.degree. C. to
25.degree. C. over a 2-hour period. Polymer is collected by
centrifugation for 45 min at 30,000.times.g at 25.degree. C.
through a cushion of 20% (w/v) sucrose in assembly buffer.
[0095] In a preferred embodiment, the tubulin/microtubules are
isolated from an animal tissue (e.g., brain tissue) according to
the methods of Hyman et al. (1991) Meth Enzy., 196: 478-485. The
brain tubulin can be modified with tetramethylrhodamine or
fluorescein N-hydroxysuccinimide ester (Molecular Probes, Inc.,
Eugene, as described by Hyman et al. (1991) supra.
[0096] Most microtubules are in a state of flux, undergoing
assembly and disassembly. In a preferred embodiment of the assays
of this invention microtubules are utilized that are stabilized as
essentially intact microtubules. This can be accomplished by using
microtubules that are naturally stable (e.g., axonemal
microtubules) or by treating the microtubules so that they are
stabilized. Methods of stabilizing microtubules are well known to
those of skill in the art and include, but are not limited to the
use of stabilizing agents such as paclitaxel and paclitaxel
derivatives (e.g., taxotere), non-hydrolyzable nucleotide (e.g.,
GTP) analogues (e.g., guanylyl-(.alpha.,.beta.)-methylene
diphosphate (GMPCPP)), and the like.
[0097] In a preferred embodiment, taxol-stabilized microtubules,
are prepared by polymerizing tubulin (2-10 mg/ml) at 37.degree. C.
for 45 minutes in BRB80 (80 mM PIPES [pH 6.8], 1 mM MgCl.sub.2, 1
mM EGTA) containing 1 mM GTP and 10% dimethyl sulfoxide (DMSO).
Taxol is then added to a concentration of 20 .mu.M.
[0098] GMPCPP-stabilized microtubules are prepared by incubating
tubulin in the above, buffer, substituting 0.5 mM GMPCPP for GTP.
The GMPCPP microtubules can be stored at room temperature without
taxol and are preferably used within 1 day after preparation.
[0099] B) Assay Reaction Mixture.
[0100] The assays of this invention are performed in a reaction
mixture that provides the components necessary for microtubule
depolymerizing or microtubule severing activity of the subject
protein (e.g., katanin, XKCM1, OP18 etc.) and that are compatible
with the enzymatic activity of the subject proteins. Typically the
reaction mixture comprises an appropriate buffer (e.g., HEPES, pH
6.5-8.0) and an energy supplying molecule such as guanosine
triphosphate (GTP) for microtubule depolymerizing proteins or
adenosine triphosphate (ATP) for severing molecules such as
katanin. One preferred assay,mixture is described in Example 1.
[0101] C) Microtubule Depolymerizing and Microtubule Severing
Agents.
[0102] As indicated above, the assays of this invention essentially
detect the activity of a test agent on a microtubule depolymerizing
or microtubule severing polypeptide. Microtubule depolymerizing
polypeptides such as OP18 (Belmont et al. (1990) Cell, 62: 579-589)
and XKCM1 (Walczak et al. (1996) Cell, 83: 37-47) increase the
frequency of catastrophes (transitions of a microtubule from a
growing to a shrinking state) and thus promote disassembly of
microtubules from their ends.
[0103] In contrast to microtubule depolymerizing proteins, other
proteins, such as katanin, promote the disassembly of microtubules
by generating internal breaks within a microtubule and are referred
to as microtubule severing proteins (See, e.g., Vale (1991) Cell
64: 827-839; Shiina et al. (1994) Science 266: 282-285; Shiina et
al. (1992) EMBO J. 11: 4723-4731; and McNally and Vale (1993) Cell,
75: 419-429).
[0104] Preferred microtubule depolymerizing proteins for the
methods of this invention include, but are not limited to XKCM1,
and OP18, while preferred microtubule severing proteins include
katanin.
[0105] i) Katanin.
[0106] Katanin, a heterodimer of 60 kDa and 80 kDa subunits
purified from sea urchin eggs, is unique among the known
microtubule and actin severing proteins in that it disrupts
contacts within the polymer lattice by using energy derived from
ATP hydrolysis (McNally and Vale (1993) Cell, 75: 419-429). Katanin
acts substoichiometrically, as one molecule of katanin can release
several tubulin dimers from a microtubule. Katanin does not appear
to proteolyze or modify tubulin, since the tubulin released from
the disassembly reaction is capable of repolymerizing (McNally and
Vale (1993) Cell, 75: 419-429). The mechanism of microtubule
severing by katanin, however, is not understood.
[0107] Katanin-catalyzed microtubule severing and disassembly could
potentially be involved in several changes in the microtubule
cytoskeleton observed in vivo. Recent studies have-shown that
katanin is concentrated at the centrosome in a
microtubule-dependent manner in sea urchin embryos (McNally et al.
(1996) J Cell Sci. 109: 561-567). One phenomenon that could require
disassembly of microtubules at the centrosome is the poleward flux
of tubulin in the mitotic spindle (Mitchison (1989) J Cell Biol.
109: 637-652). The disassembly of microtubule minus ends at the
spindle pole during poleward flux could be driven by katanin, or
katanin could simply allow depolymerization by uncapping
microtubule minus ends that are docked onto .gamma.-tubulin ring
complexes (Zheng et al. (1995) Nature 378: 578-583; Moritz et al.
(1995) Nature 378: 638-640). Another possible role for katanin at
the centrosome is in promoting the release of microtubules from
their centrosomal attachment points. Microtubules are nucleated
from .gamma.-tubulin ring complexes at the centrosome (Joshi et
al., (1992) and Moritz et al. (1995) Nature 378: 63 8-640), but
release of microtubule minus ends has been observed indirectly in
Dictyostelium (Kitanishi-Yumura et al. (1987) Cell Motil.
Cytoskeleton 8: 106-117) and directly in PtK1 cells (Keating (1997)
Proc. Natl. Acad. Sci. USA, 94: 5078-5083) and Xenopus egg extracts
(Belmont et al. (1990) Cell 62: 579-589). Finally, katanin could
accelerate the rapid disassembly of the interphase microtubule
network at the G2/M transition (Zhai et al. (1996) J. Cell Biol.
135: 201-214) by severing cytoplasmic microtubules, which would
increase the number of free microtubule ends from which
depolymerization could occur. Regardless of the particular mode of
activity, modulation of katanin activity will have profound effects
on the cell cycle.
[0108] The amino acid and nucleic acid sequences of the p60 and p80
subunits of katanin are provided in FIGS. 1A and 2A (See also SEQ
ID NO:1 and SEQ ID NO:2). It was a discovery of this invention that
the microtubule severing activity resides entirely in the p60
subunit. Thus the assays of this invention can be practiced either
with the heterodimeric katanin or with a p60 subunit alone.
[0109] The p60 and/or p80 subunits of katanin can be purified
(e.g., from sea urchin eggs, e.g., eggs from Strongylocentrotus
purpuratus) as described by McNally and Vale (1993) Cell, 75:
419-429. Alternatively, either or both subunits can be
recombinantly expressed and purified as described below and in
Example 1.
[0110] ii) XKCM1
[0111] XKCM1 (for Xenopus kinesin central motor 1) is a motor
protein essential for mitotic spindle assembly in vitro. XKCM1
localizes to centromeres and appears to regulate the polymerization
dynamics of microtubules. The isolation of an XKCM1 clone is
described by Walczak et al. (1996) Cell, 84: 37-47, and a nucleic
acid sequence of an XKCMl cDNA is provided therein and in SEQ ID
NO:3. Using this sequence information, XKCM1 can be expressed as
described below and by Walczak et al. (1996) supra.
[0112] iii) OP18
[0113] Another microtubule depolymerizing motor protein suitable
for use in the methods of this invention is OP18, also called
stathmin or stathmin/op18. OP18 is described in detail by Gradin et
al. (1998) J Cell Biol., 140(1): 131-141, by Andersen et al. (1997)
Nature, 389(6651): 640-643, by Larsson et al. (1997) Mol. Cell.
Biol., 17(9):5530-5539, and by Belmont et al. (1996) Cell, 84(4):
623-631.
[0114] iv) Other Microtubule Severing or Depolymerizing
Proteins
[0115] Other microtubule depolymerizing or severing proteins
include, but are not limited to elongation factor-1.alpha.(Shiina
et al. (1994) Science 266: 282-285) and a novel homo-oligomeric
protein described by Shiina et al. (1992) EMBO J. 11:
4723-4731.
[0116] Other microtubule depolymerizing or severing proteins can be
identified with only routine experimentation. The assays used to
identify microtubule depolymerizing or severing proteins are
identical to the assays described herein, the only difference being
that no test agent is required. A detailed example of the assay of
a microtubule severing protein (katanin) is provided in McNally and
Vale (1993) Cell, 75: 419-429. The same approach can readily be
used to identify other severing or depolymerizing proteins.
[0117] IV) Detection Methods.
[0118] Any detection method that allows detection and/or
quantification of the amount or rate of appearance of tubulin
monomers or oligomers and/or the rate of disappearance of assembled
(polymerized) microtubules can be used in the assays of this
invention. Preferred detection methods include, but are not limited
to video microscopy; DAPI fluorescence changes, fluorescence
resonance energy transfer and centrifugation.
[0119] A) Video Microscopy.
[0120] In one embodiment, microtubule depolymerization or severing
is detected by microscopy (visually or using a video or
photographic recording device). Assays involving microscopic
visualization of microtubules preferably utilize labeled (e.g.,
fluorescently labeled) microtubules. The microtubules are
preferably immobilized on a solid support (e.g., a glass slide),
and exposed to a solution containing the microtubule depolymerizing
or severing protein and an a nucleoside triphosphate. The intact
and depolymerized or severed microtubules can be directly
visualized using a microscope. Microtubule depolymerization in the
control and the assay containing the test agent can be visualized
side by side or sequentially.
[0121] The microscope can optionally be equipped with a still
camera or a video camera and may be equipped with image acquisition
and analysis software to quantify the relative abundance of intact
and fragmented microtubules.
[0122] This method can be used with essentially any label that can
be visualized in a microscope. Such labels include, but are not
limited to fluorescent labels (e.g., fluorescein, rhodamine, etc.),
colorimetric labels, and radioactive labels (with appropriate
scintillation screen), and the like. In some embodiments of the
assays of this invention, the microtubules can be visualized
without any label (e.g., via differential interference contrast
microscopy).
[0123] An illustration of the use of video microscopy to visualize
microtubule severing by katanin is provided by McNally and Vale
(1993) Cell, 75: 419-429. In this case, the microtubules are
labeled with rhodamine and the images of the severed microtubules
are captured digitally.
[0124] B) DAPI Fluorescence changes.
[0125] In another embodiment, the state of microtubule
polymerization can be determined by changes in fluorescence of DAPI
stained microtubules. It has been shown that DAPI fluorescence
intensity is higher when this dye is bound to polymerized versus
free tubulin (Heusele et al. (1987) Eur. J Biochem. 165: 613-620).
When katanin and ATP were incubated with DAPI-labeled microtubules,
a linear decrease in fluorescence intensity is observed as a
function of time, reflecting the conversion of microtubules to
tubulin.
[0126] Assay can thus be performed as described above with DAPI
labeled stabilized microtubules. The rate or amount decrease in
DAPI fluorescence is detected as described by Heusele et al. supra.
The change in fluorescence with a test agent is compared to that
observed in a negative and/or positive control reaction.
[0127] It was a surprising discovery of this invention that
tubulin, tubulin dimers, tubulin oligomers or microtubules can be
labeled with various labels such as DAPI and that the label does
not interfere with the interaction of various test agents or
cytoskeletal associated proteins with the labeled tubulin to a
degree that would prevent assaying the impact of a test agent on
microtubule polymerization, and/or depolymerization, and/or
severing. Labels that can be used include, but are not limited to
anilinonapthalene sulfonate (ANS) (e.g., Molecular Probes Catalogue
Nos: A-47, A-50, T-53, etc.), bis-ANS (Molecular Probes Catalogue
No: B-153), N-phenyl-1-naphthylene (NPN) (Molecular Probes
Catalogue No: P65), DCVJ (Molecular Probes Catalogue No: D-3923),
ruthenium red, and cresol violet.
[0128] C) Fluorescence resonance energy transfer
[0129] The degree of microtubule polymerization/depolymnerization
can also be determined by fluorescent resonance energy transfer
(FRET). Fluorescence resonance energy transfer, a phenomenon that
occurs when two fluorophores with overlapping absorption and
emission spectra are located close together (e.g., <7 nm apart)
(Stryer (1978) Ann. Rev. Biochem., 47: 819-846). FRET is a powerful
technique for measuring protein-protein associations and has been
used previously to measure the polymerization of monomeric actin
into a polymer (Taylor et al. (1981) J. Cell Biol., 89: 362-367)
and actin filament disassembly by severing (Yamamoto et al. (1982)
J. Cell Biol., 95: 711-719).
[0130] In a preferred embodiment, equimolar proportions of
differently labeled (e.g., fluorescein labeled and
rhodamine-labeled) tubulin are combined. The fluorescence is
quenched upon tubulin polymerization indicating that the
tubulin-bound fluorochromes in a microtubule come in close enough
proximity for energy transfer to occur. When the microtubule is
depolymerized or severed, a rapid unquenching of (e.g.,
fluorescein) fluorescence is observed.
[0131] The rates and/or amount of fluorescence generated by a
reaction with a test agent and a control can be compared. A
decrease in rate or amount of fluorescence in the presence of a
test agent indicates inhibitory activity on the microtubule
depolymerizing or severing protein(s).
[0132] In particularly preferred embodiment, the microtubules are
polymerized from a mixture of equal concentrations of fluorescein
and rhodamine tubulin and diluted to 600 .mu.g/ml tubulin in BRB80
containing 20 .mu.M taxol and the oxygen-depleting system
consisting of glucose oxidase (30 .mu.g/ml), catalase (100 mg/ml),
glucose (10 mM), and dithiothreitol (10 mM) (Kishino and Yanigida
(1988) Nature 334: 74-76). Aliquots 150 .mu.l of these microtubules
can be mixed (e.g., with samples of purified severing protein), and
the fluorescence from the fluorescein (excitation 492 nm; emission
518 nm) is recorded. The reaction is preferably run with a positive
and negative control. A detailed FRET assay for tubulin
polymerization is found in McNally et al. (1993) Cell, 75:
419-429.
[0133] D) Centrifugation.
[0134] Microtubule disassembly in solution can be documented in a
quantitative manner by examining the relative amounts of
sedimentable and nonsedimentable tubulin after incubation with the
severing or depolymerizing protein (e.g., katanin) and ATP or GTP.
In the absence of a modulating agent, when taxol-stabilized
microtubules are incubated tithe the katanin p80 and p60 subunits
and ATP, nonsedimentable tubulin is released from microtubule
polymer in an approximately linear manner. The rate of release
varies with microtubule depolymerizing or severing protein
concentration and will be dependent on the activity of a modulating
"test" agent if present.
[0135] In a preferred embodiment, fluorescent microtubules (e.g.,
100-300 .mu.g/ml tubulin) are incubated with the microtubule
depolymerizing or severing protein in buffer (e.g., 20 mM HEPES (pH
7.5), 2 mM MgCl.sub.2, 25 mM potassium glutamate, 0.02% Triton
X-100, 250 .mu.g/ml SBTI, and 20 .mu.M taxol or taxol derivative)
at various times. Aliquots (e.g., of 100 .mu.l) are brought to 10
mM ADP or GDP (to stop the severing or depolymerizing reaction) and
sedimented (e.g., at 228,000.times.g for 10 minutes). Supernatants
are removed, and pellets are resuspended in 100 .mu.l of buffer.
The pellets and supernatants can be brought up to 300 .mu.l (e.g.,
with BRB80), and the relative fluorescence signals in the
supernatant and the pellet are quantitated using a Perkin Elmer
L25B luminescence spectrometer.
[0136] E) Liquid Crystal Assay Systems.
[0137] In still another embodiment, binding of the microtubule
depolymerizing or severing proteins to microtubules can be detected
by the use of liquid crystals. Alternatively, it is expected that
liquid crystals can be used to monitor the state of tubulin
polymerization.
[0138] Liquid crystals can be used to amplify and transduce
receptor-mediated binding of proteins at surfaces into optical
outputs. Spontaneously organized surfaces can be designed so that
protein molecules, upon binding to ligands (e.g., microtubules)
hosted on these surfaces, trigger changes in the orientations of 1-
to 20-micrometer-thick films of supported liquid crystals, thus
corresponding to a reorientation of .about.10.sup.5 to 10.sup.6
mesogens per protein. Binding-induced changes in the intensity of
light transmitted through the liquid crystal are easily seen with
the naked eye and can be further amplified by using surfaces
designed so that protein-ligand recognition causes twisted nematic
liquid crystals to untwist (See, e.g., Gupta et al. (1998) Science,
279: 2077-2080). This approach to the detection of ligand-receptor
binding does not require labeling of the analyte, does not require
the use of electroanalytical apparatus, provides a spatial
resolution of micrometers, and is sufficiently simple that it is
useful in biochemical assays and imaging of spatially resolved
chemical libraries.
[0139] D) Synthesis or Expression of Microtubule Depolymerizing or
Severing Proteins.
[0140] i) De Novo Chemical Synthesis.
[0141] Using the information provided, herein, the microtubule
depolymerizing or severing proteins, protein subunits, or
subsequences thereof may be synthesized using standard chemical
peptide synthesis techniques. Where the desired subsequences are
relatively short, the molecule may be synthesized as a single
contiguous polypeptide. Where larger molecules are desired,
subsequences can be synthesized separately (in one or more units)
and then fused by condensation of the amino terminus of one
molecule with the carboxyl terminus of the other molecule thereby
forming a peptide bond.
[0142] Solid phase synthesis in which the C-terminal amino acid of
the sequence is attached to an insoluble support followed by
sequential addition of the remaining amino acids in the sequence is
the preferred method for the chemical synthesis of the polypeptides
of this invention. Techniques for solid phase synthesis are
described by Barany and Merrifield, Solid-Phase Peptide Synthesis;
pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. VOL 2:
Special Methods in Peptide Synthesis, Part A.; Merrifield, et al.
(1963) J. Am. Chem. Soc., 85: 2149-2156; and Stewart et al (1984)
Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford,
Ill.
[0143] ii) Recombinant Expression.
[0144] In a preferred embodiment, the microtubule depolymerizing or
severing proteins, protein subunits, or subsequences, are
synthesized using recombinant DNA methodology. Generally this
involves creating a DNA sequence that encodes the protein, placing
the DNA in an expression cassette under the control of a particular
promoter, expressing, the protein in a host, isolating the
expressed protein and, if required, renaturing the protein. DNA
encoding the microtubule depolymerizing or severing proteins,
protein subunits, or subsequences of this invention can be prepared
by any suitable method as described above, including, for example,
cloning and restriction of appropriate sequences or direct chemical
synthesis by methods such as the phosphotriester method of Narang
et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method
of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the
diethylphosphoramidite method of Beaucage et al. (1981) Tetra.
Lett., 22: 1859-1862; and the solid support method of U.S. Pat. No.
4,458,066.
[0145] Chemical synthesis produces a single stranded
oligonucleotide. This may be converted into double stranded DNA by
hybridization with a complementary sequence, or by polymerization
with a DNA polymerase using the single strand as a template. One of
skill would recognize that while chemical synthesis of DNA is
limited to sequences of about 100 bases, longer sequences may be
obtained by the ligation of shorter sequences.
[0146] Alternatively, subsequences may be cloned and the
appropriate subsequences cleaved using appropriate restriction
enzymes, The fragments may then be ligated to produce the desired
DNA sequence.
[0147] In one embodiment, the microtubule depolymerizing or
severing proteins, of this invention can be cloned using DNA
amplification methods such as polymerase chain reaction (PCR).
Thus, for example, the nucleic acid sequence or subsequence is PCR
amplified, using a sense primer containing one restriction site
(e.g., NdeI) and an antisense primer containing another restriction
site (e.g., HindIll). This will produce a nucleic acid encoding the
desired the microtubule depolymerizing or severing protein having
terminal restriction sites. This nucleic acid can then be easily
ligated into a vector containing a nucleic acid encoding the second
molecule and having the appropriate corresponding restriction
sites.
[0148] Suitable PCR primers can be determined by one of skill in
the art using the sequence information provided herein. Appropriate
restriction sites can also be added to the nucleic acid encoding
the microtubule depolymerizing or severing proteins by
site-directed mutagenesis. The plasmid containing the microtubule
depolymerizing or severing protein encoding nucleic acid is cleaved
with the appropriate restriction endonuclease and then ligated into
the vector encoding the second molecule according to standard
methods.
[0149] The nucleic acid sequences encoding the microtubule
depolymerizing or severing proteins may be expressed in a variety
of host cells, including E. coli, other bacterial hosts, yeast, and
various higher eukaryotic cells such as the COS, CHO and HeLa cells
lines and myeloma cell lines. As the microtubule depolymerizing or
severing proteins are typically found in eukaryotes, a eukaryote
host is preferred. The recombinant protein gene will be operably
linked to appropriate expression control sequences for each host.
For E. coli, this includes a promoter such as the T7, trp, or
lambda promoters, a ribosome binding site and preferably a
transcription termination signal. For eukaryotic cells, the control
sequences will include a promoter and preferably an enhancer
derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and
a polyadenylation sequence, and may include splice donor and
acceptor sequences.
[0150] The plasmids of the invention can be transferred into the
chosen host cell by well-known methods such as calcium chloride
transformation for E. coli and calcium phosphate treatment or
electroporation for mammalian cells. Cells transformed by the
plasmids can be selected by resistance to antibiotics conferred by
genes contained on the plasmids, such as the amp, gpt, neo and hyg
genes.
[0151] Once expressed, the recombinant the microtubule
depolymerizing or severing proteins can be purified according to
standard procedures of the art, including ammonium sulfate
precipitation, affinity columns, column chromatography, gel
electrophoresis and the like (See generally, R. Scopes, (1982)
Protein Purification, Springer-Verlag, N.Y.; Deutscher (1990)
Methods in Enzymology Vol. 182. Guide to Protein Purification.,
Academic Press, Inc. N.Y.). Substantially pure compositions of at
least about 90 to 95% homogeneity are preferred, and 98 to 99% or
more homogeneity are most preferred. Once purified, partially or to
homogeneity as desired, the polypeptides may then be used (e.g., as
immunogens for antibody production).
[0152] One of skill in the art would recognize that after chemical
synthesis, biological expression, or purification, the microtubule
depolymerizing or severing protein(s) may possess a conformation
substantially different than the native conformations of the
constituent polypeptides. In this case, it may be necessary to
denature and reduce the polypeptide and then to cause the
polypeptide to re-fold into the preferred conformation. Methods of
reducing and denaturing proteins and inducing re-folding are well
known to those of skill in the art (See, Debinski et al. (1993) J.
Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993)
Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal.
Biochem., 205: 263-270). Debinski et al., for example, describes
the denaturation and reduction of inclusion body proteins in
guanidine-DTE. The protein is then refolded in a redox buffer
containing oxidized glutathione and L-arginine.
[0153] One of skill would recognize that modifications can be made
to the microtubule depolymerizing or severing proteins without
diminishing their biological activity. Some modifications may be
made to facilitate the cloning, expression, or incorporation of the
targeting molecule into a fusion protein. Such modifications are
well known to those of skill in the art and include, for example, a
methionine added at the amino terminus to provide an initiation
site, or additional amino acids (e.g., polyHis) placed on either
terminus to create conveniently located restriction sites or
termination codons or purification sequences.
[0154] In a particularly preferred embodiment, the katanin
protein(s) are expressed as described in Example 1, while XKCM1 is
expressed and purified as described by Walczak et al. (1996)
supra.
[0155] V. Data Management.
[0156] In one embodiment, the assays of this invention are
facilitated by the use of databases to record assay results.
Particular with the use of large-scale screening systems, (e.g.,
screening of combinatorial libraries) data management can become a
significant issue. For example, all natural hexapeptides have been
synthesized in a single combinatorial experiment producing about 64
million different molecules. Maintenance and management of even a
small fraction of the information obtained by screening such a
library is aided by methods automated information retrieval (e.g.,
a computer database).
[0157] Such a database is useful for a variety of functions,
including, but not limited to library registration, library or
result display, library and/or result specification, documentation,
and data retrieval and exploratory data analysis. The registration
function of a database provides recordation/registration of
combinatorial mixtures and assay results to protect proprietary
information in a manner analogous to the registration/protection of
tangible proprietary substances. Library and assay result display
functions provide an effective means to review and/or categorize
relevant assay data. Where the assays utilize complex combinatorial
mixtures for test agents, the database is useful for library
specification/description. The database also provides documentation
of assay results and the ability to rapidly retrieve, correlate (or
conduct other statistical analysis), and evaluate assay data.
[0158] Thus, in some preferred embodiments, the assays of this
invention additionally involve entering test agent(s) identified as
positive (i.e., having an effect on microtubule polymerization,
and/or depolymerization, and/or severing) in a database of
"positive" compounds and more preferably in a database of
therapeutic or bioagricultural lead compounds.
[0159] The database can be any medium convenient for recording and
retrieving information generated by the assays of this invention.
Such databases include, but are not limited to manual recordation
and indexing systems (e.g., file-card indexing systems). However,
the databases are most useful when the data therein can be easily
and rapidly retrieved and manipulated (e.g., sorted, classified,
analyzed, and/or otherwise organized). Thus, in a preferred
embodiment, the signature the databases of this invention are most
preferably "automated" (e.g., electronic [e.g., computer-based])
databases. The database can be present on an individual
"stand-alone" computer system, or a component of or distributed
across multiple "nodes" (processors) on a distributed computer
systems. Computer systems for use in storage and manipulation of
databases are well known to those of skill in the art and include,
but are not limited to "personal computer systems," mainframe
systems, distributed nodes on an inter- or intra-net, data or
databases stored in specialized hardware (e.g., in microchips), and
the like.
[0160] VI. Kits for Screening for Modulators of Microtubule
Depolymerization or Microtubule Severing Agents.
[0161] In still another embodiment, this invention provides kits
for the practice of any of the methods described herein. The kits
comprise one or more containers containing one or more of the assay
components described herein. Such components include, but are not
limited to stabilized microtubules, microtubule depolymerizing or
microtubule. severing proteins or protein subunits, one or more
test agents, reaction media, solid supports (e.g., microtitre
plates) with attached components, buffers, labels, and other
reagents as described herein.
[0162] The kits may optionally include instructional materials
containing directions (i.e., protocols) for carrying out any of the
assays described herein. While the instructional materials
typically comprise written or printed materials they are not
limited to such. Any medium capable of storing such instructions
and communicating them to an end user is contemplated by this
invention. Such media include, but are not limited to electronic
storage media (e.g., magnetic discs, tapes, cartridges, chips),
optical!media (e.g., CD ROM), and the like. Such media may include
addresses to internet sites that provide such instructional
materials.
EXAMPLES
[0163] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
[0164] Katanin, a Microtubule-Severing Protein is a Novel AAA
ATPase that Targets to the Centrosome using a WD40-Containing
Subunit.
[0165] RESULTS
[0166] To begin dissecting the functional domains of katanin, we
isolated cDNA clones for the p60 and p80 subunits from cDNA derived
from sea urchin (Strongylocentrotus purpuratus) egg MRNA. After
first obtaining peptide sequence of several proteolytic fragments
from the two sea urchin katanin subunits, cDNA clones were isolated
using a combination of degenerate PCR, cDNA library screening, and
anchor-ligated PCR (see Experimental Procedures). The predicted
amino acid sequences of the cDNA clones contained 139 amino acids
(a.a.) and 306 amino acids of peptide sequences obtained by direct
microsequencing of p60 and p80 respectively.
[0167] p60 is Novel Member of the AAA ATPase Superfamily
[0168] Sequence analysis of the p60 cDNA clone revealed an open
reading frame that encodes a 516 a.a. polypeptide (FIG. 1A). A
BLAST search with the predicted p60 protein sequence revealed that
this polypeptide contains a C-terminal domain (a.a. 231-447) that
is highly conserved in the AAA ATPase superfamily (FIG. 1B)
(Confalonieri et al. (1995) BioEssays 17: 639-650). This .about.220
amino acid region contains the "Walker A" (P-loop) and "Walker B"
motifs found in many ATPases (Walker et al. (1982) EMBO J. 1:
945-95 1). AAA proteins, which contain either one or two of these
220 a.a. ATP-binding modules, constitute a large superfamily whose
members have been implicated in a variety of cellular functions
(Confalonieri et al. (1995) supra.).
[0169] Of the AAA domains entered into sequence data bases, mei-1,
a C. elegans protein required for meiosis (Clark-Maguire et al.
(1994) Genetics 13 6: 533-546), is most closely related to p60 (55%
a.a. identify, FIG. 1B). Mei-1 was discovered in a genetic screen
as a protein that is required for meiotic spindle formation, but
disappears during subsequent mitotic divisions. Interestingly, both
p60 (McNally et al. (1996) J Cell Sci. 109: 561-567) and mei- I
(Clark-Maguire et al. (1994) J. Cell Biol. 126: 199-209) are
localized to spindle poles in a microtubule-dependent manner.
However, the N-terminal half of p60 has no significant homology to
mei-1, suggesting that p60 and mei-1 may not be orthologs. BLAST
searches with p60 sequences, however, revealed several human ESTs
(expressed sequence tags) that have strong amino acid identity
outside of the AAA domain, suggesting the existence of vertebrate
homologs of p60.
[0170] p80 Contains WD40 Repeats
[0171] Sequence analysis of the sea urchin p80 cDNA clone revealed
a predicted 690 a.a. polypeptide that contains six "WD40" repeat
motifs extending from residues 1-256 (FIG. 2A). An alignment of
these repeats with two unrelated WD40 repeat-containing proteins is
shown in FIG. 2B. The WD40 repeats in several proteins have been
documented to participate in protein-protein binding interactions
(Komachi et al. (1994) Genes Dev. 8: 2857-2867; Wall et al. (1995)
Cell 83: 1047-1058). The C-terminal region of p80 (residues
257-690) did not exhibit significant amino acid identity to any
previously described protein. However, significant identity of sea
urchin p80 was observed with several human EST clones. The
sequences of these clones were used to isolate a full length human
p80 katanin homolog by PCR (see Experimental Procedures). The human
cDNA encodes a predicted 655 a.a. protein with 61% a.a. identity in
the WD40 domain (a.a. 1-256) (FIG. 2B), 23% a.a. identity in the
central 187 residues, and 54% a.a. identity in the C-terminal 164
a.a. with S. purpuratus p80 katanin (latter two regions are not
shown).
[0172] Baculovirus Expression and Molecular Structure of the
Katanin Subunits
[0173] Deciphering the roles of the two katanin subunits is
essential for understanding the enzyme's mechanism and biological
activities. However, separation of the native sea urchin p60/p80
subunits requires denaturing conditions. We therefore sought to
express the two subunits together and separately and then test
their enzymatic activities. Bacterial expression of p60 produced
largely insoluble protein, and the small amount of soluble p60 had
no microtubule-stimulated ATPase activity (data not shown).
However, using the baculovirus expression system, we obtained
soluble p60, p80, and the p60/p80 complex (each expressed with a
N-terminal His.sub.(6) tag (SEQ ID NO:14), and purified the
expressed proteins using metal affinity chromatography (FIG. 3A).
When p60 and p80 were co-expressed, the stoichiometry of the two
subunits in the purified protein was approximately equal (1.0:0.9
p60:p80 molar ratio, as determined by Coomassie staining).
Moreover, immunoprecipitation with an anti-p60 antibody led to
co-immunoprecipitation of equal quantities of p60 and p80 (FIG.
3B). These results indicate that baculovirus -expressed p60 and p80
heterodimerize, as observed with native katanin (McNally and Vale
(1993) Cell, 75: 419-429).
[0174] To examine katanin's structure, baculovirus-expressed p60,
p80, or p60/80 was adsorbed onto mica chips, and the chips were
subsequently frozen, etched, and rotary shadowed with platinum
(Heuser (1989) J. Electron Microsc. Technique 13:, 244-263; Heuser
(1983) J. Mol. Biol. 169: 155-195). The platinum-shadowed p60
appeared as a 14-16 nm ring punctuated in the center by a 3-5 nm
opening, often with what appears to be cracks radiating outward
(FIG. 4A). p80, on the other hand, appeared as .about.11 nm
particles and occasional unstructured protein aggregates; rings
were not observed (FIG. 4B). Rings were also seen for p60/p80
complexes (FIG. 4C, 4D) and native sea urchin katanin (data not
shown). Interestingly, two types of p60/p80 complexes were visible:
large .about.20 nm diameter rings with bright edges, which is
suggestive of taller complexes that extend upward from the mica
(FIG. 4D), and smaller rings of the size of p60 alone with several
p80 sized particles radiating from the central ring (FIG. 4C). The
large and small rings might represent closed and "splayed" versions
of the p60/p80 complex, respectively, which could be produced if
the complex dissociates upon mica adsorption. Both p60 and p60/p80
structures resemble the rings observed for the AAA ATPases NSF and
p97, whose dimensions are 15-17 nm (Hanson et al. (1997) Cell 90:
523-535).
[0175] p60 Katanin has Microtubule-Stimulated ATPase and Severing
Activity
[0176] With the availability of isolated p60 and p80, we then
examined whether the individual subunits have ATPase activity. The
co-expressed p60/p80 heterodimer displayed an ATP turnover rate of
0.3 ATP/sec/heterodimer; this activity was stimulated
.about.10-fold by microtubules (FIG. 5A). This basal activity and
the fold stimulation by microtubules are similar to that observed
for native sea urchin katanin (data not shown). Consistent with the
finding of an AAA domain in its sequence, p60 alone displayed a
microtubule-stimulated ATPase activity. Surprisingly, the maximal
basal and microtubule-stimulated ATPase rates of p60 were only
2-fold lower than those of the p60/p80 heterodimer (FIG. 5A). p80
itself had no detectable ATPase activity. The activation of ATPase
activity by microtubules displayed an atypical, non-hyperbolic
behavior. ATP turnover by p60 and p60/p80 was stimulated at low
concentrations of microtubules (peak at .about.2 .mu.M tubulin),
but then decreased at higher microtubule concentrations (FIG. 5A).
This same complex pattern of microtubule stimulation was also
observed for native sea urchin katanin (data not shown).
[0177] We then tested the microtubule severing activity of p60,
p80, and p60/p80 using a fluorescence microscopy assay (McNally and
Vale (1993) Cell, 75: 419-429). Both p60 and p60/p80 severed
microtubules in this assay (FIG. 5B). Broken microtubules were
observed within 1 min after introducing 0.1 .mu.M p60 or p60/p80,
and microtubules were completely disassembled after 5 min. The
reaction appeared somewhat slower with p60 alone. Microtubules
remained intact if ATP was omitted from the reaction (not shown).
In contrast, p80 was unable to sever microtubules at concentrations
5-fold higher than those used for p60 (FIG. 5B). These experiments
demonstrate that p60 alone can carry out all of the steps necessary
for coupling ATP hydrolysis to microtubule disassembly.
[0178] To better compare the microtubule severing activities of p60
and p60/p80, we developed a quantitative microtubule disassembly
assay based upon a previous finding that DAPI fluorescence
intensity is higher when this dye is bound to polymerized versus
free tubulin (Heusele et al. (1987) Eur. J. Biochem. 165: 613-620).
When katanin and ATP were incubated with DAPI-labeled microtubules,
a linear decrease in fluorescence intensity was observed as a
function of time, reflecting the conversion of microtubules to
tubulin (FIG. 5C. The loss of microtubule polymer was confirmed by
centrifugation studies, which showed an increase in
non-sedimentable tubulin with a similar time course (data not
shown). The fluorescence decrease induced by these enzymes reached
a steady-state level that was slightly higher than pure, monomeric
tubulin, suggesting that some tubulin oligomer may exist at steady
state. The rate of fluorescence decrease was proportional to p60 or
p60/p80 concentration over a 10-fold range (data not shown). When
the rates of microtubule disassembly were compared, p60 was half as
active as p60/p80 (FIG. 5C). This slower rate of microtubule
disassembly is consistent with the previously described 2-fold
decrease in ATPase activity of p60 compared with p60/p80.
[0179] The p80 WD40 Domain Targets to the Centrosome
[0180] The finding that p60 by itself can sever microtubules left
open the question of the function of the p80 katanin subunit. At
least two functional domains of p80 could be postulated. First,
since katanin is a heterodimer (McNally and Vale (1993) Cell, 75:
419-429), some part of p80 must be involved in heterodimerization
with p60. Second, because previous studies have shown that katanin
is concentrated at centrosomes in vivo (McNally et al. (1996) J.
Cell Sci. 109: 561-567), p80 could contain a domain that interacts
with a centrosomal protein to allow targeting of the katanin
holoenzyme. Because WD40 repeats have been implicated in
heterophilic protein-protein interactions (Komachi et al. (1994)
Genes Dev. 8: 2857-2867; and Wall et al. (1995) Cell 833:
10471058), the six WD40 repeats in p80 represented a good candidate
domain for participating in either dimerization or centrosome
targeting.
[0181] In order to test whether the WD40 repeats of p80 are
required for heterodimerization with p60, we deleted the entire
WD40 domain and examined whether the truncated p80
(p80.DELTA.1-302) interacted with p60 when the two polypeptides
were co-translated in a rabbit reticulocyte system. The truncated
p80 (p8.DELTA.1-302) was co-immunoprecipitated by the anti-p60
antibody only in the presence of p60 and just as efficiently as
full length p80 (FIG. 6). This finding indicates that the WD40
repeats are not required for dimerizing the two katanin subunits.
Nevertheless, it remained possible that the WD40 domain was one of
multiple, redundant p60-interacting domains. However, a C-terminal
truncation of p80 (p80 .DELTA.3 03-690) containing only the WD40
domain did not coimmunoprecipitate with p60 (FIG. 6). These results
indicate that the p80 WD40 repeats are neither necessary nor
sufficient for dimerization with p60. To determine which region of
p80 is required for interaction with p60, a p80 deletion lacking
the C-terminal 130 amino acids (p80.DELTA.560-690) was constructed
and was found not to co-inmmunoprecipitate with p60 (FIG. 6). These
findings suggest that the C-terminal 130 amino acids of p80, but
not the WD40 repeat domain, are involved in the dimerization with
p60.
[0182] To examine whether the p80 WD40 repeats bind to a protein in
the centrosome, we tested whether these repeats can target a
heterologous protein (green fluorescent protein, GFP) to the
centrosome after transient transfection in the human fibroblast
cell line, MSU1.1 (Lin et al. (1995) Int. J. Cancer 63: 140-147).
The WD40 domain of human p80 katanin was used, because it was more
likely that the human protein would interact with centrosomal
proteins in this human cell line. Immunofluorescence of MSU1.1
cells with an antibody specific for human p80 katanin (see
Experimental Procedures) showed labeling of the cytoplasm and more
concentrated staining at one or two spots that co-localized with
.gamma.-tubulin staining (FIG. 7), confirming that endogenous
katanin is concentrated at centrosomes in fibroblasts as- it is in
sea urchin embryos (McNally et al. (1996) J. Cell Sci. 109:
561-567). In contrast to the localization in sea urchin embryos,
the concentration of p80 at centrosomes in fibroblasts remained
after complete depolymerization of microtubules with nocodazole
(data not shown), suggesting that katanin is bound to the
pericentriolar material. When a fusion protein consisting of the
six WD40 repeats of human p80 katanin (a.a. 1-263) appended to the
N-terminus of green fluorescent protein (GFP) was expressed in
MSU1.1 cells, one or two foci of green fluorescence that
co-localized with .gamma.-tubulin staining was observed 2-4 hr
after transfection in addition to diffuse cytoplasmic fluorescence
(FIG. 7). Identical results were obtained in transfections of HeLa
cells (not shown). In contrast to these findings, cells transfected
with GFP alone never revealed foci of green fluorescence at
centrosomes (not shown). After longer periods of expression (8-24
hr) of p80 WD40-GFP, numerous heterogeneously-sized bright foci of
green fluorescence appeared that did not co-localize with
.gamma.-tubulin, and later, massive aggregates several .mu.m in
diameter were observed (not shown). These results indicate that the
WD40 repeats of human p80 katanin are sufficient to target GFP to
the centrosome and suggest that once the centrosome binding sites
are saturated, the additional fusion protein aggregates in the
cytoplasm.
Discussion
[0183] Katanin is a unique enzyme that couples ATP hydrolysis to
the dissociation of tubulin subunits from the microtubule lattice
(McNally and Vale (1993) Cell, 75: 419-429). Other than the motor
proteins kinesin and dynein, katanin is the only known
microtubule-associated ATPase. In this study, we have determined
the primary structure of the p60 and p80 katanin subunits and
examined the roles of the two subunits in microtubule severing and
the cellular localization of the enzyme.
[0184] Mechanism of Katanin-Mediated Microtubule Severing
[0185] Sequence analysis of p60 katanin revealed that it is a novel
member of the AAA family of ATPases. This finding suggested that
p60 might be responsible for the previously reported ATPase
activity of the native katanin dimer (McNally and Vale (1993) Cell,
75: 419-429). However, neither p60 nor p80 contained an
identifiable microtubule binding sequence, such as those found in
tau (Butner and Kirschner (1991) J. Cell Biol., 115: 717-730) or
MA.PIB (Noble et al. (1989) J. Cell Biol. 109: 3367-3376), and
therefore it was not possible to ascribe the microtubule binding
and severing activities of katanin to either subunit based upon
sequence information alone. By measuring the activities of the p60
and p80 subunits purified individually and together as a dimer, we
have found that katanin's p60 subunit exhibits both
microtubule-stimulated ATPase activity and microtubule-severing
activity in the absence of the p80 subunit. Since p60 has all
elements required for functional interactions with microtubules,
future structure-function studies on the mechanism of microtubule
severing can be focused on this single subunit. Furthermore, we
have found that p60 katanin can form rings, the dimensions and
appearance of which are similar to those reported for the AAA
proteins NSF and p97 (Hanson et al. (1997) Cell 90: 523-535). The
comparison of p60 with other AAA proteins provides clues as to how
katanin disassembles microtubules, as discussed below.
[0186] The ATPase properties of katanin show both similarities and
differences with other AAA family members. Katanin's basal ATPase
activity of 0.3 ATP/katanin/sec and maximal microtubule-stimulated
rate of 3 ATP/katanin/sec are comparable to values of 1 ATP/sec for
p97 (Peters et al. (1992) J. Mol. Biol., 223: 557-571) and 0.08
ATP/sec for recombinant NSF (Morgan et al. (1994) J. Biol. Chem.
269: 29347-29350). NSF ATPase is also stimulated two-fold upon
binding to its target protein, .alpha.- or .gamma.-SNAP (Morgan et
al. (1994) J. Biol. Chem. 269: 29347-293 50). However, katanin's
ATPase activity displays a complex stimulation by microtubules. At
low microtubule concentrations (<2 .mu.tM), ATPase activity
increases with increasing microtubule concentration, but at higher
microtubule concentrations, ATPase activity decreases until it
eventually approaches basal levels. In contrast, stimulation of
kinesin ATPase by microtubules (Gilbert et al. (1993) Biochemistry
32: 4677-4684) displays typical hyperbolic curves that reach
saturation.
[0187] At least two potential explanations could account for the
unusual ATPase behavior of katanin. One possibility is that katanin
binds microtubules at two sites, which could elevate the local
microtubule concentration by crosslinking and thereby stimulate
katanin's ATPase activity. At higher microtubule concentrations,
however, the ratio of katanin to microtubules would be lower,
resulting in a less-crosslinked network and less stimulation of
ATPase activity. In support of this idea, bundling of microtubules
by katanin has been observed by microscopy (unpublished
observations). This behavior has been seen in another
cytoskeletal-polymer stimulated ATPase, Acanthamoeba myosin 1,
which has two discrete actin binding sites: a low affinity
catalytic site and a higher affinity site not involved in catalysis
(Lynch et al. (1986) J. Biol. Chem. 261: 17156-17162).
[0188] A second explanation for katanin's complex enzymatic
behavior could involve katanin oligomerization into rings.
Rotary-shadowing EM images show oligomeric ring structures in
katanin preparations; however hydrodynamic experiments with both
native (McNally and Vale (1993) Cell, 75: 419-429) and recombinant
katanin (data not shown) suggest that the majority of the protein
is monomeric. One hypothesis is that microtubules promote p60-p60
oligomerization, and that the assembly of p60 monomers into a
higher order structure on the microtubule stimulates ATPase
activity. According to this idea, low microtubule concentrations
would facilitate multimerization, since p60 monomers would be more
likely to bind near one another on the microtubule. High
microtubule concentrations, on the other hand, would inhibit p60
assembly by sequestering p60 monomers at noncontiguous sites on the
lattice. Self assembly into rings also has been suggested as the
cause of dynamin's biphasic stimulation of GTPase activity (Tuma
and Collins (1994) J. Biol. Chem. 269: 30842-30847; and Warnock et
al. (1996) J. Biol. Chem. 271: 22310-22314). Cryo-electron
microscopy studies of the p60-microtubule complex provide a means
of testing this hypothesis.
[0189] Based upon studies of other AAA family members, katanin
oligomers/rings may prove to be important in the severing
mechanism. Although serving diverse functions, many AAA proteins
appear to share a common fuinction as nucleotide-dependent
molecular chaperones that disassemble protein complexes
(Confalonieri and Duguet (1995) supra.). The best studied AAA
member is NSF, which binds to and induces the disassembly of
ternary SNARE complexes after hydrolysis of ATP (Hanson et al.
(1995) J. Biol. Chem. 270: 1695 5-16961; and Hayashi et al. (1995)
EMBO J. 14: 2317-2325). This reaction plays a role either in
vesicle fusion and/or recycling of components in membrane
trafficking pathways. Recently, electron microscopy studies have
revealed that the NSF ring structure adopts extended and compact
conformations in the ATP-.gamma.-S and ADP states, respectively
(Hanson et al, (1997) Cell 90: 523-535). If attached at several
points to a protein complex, this transition could break apart
bonds in the SNARE complex (Hanson et al., (1997) supra.). Katanin
may work in an analogous fashion. A ring of katanin's dimensions
could potentially contact multiple tubulin sites on a microtubule,
and a structural change during ATP hydrolysis could shift the
positions of tubulin binding sites with respect to one another,
which would disrupt the microtubule lattice. Another possibility is
that katanin acts more like an ATP-regulated version of actin
severing proteins, which are thought to compete for sites at
protein-protein interfaces within the polymer. In this type of
mechanism, the AAA domain could serve as an ATP-dependent protein
clamp that binds tightly to and disrupts tubulin-tubulin interfaces
during particular steps in the ATPase cycle.
[0190] Targeting of Katanin to Centrosomes
[0191] Our studies show that p80 does not constitute an essential
element of katanin's enzymatic mechanism. The finding that p80 is
not required for microtubule-severing activity was somewhat
surprising, because all of the p60 immunoprecipitates with p80 from
sea urchin cytosol (unpublished observations). However, experiments
reported here have uncovered a role for p80 in targeting katanin to
centrosomes in vivo. This conclusion is based upon the finding that
the WD40 domain of p80 can target GFP to the centrosome in cultured
human cell lines. Because the WD40 domain cannot dimerize with
endogenous p60, the centrosomal localization must be due to direct
interaction of the WD40 domain with one or more resident
centrosomal proteins. WD40 domains are thought to form a conserved
beta propeller structure, as first determined for the beta subunits
of transducin and G.sub.i (Wall et al. (1995) Cell 83: 1047-1058;
Sondek et al. (1996) Nature 379: 369-374). However, each WD40
domain exhibits very specific heterophilic protein interactions;
exposed residues in the beta subunit of G.sub.i interact with the
alpha subunit (Wall et al. (1995) supra.), whereas the
corresponding residues in the WD40 transcription factor TUP1
mediate binding to a second transcription factor .alpha.2 (Komachi,
and Johnson (1997) Mol. Cell Biol. 17: 6023-6028). Since the G
protein beta subunits interact with multiple partner proteins (Wall
et al. (1995) Cell 83: 1047-1058; and Gaudet et al. (1996) Cell,
87: 577-588), it is also possible that the p80 katanin WD40 domain
can interact with more than one protein in vivo. p80 is the only
known centrosomal protein with a WD40 motif. The findings that
katanin has an entire subunit devoted to centrosome localization
and that this subunit is conserved between mammals and echinoderms
suggest an important role for katanin at the centrosome.
[0192] The WD40 domain of p80 katanin represents the first example
of a structural motif that targets a protein to the centrosome in
mammals, although a centrosome-targeting domain has been defined
for the Drosophila protein CP190 (Oegema et al. (1995) J. Cell
Biol. 131: 1261-1273). This provides an opportunity to identify the
centrosomal component(s) responsible for anchoring katanin. Further
information on the docking of katanin to the centrosome may provide
clues regarding katanin's role in microtubule disassembly at this
organelle.
[0193] Experimental Procedures
[0194] Peptide Microsequencing
[0195] Katanin was purified from extracts of S. purpuratus eggs
essentially as described previously (McNally and Vale (1993) Cell,
75: 419-429), except that the hydroxyapatite chromatography was
carried out using a Pharmacia HRI. 0/3 0 column packed with 20
.mu.m, ceramic hydroxyapatite beads (American International
Chemical, Natick, Mass.). Internal peptide sequences of the p60 and
p80 subunits were obtained from native sea urchin katanin as
described (Iwamatsu (1992) Electrophoresis 13: 142-147). Two
additional p80 peptides were also obtained: DASMMAM (SEQ ID NO:15)
and IQGLR (SEQ ID NO:16). p60 Cloning
[0196] A cDNA encoding a 400 bp fragment of the p60 subunit
(corresponding to a.a. 214374) was cloned from S. purpuratus first
strand cDNA using nested PCR with degenerate oligonucleotides. This
fragment was then used to screen a lambda ZAP-Express cDNA library
made from S. purpuratus unfertilized egg MRNA by hybridization.
Several independent positive clones were isolated. One clone was
completely sequenced (GENBANK accession #AF052191). p80 Cloning
[0197] An initial partial cDNA clone of p80 katanin was obtained by
screening an S. purpuratus unfertilized egg cDNA library (Wright et
al. (1991) J. Cell Biol. 113: 817-833) with an antibody specific
for p80 katanin, anti-p81.sup.aff (McNally et al. (1996) J. Cell
Sci. 109: 561-567). The insert of the initial clone was used to
isolate a longer cDNA clone (pFM18) from the same library by plaque
hybridization. A cDNA clone encoding the 5' end of p80 katanin
(pFM23) was obtained by anchor-ligated PCR (Apte and Siebert (1993)
Biotechniques 15: 890-893) using primers derived from pFM18
sequences and reverse transcription reactions utilizing S.
purpuratus unfertilized egg mRNA as template. A full-length p80
cDNA (GENBANK accession #AF052433) was generated by joining the
inserts of pFM1 and pFM23 at a common BstXI site.
[0198] BLAST searches of GENBANK with p80 sequences revealed
homology with a human infant brain cDNA (GENBANK accession #T
16102) which was obtained and sequenced. Sequences obtained from
the T 16102 clone were used to obtain multiple 3'end cDNA clones by
3' RACE from HT1080 (human fibrosarcoma) total RNA. An overlapping
cDNA clone (pFM54) containing the translation start site was
obtained by PCR amplification from an adult human hippocampal cDNA
library (Stratagene, Inc.). Sequence analysis of partial cDNAs PCR
amplified from HT1080 total RNA or from the hippocampal library
were over 98% identical in predicted a.a. sequence. The complete
DNA sequence of human p80 katanin is available from GENBANK
(accession #AF052432).
[0199] Antibody Production and Immunoprecipitation
[0200] The full-length S. purpuratus p60 cDNA coding sequence was
inserted into pMA.LC2 (New England Biolabs) and expressed as a
C-terminal fusion to maltose binding protein in E. coli. Soluble
MBP-p60 fusion protein was purified on an amylose affinity column,
eluted with maltose, and injected into rabbits (antiserum
production by BABCO, Berkeley, Calif.). To select p60-specific
antibodies that do not react with other AAA members, antibodies
recognizing the N-terminal non-AAA domain of p60 were affinity
purified on an Affi-Gel column coupled with the N-terminal residues
1- 152 of p60 fused to MBP (Harlow and Lane (1988). Antibodies: A
Laboratory Manual. (Cold Spring Harbor, N.Y.: Cold Spring Harbor
Laboratory Press)). The resulting affinity-purified antibody
recognized a single 60 kDa polypeptide in immunoblots of S.
purpuratus unfertilized egg extract.
[0201] To prepare a specific antibody to human p80 katanin, the
full length human p80 cDNA was ligated into the E. coli expression
vector, pET-28a.sup.+(Novagen) as a BamHI-XhoI fragment. The
protein was expressed and then purified in a denatured state in 8 M
urea. by nickel chelate chromatography on His-Bind Resin (Novagen).
Rabbits were immunized with polyacrylamide slices containing
SDS-PAGE resolved human p80 katanin. Resulting serum was affinity
purified with CNBr Sepharose-coupled, bacterially-expressed human
p80 katanin. The resulting affinity purified antibody recognized a
single 80 kDa polypeptide in immunoblots of SDS solubilized HeLa
cells (not shown).
[0202] For immunoprecipitations used to demonstrate association of
baculovirus-expressed S. purpuratus p60 and p80, affinity purified
anti-p60 antibodies were covalently crosslinked to protein A
Sepharose using 20 mM dimethylpimilidate (Harlow and Lane (1988)
supra.). After equilibration in TBST, 20-40 .mu.l of antibody beads
were added to katanin samples diluted in TBST containing 1-2 mg/ml
soybean trypsin inhibitor (SBTI). The immunoprecipitations were
incubated at 4.degree. C. for 1-2 hr, washed five times with 1 ml
of ice-cold TBST, and eluted in SDS-containing sample buffer.
[0203] Baculovirus Expression and Purification of Katanin
[0204] Katanin subunits were expressed using the Bac-to-Bac.TM.
baculovirus expression system (Life Technologies), a commercial
version of the site-specific transposition system for making
recombinant baculovirus (Luckow et al. (1993) J. Virology 67:
4566-4579). p60 and p80 cDNA coding sequences were each PCR
amplified (Expand polymerase, Boehringer Mannheim) and then
subcloned separately into pFastBac HT, which resulted in the fusion
of a 6xHis Ni2.sup.+ binding sequence to the N-terminus of both p80
and p60. A p60-p80 coexpression virus was made by cloning the
complete p60-FastBac HT and p80-FastBac HT coding regions into the
transfer vector, pDual. Recombinant baculoviruses were prepared
according to the Life Technologies protocol.
[0205] Sf9 cells were grown in SFM-900 II SFM (Life Technologies)
supplemented with 100.times. antibiotic/antimycotic (Life
Technologies) to 0.5.times. using the shaker culture method (Weiss
et al. (1995) pp. 79-85 in Baculovirus Expression Protocols, C. D.
Richardson, ed. Totowa, N.J.: Humana Press Inc.). Expression of
katanin subunits was performed in 11 flasks containing 200-300 ml
of media using a multiplicity of infection of 0.5-1.0 pfu/cell. The
cells were harvested at approximately 72 hr post infection by low
speed centrifugation and resuspended in lysis buffer (50 mM Tris pH
8.5, 300 mM NaCl, 2 mM MgCl.sub.2, 20 mM imidazole, 10 mM
2-mercaptoethanol, 1 mM ATP, 1 .mu.g/ml pepstatin, 1 .mu.g/ml
leupeptin, 1 .mu.g/ml aprotinin) before freezing in liquid
nitrogen, and storage at -80.degree. C.
[0206] To purify the expressed subunits, frozen cells were thawed
and DNA was sheared by two passes through a Bio-Neb Cell Disrupter
(100 psi helium, 13 1/min). Cell debris was removed by
centrifugation (40,000.times.g for 45 min). Subunits were bound in
batch to Ni.sup.2+-NTA Superflow (QIAGEN), washed [20 mM Tris pH
8.0, 1 M NaCl, 2 mM MgCl.sub.2, 40 mM imidazole, 0.02% Triton X-
100, 10 mM 2-mercaptoethanol, 0.5 mM ATP] and eluted [20 mM Tris pH
8.0, 100 mM NaCl, 150 mM imidazole, 2 mM MgCl.sub.2, 0.02% Triton
X- 100, 10 mM 2-mercaptoethanol, 100 .mu.M ATP], followed by
freezing in liquid nitrogen. Additional purification was sometimes
performed by anion-exchange chromatography. Katanin concentrations
were estimated by comparison with BSA standards using either a
commercial Bradford reagent (Bio-Rad) or by densitometric analysis
of Coomassie-stained SDS-PAGE gels with NIH-IMAGE after image
capture on a CCD-based imaging system (Foto/Analyst, Fotodyne).
[0207] Electron Microscopic Imaging
[0208] Proteins were adsorbed to mica, freeze-dried, and platinum
replicated according to established procedures (Heuser (1989) J.
Electron Microsc. Technique 13):, 244-263; Heuser (1983) J. Mol.
Biol. 169: 155-195). Sample preparation and imaging were similar to
that used in the imaging of NSF (Hanson et al. (1997) Cell 90:
523-535), except that mica flakes were washed with a buffer
consisting of 10 mM K-HEPES (pH 7.5), 2 mM MgCl.sub.2, 1 mM
nucleotide (ATP or ATP-.gamma.-S). Images were processed using
Adobe Photoshop and displayed at 300,000.times..
[0209] ATPase Assays
[0210] ATPase activity was measured by a modified malachite green
method (Kodama et al. (1986) J. Biochem. 99: 1465-1472). ATPase
reactions of 50-100 .mu.l, were carried out in a buffer previously
used for measuring the ATPase activity of native katanin [20 mM
K-HEPES pH 8.0, 25 mM potassium glutamate 2 mM MgCl.sub.2, 10%
glycerol (v/v), 0.02% Triton X-100 (w/v), 1 mg/ml BSA] (McNally and
Vale (1993) Cell, 75: 419-429), except that soybean trypsin
inhibitor (SBTI) was replaced by BSA as a carrier protein because
SBTI increased background phosphate contamination. An ATP
regenerating system, consisting of 0.5-1.0 mM phosphoenol pyruvate
and 2 units of pyruvate kinase, was included to minimize the
inhibition by ADP observed previously for native katanin (McNally
and Vale (1993) Cell, 75: 419-429). Microtubules were prepared from
bovine brain tubulin (Hyman et al. (1990) Meth. Enzymol. 196:
303-319; and Williams and Lee (1982) Meth Enzymol. 85B: 376-385).
After assembly, microtubules were sedimented (230,000.times.g; 10
min), resuspended in ATPase buffer lacking BSA, and the polymers
were resuspended by repeated passage through a 27 gauge needle.
Microtubule concentration was determined by measuring the
absorbance at 275 nm in 6 M guanidine HCl by using a molecular mass
of 110 kDa and an extinction coefficient of 1.03 ml mg.sup.-1
cm.sup.-1 (Hackney (1988) Proc. Natl. Acad Sci. USA, 85:
6314-6318). ATPase reactions were carried out at room temperature,
and were initiated by addition of katanin.
[0211] Severing Assays
[0212] Microscope-based severing assays were performed using
previously published procedures (McNally and Vale (1993) Cell, 75:
419-429), except that microtubules were immobilized by first
perfusing flow cells with a bacterially expressed kinesin mutant
that binds strongly to microtubules but is unable to hydrolyze ATP
(K560, G234A mutant; R. Vale and E. Taylor, unpublished results).
Assays were performed in 20 mM Hepes (pH 7.5), 2 mM MgCl.sub.2, 1
mM ATP with an oxygen scavenger system consisting of glucose
oxidase (220 .mu.g/ml), catalase (36 .mu.g/ml), glucose (22.5 mM),
and 2-mercaptoethanol (71.5 mM). Images were captured using a
cooled, slow-scan CCD (Photometerics) and processed using Adobe
Photoshop.
[0213] DAPI severing assays were performed using conditions where
the change in fluorescence intensity was linear with the amount of
tubulin polymer added (Heusele et al. (1987) Eur. J. Biochem. 165:
613-620). Severing reactions containing 2 .mu.M microtubules
(polymerized and resuspended in ATPase buffer as above) were
incubated with 10 .mu.M DAPI, along with 1 mM ATP, 10 mM
phosphoenol pyruvate, 250 .mu.g/ml pyruvate kinase (Boehringer
Mannheim), and 1 mg/ml BSA. The reaction volume was 80 .mu.L, and
fluorescence intensity was measured by exciting at 370 nm. and
measuring the emission at 450 mn using a model 8100 fluorimeter
(SLM Instruments) in photon counting mode.
[0214] In vitro Translation Co-Immunoprecipitation
[0215] In order to facilitate the non-radioactive detection of in
vitro translated p60 and p80, each cDNA was ligated into the vector
pCITE-4a+ (Novagen) such that the proteins would be translated in
frame with a 37 amino acid N-terminal S-Tag. In vitro synthesis of
proteins directly from plasmid DNAs was accomplished using the
Single Tube Protein System 2, T7 (Novagen). For
co-immunoprecipitation assays, p60 and p80 constructs were usually
co-expressed. However, identical results were obtained if the
constructs were expressed separately and the incubated together for
30 min at room temperature. For immunoprecipitations, lysates were
incubated on ice with Pansorbin (Calbiochem)-antibody complexes,
washed in NET buffer (50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 0.1%
Nonidet P40, 1 mM EDTA (pH 8.0), 0.25% gelatin and 0.02% sodium
azide), then resuspended in SDS-PAGE sample buffer. In vitro
translation products in both the pellets and supernatants from the
immunoprecipitations were resolved by SDS-PAGE, transferred to
nitrocellulose, probed with S-protein HRP conjugate (Novagen) and
detected by chemiluminescence.
[0216] Cell Culture and Transfections and Immunofluorescence
[0217] To allow transient expression of a human p80 WD40-GFP fusion
protein in HeLa cells, a DNA fragment containing amino acids 1-263
of human p80 katanin was generated by PCR amplification, placing a
BamHI site and a Kozak consensus at the predicted translation start
and an EcoRI site after the codon for a.a. 263. This BamHI-EcoRI
fragment was ligated into the GFP fusion vector pEGFP-N1
(Clontech).
[0218] Both MSU1.1 and HeLa cells were grown on 18 mm glass
coverslips in Optimem medium (Life Technologies) supplemented with
10% fetal bovine serum, penicillin and streptomycin. Plasmids were
transfected using Superfect Reagent (Qiagen) for 2 hr after which
coverslips were washed with PBS and placed in fresh culture medium
at 37.degree. C. with 5% CO.sub.2 for 1-24 hr.
[0219] For imaging of GFP-fluorescence and immunofluorescence with
the human p80 katanin antibody or with the .gamma.-tubulin
antibody, monoclonal GTU88 (Sigma Chemical), cells on coverslips
were fixed either in -20.degree. C. methanol or in 0.5.times. PBS,
3.7% formaldehyde, 75% methanol at 22.degree. C. for 10 min
followed by rehydration in TBST. Antibody labelling was carried out
in TBST containing 4% BSA. Images were captured with a Nikon
Microphot SA microscope, 100.times. Plan Fluor 1.3 objective,
Photometrics Quantix camera and IP Lab Spectrum software
(Scanalytics).
[0220] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
16 1 517 PRT Strongylocentrotus purpuratus misc_feature katanin p60
subunit 1 Met Ser Val Asp Glu Ile Cys Glu Asn Thr Lys Met Gly Arg
Glu Tyr 1 5 10 15 Ala Leu Leu Gly Asn Tyr Glu Thr Ser Leu Val Tyr
Tyr Gln Gly Val 20 25 30 Leu Gln Gln Ile Gln Lys Leu Leu Thr Ser
Val His Glu Pro Gln Arg 35 40 45 Lys His Gln Trp Gln Thr Ile Arg
Gln Glu Leu Ser Gln Glu Tyr Glu 50 55 60 His Val Lys Asn Ile Thr
Lys Thr Leu Asn Gly Phe Lys Ser Glu Pro 65 70 75 80 Ala Ala Pro Glu
Pro Ala Pro Asn His Gly Arg Ala Ala Pro Phe Ser 85 90 95 His His
Gln His Ala Ala Lys Pro Ala Ala Ala Glu Pro Ala Arg Asp 100 105 110
Pro Asp Val Trp Pro Pro Pro Thr Pro Val Asp His Arg Pro Ser Pro 115
120 125 Pro Tyr Gln Arg Ala Ala Arg Lys Asp Pro Pro Arg Arg Ser Glu
Pro 130 135 140 Ser Lys Pro Ala Asn Arg Ala Pro Gly Asn Asp Arg Gly
Gly Arg Gly 145 150 155 160 Pro Ser Asp Arg Arg Gly Asp Ala Arg Ser
Gly Gly Gly Gly Arg Gly 165 170 175 Gly Ala Arg Gly Ser Asp Lys Asp
Lys Asn Arg Gly Gly Lys Ser Asp 180 185 190 Lys Asp Lys Lys Ala Pro
Ser Gly Glu Glu Gly Asp Glu Lys Lys Phe 195 200 205 Asp Pro Ala Gly
Tyr Asp Lys Asp Leu Val Glu Asn Leu Glu Arg Asp 210 215 220 Ile Val
Gln Arg Asn Pro Asn Val His Trp Ala Asp Ile Ala Gly Leu 225 230 235
240 Thr Glu Ala Lys Arg Leu Leu Glu Glu Ala Val Val Leu Pro Leu Trp
245 250 255 Met Pro Asp Tyr Phe Lys Gly Ile Arg Arg Pro Trp Lys Gly
Val Leu 260 265 270 Met Val Gly Pro Pro Gly Thr Gly Lys Thr Met Leu
Ala Lys Ala Val 275 280 285 Ala Thr Glu Cys Gly Thr Thr Phe Phe Asn
Val Ser Ser Ala Ser Leu 290 295 300 Thr Ser Lys Tyr His Gly Glu Ser
Glu Lys Leu Val Arg Leu Leu Phe 305 310 315 320 Glu Met Ala Arg Phe
Tyr Ala Pro Ser Thr Ile Phe Ile Asp Glu Ile 325 330 335 Asp Ser Ile
Cys Ser Lys Arg Gly Thr Gly Ser Glu His Glu Ala Ser 340 345 350 Arg
Arg Val Lys Ser Glu Leu Leu Ile Gln Met Asp Gly Val Ser Gly 355 360
365 Pro Ser Ala Gly Glu Glu Ser Ser Lys Met Val Met Val Leu Ala Ala
370 375 380 Thr Asn Phe Pro Trp Asp Ile Asp Glu Ala Leu Arg Arg Arg
Leu Glu 385 390 395 400 Lys Arg Ile Tyr Ile Pro Leu Pro Glu Ile Asp
Gly Arg Glu Gln Leu 405 410 415 Leu Arg Ile Asn Leu Lys Glu Val Pro
Leu Ala Asp Asp Ile Asp Leu 420 425 430 Lys Ser Ile Ala Glu Lys Met
Asp Gly Tyr Ser Gly Ala Asp Ile Thr 435 440 445 Asn Val Cys Arg Asp
Ala Ser Met Met Ala Met Arg Arg Arg Ile Gln 450 455 460 Gly Leu Arg
Pro Glu Glu Ile Arg His Ile Pro Lys Glu Glu Leu Asn 465 470 475 480
Gln Pro Ser Thr Pro Ala Asp Phe Leu Leu Ala Leu Gln Lys Val Ser 485
490 495 Lys Ser Val Gly Lys Glu Asp Leu Val Lys Tyr Met Ala Trp Met
Glu 500 505 510 Glu Phe Gly Ser Val 515 2 690 PRT
Strongylocentrotus purpuratus misc_feature katanin p80 subunit 2
Met Ala Thr Lys Arg Ala Trp Lys Leu Gln Glu Leu Val Ala His Ser 1 5
10 15 Ser Asn Val Asn Cys Leu Ala Leu Gly Pro Met Ser Gly Arg Val
Met 20 25 30 Val Thr Gly Gly Glu Asp Lys Lys Val Asn Leu Trp Ala
Val Gly Lys 35 40 45 Gln Asn Cys Ile Ile Ser Leu Ser Gly His Thr
Ser Pro Val Asp Ser 50 55 60 Val Lys Phe Asn Ser Ser Glu Glu Leu
Val Val Ala Gly Ser Gln Ser 65 70 75 80 Gly Thr Met Lys Ile Tyr Asp
Leu Glu Pro Ala Lys Ile Val Arg Thr 85 90 95 Leu Thr Gly His Arg
Asn Ser Ile Arg Cys Met Asp Phe His Pro Phe 100 105 110 Gly Glu Phe
Val Ala Ser Gly Ser Thr Asp Thr Asn Val Lys Leu Trp 115 120 125 Asp
Val Arg Arg Lys Gly Cys Ile Tyr Thr Tyr Lys Gly His Ser Asp 130 135
140 Gln Val Asn Met Ile Lys Phe Ser Pro Asp Gly Lys Trp Leu Val Thr
145 150 155 160 Ala Ser Glu Asp Thr Thr Ile Lys Leu Trp Asp Leu Thr
Met Gly Lys 165 170 175 Leu Phe Gln Glu Phe Lys Asn His Thr Gly Gly
Val Thr Gly Ile Glu 180 185 190 Phe His Pro Asn Glu Phe Leu Leu Ala
Ser Gly Ser Ser Asp Arg Thr 195 200 205 Val Gln Phe Trp Asp Leu Glu
Thr Phe Gln Leu Val Ser Ser Thr Ser 210 215 220 Pro Gly Ala Ser Ala
Val Arg Ser Ile Ser Phe His Pro Asp Gly Ser 225 230 235 240 Tyr Leu
Phe Cys Ser Ser Gln Asp Met Leu His Ala Phe Gly Trp Glu 245 250 255
Pro Ile Arg Cys Phe Asp Thr Phe Ser Val Phe Trp Gly Lys Val Ala 260
265 270 Asp Thr Val Ile Ala Ser Thr Gln Leu Ile Gly Ala Ser Phe Asn
Ala 275 280 285 Thr Asn Val Ser Val Tyr Val Ala Asp Leu Ser Arg Met
Ser Thr Thr 290 295 300 Gly Ile Ala Gln Glu Pro Gln Ser Gln Pro Ser
Lys Thr Pro Ser Gly 305 310 315 320 Gly Ala Glu Glu Val Pro Ser Lys
Pro Leu Thr Ala Ser Gly Arg Lys 325 330 335 Asn Phe Val Arg Glu Arg
Pro His Thr Thr Ser Ser Lys Gln Arg Gln 340 345 350 Pro Asp Val Lys
Ser Glu Pro Glu Arg Gln Ser Pro Thr Gln Asp Glu 355 360 365 Gly Val
Lys Asp Asp Asp Ala Thr Asp Ile Lys Asp Pro Asp Ser Tyr 370 375 380
Ala Lys Ile Phe Ser Pro Lys Thr Arg Val Asp His Ser Pro Glu Arg 385
390 395 400 Asn Ala Gln Pro Phe Pro Ala Pro Leu Asp Val Pro Gly Ala
Gln Glu 405 410 415 Pro Glu Pro Phe Lys His Pro Pro Lys Pro Ala Ala
Ala Ala Ala Val 420 425 430 Ala Pro Val Ser Arg Ala Pro Ala Pro Ser
Ala Ser Asp Trp Gln Pro 435 440 445 Ala Gln Ala Asn Pro Ala Pro Asn
Arg Val Pro Ala Ala Thr Lys Pro 450 455 460 Val Pro Ala Gln Glu Val
Ala Pro Ser Arg Lys Pro Asp Pro Ile Ser 465 470 475 480 Thr Ile Ile
Pro Ser Asp Arg Asn Lys Pro Ala Asn Leu Asp Met Asp 485 490 495 Ala
Phe Leu Pro Pro Ala His Ala Gln Gln Ala Pro Arg Val Asn Ala 500 505
510 Pro Ala Ser Arg Lys Gln Ser Asp Ser Glu Arg Ile Glu Gly Leu Arg
515 520 525 Lys Gly His Asp Ser Met Cys Gln Val Leu Ser Ser Arg His
Arg Asn 530 535 540 Leu Asp Val Val Arg Ala Ile Trp Thr Ala Gly Asp
Ala Lys Thr Ser 545 550 555 560 Val Glu Ser Val Val Asn Met Lys Asp
Gln Ala Ile Leu Val Asp Ile 565 570 575 Leu Asn Ile Met Leu Leu Lys
Lys Ser Leu Trp Asn Leu Asp Met Cys 580 585 590 Val Val Val Leu Pro
Arg Leu Lys Glu Leu Leu Ser Ser Lys Tyr Glu 595 600 605 Asn Tyr Val
His Thr Ser Cys Ala Cys Leu Lys Leu Ile Leu Lys Asn 610 615 620 Phe
Thr Ser Leu Phe Asn Gln Asn Ile Lys Cys Pro Pro Ser Gly Ile 625 630
635 640 Asp Ile Thr Arg Glu Glu Arg Tyr Asn Lys Cys Ser Lys Cys Tyr
Ser 645 650 655 Tyr Leu Ile Ala Thr Arg Gly Tyr Val Glu Glu Lys Gln
His Val Ser 660 665 670 Gly Lys Leu Gly Ser Ser Phe Arg Glu Leu His
Leu Leu Leu Asp Gln 675 680 685 Leu Glu 690 3 730 PRT Xenopus
laevis misc_feature Xenopus kinesin central motor 1 (XKCM1) 3 Met
Glu Arg Leu Val Ala Thr Arg Leu Val Thr Gly Leu Ala Val Lys 1 5 10
15 Ile Met Arg Ser Asn Gly Val Ile His Asn Ala Asn Ile Thr Ser Val
20 25 30 Asn Met Asp Arg Ser Ser Val Asn Val Glu Trp Lys Glu Gly
Glu Ala 35 40 45 Asn Lys Gly Lys Glu Ile Ser Phe Ala Asp Val Ile
Ser Val Asn Pro 50 55 60 Glu Leu Leu Asp Ala Val Leu Ala Pro Thr
Asn Val Lys Glu Asn Met 65 70 75 80 Pro Pro Gln Arg Asn Val Ser Ser
Gln Asn His Lys Arg Lys Thr Ile 85 90 95 Ser Lys Ile Pro Ala Pro
Lys Glu Val Ala Ala Lys Asn Ser Leu Leu 100 105 110 Ser Glu Ser Gly
Ala Gln Ser Val Leu Arg Glu Arg Ser Thr Arg Met 115 120 125 Thr Ala
Ile His Glu Thr Leu Pro Tyr Glu Asn Glu Met Glu Ala Glu 130 135 140
Ser Thr Pro Leu Pro Ile Gln Gln Asn Ser Val Gln Ala Arg Ser Arg 145
150 155 160 Ser Thr Lys Val Ser Ile Ala Glu Glu Pro Arg Leu Gln Thr
Arg Ile 165 170 175 Ser Glu Ile Val Glu Glu Ser Leu Pro Ser Gly Arg
Asn Asn Gln Gly 180 185 190 Arg Arg Lys Ser Asn Ile Val Lys Glu Met
Glu Lys Met Lys Asn Lys 195 200 205 Arg Glu Glu Gln Arg Ala Gln Asn
Tyr Glu Arg Arg Met Lys Arg Ala 210 215 220 Gln Asp Tyr Asp Thr Ser
Val Pro Asn Trp Glu Phe Gly Lys Met Ile 225 230 235 240 Lys Glu Phe
Arg Ala Thr Met Asp Cys His Arg Ile Ser Met Ala Asp 245 250 255 Pro
Ala Glu Glu His Arg Ile Cys Val Cys Val Arg Lys Arg Pro Leu 260 265
270 Asn Lys Gln Glu Leu Ser Lys Lys Glu Ile Asp Ile Ile Ser Val Pro
275 280 285 Ser Lys Asn Ile Val Leu Val His Glu Pro Lys Leu Lys Val
Asp Leu 290 295 300 Thr Lys Tyr Leu Glu Asn Gln Ala Phe Arg Phe Asp
Phe Ser Phe Asp 305 310 315 320 Glu Thr Ala Thr Asn Glu Val Val Tyr
Arg Phe Thr Ala Arg Pro Leu 325 330 335 Val Gln Ser Ile Phe Glu Gly
Gly Lys Ala Thr Cys Phe Ala Tyr Gly 340 345 350 Gln Thr Gly Ser Gly
Lys Thr His Thr Met Gly Gly Asp Phe Ser Gly 355 360 365 Lys Ser Gln
Asn Val Ser Lys Gly Val Tyr Ala Phe Ala Ser Arg Asp 370 375 380 Val
Phe Leu Leu Leu Asp Gln Pro Arg Tyr Lys His Leu Asp Leu Asp 385 390
395 400 Val Phe Val Thr Phe Phe Glu Ile Tyr Asn Gly Lys Val Phe Asp
Leu 405 410 415 Leu Asn Lys Lys Thr Lys Leu Arg Val Leu Glu Asp Ala
Lys Gln Glu 420 425 430 Val Gln Val Val Gly Leu Leu Glu Lys Gln Val
Ile Ser Ala Asp Asp 435 440 445 Val Phe Lys Met Ile Glu Ile Gly Ser
Ala Cys Arg Thr Ser Gly Gln 450 455 460 Thr Phe Ala Asn Thr Ser Ser
Ser Arg Ser His Ala Cys Leu Gln Ile 465 470 475 480 Ile Leu Arg Arg
Gly Ser Lys Leu His Gly Lys Phe Ser Leu Val Asp 485 490 495 Leu Ala
Gly Asn Glu Arg Gly Val Asp Thr Ala Ser Ala Asp Arg Ile 500 505 510
Thr Arg Met Lys Gly Ala Glu Ile Asn Arg Ser Leu Leu Ala Leu Lys 515
520 525 Glu Cys Ile Arg Ala Leu Gly Gln Asn Lys Ser His Thr Pro Phe
Arg 530 535 540 Glu Ser Lys Leu Thr Gln Ile Leu Arg Asp Ser Phe Ile
Gly Glu Asn 545 550 555 560 Ser Arg Thr Cys Met Ile Ala Met Leu Ser
Pro Gly Phe Asn Ser Cys 565 570 575 Glu Tyr Thr Leu Asn Thr Leu Arg
Tyr Ala Asp Arg Val Lys Glu Leu 580 585 590 Ser Pro Gln Asn Ala Glu
Thr Asn Asp Asp Asn Leu Gln Met Glu Asp 595 600 605 Ser Gly Gly Ser
His Ala Ser Ile Glu Gly Leu Gln Leu Gln Asp Asp 610 615 620 Phe Leu
Leu Lys Asp Glu Glu Leu Ser Thr His Asn Ser Phe Gln Asp 625 630 635
640 Ala Leu Asn Arg Val Gly Glu Leu Glu Asp Lys Ala Val Asp Glu Leu
645 650 655 Arg Glu Leu Val Gln Lys Glu Pro Glu Trp Thr Asn Leu Leu
Gln Met 660 665 670 Thr Glu Gln Pro Asp Tyr Asp Leu Glu Asn Phe Val
Met Gln Ala Glu 675 680 685 Tyr Leu Ile Gln Glu Arg Ser Lys Val Leu
Ile Ala Leu Gly Asp Ser 690 695 700 Ile Asn Ser Leu Arg Leu Ala Leu
Gln Val Glu Glu Gln Ala Ser Lys 705 710 715 720 Gln Ile Ser Lys Lys
Lys Arg Ser Asn Lys 725 730 4 217 PRT Strongylocentrotus purpuratus
misc_feature AAA ATPase superfamily katanin p60 AAA domain 4 Val
His Trp Ala Asp Ile Ala Gly Leu Thr Glu Ala Lys Arg Leu Leu 1 5 10
15 Glu Glu Ala Val Val Leu Pro Leu Trp Met Pro Asp Tyr Phe Lys Gly
20 25 30 Ile Phe Phe Pro Trp Lys Gly Val Leu Met Val Gly Pro Pro
Gly Thr 35 40 45 Gly Lys Thr Met Leu Ala Lys Ala Val Ala Thr Glu
Cys Gly Thr Thr 50 55 60 Phe Phe Asn Val Ser Ser Ala Ser Leu Thr
Ser Lys Tyr His Gly Glu 65 70 75 80 Ser Glu Lys Leu Val Arg Leu Leu
Phe Glu Met Ala Arg Phe Tyr Ala 85 90 95 Pro Ser Thr Ile Phe Ile
Asp Glu Ile Asp Ser Ile Cys Ser Lys Arg 100 105 110 Gly Thr Gly Ser
Glu His Glu Ala Ser Arg Arg Val Lys Ser Glu Leu 115 120 125 Leu Ile
Gln Met Asp Gly Val Ser Gly Pro Ser Ala Gly Glu Glu Ser 130 135 140
Ser Lys Met Val Met Val Leu Ala Ala Thr Asn Phe Pro Trp Asp Ile 145
150 155 160 Asp Glu Ala Leu Arg Arg Arg Leu Glu Lys Arg Ile Tyr Ile
Pro Leu 165 170 175 Pro Glu Ile Asp Gly Arg Glu Gln Leu Leu Arg Ile
Asn Leu Lys Glu 180 185 190 Val Pro Leu Ala Asp Asp Ile Asp Leu Lys
Ser Ile Ala Glu Lys Met 195 200 205 Asp Gly Tyr Ser Gly Ala Asp Ile
Thr 210 215 5 213 PRT Caenorhabditis elegans misc_feature AAA
ATPase superfamily mei-1 AAA domain 5 Met Ser Leu Asp Asp Ile Ile
Gly Met His Asp Val Lys Gln Val Leu 1 5 10 15 His Glu Ala Val Thr
Leu Pro Leu Leu Val Pro Glu Phe Phe Gln Gly 20 25 30 Leu Arg Ser
Pro Trp Lys Ala Met Val Leu Ala Gly Pro Pro Gly Thr 35 40 45 Gly
Lys Thr Leu Ile Ala Arg Ala Ile Ala Ser Glu Ser Ser Ser Thr 50 55
60 Phe Phe Thr Val Ser Ser Thr Asp Leu Ser Ser Lys Trp Arg Gly Asp
65 70 75 80 Ser Glu Lys Ile Val Arg Leu Leu Phe Glu Leu Ala Arg Phe
Tyr Ala 85 90 95 Pro Ser Ile Ile Phe Ile Asp Glu Ile Asp Thr Leu
Gly Gly Gln Arg 100 105 110 Gly Asn Ser Gly Glu His Glu Ala Ser Arg
Arg Val Lys Ser Glu Phe 115 120 125 Leu Val Gln Met Asp Gly Ser Gln
Asn Lys Phe Asp Ser Arg Arg Val 130 135 140 Phe Val Leu Ala Ala Thr
Asn Ile Pro Trp Glu Leu Asp Glu Ala Leu 145 150 155 160 Arg Arg Arg
Phe Glu Lys Arg Ile Phe Ile Pro Leu Pro Asp Ile Asp 165 170 175 Ala
Arg Lys Lys Leu Ile Glu Lys Ser Met Glu Gly Thr Pro Lys Ser 180 185
190 Asp Glu Ile Asn Tyr Asp Asp Leu Ala Ala Arg Thr Glu Gly Phe Ser
195 200 205 Gly Ala Asp Val Val 210 6 215 PRT Saccharomyces
cerevisiae misc_feature AAA ATPase superfamily sug1 AAA domain 6
Ser Thr Tyr Asp Met Val Gly Gly Leu Thr Lys Gln Ile Lys Glu Ile 1
5
10 15 Lys Glu Val Ile Glu Leu Pro Val Lys His Pro Glu Leu Phe Glu
Ser 20 25 30 Leu Gly Ile Ala Gln Pro Lys Gly Val Ile Leu Tyr Gly
Pro Pro Gly 35 40 45 Thr Gly Lys Thr Leu Leu Ala Arg Ala Val Ala
His His Thr Asp Cys 50 55 60 Lys Phe Ile Arg Val Ser Gly Ala Glu
Leu Val Gln Lys Tyr Ile Gly 65 70 75 80 Glu Gly Ser Arg Met Val Arg
Glu Leu Phe Val Met Ala Arg Glu His 85 90 95 Ala Pro Ser Ile Ile
Phe Met Asp Glu Ile Asp Ser Ile Gly Ser Thr 100 105 110 Arg Val Glu
Gly Ser Gly Gly Gly Asp Ser Glu Val Gln Arg Thr Met 115 120 125 Leu
Glu Leu Leu Asn Gln Leu Asp Gly Phe Glu Thr Ser Lys Asn Ile 130 135
140 Lys Ile Ile Met Ala Thr Asn Arg Leu Asp Ile Leu Asp Pro Ala Leu
145 150 155 160 Leu Arg Pro Gly Arg Ile Asp Arg Lys Ile Glu Phe Pro
Pro Pro Ser 165 170 175 Val Ala Ala Arg Ala Glu Ile Leu Arg Ile His
Ser Arg Lys Met Asn 180 185 190 Leu Thr Arg Gly Ile Asn Leu Arg Lys
Val Ala Glu Lys Met Asn Gly 195 200 205 Cys Ser Gly Ala Asp Val Lys
210 215 7 214 PRT Escherichia coli misc_feature AAA ATPase
superfamily ftsH AAA domain 7 Thr Thr Phe Ala Asp Val Ala Gly Cys
Asp Glu Ala Lys Glu Glu Val 1 5 10 15 Ala Glu Leu Val Glu Tyr Leu
Arg Glu Pro Ser Arg Phe Gln Lys Leu 20 25 30 Gly Gly Lys Glu Pro
Lys Gly Val Leu Met Val Gly Pro Pro Gly Thr 35 40 45 Gly Lys Thr
Leu Leu Ala Lys Ala Ile Ala Gly Glu Ala Lys Val Pro 50 55 60 Phe
Phe Thr Ile Ser Gly Ser Asp Phe Val Glu Met Phe Val Gly Val 65 70
75 80 Gly Ala Ser Arg Val Arg Asp Met Phe Glu Gln Ala Lys Lys Ala
Ala 85 90 95 Pro Cys Ile Ile Phe Ile Asp Glu Ile Asp Ala Val Gly
Arg Gln Arg 100 105 110 Gly Ala Gly Leu Gly Gly Gly His Asp Glu Arg
Glu Gln Thr Leu Asn 115 120 125 Gln Met Leu Val Glu Met Asp Gly Phe
Glu Gly Asn Glu Gly Ile Ile 130 135 140 Val Ile Ala Ala Thr Asn Arg
Pro Asp Val Leu Asp Pro Ala Leu Leu 145 150 155 160 Arg Pro Gly Arg
Phe Asp Arg Gln Val Val Val Gly Leu Pro Asp Val 165 170 175 Arg Gly
Arg Glu Gln Ile Leu Lys Val His Met Arg Arg Val Pro Leu 180 185 190
Ala Pro Asp Ile Asp Ala Ala Ile Ile Ala Arg Gly Thr Pro Gly Phe 195
200 205 Ser Gly Ala Asp Leu Ala 210 8 221 PRT Saccharomyces
cerevisiae misc_feature AAA ATPase superfamily PAS1 AAA domain 8
Ile Lys Trp Gly Asp Ile Gly Ala Leu Ala Asn Ala Lys Asp Val Leu 1 5
10 15 Leu Glu Thr Leu Glu Trp Pro Thr Lys Tyr Glu Pro Ile Phe Val
Asn 20 25 30 Cys Pro Leu Arg Leu Arg Ser Gly Ile Leu Leu Tyr Gly
Tyr Pro Gly 35 40 45 Cys Gly Lys Thr Leu Leu Ala Ser Ala Val Ala
Gln Gln Cys Gly Leu 50 55 60 Asn Phe Ile Ser Val Lys Gly Pro Glu
Ile Leu Asn Lys Phe Ile Gly 65 70 75 80 Ala Ser Glu Gln Asn Ile Arg
Glu Leu Phe Glu Arg Ala Gln Ser Val 85 90 95 Lys Pro Cys Ile Leu
Phe Phe Asp Glu Phe Asp Ser Ile Ala Pro Lys 100 105 110 Arg Gly His
Asp Ser Thr Gly Val Thr Asp Arg Val Val Asn Gln Leu 115 120 125 Leu
Thr Gln Met Asp Gly Ala Glu Gly Leu Asp Gly Val Tyr Ile Leu 130 135
140 Ala Ala Thr Ser Arg Pro Asp Leu Ile Asp Ser Ala Leu Leu Arg Pro
145 150 155 160 Gly Arg Leu Asp Lys Ser Val Ile Cys Asn Ile Pro Thr
Glu Ser Glu 165 170 175 Arg Leu Asp Ile Leu Gln Ala Ile Val Asn Ser
Lys Asp Lys Asp Thr 180 185 190 Gly Gln Lys Lys Phe Ala Leu Glu Lys
Asn Ala Asp Leu Lys Leu Ile 195 200 205 Ala Glu Lys Thr Ala Gly Phe
Ser Gly Ala Asp Leu Gln 210 215 220 9 227 PRT Cricetulus
longicaudatus misc_feature AAA ATPase superfamily N-ethylmaleimide
sensitive fusion protein (NSF) AAA domai 9 Glu Lys Met Gly Ile Gly
Gly Leu Asp Lys Glu Phe Ser Asp Ile Phe 1 5 10 15 Arg Arg Ala Phe
Ala Ser Arg Val Phe Pro Pro Glu Ile Val Glu Gln 20 25 30 Met Gly
Cys Lys His Val Lys Gly Ile Leu Leu Tyr Gly Pro Pro Gly 35 40 45
Cys Gly Lys Thr Leu Leu Ala Arg Gln Ile Gly Lys Met Leu Asn Ala 50
55 60 Arg Glu Pro Lys Val Val Asn Gly Pro Glu Ile Leu Asn Lys Tyr
Val 65 70 75 80 Gly Glu Ser Glu Ala Asn Ile Arg Lys Leu Phe Ala Asp
Ala Glu Glu 85 90 95 Glu Gln Arg Arg Leu Gly Ala Asn Ser Gly Leu
His Ile Ile Ile Phe 100 105 110 Asp Glu Ile Asp Ala Ile Cys Lys Gln
Arg Gly Ser Met Ala Gly Ser 115 120 125 Thr Gly Val His Asp Thr Val
Val Asn Gln Leu Leu Ser Lys Ile Asp 130 135 140 Gly Val Glu Gln Leu
Asn Asn Ile Leu Val Ile Gly Met Thr Asn Arg 145 150 155 160 Pro Asp
Leu Ile Asp Glu Ala Leu Leu Arg Pro Gly Arg Leu Glu Val 165 170 175
Lys Met Glu Ile Gly Leu Pro Asp Glu Lys Gly Arg Leu Gln Ile Leu 180
185 190 His Ile His Thr Ala Arg Met Arg Gly His Gln Leu Leu Ser Ala
Asp 195 200 205 Val Asp Ile Lys Glu Leu Ala Val Glu Thr Lys Asn Phe
Ser Gly Ala 210 215 220 Glu Leu Glu 225 10 253 PRT
Strongylocentrotus purpuratus misc_feature katanin p80 subunit WD40
repeat region 10 Lys Arg Ala Trp Lys Leu Gln Glu Leu Val Ala His
Ser Ser Asn Val 1 5 10 15 Asn Cys Leu Ala Leu Gly Pro Met Ser Gly
Arg Val Met Val Thr Gly 20 25 30 Gly Glu Asp Lys Lys Val Asn Leu
Trp Ala Val Gly Lys Gln Asn Cys 35 40 45 Ile Ile Ser Leu Ser Gly
His Thr Ser Pro Val Asp Ser Val Lys Phe 50 55 60 Asn Ser Ser Glu
Glu Leu Val Val Ala Gly Ser Gln Ser Gly Thr Met 65 70 75 80 Lys Ile
Tyr Asp Leu Glu Pro Ala Lys Ile Val Arg Thr Leu Thr Gly 85 90 95
His Arg Asn Ser Ile Arg Cys Met Asp Phe His Pro Phe Gly Glu Phe 100
105 110 Val Ala Ser Gly Ser Thr Asp Thr Asn Val Lys Leu Trp Asp Val
Arg 115 120 125 Arg Lys Gly Cys Ile Tyr Thr Tyr Lys Gly His Ser Asp
Gln Val Asn 130 135 140 Met Ile Lys Phe Ser Pro Asp Gly Lys Trp Leu
Val Thr Ala Ser Glu 145 150 155 160 Asp Thr Thr Ile Lys Glu Trp Asp
Leu Thr Met Gly Lys Leu Phe Gln 165 170 175 Glu Phe Lys Asn His Thr
Gly Gly Val Thr Gly Ile Glu Phe His Pro 180 185 190 Asn Glu Phe Leu
Leu Ala Ser Gly Ser Ser Asp Arg Thr Val Gln Phe 195 200 205 Trp Asp
Leu Glu Thr Phe Gln Leu Val Ser Ser Thr Ser Pro Gly Ala 210 215 220
Ser Ala Val Arg Ser Ile Ser Phe His Pro Asp Gly Ser Tyr Leu Phe 225
230 235 240 Cys Ser Ser Gln Asp Met Leu His Ala Phe Gly Trp Glu 245
250 11 253 PRT Homo sapiens misc_feature putative human ortholog of
katanin p80 (Hs p80) WD40 repeat regio 11 Lys Thr Ala Trp Lys Leu
Gln Glu Ile Val Ala His Ala Ser Asn Val 1 5 10 15 Ser Ser Leu Val
Leu Gly Lys Ala Ser Gly Arg Leu Leu Ala Thr Gly 20 25 30 Gly Asp
Asp Cys Arg Val Asn Leu Trp Ser Ile Asn Lys Pro Asn Cys 35 40 45
Ile Met Ser Leu Thr Gly His Thr Ser Pro Val Glu Ser Val Arg Leu 50
55 60 Asn Thr Pro Glu Glu Leu Ile Val Ala Gly Ser Gln Ser Gly Ser
Ile 65 70 75 80 Arg Val Trp Asp Leu Glu Ala Ala Lys Ile Leu Arg Thr
Leu Met Gly 85 90 95 Leu Lys Ala Asn Ile Cys Ser Leu Asp Phe His
Pro Tyr Gly Glu Phe 100 105 110 Val Ala Ser Gly Ser Gln Asp Thr Asn
Ile Lys Leu Trp Asp Ile Arg 115 120 125 Arg Lys Gly Cys Val Phe Arg
Tyr Arg Gly His Ser Gln Ala Val Arg 130 135 140 Cys Leu Arg Phe Ser
Pro Asp Gly Lys Trp Leu Ala Ser Ala Ala Asp 145 150 155 160 Asp His
Thr Val Glu Leu Trp Asp Leu Thr Ala Gly Lys Met Met Ser 165 170 175
Glu Phe Pro Gly His Thr Gly Pro Val Asn Val Val Glu Phe His Pro 180
185 190 Asn Glu Tyr Leu Leu Ala Ser Gly Ser Ser Asp Gly Thr Ile Arg
Phe 195 200 205 Trp Asp Leu Glu Lys Phe Gln Val Val Ser Arg Ile Glu
Gly Glu Pro 210 215 220 Gly Pro Val Arg Ser Val Leu Phe Asn Pro Asp
Gly Cys Cys Leu Tyr 225 230 235 240 Ser Gly Cys Gln Asp Ser Leu Arg
Val Tyr Gly Trp Glu 245 250 12 250 PRT Homo sapiens misc_feature
TFIID WD40 repeat region 12 Lys Thr Ala Ser Glu Leu Lys Ile Leu Tyr
Gly His Ser Gly Pro Val 1 5 10 15 Tyr Gly Ala Ser Phe Ser Pro Asp
Arg Asn Tyr Leu Leu Ser Ser Ser 20 25 30 Glu Asp Gly Thr Val Arg
Leu Trp Ser Leu Gln Thr Phe Thr Cys Leu 35 40 45 Val Gly Tyr Lys
Gly His Asn Tyr Pro Val Trp Asp Thr Gln Phe Ser 50 55 60 Pro Tyr
Gly Tyr Tyr Phe Val Ser Gly Gly His Asp Arg Val Ala Arg 65 70 75 80
Leu Trp Ala Thr Asp His Tyr Gln Pro Leu Arg Ile Phe Ala Gly His 85
90 95 Leu Ala Asp Val Asn Cys Thr Arg Phe His Pro Asn Ser Asn Tyr
Val 100 105 110 Ala Thr Gly Ser Ala Asp Arg Thr Val Arg Leu Trp Asp
Val Leu Asn 115 120 125 Gly Asn Cys Val Arg Ile Phe Thr Gly His Lys
Gly Pro Ile His Ser 130 135 140 Leu Thr Phe Ser Pro Asn Gly Arg Phe
Leu Ala Thr Gly Ala Thr Asp 145 150 155 160 Gly Arg Val Leu Leu Trp
Asp Ile Gly His Gly Leu Met Val Gly Glu 165 170 175 Leu Lys Gly His
Thr Asp Thr Val Cys Ser Leu Arg Phe Ser Arg Asp 180 185 190 Gly Glu
Ile Leu Ala Ser Gly Ser Met Asp Asn Thr Val Arg Leu Trp 195 200 205
Asp Ala Ile Lys Ala Phe Glu Asp Leu Glu Thr Asp Asp Phe Thr Thr 210
215 220 Ala Thr Gly His Ile Asn Leu Pro Glu Asn Ser Gln Glu Leu Leu
Leu 225 230 235 240 Gly Thr Tyr Met Thr Lys Ser Thr Pro Val 245 250
13 251 PRT Thermomonospora curvata misc_feature putative
serine/threonine kinase PkwA WD40 repeat region 13 Ala Ser Gly Asp
Glu Leu His Thr Leu Glu Gly His Thr Asp Trp Val 1 5 10 15 Arg Ala
Val Ala Phe Ser Pro Asp Gly Ala Leu Leu Ala Ser Gly Ser 20 25 30
Asp Asp Ala Thr Val Arg Leu Trp Asp Val Ala Ala Ala Glu Glu Arg 35
40 45 Ala Val Phe Glu Gly His Thr His Tyr Val Leu Asp Ile Ala Phe
Ser 50 55 60 Pro Asp Gly Ser Met Val Ala Ser Gly Ser Arg Asp Gly
Thr Ala Arg 65 70 75 80 Leu Trp Asn Val Ala Thr Gly Thr Glu His Ala
Val Leu Lys Gly His 85 90 95 Thr Asp Tyr Val Tyr Ala Val Ala Phe
Ser Pro Asp Gly Ser Met Val 100 105 110 Ala Ser Gly Ser Arg Asp Gly
Thr Ile Arg Leu Trp Asp Val Ala Thr 115 120 125 Gly Lys Glu Arg Asp
Val Leu Gln Ala Pro Ala Glu Asn Val Val Ser 130 135 140 Leu Ala Phe
Ser Pro Asp Gly Ser Met Leu Val His Gly Ser Asp Ser 145 150 155 160
Thr Val His Leu Trp Asp Val Ala Ser Gly Glu Ala Leu His Thr Phe 165
170 175 Glu Gly His Thr Asp Trp Val Arg Ala Val Ala Phe Ser Pro Asp
Gly 180 185 190 Ala Leu Leu Ala Ser Gly Ser Asp Asp Arg Thr Ile Arg
Leu Trp Asp 195 200 205 Val Ala Ala Gln Glu Glu His Thr Thr Leu Glu
Gly His Thr Glu Pro 210 215 220 Val His Ser Val Ala Phe His Pro Glu
Gly Thr Thr Leu Ala Ser Ala 225 230 235 240 Ser Glu Asp Gly Thr Ile
Arg Ile Trp Pro Ile 245 250 14 6 PRT Artificial Sequence (His)6 or
6xHis tag 14 His His His His His His 1 5 15 7 PRT Artificial
Sequence katanin p80 peptide 15 Asp Ala Ser Met Met Ala Met 1 5 16
5 PRT Artificial Sequence katanin p80 peptide 16 Ile Gln Gly Leu
Arg 1 5
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