Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-11-12
2002-09-17
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C424S130100, C424S139100, C424S185100, C424S192100, C435S070100, C435S070200, C435S071100, C436S501000, C436S503000, C436S518000, C436S528000, C436S547000, C530S350000, C530S387100
Reexamination Certificate
active
06451541
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.
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
Sc
(prion protein, ‘Sc’ for scrapie), a posttranslationally modified form of the proteinase K sensitive host encoded PrP
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 &bgr;-sheet content for PrP
Sc
as compared to a high &agr;-helix content in PrP
c
(17, 31, 38). Structural predictions of PrP
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 &agr;-helices (24). It has been suggested that prion propagation involves the conversion of &agr;-helical domains in PrP
c
into &bgr;-sheets in PrP
Sc
(26, 30, 31). The in vitro conversion of PrP
c
into PrP
Sc
was demonstrated employing a proteinase K resistance assay (28). A modified model was recently suggested according to which PrP
c
must be partially unfolded and refolded into PrP
Sc
under the direction of an oligomeric PrP
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).
So far, there is no method described allowing the straightforward detection or isolation of natural prion proteins. The isolation of PrP
c
described in the prior art (31) is time consuming, ineffective and yields only minimum amounts of protein. The isolation of PrP
sc
described in the prior art (31, 35, 64) is also time consuming and ineffective and the purity of the PrP
sc
is speculative. Furthermore, up to now it was not possible to discriminate between the cellular isoform PrP
c
and the isoform PrP
sc
or PrP27-30, which is a prerequisite for the development of a simple and reliable assay for diagnosing a transmissible spongiform encephalopathy.
Tatzeh et al., PNAS USA 92 (1995), 2944-2984, investigate proteins that might feature in the conversion of the cellular prion protein (PrP
c
) into the scrapie isoform (PrP
sc
). It was found that in scrapie-infected N2a cells the expression and subcellular translocation of specific heat shock proteins was altered. However, said document does not disclose that chaperones like the heat shock proteins specifically bind to prion proteins and, thus, can be used to detect or isolate prion proteins.
Therefore, the technical problem underlying the present invention is to provide a simple method for the efficient isolation of prion proteins and the detecion of said proteins, preferably in a way that allows for discrimination between different isoforms of PrP.
The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
Thus, the present invention relates to 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.
In addition, the present invention relates to 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.
When carrying out experiments in order to identify proteins capable of interacting with PrP
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
c
was performed using recombinant prion GST-fusion peptides. The results show that a GST-PrP
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). Specificity of the PrP
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
c
and recombinant full-length Hsp60 as well as GroEL. This result shows that the PrP
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.
Identification of the interaction site between amino acid 180 and amino acid 210 by mapping of PrP
c
peptides showed that binding of Hsp60 to PrP
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 &agr;-helical domains H3 (amino acids 178 to 191) and H4 (amino acids 202 to 218) (24). The conversion of &agr;-helical regions into &bgr;-sheets of PrP are thought to be responsible for PrP
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
Sc
as has been shown for the interaction of the yeast prion-like factor [psi
+
] 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
c
to PrP
Sc
amyloids e.g. by trapping misfolded forms of PrP
c
.
More recent data suggested th
Edenhofer Frank
Rieger Roman
Weiss Stefan
Winnacker Ernst-Ludwig
Roylance Abrams Berdo & Goodman L.L.P.
Swartz Rodney P
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