U.S. patent application number 10/106825 was filed with the patent office on 2005-10-13 for chaperones capable of binding to prion proteins and distinguishing the isoforms prpc and prpsc.
Invention is credited to Edenhofer, Frank, Rieger, Roman, Weiss, Stefan, Winnacker, Ernst-Ludwig.
Application Number | 20050227287 10/106825 |
Document ID | / |
Family ID | 8222782 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050227287 |
Kind Code |
A1 |
Winnacker, Ernst-Ludwig ; et
al. |
October 13, 2005 |
Chaperones capable of binding to prion proteins and distinguishing
the isoforms PrPc and PrPSc
Abstract
The present invention relates to methods for the detection or
isolation of prion proteins by use of chaperones specifically
binding to said proteins. The invention further relates to a method
for in-vitro diagnosis of a transmissible spongiform encephalopathy
and to pharmaceutical compositions, preferably for the prevention
or treatment of said disease.
Inventors: |
Winnacker, Ernst-Ludwig;
(Munchen, DE) ; Weiss, Stefan; (Munchen, DE)
; Edenhofer, Frank; (Munchen, DE) ; Rieger,
Roman; (Weilheim, DE) |
Correspondence
Address: |
DLA PIPER RUDNICK GRAY CARY US, LLP
1625 MASSACHUSETTS AVENUE, NW
SUITE 300
WASHINGTON
DC
20036-2247
US
|
Family ID: |
8222782 |
Appl. No.: |
10/106825 |
Filed: |
March 27, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10106825 |
Mar 27, 2002 |
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09180652 |
Nov 12, 1998 |
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6451541 |
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09180652 |
Nov 12, 1998 |
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PCT/EP97/02444 |
May 13, 1997 |
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Current U.S.
Class: |
435/7.1 ;
435/23 |
Current CPC
Class: |
A61P 25/28 20180101;
C07K 14/47 20130101; G01N 2800/2828 20130101; G01N 33/6896
20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/007.1 ;
435/023 |
International
Class: |
G01N 033/53; C12Q
001/37 |
Foreign Application Data
Date |
Code |
Application Number |
May 14, 1996 |
DE |
EP 96 107 677.5 |
Claims
1. A method for the detection of a prion protein comprising the
steps of: (a) contacting a probe suspected to contain a prion
protein with a chaperone, and (b) determining whether a prion
protein binds to the chaperone.
2. A method for the isolation of a prion protein comprising the
steps of: (a) contacting a probe containing a prion protein with a
chaperone, and (b) isolating the chaperone-bound protein from the
chaperone.
3. The method of claim 1 or 2, wherein a fragment, analogue or
derivative of said chaperone which is capable of binding the prion
protein is used.
4. The method of any one of claims 1 to 3, wherein the chaperone is
Hsp60 or GroEL.
5. The method of any one of claims 1 to 4, wherein the chaperone is
a recombinant protein.
6. The method of claim 5, wherein the chaperone is part of a fusion
protein.
7. The method of claim 6, wherein the chaperone is part of a fusion
protein with glutathione-S-transferase, FLAG, Oligohistidin, GFP,
CBP, MBP, BTag or S-peptide (ribonuclease A).
8. The method of any one of claims 1 to 7, wherein the prion
protein is PrP.sup.c and/or an isoform of PrP.sup.c.
9. The method of claim 8, wherein the prion protein isoform is the
isoform PrP.sup.sc or a fragment, analogue or derivative
thereof.
10. The method of claim 8 or 9, wherein the prion protein is the
processed form PrP.sup.c23-231 and/or the isoform PrP.sup.sc is the
derivative PrP27-30 or a fragment thereof.
11. The method of any one of claims 1 to 10, wherein the chaperone
is detectably labelled.
12. The method of claim 12, wherein the detectable label is
selected from a radioisotope, a fluorescent compound, a colloidal
metal, a chemiluminescent compound, a bioluminescent compound, a
phosphorescent compound or an enzyme.
13. The method of any one of claims 1 to 10, wherein the chaperone
is bound to a solid phase.
14. The method of claim 13, wherein the solid phase is
gluthathione-sepharose, anti-FLAG-antibody, IMAC-Ni.sup.2+,
anti-GFP-antibody, anti-BTag-antibody, Calmodulin, S-protein 104 aa
or maltose.
15. The method of claim 13 or 14, wherein the chaperone is part of
a matrix contained within an affinity chromatography column and
wherein step (b) is modified in such a way that (i) the probe
suspected to contain the prion protein is passed through the
column, (ii) after washing, the prion protein is eluted from the
column, optionally by a change in pH or ionic strength and
collected; and (iii) optionally the collected prion protein is
further purified.
16. The method of claim 13 or 14, wherein isolation of the prior
protein is carried out as a batch process.
17. The method of any one of claims 1 to 16, wherein the probe is
from tissue, preferably brain, ileum, cortex, dura mata, purcinje
cells, lymphnodes, nerve cells, spleen, tonsils, muscle cells,
placenta, pancreas eyes, backbone marrow or peyer'sche plaques.
18. The method of any one of claims 1 to 16, wherein the probe is
from a body fluid.
19. The method of claim 18, wherein the body fluid is blood,
cerebrospinal fluid, semen or milk.
20. The method according to any one of claims 1 to 19 for the
in-vitro diagnosis of a transmissible spongiform encephalopathy,
wherein step (b) is modified in such a way that the differences in
the strength of the binding of the chaperone to PrP.sup.c and an
isoform of PrP.sup.c, respectively, preferably PrP.sup.sc, are used
to determine whether an isoform of PrP.sup.c is present in the
probe or not.
21. A complex of a chaperone and a prion protein as defined in any
one of claims 1 to 20.
22. A composition for the detection and/or isolation of a prion
protein comprising a chaperone as defined in any one of claims 1 to
20.
23. A diagnostic composition comprising a chaperone as defined in
any one of claims 1 to 20.
24. A pharmaceutical composition comprising a chaperone as defined
in any one of claims 1 to 20.
25. A pharmaceutical composition comprising a substance that
inactivates the chaperone as defined in any one of claims 1 to
20.
26. The pharmaceutical composition of claim 25, wherein said
substance is an antibody, preferably a monoclonal antibody.
27. The pharmaceutical composition of any one of claims 23 to 25,
for the prevention or treatment of a transmissible spongiform
encephalopathy.
28. The pharmaceutical composition of claim 27, wherein the
transmissible spongiform encephalopathy is Scrapie, bovine
spongiform encephalopathy (BSE), Creutzfeldt-Jacob Disease (CJD),
Gerstmann-Stru.beta.ler-Scheinker- -Syndrome (GSS), Kuru, fatal
familial insomnia (FFI) or transmissible mince encephalopathy
(TME).
Description
[0001] The present invention relates to methods for the detection
or isolation of prion proteins by use of chaperones specifically
binding to said proteins.
[0002] The invention further relates to a method for in-vitro
diagnosis of a transmissible spongiform encephalopathy and to
pharmaceutical compositions, preferably for the prevention or
treatment of said disease.
[0003] Transmissible spongiform encephalopathies (TSEs) are
neurodegenerative diseases such as scrapie of sheep, bovine
spongiform encephalopathy (BSE) of cattle and Creutzfeldt-Jakob
disease (CJD) of man (34). Infectious preparations derived from
infected brains are resistant to ultraviolet and ionizing radiation
as well as other procedures which inactivate nucleic acids
indicating that nucleic acids may not be required for infectivity.
Purification of infectious preparations from brains revealed the
presence of a protein required for infectivity (36). These
experimental observations led to the `protein only` hypothesis,
which proposes that proteinaceous infectious particles (`prions`)
are responsible for the transmission of TSEs (3, 4, 36). Prions
consist mainly of a protease resistant protein designated
PrP.sup.Sc (prion protein, `Sc` for scrapie), a posttranslationally
modified form of the proteinase K sensitive host encoded PrP.sup.c
(`c` for cellular) (8, 9, 11, 34). Both isoforms share the same
amino acid sequence, but differ in their secondary structure (31,
42). Circular Dichroism (CD) and Fourier Transform Infrared (FTIR)
spectroscopy revealed a significantly higher .beta.-sheet content
for PrP.sup.Sc as compared to a high .alpha.-helix content in
PrP.sup.c(17, 31, 38). Structural predictions of PrP.sup.c led to a
model which proposed that four domains between amino acid residues
109 to 122, 129 to 141, 178 to 191 and 202 to 218 form
.alpha.-helices (24). It has been suggested that prion propagation
involves the conversion of .alpha.-helical domains in PrP.sup.c
into .beta.-sheets in PrP.sup.Sc (26, 30, 31). The in vitro
conversion of PrP.sup.c into PrP.sup.Sc was demonstrated employing
a proteinase K resistance assay (28). A modified model was recently
suggested according to which PrP.sup.c must be partially unfolded
and refolded into PrP.sup.Sc under the direction of an oligomeric
PrP.sup.Sc seed (29). This model provides explanations for scrapie
species barriers (27) and strain-specific properties of prions (7).
In addition, experiments employing transgenic mice led to the
proposal that prion propagation requires a species-specific
macromolecule designated `protein X` (43).
[0004] So far, there is no method described allowing the
straightforward detection or isolation of natural prion proteins.
The isolation of PrP.sup.c described in the prior art (31) is time
consuming, ineffective and yields only minimum amounts of protein.
The isolation of PrP.sup.sc described in the prior art (31, 35, 64)
is also time consuming and ineffective and the purity of the
PrP.sup.sc is speculative. Furthermore, up to now it was not
possible to discriminate between the cellular isoform PrP.sup.c and
the isoform PrP.sup.sc or PrP27-30, which is a prerequisite for the
development of a simple and reliable assay for diagnosing a
transmissible spongiform encephalopathy.
[0005] Therefore, the technical problem underlying the present
invention is to provide a simple method for the efficient isolation
of prion proteins and the detection of said proteins, preferably in
a way that allows for discrimination between different isoforms of
PrP.
[0006] The solution to said technical problem is achieved by
providing the embodiments characterized in the claims.
[0007] Thus, the present invention relates to a method for the
detection of a prion protein comprising the steps of:
[0008] (a) contacting a probe suspected to contain a prion protein
with a chaperone, and
[0009] (b) determining whether a prion protein binds to the
chaperone.
[0010] In addition, the present invention relates to a method for
the isolation of a prion protein comprising the steps of:
[0011] (a) contacting a probe containing a prion protein with a
chaperone, and
[0012] (b) isolating the chaperone-bound protein from the
chaperone.
[0013] When carrying out experiments in order to identify proteins
capable of interacting with PrP.sup.c it was surprisingly found
that chaperones are capable of specifically binding to prion
proteins. The specificity of the observed in vivo interactions was
confirmed by in vitro binding studies employing recombinant prion
proteins. Mapping of the interaction site between the molecular
chaperones and PrP.sup.c was performed using recombinant prion
GST-fusion peptides. The results show that a GST-PrP.sup.c fusion
protein binds specifically to Hsp60 in an S. cerevisiae environment
as well as in vitro. The Hsp60 family is one of the best
characterized members of the molecular chaperones which mediate
ATP-dependent folding of polypeptide chains (13, 18, 22, 23) and
which are widely distributed and conserved between prokaryotes and
mammals. Human Hsp60 (544 amino acids) is proposed to form
tetradecameric complexes in vivo as shown in the crystal structure
of the prokaryotic homologue GroEL (10). The cDNAs isolated by a
two-hybrid screen in S. cerevisiae (15, 19, 21) encode N-terminally
truncated proteins of 399, 317 and 246 amino acids in length,
comprising at least in part the apical domain of the Hsp60 monomer.
This apical domain contains several amino acid residues which
specifically mediate peptide binding in the case of GroEL (14).
[0014] Specificity of the PrP.sup.c/Hsp60 interaction in vivo was
confirmed employing the `false baits` LexA-bicoid and LexA-NFI/CTF2
as well as authentic LexA and LexA-GST. The interaction was
confirmed in vitro using recombinant GST-PrP.sup.c and recombinant
full-length Hsp60 as well as GroEL. This result shows that the
PrP.sup.c/Hsp60 interaction does not involve additional factors and
that thus, chaperones can be used for the detection and isolation
of prion proteins. The recombinant rPrP27-30 (47) represents the
proteinase K sensitive isoform of the proteinase K resistant core
PrP27-30 isolated from scrapie preparations. The results of the in
vitro interaction between rPrP27-30 and Hsp60 reveal that the core
region of PrP (amino acids 90 to 231) is sufficient for binding to
Hsp60.
[0015] Identification of the interaction site between amino acid
180 and amino acid 210 by mapping of PrP.sup.c peptides showed that
binding of Hsp60 to PrP.sup.c occurs within a highly conserved
region of the prion protein containing amino acids 180, 198, 200
and 210. Mutation of these residues segregate with inherited prion
diseases in humans (33). In addition, the chaperone-binding
fragment GST::P180-210 contains at least in part the two putative
.alpha.-helical domains H3 (amino acids 178 to 191) and H4 (amino
acids 202 to 218) (24). The conversion of .alpha.-helical regions
into .alpha.-sheets of PrP are thought to be responsible for
PrP.sup.Sc formation. There are several possibilities to suggest a
possible physiological relevance of the Hsp60/PrP interaction. (i)
Hsp60 might be involved in the propagation of PrP.sup.Sc as has
been shown for the interaction of the yeast prion-like factor
[psi.sup.+] with the molecular chaperone Hsp104 (12, 50). Based on
studies with transgenic mice, it has been suggested recently that a
species-specific macromolecule, designated `protein X`,
participates in prion formation (43). Protein X was proposed to
function as a molecular chaperone facilitating the transformation
of PrP isoforms. This unknown factor `X` might in fact be Hsp60.
(ii) Alternatively, Hsp60 could prevent aggregation of PrP.sup.c to
PrP.sup.Sc amyloids e.g. by trapping misfolded forms of
PrP.sup.c.
[0016] More recent data suggested that so-called "chemical
chaperones" such as glycerol, trimethylamine N-oxide (TMAO), and
dimethylsulfoxide (DMSO) interfere with PrP.sup.Sc formation by
stabilizing the .alpha.-helical conformation of PrP.sup.c. (67)
[0017] The detection or isolation of prion proteins by the methods
of the invention relates to recombinantly produced prion proteins
or prion proteins from natural sources. Prion proteins can be
extracted from natural sources, for example, by the method
described in (31, 64); involving suspending tissue in sucrose,
homogenization and clarification by centrifugation.
[0018] Contacting the probe suspected to contain a prion protein
with a chaperone can be carried out by known methods, for example,
with the chaperone being in solution or being immobilized, for
example on a matrix such as a gel or a resin for chromatography
(66).
[0019] Contacting of the probe with the chaperone and analyzing of
the complex chaperone-prion protein can also be carried out as
described in the Examples below.
[0020] Suitable chaperones which specifically bind to prion
proteins can be inter alia determined by the person skilled in the
art by assaying the binding of a particular chaperone to prion
proteins as described in the Examples, below.
[0021] In one preferred embodiment of the method of the invention,
a fragment, analogue or derivative of said chaperone is used which
is still capable of binding the prion protein.
[0022] As used herein, the term "derivative" refers to such
derivatives which may be prepared from the functional groups which
occur at side chains on the residues or the N- or C-terminus
groups, by means known in the art.
[0023] The term "fragment" relates to any fragment of the chaperone
which still has the capability to interact with the prion protein
and such a fragment can be prepared by techniques known to the
person skilled in the art.
[0024] In another preferred embodiment of the invention, the
chaperone used in the method for detection and/or isolation of a
prion protein is Hsp60 or GroEL.
[0025] In a further preferred embodiment the chaperone is a
recombinant protein, i.e., the chaperone is produced by recombinant
DNA technology, namely by expression from a cloned DNA
sequence.
[0026] In a still further preferred embodiment, the chaperone is
part of a fusion protein, which can comprise, besides the chaperone
a protein or preferably, a protein domain which confers to the
fusion protein a specific binding capacity. Preferably, the
recombinant chaperone is fused to glutathione-S-transferase.
[0027] Any prion protein, isoform, fragment or derivative of such
prion protein or mixture of said substances can be detected or
isolated by the method of the invention as long as it is capable of
being bound by the chaperone. In a preferred embodiment the prion
protein to be detected or isolated is the prion protein PrP.sup.c
and/or an isoform of PrP.sup.c. Preferably the prion protein
isoform is the isoform PrP.sup.sc or a fragment or derivative
thereof.
[0028] In a further preferred embodiment the prion protein is the
processed form PrP.sup.c23-231 comprising amino acids 23 to 231 of
PrP.sup.sc and/or the isoform PrP.sup.sc is the N-terminally
truncated derivative PrP27-30 or a fragment thereof.
[0029] As already stated above, for determining whether a prion
protein was bound to a chaperone, the chaperone can be in solution
or be attached to a solid phase.
[0030] Following incubation of both participants in solution, the
interaction can be proved by co-immunoprecipitation (51) followed
by Western Blotting (44) employing a PrP specific antibody as
described, for example, in (20), a chaperone-specific antibody or
an antibody directed against one of the Tags, i.e. GST-antibody,
FLAG-antibody, BTag-antibody, antibody directed against the
calmodulin binding protein, the S-peptide, the
maltose-binding-protein, oligohistidin and the green fluorescent
protein (GFP). Furthermore, the interaction can, for example, be
proved by (i) crosslinking employing reagents such as
dimethylsuberimidate (52), (ii) by affinity chromatography (66) by
adding the immobilized ligand directed against one of the tags
fused to one of the both partners (Criss-Cross interactions), or
(iii) by analyzing the complex by a non-denaturing polyacrylamid
gel (53) or by a size exclusion chromatography which is mostly
HPLC/FPLC (54).
[0031] In a preferred embodiment, the chaperone is in solution and
detectably labelled. The person skilled in the art will know
suitable labels or will be able to ascertain such labels using
routine experimentation. Preferably the detectable label is
selected from a radioisotope, a fluorescent compound, a colloidal
metal, a chemiluminescent compound, a bioluminescent compound, a
phosphorescent compound or an enzyme.
[0032] Alternatively, the chaperone is bound to a solid phase for
the detection and/or isolation of a prion protein. Suitable
materials are known to the person skilled in the art and include,
for example, a gel or a resin (Sepharose, agarose, nitrocellulose,
dynabeads.RTM., polystyrene etc.).
[0033] In a preferred embodiment, the solid phase is a matrix
comprising glutathione, such as glutathione-sepharose. The protein
domain used for binding of the chaperone to a matrix can also be an
oligohistidine (55), Calmodulin binding peptide (CBP) (56),
S-peptide (ribonuclease A) (57), FLAG (58), green-fluorescent
protein (GFP, 65), BTag (59), or maltose-binding-protein (MBP; 61).
The tagged chaperone can be immobilized to gluthathione,
IMAC-Ni.sup.2+, Calmodulin, S-protein 104 aa (57),
anti-FLAG-antibodies, anti GFP-antibodies, BTag-antibodies (59) or
maltose (60).
[0034] Alternatively, coupling of the chaperone itself by the
fusion protein can be done via thiol-groups of non-oxidized
cysteins or, alternatively, via free lysine or .alpha.-amino groups
to cyanogen bromide agarose or .alpha.-hydroxy succinimide
activated agarose (63).
[0035] Regarding the interaction of the prion proteins with
chaperones where one of the compounds is immobilized, the
interaction can be determined by IASYS (FISONS). Protein-protein
interactions can be detected and measured by biosensors, which use
the evanescent field to probe biomolecular mass and concentration
close to the probe surface. Alternatively, such interaction can be
determined by far western blovaffinity blot (62): the prion-protein
either tagged or untagged or the chaperone either tagged or
untagged are blotted onto a membrane such as nitrocellulose or
PVDF. The other interaction partner either the tagged/untagged
prion protein or the tagged/untagged chaperone in solution is
incubated with the protein associated membrane. Interaction is
confirmed by addition of an antibody directed against the protein
in solution itself or the tag fused to the protein (62).
[0036] In a still further preferred embodiment of the method of the
invention for isolating prion proteins, the chaperone is part of a
matrix contained within an affinity chromatography column (63, 66)
and step (b) is modified in such a way that
[0037] (i) the probe suspected to contain the prion protein is
passed through the column,
[0038] (ii) after washing, the prion protein is eluted from the
column, optionally by a change in pH or ionic strength and
collected; and
[0039] (iii) optionally the collected prion protein is further
purified.
[0040] By this kind of affinity chromatography which is, for
example, describes in (55, 63, 66), impurities contained in the
prion protein preparation are passed through the column. The prion
protein(s) will be bound to the column by the chaperone. Suitable
conditions for allowing the specific binding of the prion protein
to the column and for eluting the prion protein from the gel can be
determined by the person skilled in the art and are, for example,
described in (47, 48).
[0041] In an alternative embodiment, the isolation of the prion
protein is carried out as a batch process according to standard
procedures or, for example, by using a modified version of the
procedure described in the Examples, below, wherein instead of the
prion protein, the chaperone is attached to glutathione-Sepharose
beads, for example, gluthathione-Sepharose 4B beads.
[0042] Prion proteins isolated and purified according to the method
of the invention can be used, for example, as immunogen for raising
antibodies, as active component of pharmaceutical compositions or
for the development of diagnostic assays, such as ELISA.
[0043] The probe can be obtained from various organs, preferably
from tissue, for example brain, ileum, cortex, dura mata, purcinje
cells, lymphnodes, nerve cells, spleen, tonsils, muscle cells,
placenta, pancreas eyes, backbone marrow or peyer'sche plaques or
from a body fluid, preferably from blood, cerebrospinal fluid,
semen or milk.
[0044] As is evident from the results presented in Example 6,
binding of the chaperone GroEL is stronger to rPrP27-30 compared to
PrP.sup.c (see lane 3 of FIG. 2B versus lane 2 of FIG. 2C). These
results were confirmed by further titration experiments with GroEI
and Hsp60 (data not shown). Thus, determining the strength of
binding of a chaperone with the prion protein in a probe by
comparing it with the strength of binding of the same chaperone
with PrP.sup.sc (or rPrP27-30) and PrP.sup.c standards allows the
determination of whether a prion protein indicative for
transmissible spongiform encephalopathy (TSE) is contained in a
sample.
[0045] Accordingly, a further preferred embodiment of the invention
relates to a method for the in-vitro diagnosis of a transmissible
spongiform encephalopathy, wherein step (b) is modified in such a
way that the differences in binding of the chaperone to PrP.sup.c
and an isoform of PrP.sup.c, respectively, preferably PrP.sup.sc,
are used to determine whether an isoform of PrP.sup.c is present in
the probe or not.
[0046] The present invention furthermore provides a complex of the
chaperone and a prion protein and, in addition, a composition for
the detection and/or isolation of a prion protein comprising a
chaperone as defined above.
[0047] Furthermore, the present invention relates to a diagnostic
composition comprising the chaperones as defined above. Such
compositions may contain additives commonly used for diagnostic
purposes. Said compositions can be used for the diagnosis of
transmissible spongiform encephalopathies by applying the approach
described above, wherein a probe taken from a body is incubated
with a chaperone and the strength of binding of the chaperone to
the prion protein contained in the probe is determined. In the case
that brain is used as a probe, diagnosis is often carried out post
mortem but is, in certain cases, also possible on the living
organism (biopsy). In the case that blood, milk or cerebrospinal
fluid is used as a probe, diagnosis is possible for living
individuals.
[0048] In another embodiment, the present invention relates to a
pharmaceutical composition comprising a chaperone as defined above
or, alternatively, comprising a substance that inactivates said
chaperone. Such compositions can optionally comprise
pharmaceutically acceptable carriers.
[0049] Since, for example, chaperons like Hsp60 are assumed to be
capable of preventing the aggregation of PrP.sup.c to PrP.sup.sc,
it might be possible to block the conversion of the isoform
PrP.sup.c into the prion associated isoform PrP.sup.sc by
administration of such chaperones which specifically bind prion
proteins and, thus, to prevent or treat transmissible spongiform
encephalopathy.
[0050] On the other hand, it might be possible that chaperones are
involved in the transformation of PrP.sup.c to PrP.sup.sc. Thus,
blocking such transformation by the administration of agents which
specifically inactivate such chaperones which specifically interact
with prion proteins could also be helpful for the treatment or
prevention of transmissible spongiform encephalopathies. Such
substances can be selected by the person skilled in the art by
routine experimentation and include ligands that bind to the
chaperone, thus preventing the interaction of the chaperone with
the prion protein. Examples of such ligands are antibodies,
preferably monoclonal antibodies, or a fragment of a protein which
a domain responsible for binding to the chaperone, e.g. a fragment
of PrP.sup.c containing amino acids 180 to 210.
[0051] Preferably, said compositions are used for the prevention or
treatment of transmissible spongiform encephalopathy, for example,
Scrapie, bovine spongiform encephalopathy (BSE), Creutzfeld-Jacob
Disease (CJD), Gerstmann-Stru.beta.ler-Scheinker-Syndrome (GSS),
Kuru, fatal familial insomnia (FFI) or transmissible mink
encephalopathy (TME).
LEGENDS TO THE FIGURES
[0052] FIG. 1: Identification of PrP.sup.c23-231/Hsp60 Interaction
Employing the Two-Hybrid System.
[0053] Two different phenotypes confirm this interaction. Yeast
cells containing the reporter plasmid pSH18-34 were cotransformed
with the pJG4-5 plasmid carrying the cDNA clone encoding for Hsp60
(amino acids 146-544) and the bait plasmids pSH2-1 (row 1),
pSH2-1-GST (row 2), pSH2-1-GST-PrP.sup.c (row 3), pSH2-1-NFI/CTF2
(row 4) (49), pEG202-bicoid (row 5) (21) and pSH2-1-PrP.sup.c-GST
(row 6). Five of each transformants were resuspended in TE, dotted
on galactose plates either supplemented with X-Gal (A) or leucine
deficient (B) and incubated at 30.degree. C. for 5 days.
[0054] FIG. 2.: Immunoblot Analysis of Pull-Down Assays to
Demonstrate the In Vitro Interaction of Hsp60 and GroEL in the
Presence of PrP Fused to GST.
[0055] Numbers on the left side indicate size in kDa. (A)
Recombinant GST (1 mg), GST-rPrP27-30 (2 mg) as well as
GST-PrP.sup.c (2 mg) immobilized on glutathione-Sepharose were
incubated with 10 mg Hsp60. After centrifugation beads were washed
and resuspended in sample buffer. 4 ml each of GST-PrP.sup.c (lane
2), GST-rPrP27-30 (lane 3) and GST (lane 4) as well as 200 ng Hsp60
as a control (lane 1) were analyzed by SDS-PAGE (12.5%) and
immunoblotting (PVDF) employing a monoclonal mouse anti-Hsp60
antibody and chemiluminescence detection. (B) Recombinant GST (1
mg) and GST-PrP.sup.c (2 mg) immobilized on glutathione-Sepharose
as well as glutathione-Sepharose alone were incubated with 25 mg
GroEL. After washing beads were resuspended in sample buffer. 4 ml
each of a 1:1 slurry of beads (lane 2), GST-PrP.sup.c (lane 3) and
GST (lane 4) as well as 2 mg GroEL as a control (lane 1) were
analyzed on a 12.5% SDS gel and blotted on a NC membrane. Protein
detection was performed employing an anti-GroEL antibody and
chemiluminescence. (C) Recombinant GST (1 mg) as well as
GST-rPrP27-30 (2 mg) immobilized on glutathione-Sepharose were
incubated with 25 mg GroEL. After washing beads were resuspended in
sample buffer. 4 ml each of GST-rPrP27-30 (lane 2) and GST (lane 3)
as well as 1 mg GroEL as a control (lane 1) were analyzed by
SDS-PAGE and immunoblotting employing an anti-GroEL antibody and
chemiluminescence.
[0056] FIG. 3: Mapping the PrP.sup.c/GroEL Interaction Site Using
Fragments of PrP.sup.c as Fusions with GST.
[0057] (A) Six fragments of PrP.sup.c were designed on the basis of
biochemical predictions such as hydrophilicity, antigenicity and
secondary structures and represent amino acids 23 to 52, amino
acids 53 to 93, amino acids 90 to 109, amino acids 129 to 175,
amino acids 180 to 210 and amino acids 218 to 231 (48). (B) Mapping
analysis of the PrP/Hsp60 interaction site using the six GST fused
PrP fragments. 2 mg each of the fragments bound to
glutathione-Sepharose were incubated with 10 mg Hsp60. The beads
were washed and resuspended in sample buffer. 4 ml each of the
fragments GST::P23-52 (lane 1), GST::P53-93 (lane 2), GST::P90-109
(lane 3), GST::P129-175 (lane 4), GST::P218-231 (lane 5) and
GST::P180-210 (lane 6) were analyzed on a 12.5% SDS gel and blotted
onto a PVDF membrane followed by development employing an
anti-Hsp60 antibody and chemiluminescence. (C) Mapping analysis of
the PrP/GroEL interaction site. 2 mg each of the fragments bound to
glutathione-Sepharose were incubated with 10 mg GroEL. The beads
were washed and resuspended in sample buffer. 4 ml each of the
fragments GST::P23-52 (lane 1), GST::P53-93 (lane 2), GST::P90-109
(lane 3), GST::P129-175 (lane 4), GST::P218-231 (lane 5) and
GST::P180-210 (lane 6) were analyzed on a 12.5% SDS gel and blotted
(PVDF). GroEL was detected by chemiluminesence using an anti-GroEL
antibody.
[0058] FIG. 4: Co-Expression of FLAG-tagged Hsp60 and GST-tagged
prion proteins from Syrian golden hamster in the baculovirus
system.
[0059] Cell lysates were analyzed on 12.5% SDS gel. Western
Blotting employing an anti-Hsp60 antibody (Sigma Catalogue # H
4149) antibody shows decreased levels of FLAG::Hsp60 when
co-expressed with GST::haPrP.sup.c23-231 (lane 3) and
GST::haPrp90-231 (lane 4) compared to FLAG::Hsp60 expression (lane
1) and co-expression of GST control (lane 2).
[0060] The following examples illustrate the invention:
EXAMPLE 1
Construction of Vectors
[0061] Construction of yeast vectors. Cloning procedures were
performed as described previously unless otherwise stated (40). The
shuttle vectors pSH2-1 and pEG202, which direct the synthesis of
different LexA hybrids (amino acids 1-87 and amino acids 1-202)
(19, 21), were used to construct the LexA fusion `baits`.
[0062] (i) Construction of pSH2-1/pEG202-GST. A 666-bp DNA fragment
coding for glutathione S-transferase (GST) was amplified by PCR
(39) from the cDNA clone pAcSG2T::PrP.sup.c23-231 (48). The
fragment was subcloned into plasmids pSH2-1 and pEG202 using the
EcoRI/BamHI restriction sites, resulting in the vectors
pSH2-1/pEG202-GST.
[0063] (ii) Construction of pSH2-1/pEG202-PrP.sup.c. A 646-bp DNA
fragment containing nucleotides coding for amino acids 23 to 231 of
the Syrian golden hamster PrP.sup.c protein was amplified by PCR
from the cDNA clone pAcSG2T::PrP.sup.c23-231. The PrP.sup.c
cassette was cloned via EcoRI/BamHI restriction sites into vectors
pSH2-1 and pEG202, yielding pSH2-1/pEG202-PrP.sup.c.
[0064] (iii) Construction of pSH2-1/pEG202-GST-PrP.sup.c. A 646-bp
DNA fragment coding for amino acids 23 to 231 of the PrP.sup.c
protein was amplified by PCR from the cDNA clone
pAcSG2T::PrP.sup.c23-231. The PrP.sup.c fragment was cloned via
BamHI/SalI restriction sites into vector pSH2-1-GST, yielding
pSH2-1-GST-PrP.sup.c. The GST-PrP.sup.c cassette was excised from
this vector using the EcoRI and SalI restriction sites and cloned
into pEG202, resulting in pEG202-GST-PrP.sup.c.
[0065] (iv) Construction of pSH2-1-PrP.sup.c-GST. The
PrP.sup.c23-231 cassette was amplified by PCR from
pAcSG2T::PrP.sup.c23-231 and cloned into pSH2-1-GST via the EcoRI
restriction site resulting in pSH2-1-PrP.sup.c-GST.
[0066] Correct orientation, reading frames and sequences of the PCR
amplified fragments were confirmed by dideoxy sequencing (41).
Example 2
Expression of the `Bait` Protein LexA-GST-PrP.sup.c in S.
cerevisiae Strain EGY48
[0067] For `two-hybrid` screening, a fusion protein `bait`
consisting of the bacterial repressor LexA binding domain (19, 21)
and the Syrian Golden Hamster prion protein PrP.sup.c23-231
(aa=amino acids 23 to 231, referred to as PrP.sup.c) (5) fused to
glutathione S-transferase (GST) was tested. As reported recently,
fusion with GST significantly enhances the solubility and stability
of recombinant Prp.sup.c (47, 48). Cells of the yeast strain EGY48
were cotransformed with the reporter plasmid pSH18-34 and the bait
plasmids and tested for their intrinsic ability to activate the
reporter system. The pSH-GST-PrP.sup.c construct showed a low level
of intrinsic activation. Expression of the LexA-GST-PrP.sup.c
fusion protein in S. cerevisiae was confirmed by immunoblotting
employing a polyclonal anti-PrP antibody (20) directed against aa
95 to 110 (data not shown).
Example 3
Identification of PrPC/Hsp60 Interaction by the Two-Hybrid
Screen
[0068] Detailed procedures for using the yeast two-hybrid system
have been detailed previously (1, 6, 19, 21). S. cerevisiae strain
EGY48 (MATa ura3 his3 trp1 LEU2::LexAop6-LEU2), which carries a
chromosomal insertion of LexA binding sites upstream of the
LEU2-gene was used as the recipient host (19, 21). In brief, yeast
strain EGY48 was transformed with the reporter plasmid pSH18-34
containing a LexA controlled lacZ gene as a second reporter. S.
cerevisiae cells were cotransformed with the `bait`-plasmid and
pJG4-5 containing a HeLa cDNA library fused to the acidic B42
transactivation domain (19, 21). The cDNA insert of the pJG4-5
plasmid is controlled by a galactose inducible promoter. Therefore,
interaction between the two hybrids occurs only in the presence of
galactose. 2000 Colonies able to grow in the absence of leucine
(first reporter gene) were dotted on galactose plates supplemented
with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) and
screened for b-galactosidase production (blue color, second
reporter gene).
[0069] cDNAs of 55 positive clones were recovered from 5 ml S.
cerevisiae cell cultures. Cells were incubated at 30.degree. C. for
2 days, harvested by centrifugation (3000 rpm, 10 min at 4.degree.
C.) and washed (1 M sorbitol, 0.1 M EDTA, pH 8.0). After
resuspension in SCE (1 M sorbitol, 0.1 M sodium citrate, pH 5.8, 10
mM EDTA, 0.1 M b-mercaptoethanol) the cells were incubated with 40
ml Lyticase (5 U/ml, Sigma) for 1 h at 37.degree. C. After
centrifugation, cells were resuspended in TE-lysis buffer (50 mM
Tris/HCl, pH 7.4, 20 mM EDTA containing 1% SDS) and incubated for
30 min at 65.degree. C. The lysate was phenol extracted, the DNA
ethanol precipitated and resuspended in TE. The DNA was transformed
in E. coli strain KC8 which enables the selection of pJG4-5
plasmids by ampicillin resistance and complementation of its
tryptophan auxotrophy (19, 21). As control experiments the plasmids
were retransformed in EGY48 and the transformants tested for
b-galactosidase production and for their Leu.sup.+-phenotype. Five
retransformants were dotted on corresponding plates, incubated for
5 days at 30.degree. C. and finally sequenced. cDNA inserts were
sequenced with the T7-sequencing kit (Pharmacia) based on the
dideoxy method (41). Homology searches for the cDNA sequences were
performed at the National Center for Biotechnology Information
using the BLAST network service (http://www.ncbi.nim.nih.gov/-
Recipon/bs_seq.html). Approx. 20% (corresponding to nine cDNAs)
encoded heat shock protein 60 (Hsp60). The isolated Hsp60 cDNAs
encode for three N-terminally truncated proteins with different
lengths starting at position aa 146, aa 228 and aa 298,
respectively (EMBL M34664). All of them contain parts of the
putative peptide binding domain of Hsp60 (14).
Example 4
Test for Specificity of the PrP.sup.c/Hsp60 Interaction
[0070] Specificity of the observed in vivo interaction between
PrP.sup.c and Hsp60 (FIG. 1, row 3) was demonstrated by several
recloning experiments. In particular, it was shown that the inverse
fusion PrP.sup.c::GST (FIG. 1, row 6), as well as authentic
PrP.sup.c lacking GST (data not shown) strongly interact with
Hsp60. In contrast, LexA-GST (FIG. 1, row 2), authentic LexA (FIG.
1, row 1), as well as the two `false-baits` LexA-NFI/CTF2 (47)
(FIG. 1, row 4) and LexA-bicoid (FIG. 1, row 5) showed no
interaction with Hsp60.
Example 5
Recombinant Hsp60 Binds Specifically to PrP.sup.c23-231 and
rPrP27-30 In Vitro
[0071] PrP.sup.c23-231 represents the mature form of the cellular
prion protein. Scrapie prion isolates consist mainly of the
protease-resistant core which is 27 30 kDa in size (referred to as
PrP27-30) (35, 42), comprising amino acids 90 to 231. We employed
recombinant GST fusion proteins bound to glutathione Sepharose
beads to confirm the interaction with recombinant full-length Hsp60
in vitro. GST as well as PrP.sup.c23-231 and rPrP27-30 fused to GST
(47, 48) were immobilized and incubated with Hsp60. Hsp60 was
detected in the presence of GST-PrP.sup.c (FIG. 2A, lane 2), and
GST-rPrP27-30 (FIG. 2A, lane 3) but not with GST alone (FIG. 2A,
lane 4). Another human chaperone, Hsp70, did not interact with any
of these proteins (data not shown) demonstrating that the
interaction of PrP.sup.c with Hsp60 is highly specific.
[0072] Proteins and antibodies. GST, GST::PrP.sup.c23-231, as well
as the GST::PrP.sup.c fragments GST::P23-52, GST::P53-93,
GST::P90-109, GST::P129-175, GST::P180-210 and GST::P218-231 were
prepared as described (48). GST::rPrP27-30 (aa 90 to 231 of the
Syrian Golden Hamster prion protein) was expressed in and purified
from E. coli and from the baculovirus expression system (47). GroEL
and anti-rabbit-IgG-POD as well as anti-mouse-IgG-POD were obtained
from Boehringer Mannheim. Recombinant human Hsp60 was provided by
StressGen and monoclonal mouse anti-Hsp60 was obtained from
Sigma.
[0073] SDS PAGE and immunoblotting. Protein samples were analyzed
on 12.5% SDS Phastgels (Pharmacia) as described previously (48).
Rainbow marker (RPN 756, Amersham) was used as a size standard.
Following electrophoresis, gels were blotted onto nitrocellutose
(NC, Schleicher & Schuell) or polyvinyldifluoride membranes
(PVDF, Millipore) (40 min. at 70.degree. C.). The blots were
incubated with a polyclonal anti-GroEL or anti-PrP antibody at
1:800/1:400 dilutions. Incubation steps were performed as described
previously (44). Antibody detection was performed by
chemiluminescence (ECL system, Amersham) or in the presence of DAB
(Sigma).
Example 6
Binding of the Bacterial GroEL to PrP.sup.c23-231 and rPrP27-30 In
Vitro
[0074] To investigate whether GroEL, the prokaryotic homologue of
the Hsp60 family is also capable of binding to PrP.sup.c,
recombinant GroEL in corresponding in vitro binding experiments was
employed. Pull-down assays were performed by equilibrating
glutathione-Sepharose 4B beads (Pharmacia) loaded with GST or the
GST fusion protein in refolding buffer (RF) (32) including 0.5%
Triton-X-100. The equilibrated beads were incubated with an up to
10 fold molar excess of GrOEL or Hsp60 (monomer) at room
temperature in the presence of RF including 0.5% Triton-X-100.
After centrifugation (2500 rpm, 10 min) the beads were washed with
RF and analyzed on a 12.5% SDS Phastgel, blotted and probed for the
presence of GroEL or Hsp60.
[0075] GroEL was found to exhibit specificity in the interaction
with PrP.sup.c (FIG. 2B, lane 3) and rPrP27-30 (FIG. 2C, lane 2)
fused to GST whereas no binding occurs in the presence of GST alone
(FIG. 2B, lane 4 and FIG. 2C, lane 3). However, the strength of
binding is stronger for rPrP27-30 compared to PrP.sup.c.
Example 7
Mapping of the Interaction Site for Hsp60 and GroEL within PrP
[0076] To obtain a comprehensive map of the PrP.sup.c-binding site
on the molecular chaperones six fragments of PrP fused to GST were
employed and their ability to bind Hsp60 and GroEL was tested.
These peptides were designed on the basis of biochemical
predictions regarding hydrophilicity, antigenicity and secondary
structure (48) and represent aa 23 to 52, aa 53 to 93, aa 90 to
109, aa 129 to 175, aa 180 to 210 and aa 218 to 231 (FIG. 3A). The
immobilized peptides were incubated with Hsp60 and GroEL,
respectively. This mapping analysis identified Hsp60 (FIG. 3B, lane
6) and GroEL (FIG. 3C, lane 6) only in the presence of
GST::P180-210, demonstrating that it is only the PrP region
represented by amino acids 180 to 210 which interacts with the
molecular chaperone.
Example 8
Downregulation of Hsp60 in the Presence of Hamster PrP.sup.c23-231
in the Fusion with GST
[0077] FLAG tagged Hsp60 has been synthesized as a 61 kDA protein
in the baculovirus system (FIG. 4, lane 1). Co-expression of
GST::rPrP.sup.c (rPrP23-231) (lane 3) and GST::rPrP27-30
(rPr90-231) (lane 4) downregulates FLAG::Hsp60 expression, whereas
co-expression of GST does not affect expression of FLAG::Hsp60
(lane 2).
[0078] Co-infection was carried out followed by Western Blot
analysis. Total protein was harvested from baculovirus infected
insect cells by standard methods and analyzed by SDS-PAGE. The
following Western Blot employed an anti-Hsp60 antibody (Sigma # H
4149).
[0079] The downregulation of Hsp60 in the presence of PrP.sup.c can
occur either on transcription or translational level.
Alternatively, PrP could trigger proteolytic degradation of Hsp60.
Finally, PrP.sup.c could lead to an increased secretion process of
Hsp60. The presence of Hsp60 in the culture medium would prove this
hypothesis. Downregulation of Hsp60 in the presence of PrP can
account for a direct PrP/Hsp60 interaction which leads to an
increased downregulation of Hsp60. Application of PrP-peptides
spanning parts of the prion protein can identify the region of the
prion protein which is responsible for the downregulation of Hsp60
expression.
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References