U.S. patent application number 12/854608 was filed with the patent office on 2012-05-31 for methods for identifying factors that control the folding of amyloid proteins of diverse origin.
This patent application is currently assigned to ARCH DEVELOPMENT CORPORATION. Invention is credited to Susan Lindquist.
Application Number | 20120135435 12/854608 |
Document ID | / |
Family ID | 26749757 |
Filed Date | 2012-05-31 |
United States Patent
Application |
20120135435 |
Kind Code |
A1 |
Lindquist; Susan |
May 31, 2012 |
Methods for Identifying Factors That Control the Folding of Amyloid
Proteins of Diverse Origin
Abstract
The present invention provides a yeast cell based system for
determining factors that control the folding of amyloid proteins of
diverse origins. Further the present invention provides methods of
using such a system to screen for reagents that affect amyloid
formation, a process that is integral to several devastating human
disease including Creutzfeld-Jacob disease (CJD), fatal familial
insomnia (FFI), Gertsmann-Straussler-Scheinker (GSS) syndrome, and
kuru. The system of the present invention provides a rapid
screening system to quickly and cheaply identify reagents that
affect the folding and aggregation properties of the target
protein.
Inventors: |
Lindquist; Susan; (Chestnut
Hill, MA) |
Assignee: |
ARCH DEVELOPMENT
CORPORATION
Chicago
IL
|
Family ID: |
26749757 |
Appl. No.: |
12/854608 |
Filed: |
August 11, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09207649 |
Dec 8, 1998 |
7799535 |
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12854608 |
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60069168 |
Dec 9, 1997 |
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60084824 |
May 8, 1998 |
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Current U.S.
Class: |
435/8 ;
435/29 |
Current CPC
Class: |
G01N 33/6896 20130101;
C12Q 1/025 20130101 |
Class at
Publication: |
435/8 ;
435/29 |
International
Class: |
C12Q 1/66 20060101
C12Q001/66; G01N 21/64 20060101 G01N021/64; C12Q 1/02 20060101
C12Q001/02 |
Goverment Interests
[0001] The U.S. government owns rights in the present invention
pursuant to grant number NIH GM 25874 from the National Institutes
of Health.
Claims
1. A method of identifying a candidate substance that inhibits the
aggregation of an aggregate-prone amyloid protein, comprising: (a)
contacting a yeast cell that expresses an aggregate-prone amyloid
protein with said candidate substance under conditions effective to
allow aggregated amyloid formation; and (b) determining the ability
of said candidate substance to inhibit the aggregation of the
aggregate-prone amyloid protein.
2. The method of claim 1, wherein the aggregate-prone amyloid
protein comprises a Sup35 or URE3 polypeptide.
3. The method of claim 1, wherein the aggregate-prone amyloid
protein comprises a PrP or .beta.-amyloid polypeptide.
4. The method of claim 1, wherein the aggregate-prone amyloid
protein is a chimeric protein.
5. The method of claim 4, wherein the chimeric protein comprises at
least the N-terminal domain of Sup35.
6. The method of claim 4, wherein the chimeric protein comprises at
least an aggregate forming domain of a mammalian amyloid
polypeptide.
7. The method of claim 4, wherein the chimeric protein comprises at
least an aggregate forming domain of an aggregate-prone amyloid
protein operably attached to a detectable marker protein.
8. The method of claim 7, wherein said marker protein is green
fluorescent protein or luciferase.
9. The method of claim 7, wherein said marker protein is a
drug-resistance marker protein.
10. The method of claim 7, wherein said marker protein is a hormone
receptor.
11. The method of claim 10, wherein said hormone receptor is a
glucocorticoid receptor.
12. The method of claim 6, wherein the mammalian amyloid
polypeptide is PrP or .beta.-amyloid.
13. The method of claim 12, wherein the chimeric protein comprises
as least about amino acids 1-42 of .beta.-amyloid protein.
14. The method of claim 4, wherein the chimeric protein comprises
Sup35 in which the N-terminal domain has been replaced by amino
acids 1-42 of .beta.-amyloid protein.
15. The method of claim 1, wherein any aggregation of the
aggregate-prone amyloid protein is detected by the ability of the
aggregated protein to bind Congo Red.
16. The method of claim 1, wherein any aggregation of the
aggregate-prone amyloid protein is detected by increased protease
resistance of the aggregated protein.
17. The method of claim 1, wherein the aggregate-prone amyloid
protein is labeled.
18. The method of claim 17, wherein the label is a radioactive
isotope, a fluorophore, or a chromophore.
19. The method of claim 18, wherein the label is .sup.35S.
20. The method of claim 18, wherein the fluorophore comprises a
green fluorescent protein polypeptide.
21. The method of claim 1, wherein any aggregation is determined by
the presence of a [PSI+] phenotype.
22. The method of claim 1, wherein said yeast cell overexpresses
Hsp104.
23. A method of identifying a candidate substance for therapeutic
activity against an amyloidogenic disease in an animal, said method
comprising: (a) contacting a yeast cell that expresses an
aggregate-prone amyloid protein with said candidate substance under
conditions effective to allow amyloid formation; and (b)
determining the ability of said candidate substance to inhibit
aggregation of the aggregate-prone amyloid protein, wherein the
ability to inhibit aggregation is indicative of therapeutic
activity.
24. The method of claim 23, wherein the aggregate-prone amyloid
protein comprises a PrP, .beta.-amyloid, Sup35, or URE3
polypeptide.
25. The method of claim 23, wherein the protein is a chimeric
protein.
26. The method of claim 25, wherein the chimeric protein comprises
a Sup35 polypeptide.
27. The method of claim 25, wherein the chimeric protein comprises
a mammalian amyloid polypeptide.
28. The method of claim 27, wherein the mammalian amyloid
polypeptide is PrP or .beta.-amyloid.
29. The method of claim 23, wherein any aggregation of the
aggregate-prone amyloid protein is detected by the ability of the
aggregation to bind Congo Red.
30. The method of claim 23, wherein the aggregate-prone amyloid
protein is labeled.
31. The method of claim 30, wherein the label is a radioactive
isotope, a fluorophore, or a chromophore.
32. The method of claim 31, wherein the label is .sup.35S.
33. The method of claim 31, wherein the fluorophore comprises a
green fluorescent protein polypeptide.
34. The method of claim 23, wherein the aggregation of the
aggregate-prone amyloid protein is determined by the presence of a
[PSI+] phenotype.
35. The method of claim 23, wherein the disease is selected from
the group consisting of Alzheimer's disease, scrapie, spongiform
encephalopathy in a mammal, kuru, Creutzfeldt-Jakob disease,
Gestmann-Strausser-Scheinker disease, or fatal familial
insomnia.
36. The method of claim 35, wherein the mammal is bovine, feline, a
mink, deer, elk, a mouse, a hamster, an ape, a monkey, or human.
Description
1.0 BACKGROUND OF THE INVENTION
[0002] The present application is a continuation of provisional
application Ser. No. 60/069,168 filed Dec. 9, 1997, and provisional
application Ser. No. 60/069,168, filed May 8, 1998, the entire
disclosure of each of which is incorporated herein by reference
without disclaimer.
1.1 Field of the Invention
[0003] The present invention relates generally to the fields of
genetics and cellular biology. More particularly, it concerns a
yeast based system for the determination of compounds that affect
amyloid formation. The present invention relates to the
determination of compounds that affect the amyloid associated with
Alzheimer's disease, Transmissible spongiform encephalopathies
(TSEs), and several rare human neuropathies: Creutzfeld-Jacob
disease (CJD), fatal familial insomnia (FFI),
Gertsmann-Straussler-Scheinker (GSS) syndrome, and kuru.
1.2 Description of Related Art
[0004] 1.2.1 Yeast Prions
[0005] Recently, a novel mode of inheritance has been discovered in
Saccharomyces cerevisiae (Wickner, 1994; Lindquist, 1997).
Phenotypes transmitted by two dominant, cytoplasmically inherited
genetic elements, [PSI+] and [URE3], seem to depend upon the
inheritance of altered protein structures, rather than altered
nucleic acids. The "protein-only" hypothesis for their inheritance
led these elements to be called "yeast prions" (Wickner, 1994). The
term "prion" was first coined to describe the infectious agent
hypothesized to cause mammalian spongiform encephalogathies (TSEs)
by a "protein only" mechanism: a normal cellular protein
(PrP.sup.C) adopts an altered conformation (PrP.sup.Sc) and
interacts with other PrP.sup.C proteins to change their
conformation as well (Prusiner, 1996).
[0006] The yeast [PSI+] element, the subject of the inventor's
work, does not generally kill cells. It reduces the fidelity of
ribosome translation termination and thereby suppresses nonsense
codons (Lindquist, 1997). This phenotype is thought to result from
a change in the state of the translation-termination factor, Sup35,
that interferes with its normal function. In [psi-] cells, Sup35 is
protease sensitive and is mostly soluble; in [PSI+] cells, Sup35
has increased protease resistance and is mostly aggregated
(Paushkin et al., 1996; Patino et al., 1996; Paushkin et al.,
1997). "Aggregate" is used in a general sense; Sup35 may be
polymerized into an amyloid-like structure, or coalesced in a less
ordered state. When pre-existing Sup35 is in the aggregated state,
newly made Sup35 aggregates too, causing a self-perpetuating loss
of function in the termination factor and a heritable change in
translational fidelity (Patino et al., 1996; Paushkin et al.,
1997).
[0007] [PSI+] depends upon the chaperone protein Hsp104. The first
known function of Hsp104 was in thermotolerance in yeast, where it
increases survival after exposure to extreme temperatures up to
1000-fold (Sanchez and Lindquist, 1990). It does so by promoting
the reactivation of proteins that have been damaged by heat and
have begun to aggregate (Parcell et al., 1994). At normal
temperatures, Hsp104 overexpression cures cells of [PSI+]. Sup35
becomes soluble and the fidelity of translation termination is
restored. This state is heritable, even when overexpression of
Hsp104 ceases (Chernoff et al., 1995). Because the only known
function of Hsp104 is to alter the conformational state of other
proteins, these observations provide a strong argument that [PSI+]
is indeed based upon a heritable (self-perpetuating) change in the
conformational state of Sup35.
[0008] Surprisingly, deletions of HSP104 also cure cells of [PSI+],
and Sup35 is soluble in such cells as well (Patino et al., 1996;
Chernoff et al., 1995). This is very different from heat-induced
aggregates, which remain insoluble in hsp104 deletion strains.
Clearly, the relationship between Hsp104 and [PSI+] is more complex
than the relationship between Hsp104 and thermotolerance.
[0009] 1.2.2 Human Prions
[0010] The family of transmissible spongiform encephalopathies
(TSEs) include scrapie in sheep, bovine spongiform encephalopathy
(BSE) or "mad cow disease" in cattle, and several rare human
neuropathies: Creutzfeld-Jacob disease (CJD), fatal familial
insomnia (FFI), Gertsmann-Straussler-Scheinker (GSS) syndrome, and
kuru (Caughey and Chesebro, 1997; Prusiner, 1996). A central event
in TSE pathogenesis is the accumulation in the nervous system of an
abnormally-folded version (PrP.sup.Sc) of a normal cellular
protein, PrP.sup.C. Griffith first proposed a "protein-only" model
to explain the unconventional behavior of the infectious TSE agent
(Griffith, 1967). Indeed, the "prion", a term by which the agent is
popularly known today, appears to be almost entirely proteinaceous:
consisting primarily of PrP.sup.Sc (Caughey and Chesebro, 1997;
Prusiner, 1996).
[0011] Several lines of evidence show that PrP.sup.C is
conformationally distinct from PrP.sup.Sc, although both molecules
derive from the same primary sequence and have no detectable
post-translational differences (Caughey and Chesebro, 1997;
Prusiner, 1996; Caughey et al., 1991; Pan et al., 1993; Riek et
al., 1996). The conversion of PrP.sup.C to PrP.sup.Sc appears to
involve direct interactions of PrP.sup.C with pre-existing
PrP.sup.Sc (Caughey and Chesebro, 1997; Prusiner, 1996; Kocisko et
al., 1994). However, the exact mechanism underlying conversion is
not known. Genetic and inhibitor studies have suggested that other
cellular factors may influence TSE pathogenesis or serve as
regulators of disease (Kenward et al., 1996; Talzelt et al., 1996;
Carlson et al., 1988; Caughey et al., 1994; Telling et al., 1995;
Edenhofer et al., 1996). None have been conclusively identified;
however, cellular osmolytes (sometimes called chemical chaperones;
Caughey and Raymond, 1991) and protein chaperones have been
frequently speculated to be among them (Kenward et al., 1996;
Caughey et al., 1994; Telling et al., 1995; Edenhofer et al.,
1996).
2.0 SUMMARY OF THE INVENTION
[0012] The chaperone protein Hsp104 controls the genetic behavior
of a mysterious yeast prion-like element known as [PSI+]. The
chaperone Hsp104 controls the aggregation of Sup35, the protein
determinant of [PSI+].
[0013] The present invention includes, but is not limited to, the
following features:
[0014] 1) The protein Sup35 forms amyloid-like protein fibers in
vitro. This is a property shared by other amyloidogenic proteins
that cause human disease.
[0015] 2) The yeast protein Hsp104 affects the behavior of Sup35 in
vitro. It also affects the behavior of PrP (the mammalian prion
protein) in vitro in a similar manner and interacts in a specific
manner with .beta.-amyloid peptide 1-42 (Alzheimer's disease
peptide).
[0016] 3) When mammalian PrP is expressed in yeast cells, its
folding state depends upon the Hsp104 protein. This is the final
element that establishes that yeast can provide an excellent model
system for studying factors that affect the folding properties of
human disease proteins that have an amyloidogenic character.
[0017] In important embodiments of the present invention, this
yeast system is used in methods of identifying a candidate
substance that inhibits the aggregation of an aggregate-prone
amyloid protein. Such methods comprise contacting a yeast cell that
expresses an aggregate-prone amyloid protein with the candidate
substance under conditions effective to allow aggregated amyloid
formation, and determining the ability of the candidate substance
to inhibit the aggregation of the aggregate-prone amyloid
protein.
[0018] The term "aggregate-prone amyloid protein" is meant to be
any protein that is able to form an amyloid or amyloid-like
deposit. Amyloid or amyloid like deposits are generally insoluble
fibrillary material. Although many proteins are capable of
aggregating at high concentrations, aggregate prone amyloid
proteins are able to, and often do, aggregate under physiological
conditions, such as inside of a cell. Aggregate-prone amyloid
proteins include yeast proteins, such as Sup35 and URE3, and
mammalian proteins, such as PrP and .beta.-amyloid polypeptide. The
inventors contemplate that a protein of essentially any origin may
be used in the present invention.
[0019] In some preferred embodiments, the aggregate-prone amyloid
protein is a chimeric protein. By "chimeric protein" it is meant
that the protein comprises polypeptides that do not naturally occur
together in a single protein unit. Preferred chimeric proteins
comprises at least the N-terminal domain of Sup35. This domain has
been found to form aggregates in yeast and in vitro and is capable
of causing the aggregation of chimeric proteins comprising this
domain. Other preferred chimeric proteins include comprises at
least an aggregate forming domain of a mammalian amyloid
polypeptide, such as at least amino acids 1-42 of the
.beta.-amyloid protein or at least the aggregate forming domain of
PrP. In an important embodiment, the chimeric protein comprises
Sup35 in which the N-terminal domain has been replaced by amino
acids 1-42 of .beta.-amyloid protein.
[0020] In other embodiments, the chimeric protein comprises at
least an aggregate forming domain of an aggregate-prone amyloid
protein operably attached to a detectable marker protein. By
"operably attached" it is meant that the aggregate forming domain
and the marker protein are attached such that the chimeric protein
maintains the ability to aggregate and the marker protein maintains
the property of allowing detection of aggregation of the chimeric
protein. For example, the Sup35 N-terminal domain is operably
linked to the green fluorescent protein when this polypeptide is
capable of aggregating and the aggregated protein maintains the
ability of green fluorescent protein to fluoresce.
[0021] In other embodiments, the aggregation of the chimeric
protein leads to loss of function of the marker protein. When the
marker protein is an enzyme, aggregation of the marker protein
leads to loss of the enzymatic activity of the marker protein. That
is to say that the enzymatic marker protein maintains its enzymatic
activity when not aggregated, but aggregation leads to the loss of
enzymatic activity. Loss of activity may be due to an alteration of
the structure of the marker protein or may be due to sequestering
of the protein in the aggregates away form the substrate or
cofactors. The enzyme may be luciferase, confer drug resistance,
or, in preferred embodiments, have the translation termination
activity of the Sup35 protein. In other embodiments, the marker
protein is a hormone receptor, such as the glucocorticoid
receptor.
[0022] The inventors contemplate that the aggregation of the
aggregate-prone amyloid protein may be detected in a number of
ways. Including the methods described above, the aggregate may be
detected by staining with Congo Red. In other embodiments, the
aggregation is detected by the characteristic protease resistance
of the aggregated protein. The ability to detect the protein may be
increased by labeling the aggregate-prone amyloid protein. Labels
useful in the detection of the aggregate-prone amyloid protein
include radioactive isotope labels, such as .sup.35S, fluorophores,
such as green fluorescent protein, or chromophores. In some
preferred embodiments, aggregation is determined by the presence of
a [PSI+] phenotype.
[0023] Although overexpression of the chimeric protein comprising
the aggregate-prone domain of an aggregate-prone amyloid protein
often leads to aggregation of the chimeric protein, the inventor
has found that this aggregation is dependent on expression of heat
shock proteins, such as Hsp104. Therefore, conditions effective to
allow amyloid formation may involve modulating the expression of
Hsp104 in the yeast cell. In preferred embodiments, the yeast cell
overexpresses Hsp104.
[0024] In other embodiments of the present invention, the yeast
systems are used to identify candidate substances for therapeutic
activity against an amyloidogenic disease in an animal. These
methods comprise contacting a yeast cell that expresses an
aggregate-prone amyloid protein with said candidate substance under
conditions effective to allow amyloid formation and determining the
ability of said candidate substance to inhibit aggregation of the
aggregate-prone amyloid protein. Thus, the ability to inhibit
aggregation is indicative of therapeutic activity.
[0025] Amyloidogenic diseases in animals include Alzheimer's
disease, scrapie, spongiform encephalopathy in a mammal, kuru,
Creutzfeldt-Jakob disease, Gestmann-Strausser-Scheinker disease, or
fatal familial insomnia. In preferred embodiments, one would
express a protein comprising the aggregate forming domain of the
etiological agent of a particular disease in the yeast system to
identify therapeutic compounds for that particular disease.
Therefore, in determining therapeutic compounds for Alzheimer's
disease, one would use a yeast system comprising at least amino
acids 1-42 of the .beta.-amyloid protein.
[0026] Likewise, in determining therapeutic compounds for scrapie,
spongiform encephalopathy in a mammal, kuru, Creutzfeldt-Jakob
disease, Gestmann-Strausser-Scheinker disease, or fatal familial
insomnia, one would use a yeast system comprising the
aggregate-forming domain of PrP. In preferred embodiments, the
mammalian encephalopathy is bovine, feline, a mink, deer, elk, a
mouse, a hamster, an ape, a monkey, or human.
[0027] Although many alternative forms of the PrP gene exist, the
inventor contemplates that the expression in the yeast of a gene
encoding the form linked to a specific disease is preferred for
finding therapeutic agents for that disease. For example, for
finding therapeutic agents for scrapie, it is preferred that
proteins comprising aggregate forming domain of the goat or sheep
PrP protein are expressed in the yeast. Of course, due to
similarities between the PrP proteins, and even between the
different types amyloid proteins, a therapeutic agent for one
amyloidogenic disease may have therapeutic activity for one or more
other amyloidogenic diseases.
[0028] As used herein "a" or "an" will be understood to mean one or
more.
3.0 BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0030] FIG. 1A, FIG. 1B and FIG. 1C. Specificity of circular
dichroism spectral shifts with Hsp104 and Sup35. Right, predicted
(--) and actual (-) spectra of mixed proteins. Left, individual
spectra used to generate predicted spectra. (A) Sup35 in LSB1 (low
salt buffer) with aldolase or Hsp70. (B) Hsp104 and aldolase or IgM
in LSB1. (C) Top, Hsp104 and Sup35 in LSB1. Bottom, Hsp104 and
Sup35 in HSB (high salt buffer). Data, buffer spectra subtracted,
are presented in millidegrees because the possibility of proteins
partitioning out of solution invalidates molar ellipticity
calculations. Hsp104, Hsp70, aldolase, or the buffer in which they
were prepared was directly added to Sup35 at a .about.1:2.5
gram-weight ratio. Reactions were incubated for 1 hr with 1 mM ATP
at 37.degree. C. and spectra were then recorded at 25.degree.
C.
[0031] FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D. Specificity of
circular dichroism spectral shifts with Hsp104 and rPrP. Predicted
(--), actual (-) and individual spectra as in FIG. 1. rPrP was
prepared and folded into either .beta. sheet or .alpha. helical
forms. (A) Hsp104 and rPrP.beta.; (B) GroEL and rPrP.beta.; (C)
Aldolase and rPrP.beta.; (D) Hsp104 and rPrP.alpha.. Proteins and
rPrP were mixed (each at 0.5 mg/ml) with each other in LSB2.
Reactions were incubated at 37.degree. C. for 1 hr, diluted 5-fold
with cold water, and spectra were measured at 12.degree. C.
[0032] FIG. 3A and FIG. 3B. Effects of mixing PrP peptides with
Hsp104. (A) Location of peptides used in this study. Peptides
prepared as in (Zhang et al., 1995) were derived from the hamster
PrP sequence except for peptide K, derived from mouse PrP. (B)
Effects of PrP peptides on the ATPase activity of Hsp104. Bars
extend from the value obtained for Hsp104 without added
peptide.
[0033] FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D. Specificity of
circular dichroism spectral shifts with different PrP peptides.
Predicted (--), actual (-) and individual spectra as in FIG. 1.
Reactions were performed as in FIG. 2 except that the buffer used
was 20 mM Tris, 10 mM MgSO.sub.4, 50 mM KCl, 1.25 mM ATP at pH 7.5
to approximate ATPase assay conditions.
[0034] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E and FIG. 5F.
Effects of chaperones on cell-free conversion of .sup.35S-PrP.sup.C
to its protease-resistant form.
[0035] FIG. 5A. Conversions (as percent of total
.sup.35S-PrP.sup.C) obtained after 24 hours either with PrP.sup.Sc
or without PrP.sup.Sc (100 ng), but with the indicated chaperones
(each at 5 .mu.M, with 5 mM ATP), using the standard assay
described in methods. In indicated reaction (second from left),
PrP.sup.Sc was partially-denatured with guanidinium hydrochloride
(GdnHCl). Identical results were obtained, over a broad range of
chaperone concentrations, with or without ATP.
[0036] FIG. 5B. Conversions performed as in A, with the addition of
untreated PrP.sup.Sc. Mean values are from 3-6 studies, with
standard errors. Buffers for storing various chaperones differed
slightly in salt and glycerol content, but none affected
conversion.
[0037] FIG. 5C. Concentration-dependent effects of chaperones in
promoting conversion with untreated PrP.sup.Sc. Other Hsps tested
as in FIG. 5A.
[0038] FIG. 5D. SDS-PAGE phosphorimage of .sup.35S-PrP.sup.C
products from representative conversion reactions obtained with 3
ng .sup.35S-PrP.sup.C and increasing amounts of PrP.sup.Sc (3-1000
ng). One tenth of each reaction was left untreated (-PK); the
remainder was digested with proteinase K (+PK). GroEL and GroES
were at 1 .mu.M. When indicated, PrP.sup.Sc was partially-denatured
with GdnHl. PrP.sup.Sc fold represents the ratio of
PrP.sup.Sc:.sup.35S-PrP.sup.C in the reaction.
[0039] FIG. 5E. ATP dependence of GroEL-mediated conversions.
SDS-PAGE phosphorimages of representative conversion reactions
obtained with untreated PrP.sup.Sc and GroEL (WT and mutant D87K),
with or without ATP. Both proteinase K-treated (+PK; bottom) and
untreated samples (-PK, one-fifth sample; top) are shown.
[0040] FIG. 5F. .sup.35S-PrP.sup.GPI- conversions with or without
chaperones. Reactions contained either untreated PrP.sup.Sc or
GdnHCl-treated PrP.sup.Sc, and a variant PrP missing the GPI
anchor, .sup.35S-PrP.sup.GPI-. S-PrP.sup.GPI- and
.sup.35S-PrP.sup.C preparations are compared (right panel): UG
unglycosylated, MG monoglycosylated, and DG diglycosylated PrP
species as indicated.
[0041] FIG. 6A, FIG. 6B and FIG. 6C. Time course of conversion with
or without chaperone.
[0042] FIG. 6A. Appearance of PrP-res at 2, 6, 24, and 48 hours, in
reactions treated with proteinase K, analyzed by quantitative
phosphorimaging of SDS-PAGE. Mean values from three independent
measurements, with standard errors.
[0043] FIG. 6B. Pelletable .sup.35S-PrP determined by quantitative
phosphorimaging of SDS-PAGE. At the indicated times, .sup.35S-PrP
reaction products were centrifuged at 15,000.times.g for 30 minutes
at 22.degree. C. After separating the supernatant fraction (S), the
pelletable fraction (P) was resuspended in conversion buffer, and
both fractions were prepared for SDS-PAGE. Mean values from three
independent studies, with standard errors.
[0044] FIG. 6C. Protease-resistant .sup.35S-PrP in pellet (P) and
supernatant (S) fractions quantified from SDS-PAGE phosphorimages
of 24 hour reactions. Average of two independent studies.
[0045] FIG. 7A, FIG. 7B, FIG. 7C and FIG. 7D. Combined effects of
chaperones and partially-denatured PrP.sup.Sc on conversion.
[0046] FIG. 7A. Conversions obtained with partially-denatured
PrP.sup.Sc (4M urea pre-treatment) with buffer alone, or with the
indicated chaperones and control proteins (each at 5 .mu.M). Mean
values from 3-6 independent measurements, with standard errors.
[0047] FIG. 7B. SDS-PAGE phosphorimage of representative conversion
reactions obtained with untreated PrP.sup.Sc (0) or PrP.sup.Sc
partially denatured in the presence of increasing urea
concentrations (1-5 M), with or without chaperone (Hsp104 or GroEL,
3 .mu.M). Only proteinase K-treated (+PK) samples are shown.
[0048] FIG. 7C. SDS-PAGE phosphorimage of representative conversion
reactions obtained with Hsp104 (WT or mutant KT218), with or
without ATP, and untreated or partially-denatured PrP.sup.Sc (4M
urea pre-treatment). Only proteinase K-treated samples (+PK) are
shown.
[0049] FIG. 7D. SDS-PAGE phosphorimage of representative conversion
reactions obtained with partially-denatured PrP.sup.Sc (4M urea
pre-treatment), with or without ATP, and with or without GroEL (WT
or mutant D87K). Both proteinase K-treated (+PK; bottom) and
untreated samples (-PK, one-fifth sample; top) are shown.
[0050] FIG. 8. Conversion of .sup.35S-PrP.sup.C in the presence of
chemical chaperones. SDS-PAGE phosphorimages of representative
conversion reactions obtained with partially-denatured PrP.sup.Sc
(4M urea pre-treatment) in the presence of increasing
concentrations of DMSO, glycerol, sucrose, or trehalose.
4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0051] The invention demonstrates that yeast cells provide a system
in which the folding of amyloidogenic proteins from diverse
organisms is subject to manipulation. The most immediate
application is the use of yeast cells to screen for reagents that
affect amyloid formation, a process that is integral to several
devastating human diseases. Screening for agents that affect these
disease factors is very expensive and time consuming in animal
models and cultured cells. Yeast will provide a rapid first
screening system to quickly and cheaply identify reagents that
affect the folding and aggregation properties of the target
protein. These can then be screened by conventional methods to
determine which are therapeutically applicable.
[0052] The inventor has found that Hsp104 controls the behavior of
a factor that alters a particular physiological property of yeast
cells in a heritable way. This change in physiology was shown to be
associated with a heritable change in the aggregation state of a
particular protein, Sup35, that is controlled by genetic
manipulation of Hsp104. Subsequently, the inventor demonstrated
that Sup35 has a very unusual biochemical property that it shares
with certain human disease proteins. Specifically it forms amyloid
fibers that stain with the dye Congo Red and shows apple green
birefringence. Staining with this dye is a common diagnosis for
human amyloid diseases (Glover et al., 1997; incorporated herein by
reference).
[0053] The present invention is based, in part, on the inventor's
discovery that, in a purified system in vitro, Hsp104 affects the
folding state of the yeast amyloidogenic protein Sup35. Moreover,
it also affects the folding state of a mammalian amyloidogenic
protein, the prion protein known as PrP. The yeast protein was also
shown to interact in a highly specific manner with another
mammalian amyloid protein, .beta.-amyloid peptide 1-42 (Alzheimer's
disease peptide). The inventor has established that the folding
state of the mammalian PrP protein, when expressed in yeast,
depends upon the same type of manipulations that the folding of the
yeast amyloid Sup35 depends upon. This establishes that yeast
provides a surprisingly advantageous and widely applicable system
for testing factors that affect the folding and amyloidogenic
properties of mammalian disease proteins (Schirmer and Lindquist,
1997; DebBurman et al., 1997).
4.1 Methods of Screening and Selecting Amyloid Formation
[0054] The inventor contemplates that the formation of amyloid
fibers may be detected by a number of mechanisms. In some
embodiments, the aggregation may be detected by its ability to bind
Congo Red and show apple green birefringence under polarized light
(Baker et al., 1994; Guiroy et al., 1993; Gasset et al., 1992;
Tashima et al., 1986; Bockman et al., 1985; Bendheim et al., 1984;
Prusiner et al., 1983). However, in other embodiments, the
aggregation is detected indirectly. For example, in embodiments
comprising the Sup35 aggregation domain (N-terminal domain), the
physiologically important C-terminal domain may be sequestered in
the cell by the addition of the endogenous Sup35 protein into the
aggregation, causing a change in phenotype of the cell. Thus,
aggregation may be detected by the presence of the [PSI+] phenotype
in the yeast cells. Depending upon how much of the Sup35 comprising
protein is expressed and aggregated in the yeast, this phenotype is
characterized by an increase in nonsense suppression, lesser
aggregation, or cell death, higher aggregation.
[0055] Chernoff et al. (1995) used a color test for the [PSI+]
phenotype. In this test, a adel-14 strain was used. In this strain,
the adel-14 nonsense mutation is suppressed in the presence of the
[PSI+] phenotype. This leads to white colored colonies. In the
absence of the [PSI+] phenotype, this strain has a red color. This
test provides a screen for the [PSI+] phenotype. Therefore, the
ability of conditions or compositions to affect the [PSI+]
phenotype may be detected by their ability to affect a color change
in this [PSI+]/adel-14 strain.
[0056] In some preferred embodiments, the [PSI+] phenotype kills
the yeast cell. Such cells are particularly useful in screening for
the [PSI+] phenotype. For example, yeast expressing a chimeric
protein comprising the .beta.-amyloid peptide (1-42) and the Sup35
C-terminal domain have a [PSI+] phenotype that leads to cell death.
The inventor contemplates that such cells are an excellent system
for screening candidate compounds for their ability to inhibit
.beta.-amyloid aggregation, because only yeast grown in the
presence of compounds that inhibit or reverse the [PSI+] phenotype
will survive.
[0057] The inventor has shown that chimeric proteins comprising an
aggregate prone domain have prion properties. For example, in a
yeast expressing a chimeric protein comprising the N-terminal
domain of Sup35 and GFP, the GFP was shown to aggregate. This same
result was seen in a yeast strain expressing a chimeric protein
comprising the N-terminal domain of Sup35 and GFP but that lacked
expression of the N-terminal domain of the endogenous Sup35. This
shows the aggregation of the chimeric protein was independent of
the endogenous protein comprising the aggregate prone domain.
Furthermore, chimeric proteins comprising GFP may be particularly
useful in methods of screening agents that prevent aggregation, as
the fluorescence pattern GFP is quickly and easily screened.
[0058] The inventor contemplates that, because chimeric proteins
comprising an aggregate prone domain take on prion-like properties
in yeast, such proteins are useful in developing screens or
selections for the presence of aggregation. When a chimeric protein
comprising an aggregate prone domain, such as the N-terminal domain
of Sup35, and another polypeptide, such as luciferase or the
glucocorticoid receptor, is expressed in yeast under conditions
that lead to aggregation, aggregation of the chimeric protein leads
to changes in the activities of the other polypeptide. Therefore,
in yeast cells comprising the Sup35 aggregate prone domain and
luciferase, the presence of aggregation can be detected by the loss
of luciferase activity in the cells. In other preferred
embodiments, the chimeric protein comprises an aggregate prone
domain and a drug-resistance marker. In such embodiments,
aggregation leads to antibiotic sensitivity.
4.2 Amyloid Diseases
[0059] In an important embodiment, the present invention is a
screen for compounds that are therapeutic for amyloid diseases. The
inventors contemplate that, by using polypeptides comprising the
etiological agent of the amyloid disease, the methods of the
present invention may be used to find therapeutic compounds for
essentially any amyloid disease. A number of amyloid diseases occur
in mammals and are discussed herein.
[0060] A number of neurodegenerative diseases in mammals have been
linked to the aggregation of the product of the PrP gene (prion
protein). Such diseases include scrapie in sheep and goats, mad cow
disease (bovine spongiform encephalopathy), transmissible mink
encephalopathy, chronic wating disease in captive mule deer and
elk, feline spongiform encephalopathy, and prion diseases of other
animals including mice, hamsters, nyala, greater kudu, eland,
gembok, arabian oryx. Of course, prion diseases are also seen in
apes, monkeys, and humans.
[0061] In humans, as in many animals, prion diseases can be
sporadic, inherited, or may be brought on by inoculation with
infectious prion particles. Common names of prion diseases in
humans are kuru, Creutzfeld-Jakob disease (CJD),
Gerstmann-Straussler-Scheinker (GSS), and fatal familial insomnia
(FFI). The classification of human prion diseases is based on
clinical and neuropathological findings (Prusiner, 1996;
incorporated herein by reference).
[0062] Prion diseases resulting from the horizontal transmission of
infectious prions are iatrogenic CJD and kuru. Inherited forms GSS,
fanilial CJD, and FFI have all been associated with oneor more
mutations in the protein coding region of the PrP gene (Bertoni et
al., 1992; Dlouhy et al., 1992; Doh-ura et al., 1989; Gabizon et
al., 1993; Goldfarb et al., 1990; Goldfarb et al., 1991; Goldfarb
et al., 1992; Goldgaber et al., 1989; Hsiao et al., 1989; Kitamoto
et al., 1993a; Kitamoto et al., 1993b; Medori et al., 1992;
Petersen et al., 1992; Poulter et al., 1992). Sporadic forms of
prion disease in humans comprise most cases of CJD and some cases
of GSS (Masters et al., 1978).
[0063] Regardless of the origin of the prion diseases, all have
been associated with abnormal folding of the cellular protein
PrP.sup.c into a protease resistant form, PrP.sup.sc, that
aggregates (Oesch et al., 1985; Bolton et al., 1982; McKinley et
al., 1982; Bolton et al., 1984; Prusiner et al., 1984; Bolton et
al., 1987).
[0064] Another amyloid disease in humans is Alzheimer's disease
(AD). One of the key events in AD is the deposition of amyloid as
insoluble fibrous masses (amyloidogenesis) resulting in
extracellularneuritic plaques and deposits around the walls of
cerebral blood vessels (WO 96/39834; incorporated herein by
reference). The main component of amyloid is a 4.1-4.3 kDa peptide,
called .beta.-amyloid, that is part of a much longer amyloid
precursor protein APP (Muller-Hill and Beyreuther, 1989). Peptides
containing the sequence 1-40 or 1-42 of .beta.-amyloid and shorter
derivatives can form amyloid-like fibrils in the absence of other
protein (Pike et al., 1993).
[0065] The inventors have shown that proteins comprising PrP and
.beta.-amyloid polypeptides are capable of forming aggregates in a
yeast based system. Thus, this system provides a mechanism of
testing compounds for their ability to inhibit the aggregation of
these polypeptides in an inducible, yeast-based system. Such
compounds may be used as amyloid disease therapeutic compounds.
5.0 EXAMPLES
[0066] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
5.1 Example 1
Interactions of the Chaperone Hsp104 with Yeast Sup35 and Mammalian
PrP
[0067] A critical missing link in the "protein-only" hypothesis for
[PSI+] inheritance is any evidence that Hsp104 actually interacts
directly with Sup35. Indeed, little is known about the interaction
of Hsp104 with any substrate, as the heat-denatured aggregates that
constitute its other likely in vivo substrates are inherently
difficult to study. Here the inventor provides evidence for a
highly specific interaction in vitro between Hsp104 and Sup35. This
interaction produces a change in protein structure and inhibits the
ATPase activity of Hsp104. The inventor also reports that Hsp104
interacts in a remarkably similar way with mammalian PrP, the
protein determinant of the neurodegenerative "prion" diseases
(Prusiner, 1996; Caughey and Chesebro, 1997), and with
.beta.-amyloid peptide (Glenner and Wong, 1984).
[0068] 5.1.1 Materials and Methods
[0069] 5.1.1.1 Protein and Peptide Preparation
[0070] Hsp104 (prepared as in 12), Hsp70 (obtained from J. Glover),
aldolase (Pharmacia), and IgM (Rockland) were stored in 20 mM Tris
pH 8.0, 2 mM EDTA, 1.4 mM .beta.-mercaptoethanol, 5% glycerol, 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). Sup35 and the
fragment MN (purified as in 13) were dialyzed against HSB (high
salt buffer, 20 mM HEPES pH 7.5, 10 mM MgCl.sub.2, 140 mM KCl, 15
mM NaCl freshly supplemented with 5 mM mercaptoethanol and 1 mM
AEBSF) to remove imidazole, and then dialyzed against either HSB or
LSB (low salt buffer, 10 mM MES pH 6.5, 10 mM MgSO.sub.4).
Concentrations were determined by the Bradford assay with BSA
(bovine serum albumin) as a standard. Concentrations of PrP
(prepared and folded into either B sheet or a helical forms;
Mehlhorn et al., 1996; Zhang et al., 1997), PrP peptides (as in
16), and .beta.-amyloid (Sigma) were determined spectroscopically
using calculated extinction coefficients.
[0071] 5.1.1.2 ATPase Assays
[0072] PrP peptides (1 mM resuspended in H.sub.2O were assayed in
40 mM Tris pH 7.5, 175 mM NaCl, 5 mM MgCl.sub.2, and 5 mM ATP in a
25 .mu.l reaction volume containing 1 .mu.g of Hsp104. Peptides A,
G, H, and K were resuspended in dimethyl sulfoxide (DMSO) which was
also added to controls containing Hsp104 alone. Effects of other
proteins on Hsp104's ATPase activity were measured in LSB or HSB.
Phosphate released (mean and standard deviation of at least 3
independent reactions) after 8 minutes at 37.degree. C. was
measured with Malachite Green (Lanzetta et al., 1979).
[0073] 5.1.1.3 Spectropolarimetry
[0074] Hsp104, Hsp70, aldolase, IgM or storage buffer were added to
Sup35 or rPrP in the buffers indicated. When aldolase and IgM were
tested as substrates of Hsp104, they were first dialyzed against
HSB and subsequently LSB, so that their treatment matched that of
Sup35. In LSB Sup35 solutions were somewhat cloudy, suggesting some
aggregation, but little or no protein precipitated to the bottom of
cuvettes during analysis. Chaperones and control proteins were
added to Sup35 at a .about.1:2.5 gram-weight ratio (e.g. Sup35 at
.about.0.4 mg/ml and Hsp104 at 0.15 mg/ml). Reactions were
incubated for 1 hr with 1 mM ATP at 37.degree. C., and transferred
to a 0.1 mm path-length cuvette. Spectra were recorded at
25.degree. C. in a Jasco 715 spectropolarimeter (bandwidth 1.0 nm,
response time 16 sec, speed 20 nm/min, step resolution 0.2 nm,
accumulations 4).
[0075] Proteins and rPrP were mixed with each other (each at 0.5
mg/ml) or with the appropriate storage buffer in LSB2: 20 mM
phosphate buffer pH 6.5, 10 mM MgSO.sub.4, 1.25 mM ATP. PrP
peptides were mixed with Hsp104 in 20 mM Tris buffer containing 10
mM MgSO.sub.4, 50 mM KCl, and 1.25 mM ATP at pH 7.5 to approximate
ATPase assay conditions. After 1 hr at 37.degree. C., reactions
were diluted 5-fold with cold water, and spectra were measured at
12.degree. C. as above. (A larger spectral shift was observed with
these conditions for peptide F, presumably because the structural
changes obtained with this peptide are unstable at higher
temperatures. Although temperature had little effect on the spectra
obtained with other peptides or rPrP, for consistency, 12.degree.
C. was used for all).
[0076] 5.1.1.4 Congo Red Dye Binding Assays
[0077] Reaction conditions were as for CD studies with the addition
of Congo red to a final concentration of 10 .mu.M. After 30 min at
25.degree. C., absorbances at 320, 477, and 540 nm were determined.
Congo red dye binding was measured using the equation
[(OD.sub.540/25,295)-(OD.sub.477/46,306)] (Klunk et al., 1989).
[0078] 5.1.2 Results
[0079] 5.1.2.1 Circular Dichroism Of Hsp104 And Sup35 Mixtures
[0080] Attempts to detect an interaction between Sup35 and Hsp104
by co-immunoprecipitation or by affinity chromatography with
immobilized Hsp104 were unsuccessful suggesting that if Hsp104
interacts with Sup35, this interaction is weak, transient, or
depends upon unique conditions, conformations, or cofactors. Since
changes in the expression of Hsp104 lead to changes in the physical
state of Sup35 in vivo, as an alternative mechanism for probing
interactions between these proteins, the inventor discovered that
changes in state could be detected by circular dichroism when
purified Hsp104 and Sup35 were mixed in vitro. If two proteins do
not interact, or if they interact without a substantial change in
secondary structure, the CD spectrum of their mixture should equal
that predicted from the simple addition of their individual
spectra.
[0081] When either Sup35 or Hsp104 was mixed with any of several
control proteins--aldolase, immunoglobulins (IgG and IgM),
.alpha.-2 macroglobulin, apoferritin, and
.alpha.-lactalbumin--observed spectra matched the predicted spectra
(FIG. 1A and FIG. 1B). These control proteins encompass a wide
variety of structural features, including proteins that are largely
.alpha.-helical or .beta.-sheet, monomeric or oligomeric, large or
small. Furthermore, spectral shifts observed when another
chaperone, Hsp70, was mixed with Sup35 were small (FIG. 1A).
[0082] In contrast, when Hsp104 and Sup35 were mixed, the observed
spectrum differed dramatically from the predicted spectrum (FIG.
1C, top right). Thus, these two proteins interacted in a highly
specific manner to produce a change in the physical state of one or
both proteins. ATP is required for some Hsp104 functions (Parcell
et al., 1994; Schirmer et al., 1996), but was not required for the
change in CD spectrum with Sup35 and Hsp104. However, ATP markedly
increased the rate at which this change occurred (Table 1).
TABLE-US-00001 TABLE 1 ATP affects rate of CD change Time, min -ATP
+ATP 0 2.9 2.2 3 7.9 15.2 6 9.9 17.2 10 10.9 18.2 15 11.8 18.8 20
12.4 19.2 30 13.3 19.8 72 16.2 21.9 The difference between the
actual spectrum and the predicted spectrum at 225 nm for each
timepoint is presented in millidegrees. In each of three separate
studies, the spectral change in mixtures of Sup35 and Hsp104
proceeded more rapidly with ATP than without ATP, although the
absolute rates varied, most likely due to differences in the Sup35
preparations.
[0083] The interaction between Hsp104 and Sup35 apparently depended
upon the structural state of Sup35. When Sup35 was dialyzed against
low salt buffer at pH 6.5 (LSB, FIG. 1C, top left, solid line) or a
higher salt buffer at pH 7.5 (HSB, FIG. 1C, bottom left, solid
line) a difference in the CD spectra indicated that the protein was
in a different structural state. When Hsp104 was added, the actual
CD spectrum deviated from the predicted spectrum only when Sup35
had been dialyzed in LSB (FIG. 1C, compare right panels). Mixtures
of Sup35 and several control proteins showed no deviation from
predicted spectra in LSB or HSB. Similarly, control proteins mixed
with Hsp104 showed no spectral shifts in either buffer. Moreover,
the CD spectrum of Hsp104 itself did not change with the buffer
(FIG. 1C, left panels, dashed line).
[0084] 5.1.2.2 Sup35 Aggregation
[0085] In vivo, the inheritance of [PSI+] is associated with the
partitioning of Sup35 into aggregates, a change in state that
requires Hsp104 (Paushkin et al., 1996; Patino et al., 1996;
Chernoff et al., 1995). In vitro, Sup35 forms highly ordered,
amyloid-like fibers after prolonged incubations in the absence of
Hsp104 (Glover et al., 1997). In CD studies the proteins did not
precipitate to the bottom of the cuvette or exhibit significant
binding to the walls of the tube. However, the upward shift in the
spectrum might be due, at least in part, to a partitioning of
protein from solution while it remains in suspension (We and Chen,
1989).
[0086] To determine whether the interaction between Hsp104 and
Sup35 detected by CD analysis in vitro is related to the biological
interaction between the two proteins in vivo, the inventor
investigated their association and changes in protein aggregation.
Solutions containing mixtures of Sup35 and Hsp104 invariably
scattered more light at 320 nm (typically .about.30% more) than the
simple sum of light scattering by each protein alone. An increase
in Congo red dye binding was also detected by the characteristic
spectral shift that occurs when this dye binds amyloid proteins
(Klunk et al., 1989).
[0087] 5.1.2.3 Effects of Sup35 on the ATPase Activity of
Hsp104
[0088] When other members of the HSP100 (clp) family are incubated
with substrates, the rate at which they hydrolyze ATP is increased
(Maurizi et al., 1994; Hwang et al., 1988; Wawrzynow et al., 1995).
Thus, changes in the ATPase activity of Hsp104 provide another
method for detecting an interaction with Sup35. When assayed in
HSB, in which no CD changes were observed, Sup35 weakly stimulated
the ATPase activity of Hsp104 (Table 2). Surprisingly, in LSB, in
which CD changes were observed, Sup35 strongly inhibited the ATPase
activity of Hsp104.
TABLE-US-00002 TABLE 2 Effects of proteins and peptides on the
ATPase activity of Hsp104 HSB LSB Hsp104 alone 1.0 +/- 0.05 1.0 +/-
0.1 Sup35 1.2 +/- 0.1 0.6 +/- 0.1 N-term Sup35 1.2 +/- 0.1 0.7 +/-
0.1 PrP.beta. 1.2 +/- 0.1 0.6 +/- 0.05 .beta.-amyloid 1-42 0.8 +/-
0.05 0.3 +/- 0.05 .beta.-amyloid 1-40 1.1 +/- 0.05 0.5 +/- 0.1
reverse amyloid 40-1 1.1 +/- 0.1 0.8 +/- 0.1 aldolase 1.1 +/- 0.05
1.0 +/- 0.1 BSA 1.0 +/- 0.05 1.0 +/- 0.05 apoferritin 1.0 +/- 0.1
1.0 +/- 0.05 IgM 1.1 +/- 0.05 1.1 +/- 0.05 Hsp104 ATPase activity
was measured in HSB or LSB1 and is presented as the activity of
Hsp104 with protein divided by the activity of Hsp104 in buffer
alone. Within individual studies very little variance was observed;
however, even with the results from three different preparations of
Sup35 averaged here, only ~10% variability was observed.
[0089] Previous studies identified the N-terminal domain of Sup35
as the essential "priori-determining" region (Ter-Avanesyan et al.,
1993). This domain is also responsible for the formation of
self-seeded amyloid fibrils by Sup35 in vitro. (Glover et al.,
1997; King et al., 1997). The ATPase activity of Hsp104 was
inhibited by this domain to an extent similar to that observed with
Sup35 itself (Table 2).
[0090] 5.1.2.4 Effects Of Other Amyloids on the ATPase Activity of
Hsp104
[0091] The expansion of the mammalian prion hypothesis to the yeast
[PSI+] element was initially based upon genetic arguments. PrP and
Sup35 are unrelated in sequence and in biological function
(Wickner, 1994; Lindquist, 1997; Chernoff et al., 1995).
Nonetheless, the capacity for both proteins to form amyloid-like
aggregates (Glover et al., 1997; Prusiner et al., 1983; Gasset et
al., 1992) suggests an underlying biochemical similarity between
them. The inventor investigated whether this similarity would
extend to shared molecular features in the two proteins that allow
recognition by Hsp104.
[0092] The change in state of mammalian PrP associated with TSEs is
characterized by increased .beta.-sheet content and protease
resistance in amino-acid segment 90 to 231 (Prusiner et al., 1982;
Caughey et al. 1991). A recombinant hamster protein corresponding
to this segment, in a 13 sheet-rich conformation, rPrP.beta.
(Mehlhorn et al., 1996; Zhang et al., 1997), produced the same
unexpected effect on the ATPase activity of Hsp104 as did Sup35
(Table 2).
[0093] The inventor also tested another amyloidogenic peptide,
.beta.-amyloid 1-42, a fragment often found in the neural plaques
associated with Alzheimer's disease (Glenner and Wong, 1984).
Again, the ATPase activity of Hsp104 was inhibited (Table 2). Less
inhibition was observed with a less amyloidogenic derivative,
.beta.-amyloid 1-40, still less with a peptide containing the same
amino acids in the reverse order, and no inhibition was observed
with a wide variety of control proteins (Table 2). Thus, the
unexpected inhibitory effects of these three amyloidogenic
polypeptides on Hsp104's ATPase activity are specific and strongly
suggest an underlying biochemical similarity between them.
[0094] 5.1.2.5 Circular Dichroism of Hsp104 and PrP Mixtures
[0095] When Hsp104 was mixed with rPrP.beta. (Mehlhorn et al.,
1996; Zhang et al., 1997), the CD spectrum of the solution differed
dramatically from the spectrum predicted by the addition of
individual spectra (FIG. 2A, right). This result was very
reproducible in both degree and effect, with two different
preparations of rPrP and two of Hsp104. When rPrP.beta. was mixed
with several other chaperones, only GroEL (Hsp60) yielded a
substantial spectral shift (FIG. 2B right). Other chaperones
(Cdc37, Hsp90, Hsp70), as well as some non-chaperone proteins
(apoferritin, .beta.-galactosidase, .alpha.2-macroglobulin, and
.alpha.-lactalbumin) yielded spectral shifts with PrP, but they
were much smaller than those observed with Hsp104 and GroEL.
Finally, when rPrP.beta. was mixed with BSA or aldolase (FIG. 2C
right), predicted and actual spectra were virtually identical.
[0096] As with Sup35, the interaction between Hsp104 and rPrP
depended upon the structural state of rPrP. When rPrP was
pre-incubated under conditions (Mehlhorn et al. 1996; Zhang et al.,
1997) that promote an .alpha.-helical conformation (rPrP.alpha.)
rather than a B sheet-rich conformation (rPrP.beta.), and mixed
with Hsp104, the actual spectrum matched the predicted spectrum
(FIG. 2D, right). The .alpha.-helical and .beta. sheet-rich forms
of rPrP, once acquired, were stable after transfer to the same
buffer. Since they were in the same buffer when mixed with Hsp104,
the different results obtained with rPrP.alpha. and rPrP.beta. can
be attributed to an effect of substrate structure on interaction
with Hsp104, rather than to an effect of buffer.
[0097] 5.1.2.6 Correlation Between Structural Transitions and
ATPase Inhibition with PrP Peptides
[0098] Since for both Sup35 and PrP, the inhibition of Hsp104's
ATPase activity occurred under the same conditions where a spectral
shift occurred, the inventor postulated that these amyloidogenic
proteins might inhibit the ATPase activity of Hsp104 by coupling it
to a major change in structure. To investigate this possibility
further, the inventor took advantage of various PrP peptide
derivatives (FIG. 3A) and the structural transitions of both PrP
and these derivatives (Mehlhorn et al. 1996; Zhang et al., 1997;
Zhang et al., 1995; Gasset et al., 1992; Nguyen et al., 1995).
Several peptides from the amino-terminal region had little or no
effect on the ATPase activity of Hsp104 (FIG. 3B, peptides A and
B); a peptide corresponding to amino acids 90-145 strongly
stimulated ATP hydrolysis by Hsp104 (FIG. 3B, peptide F); several
peptides derived from the carboxy-terminus inhibited it (FIG. 3B,
peptides G to K).
[0099] Peptides with different effects on the ATPase activity of
Hsp104 were then tested for spectral shifts in the presence of
Hsp104. When peptide B was mixed with Hsp104, the CD spectrum was
equivalent to that predicted from the addition of the individual
spectra (FIG. 4A). The actual and predicted spectra of Hsp104 and
peptide F were not identical, but the deviation was small (FIG.
4B). In contrast, the spectra obtained from mixing Hsp104 with the
carboxyl-terminal peptides G and J were very different from the
predicted spectra (FIG. 4C and FIG. 4D). Thus, the PrP peptides
that inhibited the ATPase activity of Hsp104 yielded the strongest
spectral shift.
[0100] 5.1.3 Discussion
[0101] The dependence of [PSI+] on the protein chaperone Hsp104
provides one of the strongest genetic arguments that the
inheritance of a phenotypic trait can be due to the inheritance of
a change in protein conformation, in this case, the conformation of
Sup35 (Paushkin et al., 1996; Patino et al., 1996; Chernoff et all,
1995). The validity of this argument rests on two assumptions, 1)
that Hsp104 and Sup35 interact directly, and 2) that this
interaction influences the physical state of Sup35. Here the
inventor provides evidence in support of both. Remarkably, very
similar results were obtained with PrP, the mammalian protein whose
altered conformation is thought to propagate the transmissible
spongiform encephalopathies (Prusiner, 1996; Caughey and Chesebro,
1997). .beta.-amyloid, the peptide whose deposition in amyloids is
thought to contribute to Alzheimer's disease (Glenner and Wong,
1984) also interacted with Hsp104 in a similar manner. These
findings reveal an underlying biochemical similarity between these
otherwise unrelated proteins.
[0102] Circular dichroism studies provided one line of evidence for
the direct interaction of Hsp104 with Sup35 and with PrP. The
actual spectra observed when Hsp104 is mixed with either of these
proteins is different from the spectra predicted by the simple
addition of their individual spectra. These spectral shifts are
highly specific. When control proteins, encompassing a wide variety
of structural features, are mixed with Hsp104, actual spectra match
the predicted spectra. Further, when Sup35 or rPrP.beta. are mixed
with control proteins (including other chaperones) spectral
deviations are relatively small, or undetectable (except in the
case of rPrP.beta. and GroEL). Finally, the interactions of Hsp104
with Sup35 and PrP themselves appear to depend upon the initial
structural states of Sup35 and PrP.
[0103] Currently, producing different structural states of Sup35
depends upon using different buffers and, although these buffers
did not influence Hsp104's CD spectrum, they might influence
Hsp104's interaction with Sup35. However, in the case of rPrP
distinct conformational states, once established, are stable on
transfer to the same buffer (Mehlhorn et al. 1996; Zhang et al.,
1997). A large spectral shift occurs with rPrP.beta., a .beta.
sheet-rich, multimeric conformation (Zhang et al., 1997) thought to
be associated with TSE diseases, but not with rPrP.alpha., an
.alpha. helix-rich, monomeric conformation thought to mimic the
normal cellular form.
[0104] The ability of both Sup35 and PrP to inhibit the ATPase
activity of Hsp104 provides independent evidence for an interaction
between these proteins. The same specificity was observed as with
CD: 1) control proteins do not inhibit the ATPase activity of
Hsp104, 2) Sup35 inhibits it under the conditions that lead to a
change in CD spectrum, but not under the conditions where no change
in CD spectrum occurred, and 3) rPrP.beta. also inhibited it under
the conditions that lead to a change in the CD spectrum. Studies
with a series of peptides spanning the PrP sequence provide another
link between the Hsp104::substrate interactions that lead to
structural transitions and those that inhibit ATPase activity. The
strongest inhibition in Hsp104's ATPase activity occurred with the
peptides that produced the strongest CD shifts.
[0105] The inhibition of Hsp104's ATPase activity was itself
surprising. Interactions between other HSP100 proteins and their
substrates generally stimulate the chaperone's ATPase activity
(Maurizi et al., 1994; Hwang et al., 1988; Wawrzynow et al., 1995).
At least some of these interactions, however, seem to involve less
dramatic structural transitions (Schirmer et al., 1996). For
example, ClpA (an E. coli relative of Hsp104) converts the RepA
protein from diers to monomers (Wickner et al., 1994). Both ClpA
and Hsp104 are hexameric proteins with multiple ATP binding sites
and, presumably, multiple substrate binding sites. Perhaps the
structural transitions of more complex, amyloidogenic substrates
involve more coupled or "concerted" work from the chaperone and
this inhibits its free-running ATPase activity.
[0106] It is striking that this .beta.-amyloid peptide also
inhibited the ATPase activity of Hsp104. .beta.-amyloid, Sup35, and
PrP differ in size and biological function and have unrelated
sequences (except for weak homology in a few oligopeptide repeats
of Sup35 and PrP). Yet, all share the capacity to assemble into
amyloid-like aggregates (Glenner and Wong, 1984; Glover et al.,
1997; Prusiner et al., 1983). The [PSI+] genetic trait is linked to
the aggregation of Sup35; the pathologies of TSEs and Alzheimer's
disease are generally associated with the aggregation of PrP and
.beta.-amyloid respectively (Prusiner, 1996; Caughey and Chesebro,
1997; Glenner and Wong, 1984). Presumably, it is the shared
capacity for such conformational transitions that leads to
recognition by Hsp104.
5.2 Example 2
Chaperone-Supervised Conversion of Prion Protein to its
Protease-Resistant-Form
[0107] Shown in this example is an assessment of whether or not
molecular chaperones, whose known functions are to alter the
conformational states of proteins (Hartl, 1996; Buchner, 1996;
Parsell and Lindquist, 1993), regulate the conversion of PrP.sup.C
to PrP.sup.Sc '. To test for chaperone involvement, the inventor
used a cell-free assay, wherein metabolically-labeled
.sup.35S-PrP.sup.C, purified from cultured cells in an acid-treated
state, is converted to a conformational state characteristic of
PrP.sup.Sc (Kocisko et al., 1994; Caughey et al., 1995). In this
altered state, PrP is aggregated and a specific portion of the
molecule is highly resistant to proteolysis.
[0108] This simple in vitro conversion reaction faithfully
recapitulates several salient TSE features. First, like
experimental TSEs, in vitro conversion of PrP.sup.C to its
protease-resistant form requires pre-existing PrP.sup.Sc (Kocisko
et al., 1994; Caughey et al., 1995; Bessen et al., 1995; Kocisko et
al., 1995). Secondly, strain-specific PrP.sup.Sc protease digestion
properties, specifically those associated with two mink TSE
strains--hyper and drowsy, were precisely propagated from
PrP.sup.Sc to radiolabeled PrP.sup.C in this assay (Bessen et al.,
1995). Thirdly, the known in vivo bathers to transmitting TSEs
between different species were reflected well in the efficiencies
of in vitro conversion (Kocisko et al., 1995; Raymond et al.,
1997). Lastly, this cell-free assay modeled accurately another in
vivo TSE barrier, based on genetic polymorphisms in PrP, which
render sheep either highly susceptible, moderately susceptible, or
resistant to scrapie (Bossers et al., 1997).
[0109] Together, these studies provide substantial evidence that in
vitro converted, protease-resistant PrP is either authentic
PrP.sup.Sc or has a very similar conformation. The in vitro
converted material is operationally referred to herein as
protease-resistant PrP (PrP-res).
[0110] Here, the inventor provides the first evidence that
molecular chaperones can regulate conformational transitions in
PrP. Two protein chaperones, GroEL and Hsp104, promoted in vitro
conversion, in contrast, the chemical chaperones, sucrose,
trehalose, and DMSO inhibited it. Importantly, the inventor's
results with chaperones demonstrate that in vitro converted PrP-res
is a bonafide conformationally-altered PrP molecule. Chaperones
provide new understanding of the nature of PrP intermediates
involved in PrP conversion, and provide evidence that the
conversion process has two steps. The ability of chaperone-like
molecules to supervise PrP.sup.Sc formation in TSEs in vivo means
that these molecules represent important clinical targets to combat
this dreaded disease.
[0111] 5.2.1 Materials and Methods
[0112] 5.2.1.1 Chaperone Proteins
[0113] Yeast Hsp40 (Ydj1), Hsp70 (ssa1/ssa2), and Hsp104 (WT and
mutant) were purified as previously described (Cyr et al., 1992;
Zeigelhoffer et al., 1995; Parsell et al., 1993) and were obtained
from J. R. Glover and Y. Kimura. Bacterial GroES and GroEL (WT and
mutant) were obtained by A. L. Horwich. Hsp26 was obtained from T.
Suzuki and E. Vierling, and Yeast Hsp90 was obtained by J.
Buchner.
[0114] 5.2.1.2 Chaperone Folding Assays
[0115] Hsp104 promoted the refolding of kinetically-trapped
denatured luciferase, but only when Hsp40, Hsp70 and ATP were also
present (J. R. Glover and S. Lindquist, manuscript in preparation).
The function of other chaperones were assessed using previously
published procedures. GroEL and GroES activities were measured by
the refolding of denatured rhodanese (Mendosa et al., 1991); Hsp90
suppressed the aggregation of .beta.-galactosidase (Freeman and
Morimoto, 1996); Hsp26 activity was measured by the suppression of
aggregation of malate dehydrogenase (Lee et. al., 1995).
[0116] 5.1.1.3 PrP Purification
[0117] PrP.sup.Sc was purified from hamsters infected with 263K
strain of scrapie as previously described (Kocisko et al., 1994).
Hamster .sup.35S-PrP.sup.C and .sup.35S-PrP.sup.-GPI- proteins were
purified from cultured cells by a procedure described in B. Caughey
et al., (1996) (Caughey et al. 1995), except that radiolabeled
proteins were eluted with 0.1M acetic acid at 22.degree. C. for 30
minutes and stored at 4.degree. C. before use. To obtain
non-glycosylated .sup.35S-PrP.sup.C, cultured cells were
pre-incubated and .sup.35S-labeled in the presence of 2 .mu.g/ml
tunicamycin (Boehringer-Mannheim), an inhibitor of glycosylation
(Caughey et al., 1995).
[0118] 5.2.1.4 Cell-Free PrP Conversion
[0119] Unless otherwise stated, all reactions were performed using
the same modification of a published procedure (Caughey et al.,
1995). .sup.35S-PrP.sup.C (20,000 cpm .about.3 ng) denatured in
0.1M acetic acid was diluted into 1.times. conversion buffer (CB:
50 mM sodium citrate-HCl, pH 6.0, supplemented with 1% N-lauryl
sarkosine). PrP.sup.Sc (100 ng) was incubated with
.sup.35S-PrP.sup.C (20 .mu.l volume) at 37.degree. C. for 24 hours.
When indicated, PrP.sup.Sc was pretreated for one hour with either
2M GdnHCl at 37.degree. C. or 4M urea at 22.degree. C.; in
conversion reactions, GdnHCl and urea were present at 0.2M and 0.4M
respectively. In chaperone-mediated conversions, chaperones (1
.mu.M, unless otherwise stated) were added to CB prior to the
addition of .sup.35S-PrP.sup.C and PrP.sup.Sc. Reactions with
chaperones contained 10 mM MgCl.sub.2, 1.5 mM NaCl, and 140 mM KCl,
and unless otherwise stated, 5 mM ATP. All reactions with ATP
included an ATP regenerating system containing 20 mM
phosphocreatine and 10 .mu.g/ml creatine phosphokinase. These
supplements did not affect PrP conversion.
[0120] For each reaction, one-tenth to one-fifth of the sample was
left untreated for determination of percent conversion of
.sup.35S-PrP.sup.C to PrP-res (Caughey et al., 1995). The remainder
was digested with proteinase K (PK; 80 .mu.g/ml) for 1 hour at
37.degree. C., and both PK-untreated and PK-treated samples were
prepared for SDS-PAGE (Caughey et al., 1995). .sup.35S-PrP products
were visualized in dried gels by phosphorimaging and quantified
with ImageQuant software (Molecular Dynamics).
[0121] 5.2.2 Results
[0122] 5.2.2.1 Chaperones Alone do not Convert PrP.sup.C to
PrP-res
[0123] The inventor first examined the ability of major cellular
chaperones GroES (Hsp10), Hsp26, Hsp40, GroEL (Hsp60), Hsp70,
Hsp90, and Hsp104, to promote .sup.35S-PrP.sup.C conversion in the
absence of PrP.sup.Sc. These chaperones were chosen because they
employ different mechanisms to affect the conformation and physical
state of other proteins (Hartl, 1996; Buchner, 1996; Parsell and
Lindquist, 1993). In separate studies, these same chaperone
preparations functioned appropriately in a variety of protein
folding assays. Yet, over a broad range of concentrations, alone
and in various combinations, with (FIG. 5A) or without ATP, none of
these chaperones promoted the conversion of PrP.sup.C to PrP-res,
in the absence of PrP.sup.Sc. This observation strongly underscores
the importance of pre-existing PrP.sup.Sc in the conversion of
PrP.sup.C.
[0124] 5.2.2.2 GroEL Promotes Conversion in Reactions Nucleated
with Untreated PrP.sup.Sc
[0125] Next, the inventor determined whether chaperones influenced
.sup.35S-PrP.sup.C conversion in the presence of PrP.sup.Sc. To
date, efficient in vitro conversion of PrP.sup.C to PrP-res has
usually required partial chemical denaturation of PrP.sup.Sc (left
bars, FIG. 5A; Edenhofer et al., 1996; Freeman and Morimoto, 1996).
Untreated and completely denatured PrP.sup.Sc (6M GdnHCl
pretreatment) have little (FIG. 5D) and no converting ability
respectively (Kocisko et al., 1994; Caughey et al., 1995). The
inventor first asked if chaperones influenced conversion with
PrP-res that was not subjected to partial denaturation. Several
chaperones produced reproducible, but very small increases in
conversion (FIG. 5B and FIG. 5D). One, however, facilitated
conversion at a high level (FIG. 5A and FIG. 5B). With GroEL,
typically 25-30%, and occasionally 50-100%, of .sup.35S-PrP.sup.C
converted.
[0126] Notably GroEL not only reduced by 10-fold the quantity of
PrP.sup.Sc required for detectable conversion, but also increased
by more than 10-fold the maximal levels of conversion attained,
compared to reactions nucleated with the same preparation of
untreated PrP.sup.Sc, but no GroEL (FIG. 5D). These effects of
GroEL were dose-dependent (FIG. 5C).
[0127] 5.2.2.3 GroEL Effects Require ATP, but not GroES
[0128] GroEL-promoted protein folding usually, but not always,
requires the co-chaperone GroES and ATP (Hartl, 1996; Buchner,
1996). PrP conversion was not observed in the absence of ATP (FIG.
5E). Moreover, two point mutants of GroEL, which block release of
substrate (D87K and 337/349; Kocisko et al., 1994) strongly reduced
conversion (FIG. 5E). Surprisingly, however, the stimulating
effects of GroEL on .sup.35S-PrP.sup.C conversion were consistently
eliminated by GroES (FIG. 5D). This inhibition was due to an effect
of GroES on GroEL, rather than on PrP, because GroES did not
inhibit the denaturant-promoted conversion of .sup.35S-PrP.sup.C
that occurs in the absence of GroEL.
[0129] 5.2.2.4 Post-Translational PrP Modifications Modestly Affect
Chaperone-Promoted Conversions
[0130] The inventor used a PrP mutant that lacks the
glycosylphosphatidylinositol (GPI) anchor (PrP.sup.GPI-; Edenhofer
et al., 1996; Freeman and Morimoto, 1996) and accumulates in mono-
and unglycosylated form (FIG. 5F, right), to determine if these
natural modifications affect chaperone-mediated conversion. Again,
of the various chaperones tested, GroEL was the only one that
efficiently stimulated conversion in the presence of untreated
PrP.sup.Sc (FIG. 5F); and, once again, GroEL-promoted effects were
ablated in the absence of ATP and inhibited by GroES (FIG. 5F).
With this form of PrP, however, conversion was more efficient
(typically 30-40%).
[0131] Moreover, conversion was also achieved with a combination of
Hsp104, Hsp70, and Hsp40, albeit less consistently and less
strongly than with GroEL (FIG. 5F). Results similar to those
obtained with PrP.sup.GPI-, were also obtained with unglycosylated
.sup.35S-PrP.sup.C purified from cells cultured with tunicamycin.
Therefore, the ability of the chaperones to mediate the conversion
of .sup.35S-PrP.sup.C to PrP-res was modestly facilitated by the
absence of N-linked sugars or the GPI-anchor.
[0132] 5.2.2.5 Conversion Kinetics Reveal a Two-Step Process
[0133] When .sup.35S-PrP.sup.C converts to PrP-res, it becomes
associated with PrP.sup.Sc, which is a pelletable aggregate
(Caughey et al., 1995; Bessen et al., 1997). To gain insight into
the chaperone-mediated conversion process, the inventor analyzed
the kinetics of conversion, monitoring both protease-resistance and
insolubility. GroEL promoted the acquisition of both of these
signature features of PrP.sup.Sc in .sup.35S-PrP (FIG. 6A and FIG.
6B). In reactions driven with untreated PrP.sup.Sc and GroEL,
protease resistance was acquired at a pace similar to that observed
in reactions nucleated with partially-denatured PrP.sup.Sc, in the
absence of GroEL (FIG. 6A). Moreover, in both sets of reactions,
protease-resistant radioactivity was found only in pelletable
material (FIG. 6C).
[0134] Surprisingly, however, when the rate at which .sup.35S-PrP
became insoluble was examined, the chaperone-driven reaction showed
very different kinetics than those driven by partially-denatured
PrP.sup.Sc. No pelletable radioactivity was detected at two hours
in reactions driven by partially-denatured PrP.sup.Sc (FIG. 6A and
FIG. 6B). In striking contrast, in chaperone-driven reactions, the
conversion of PrP to a pelletable form was virtually complete in
two hours. This occurred long before .sup.35S-PrP converted to its
characteristic protease-resistant form (FIG. 6A and FIG. 6B). This
pelleting of .sup.35S-PrP.sup.C was almost certainly due to an
association with pre-existing PrP.sup.Sc, because in parallel
reactions with GroEL, but without PrP.sup.Sc, most
.sup.35S-PrP.sup.C remained soluble (FIG. 6B).
[0135] 5.2.2.6 In Reactions Nucleated With Partially-Denatured
PrP.sup.Sc, Hsp104 also Promotes Conversion
[0136] Another chaperone was effective in reactions seeded with
partially-denatured PrP.sup.Sc. For these reactions, a milder
denaturant, urea, was used because some chaperones are sensitive to
inhibition by GdnHCl (Todd and Lorimor, 1995). Moreover, the lower
basal rate of conversion obtained with urea (FIG. 7A, buffer)
allowed the inventor to test the ability of other chaperones to
either inhibit or stimulate conversion.
[0137] None inhibited (FIG. 7A). Several stimulated, but only to a
small degree (FIG. 7A). Strikingly, under these conditions, in
addition to GroEL, Hsp104 strongly stimulated conversion (FIG. 7A).
With Hsp104, typically 20-30%, occasionally more than 50% of total
.sup.35S-PrP.sup.C converted. The stimulatory effects of Hsp104
required partial denaturation of PrP.sup.Sc, with pre-treatments in
3-4 M urea being optimal (FIG. 7B).
[0138] 5.2.2.7 Folded State of PrP.sup.Sc Governs Properties of
Chaperone-Promoted Conversion
[0139] While some Hsp104 functions require ATP (Parsell and
Lindquist, 1993; Schirmer et al., 1996), in these reactions
nucleotide was somewhat stimulatory, but was not required (FIG.
7C). Furthermore, two ATPase-deficient Hsp104 mutants (KT218 and
KT620; Pan et al., 1993) promoted .sup.35S-PrP.sup.C conversion
nearly as well as wild-type Hsp104 (FIG. 7C).
[0140] Remarkably, the use of partially-denatured PrP.sup.Sc
changed the character of conversions promoted by GroEL as well.
These conversions lost ATP-dependence (FIG. 7D). Moreover, they
became refractory to GroES inhibition (FIG. 7A). Thus,
chaperone-mediated conversions are mechanistically distinct in
reactions nucleated with partially-denatured PrP.sup.Sc, and those
nucleated by untreated PrP.sup.Sc.
[0141] 5.2.2.8 Chemical Chaperones Inhibit Conversion
[0142] The inventor also tested the effects of several small
organic molecules (or chemical chaperones) known to affect protein
folding: sucrose, glycerol, trehalose, DMSO and the cyclodextrin
compounds (Talzelt et al., 1996; Welch and Brown, 1996; Yancey et
al., 1982). None of the compounds tested affected
.sup.35S-PrP.sup.C conversions in reactions without PrP.sup.Sc, nor
in reactions seeded with untreated PrP.sup.Sc.
[0143] In reactions seeded with partially denatured PrP.sup.Sc,
DMSO had a complex dose-dependent effect, intermediate levels
(1-3%) stimulated conversion 2-3 fold and higher levels (up to 30%)
virtually eliminated conversion (FIG. 8). Glycerol (FIG. 8) and
cyclodextrin compounds (.alpha.-,.beta.-,.gamma.-forms) had no
effect. Sucrose and trehalose inhibited conversion. This inhibition
was observed only at high concentrations, but is physiologically
relevant because these osmolytes are known to accumulate to such
levels in vivo under stressful conditions (Yancey et al.,
1982).
[0144] 5.2.3 Discussion
[0145] Recently, protein chaperones and small organic molecules
have figured prominently among cellular factors speculated to
influence conversion of PrP.sup.C to PrP.sup.Sc (Kenward et al.,
1996; Talzelt et al., 1996; Telling et al., 1995; Edenhofer et al.,
1996). In scrapie-infected cells, some of the same organic
molecules the inventor tested have been shown to reduce the rate of
PrP.sup.Sc formation (Talzelt et al., 1996). The inventor provides
the first evidence that protein chaperones and small organic
molecules can directly affect conformational transitions of PrP.
The inventor's findings also provide the first direct demonstration
that chaperone Hsp104 can alter the conformation state of another
protein.
[0146] In studying the conversion of PrP.sup.C to PrP-res, the
inventor employed previously characterized chaperones from bacteria
and the eukaryotic cytosol because protein chaperones have not yet
been identified in compartments where PrP.sup.C converts to
PrP.sup.Sc. Indeed, the site where conversion occurs is still
unclear. WT PrP.sup.C is thought to convert extracellularly, within
endosomes, or in caveolae (Caughey and Raymond, 1991; Borchelt et
al., 1992; Vey et al., 1996). Mutant PrP, proposed to model
inherited TSEs, can acquire certain PrP.sup.Sc-like properties
spontaneously in the ER/Golgi complex (Daude et al., 1997). Of the
chaperones the inventor tested, only GroEL and Hsp104 affected
conversion. The inventor's results indicate that such chaperone
interactions in vivo are likely to be highly specific. Clearly, the
elucidation of PrP chaperone interactions in vivo are of great
import as they provide potential targets for therapeutic
intervention.
[0147] The inventor's discoveries provide a unifying biochemical
connection between mammalian TSEs (the so called "prion" diseases)
and [PSI+], a novel genetic element in yeast (sometimes called a
"yeast prion"; Wickner, 1994). The proposed "mammalian prion"
determinant PrP.sup.Sc, and the "yeast prion" determinant Sup35 are
functionally unrelated and share no sequence identity. Moreso,
[PSI+] produces a heritable change in metabolism rather than a
lethal infection. However, both mammalian and yeast "prions"
apparently share a common mode of transmission based upon
self-propagating changes in protein conformation (Glover et al.,
1997; Chernoff et al., 1995; Patino et al., 1996).
[0148] Among yeast chaperones, the striking specificity of Hsp104
for PrP conversions, and its known in vivo specificity in
regulating [PSI+] (Wickner, 1994; Glover et al., 1997; Chernoff et
al., 1995; Patino et al., 1996) suggest that conformations of PrP
and Sup35 share an underlying biochemical similarity that allows
for recognition by particular chaperones and prion-like
conformational transitions. The present invention evidences the
specific interactions of Hsp104 with PrP and Sup35 proteins using
circular dichroism and ATP hydrolysis measurements.
[0149] The standard for testing the disease properties of PrP is to
determine its protease resistance pattern. The protease resistant
form is associated with disease. The inventor has shown that PrP
when produced in yeast is protease resistant and that this protease
resistant state depends upon the Hsp104 protein. Thus the
protease-resistant character of PrP, which is the hallmark of its
disease potential, is affected by manipulation of yeast cells. The
inventor also shows that these same manipulations of yeast cells
alter the folding state and protease resistance of a yeast amyloid
protein. Together these results establish that manipulating yeast
cells provides a general mechanism for studying factors that
control the folding of amyloidogenic proteins. Amyloid proteins are
important causative factors in many human diseases. Thus, the
presently disclosed, inventive use of yeast cells provides an
excellent system for testing and manipulating the folding state of
amyloid proteins, which is useful in identifying therapeutic
agents.
[0150] All of the methods and apparatus disclosed herein can be
made and executed without undue experimentation in light of the
present disclosure. While the compositions and methods of this
invention have been described in terms of preferred embodiments, it
will be apparent to those of skill in the art that variations may
be applied to the methods and apparatus and in the steps or in the
sequence of steps of the method described herein without departing
from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents which are
both chemically and physiologically related may be substituted for
the agents described herein while the same or similar results would
be achieved. All such similar substitutes and modifications
apparent to those skilled in the art are deemed to be within the
spirit, scope and concept of the invention.
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