WO2001019858A2 - A family of ubiquitin-like proteins binds the atpase domain of hsp70-like stch - Google Patents

Abstract

The present invention relates to the discovery of a domain on chaperone proteins that binds to ubiquitin-like proteins, including a novel protein Chap1/DSK2 (Ubiquilin-2). Novel biological tools, prophylactics, therapeutics, diagnostics, and methods of use of the foregoing are also disclosed.

Description

A FAMILY OF UBIQUITY-LIKE PROTEINS BINDS THE ATPASE DOMAIN OF HSP70-LIKE STCH

FIELD OF THE INVENTION The present invention relates to the discovery of a domain on chaperone proteins that binds to ubiquitin-like proteins, including a novel protein Chap1/DSK2 (Ubiquilin-2). Novel biological tools, prophylactics, therapeutics, diagnostics, and methods of use of the foregoing are also disclosed.

BACKGROUND OF THE INVENTION The Hsp70-iike gene family encodes a group of related protein chaperones that are required for the viability of all living organisms. (Hartl, Nature, 381:571 -80 (1996)). The structure of all Hsp70 proteins is similar and consists of a highly conserved amino-terminal ATPase domain that can be proteolytically cleaved from the full-length product as an enzymatically active 45 kDa fragment. (Flaherty et al.. Nature, 346:623-8 (1990)). The less well-conserved 25 kDa carboxy-terminal domain performs the common function of reversibly capturing nascent or denatured cellular polypeptides with exposed hydrophobic residues to initiate a wide range of protein processing events. (Hartl, Nature, 381 :571-80 (1996); Pelham, Cell, 46:959-61 (1986); and Flynn et al., Nature, 353:726-30 (1991)). The complexity of the activity of Hsp70 members has become increasingly apparent with the recognition that these proteins interact with other co-chaperones, including Hsp40, Hsp90, and Stil/Hop, to form a functional unit. (Chang et al., Mol Cell Biol, 17:318-25 (1997)). in addition, a group of non-chaperone "Hsp70 interacting proteins" has been identified, which are required for regulating protein folding and/or ATPase activity. For example a tetratricopeptide repeat (TPR) protein, designated Hip, binds and stabilizes the ADP bound state of Hsc70. (Hohfeld et al., Cell, 83:589-98 (1995)).

Similarly, Bag-1 /Rap46, HspBPI , auxillin, and Chip, have recently been identified as additional Hsp70-associated products that are proposed to regulate chaperone function under selected cellular conditions. (Takayama et al.,Embo J, 16:4887-96 (1997); Raynes and Guerriero, J Biol Chem, 273:32883-8 (1998); Ballinger et al., Mol Cell Biol, 19:4535-45 (1999); and Jiang et al., J Biol Chem, 272:6141-5 (1997)). In contrast to the bi-functional structure of Hsp70 family members, a novel gene product was isolated, designated Stch, which encodes the "core ATPase" domain of Hsp70 but lacks the peptide binding domain. (Otterson et al., Embo J, 13:1216-25 (1994)). The truncated structure of Stch is conserved in C. elegans, rat, and human tissues where the Stch product was observed to resemble a proteolytically cleaved N-terminal ATPase fragment of Hsc70/Hsp70. (Otterson and Kaye, Gene, 199:287-92 (1997)). Consistent with these observations, the human Stch protein was shown to exhibit basal ATPase activity that was independent of peptide stimulation. At present, the understanding of how chaperones, like Stch, modulate cellular events such as protein folding and unfolding, protein transport and assembly into macromolecular complexes, protein degardation, apoptosis, and mitotic spindle activity is in its infancy. BRIEF SUMMARY OF THE INVENTION The present invention concerns the discovery of the Chap1/Dsk2 gene (also referred to as Chapl or Ubiquilin-2) and the Chap1/Dsk2 protein (also referred to as Chapl or Ubiquilin-2), a polypeptide that binds to a specific domain of Stch, a member of the family of chaperone proteins. The Chapl /Dsk2 gene was cloned and sequenced in its entirety and the cDNA sequence (GenBank acession No. AF189009) is provided in FIGURE 5 (SEQ. ID. No. 1). The protein encoded by Chap1/Dsk2 (i.e., Chap1/Dsk2) is provided in FIGURE 1 (SEQ. ID. No. 2). Chap1/Dsk2 and Chap1/Dsk2 were discovered during two-hybrid screening experiments designed to determine the role of the Stch product in regulating protein processing. Accordingly, in these experiments, multiple overlapping human cDNA clones that encode distinct, ubiquitin- related proteins that bound efficiently to a conserved 20 amino acid region within the Stch ATPase domain were isolated. Analysis of the Chap1/Dsk2 gene showed that it is a homologue of the S. cerevisiae DSK2 gene, which, together with RAD23, are important for the proper organization of the yeast mitotic spindle and transit through mitosis. (Biggins et al., J Cell Biol, 133:1331-46 (1996)). In contrast, another ubiquitin-related protein identified in the screen, Chap2, represents the human Bat3 protein (Banerji et al., Proc Natl Acad Sci U S A, 87:2374-8 (1990)) and is a homologue of the Xenopus Scythe protein, which is essential for reaper-induced apoptosis. (Thress et al., Embo J, 17:6135-43 (1998)). The discovery that ubiquitin-linked proteins bind to a conserved peptide motif within the 'core ATPase' of chaperones like Stch provides evidence that Hsp70 family members modulate specialized cellular events including, but not limited to, degradation pathaways involving the proteasome, apoptosis, and mitosis, by forming a chaperone/ubiquitin-linked protein complex.

Several aspects of the invention concern the modulation of the formation of a chaperone/ubiquitin-linked protein complex. For example, embodiments include a biological complex comprising Chapl or Chap2 and one or more chaperone proteins. These complexes can also have other proteins associated including, but not limited to, cytoskeletal proteins, specifically proteins found in the brain that undergo rapid degradation or otherwise signal apoptosis. Methods of modulating the assembly of such complexes are also embodiments.

One method of the invention involves administering a chaperone protein or fragment of a chaperone protein or a nucleic acid encoding these molecules. Desirably, modulation of the assembly of a chaperone/ubiquitin-linked protein complex is accomplished by administering a fragment of a chaperone that is involved in binding to a ubiquitin- linked protein (e.g., regions in the ATPase domain of the chaperone). A preferred approach for modulating the assembly of a biological complex comprising a chaperone and Ubiquilin-2, for example, involves administering a fragment of Stch, the Ubiquilin-2 protein, fragments of Ubiquilin-2, and fragments of other chaperones involved in binding to Ubiquilin-2. Additionally, the modulation of a biological complex comprising a ubiquitin-linked protein and a chaperone can be accomplished by providing, nucleic acids that encode the polypeptides above.

Other embodiments include cells that have the nucleic acids encoding ubiquitin-linked proteins (e.g., Chap 1 and Chap2 or fragments thereof), cells that express ubiquitin-linked proteins, antibodies that recognize these polypetides, and software and hardware that have nucleotide or polypeptide information or protein modeling information corresponding to these sequences, as well as, data from Chapl characterization assays and diagnostic profiles. Additionally, nucleic acids that complement nucleic acids encoding Chapl or fragments of Chapl and cells that have these sequences are embodiments of the invention. Another aspect of the invention includes the use of therapeutic or prophylactic agents (e.g., Chapl, fragments of Chapl, fragments of Stch involved in binding to a chaperone, or nucleic acids encoding these compositions) to modulate Chapl -mediated adhesion to a chaperone. Further, methods of discovering such agents including approaches in rational drug design and combinatorial chemistry are also embodiments.

Still more embodiments include biotechnological tools, diagnostic assays, diagnostic kits, and methods of use of the foregoing. For example, multimeric and multimerized Chapl, fragments of Chapl, fragments of Stch, and nucleic acids encoding these sequences or complementary sequences are used as biotechnological tools or diagnostic reagents. Diagnostic assays preferably measure the concentration or expression level of Chapl or nucleic acid encoding Chapl in tested subjects and compare these values to those obtained from healthy individuals or individuals that are suffering from a disease associated with an abnormal expression of Chapl (e.g. cancer). These values can be recorded (Chapl disease-state profiles). These Chapl disease-state profiles can be recorded on software and hardware and can be used to analyze disease-state profiles of tested subjects so as to identify the presence or prevalence of a human disease associated with an abnormal expression or level of Chapl. Desirably, measurements of the concentration or expression level of Chapl or nucleic acids encoding Chapl are made from blood. These disease- state profiles are invaluable tools for the prognosis, diagnosis, and treatment of Chapl -related diseases. Pharmaceuticals having Chapl or fragments of Chapl or nucleic acids encoding these polypeptides or agents that interact with Chapl are also embodiments of the invention. Additionally, pharmaceuticals having Stch or fragments of Stch or other chaperone proteins or fragments of chaperone proteins are embodiments. Desirably, fragments of other chapperone proteins comprise regions of the ATPase domain that bind to Chap 1. Further, methods of treatment and prevention of a Chapl -related disease, specifically cancers, auto-immune disorders, or neurodegenerative diseases such as Alzheimers disease and others, using these pharmaceuticals are provided. The pharmaceuticals of the invention can also include carriers and other agents that promote delivery of the active ingredients. Methods of treatment and prevention of Chapl -related disease, involve identifying a subject in need of an agent that modulates the association of Chapl with a chaperone and administering to said subject a therapeutically effective dose of an agent that modulates adhesion of Chapl to a chaperone.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 (A) shows the amino acid sequence of the human Chapl /Dsk2 (Ubiquilin-2) protein (see also SEQ. ID. No. 2). The conserved ubiquitin domain (from residues 33 to 101) is over-lined and is preceded by a 21 -residue leader (potentially, mitochondrial import) peptide; the conserved UBA domain is over-lined by a broken line; the domain resembling the C-terminal region of S. cerevisiae Stil and rat Hip is indicated by an open rectangle. (B) illustrates the alignment between the yeast DSK2 gene (yDsk2) and Chap1/Dsk2, showing duplication of the human homologue. The ubiquitin domain is depicted as a black rectangle, the UBA domain (aa 326-369 in yDsk2; aa 578-620 in Chapl /Dsk2) is identified, and the minimal sequence for Stch binding (aa 319-520) is shown as a stippled rectangle.

FIGURE 2 illustrates the observation that the human Chap1/Dsk2 gene suppresses the G2/M arrest phenotype of dsk2 rad23 . Strain MY5156 (dsk2 rad23 ) was transformed with the indicated plasmids. Cultures were grown in SC-Ura galactose medium until early logarithmic phase at 30°C and were shifted to 37°C for 10 hours. Cells were fixed and DAPI stained cells were counted. The numbers shown represent the percentages of the various phenotypes ( > 100 cells were counted). Cell types shown are (left to right): unbudded G1 phase cells, small-budded S/G2 phase cells, large-budded G2/M phase cells, and large-budded post-mitotic cells.

FIGURE 3shows the results of Chap-1 characterization assays performed with various fragments of Stch. Chapl /Dsk2 binds to a conserved peptide at the carboxy-terminal end of the ATPase domain. (A) provides a diagram of Stch peptide clones analyzed. The notation "+ + " indicates strongly positive B-galactosidase activity and abundant growth on SC-His media, whereas the notation "-" indicates an absence of Ξ-galactosidase activity and no growth on SC-His plates after 4 day incubation. (B) provides a partial amino acid sequence alignment showing human Stch (hStch), human BiP/Grp78 (hBip), and the S. cerevisiae Hsp70 product SSA4. The terminal, conserved region of the Hsp70-like ATPase domain is boxed with the corresponding Stch clones over-lined.

FIGURE 4shows a group of proteins containing ubiquitin-like domains that bind to the ATPase domain of Hsp70-like proteins. The N-terminal ubiquitin-like domain is indicated by a black rectangle. The ubiquitin associated domain (Uba) is indicated by a hatched rectangle. The region resembling the C-terminal domain of Stil/Hip is indicated by a stippled box. Only one isoform of Bag1/Rap46 is shown.

FIGURE 5shows the nucleotide sequence encoding Chapl (See also SEQ. ID. No 1).

DETAILED DESCRIPTION OF THE INVENTION The Stch gene encodes an Hsp70-like molecule that retains ATPase activity, but lacks a carboxyl-terminal peptide-biπding domain. We identified a short region within Stch that is conserved in the ATPase domain of all Hsp70 members and that binds a family of ubiquitin-like genes, including a novel protein called Chapl (also referred to as

Ubiquilin-2). Desirable fragments of Stch that bind to a ubiquitin-like gene include the polypeptide sequence of clone 7 and preferable fragments of Stch include the boxed region shown in FIGURE 3. More preferable fragments, however, include the approximately 20 amino acid residues of clone 7 that are not found in clone 8.

During our analysis, of Chap 1 and Chapl , we discovered that regions of this protein are homologous to others found in the art. For example, it was found that the human Chapl gene is a duplicated homologue of the yeast Dsk2 gene that participates in the organization of the spindle pole body during mitosis and is required, with RAD23, for proper transit through the G2/M phase of the cell cycle. Further, we discovered that Chapl shares homology with an identified partial sequence referred to as HRIHFB2157. (Ueki et al., Nature Biotech 16:1338-1342 (1998)).

We have demonstrated that the human Chap1 /Dsk2 cDNA efficiently restores viability and suppresses the G2/M arrest phenotype of dsk2 rad23 S. cerevisiae mutants. In contrast, another ubiquitin-like protein identified by our Stch screen was the human Chap2 gene. This gene is a homologue for Bat3/Scythe, which is an essential component of reaper-induced apoptosis in xeπopus egg extracts. Both Chap1/Dsk2 and Bat3/Scythe bind to the same 20-amino acid peptide within Stch and both encode N-terminal ubiquitin-like domains, which are not required for Stch binding. In addition, Chapl /Dsk2 contains a C-terminal ubiquitin-associated (Uba) domain and two distinct repeats of Stil-like sequences that are also conserved in the chaperone-binding proteins, Hip and pδO/Stil/Hop. These findings, together with the observation that the ubiquitin-like, anti-apoptosis Bag1/Rap46 protein binds to the ATPase domain of 70 kDa heat shock proteins, provide evidence that members of the Hsp70-like ATPase family can regulate cell cycle, cell death, and proteasome-mediated protein degradation.

Additionally, we mapped the location of the Ubiquilin-2 gene to chromosome X, between NIB1132(12.44 cR) and WI-9877 (9.8 cR), which places the markers within the human chromosome bands Xp11.23 and Xp1 1.1.

Alterations in protein processing and degradation have been previously associated with human neurodegenerative disorders and defects in Ubiquilin-2 may be linked to the wide range of πeuropsychiatric disorders that have been localized to Xp11. (Reyπiers et al., Am J. Human Genetics 65:1406 (1999).

Embodiments of the invention include software and hardware comprising nucleic acid sequences encoding Chapl or fragments of Chapl (Chapl ) or complements of these sequences and protein sequences corresponding to

Chapl and fragments of Chapl . Preferred software and hardware have nucleic acid sequences that encode fragments of Chapl that bind to a chaperone protein. Additionally, the software and hardware of the invention include embodiments that provide disease-state profiles that have information such as concentrations and expression levels of Chapl (e.g., mRNA) or Chapl detected in biological samples from healthy subjects, as well as, subjects suffering from a Chapl -related disease. The software and hardware embodiments of the invention are also used to further characterize Chapl (e.g., to develop protein models of Chapl, to identify homologous proteins, and to identify agents that interact with Chapl) and to provide diagnostic and prognostic information that allows for the determination of the disease state of a tested individual.

Nucleic acids encoding full-length Chapl or nucleic acids encoding fragments of Chapl are embodiments of the invention. Preferred nucleic acid embodiments include nucleic acids encoding fragments of Chapl that bind to a chaperone. Further, nucleic acids encoding regions of Stch involved in binding to a chaperone protein are within the scope of the invention. Additionally, the nucleic acid embodiments of the invention include nucleic acids or derivatives thereof that are complementary to full-length Chapl or fragments of Chapl (e.g., antisense oligonucleotides and ribozymes). Preferred complementary nucleic acids of the invention include nucleic acids or derivatives thereof that are complementary to fragments of Chapl that have a nucleotide sequence that encodes a polypeptide that binds to a chaperone. The nucleic acid embodiments can be manufactured as monomeric, multimeric, and multimerized agents. The nucleic acid embodiments also include vectors, plasmids, and recombinant constructs having nucleic acids encoding full-length Chapl, fragments of Chapl, and fragments of Stch that bind to a chaperone protein. Additional embodiments are vectors, plasmids, and recombinant constructs having nucleic acids complementary to the full-length Chapl or fragments of Chapl. Cells having the nucleic acid embodiments of the present invention, including cells in animals having a nucleic acid embodiment created by genetic engineering (e.g., cells in a transgenic animal or an oocyte), are within the scope of aspects of the invention.

Protein-based embodiments include full-length Chapl, fragments of Chapl, and fragments of Stch that bind to a chaperone protein. Preferred protein-based embodiments include fragments of Chapl that have an amino acid sequence that encode a polypeptide that binds to a chaperone. Further, the protein-based embodiments include protein derivatives or modifications of Chapl or fragments of Chapl including, but not limited to peptidomimetics. The protein-based embodiments can be manufactured as monomeric, multimeric, and multimerized agents. Cells having the protein-based embodiments of the present invention, including cells in animals having a protein-based manufacture of the present invention (e.g., cells in a transgenic animal or an oocyte), are within the scope of aspects of the invention. In some embodiments, the polypeptides of the invention are used to generate antibodies. Preferred embodiments include polyclonal and monoclonal antibodies that recognize epitopes corresponding to regions of Chapl that are involved in binding to a chaperone. These antibodies have application in biological assays, therapeutics, and can be used to diagnose human disease by identifying the presence of Chapl in a biological sample.

Several types of assays that provide information about Chapl or the formation of the Chapl -chaperone complex are embodiments of the invention. These assays are collectively referred to as "Chapl characterization assays". One type of Chapl characterization assay concerns measuring the ability of Chapl or fragments thereof to bind to a chaperone or fragments of a chaperone protein. For example, methods of performing Chapl characterization assays are provided, in which a chaperone or Chapl are disposed on a support and are subsequently contacted with a ligand (e.g., Chapl or a chaperone or a fragment thereof, depending on the support-bound molecule) and Chapl- mediated adhesion is determined. A similar binding assay can be employed in the presence of an inhibiting or enhancing molecule (a "modulator") such as a peptide or peptidomimetic (collectively referred to as a "peptide agent") or a chemical. The supports in these assays can be conventional resins, plastics, lipids, and membranes.

In some aspects, the modulation of Chapl -mediated adhesion is accomplished by using a modulator that is a nucleic acid embodiment. For example, a construct encoding Chapl is transfected into cells so as to raise the concentration of Chapl and thereby promote Chapl -mediated adhesion to a chaperone or, alternatively, a construct encoding a nucleic acid that is complementary to a nucleic acid encoding Chapl (e.g., an antisense inhibitor or a ribozyme) is used to reduce the concentration of Chapl and thereby inhibit Chapl -mediated adhesion to a chaperone. Further, in some embodiments, nucleic acids encoding wild-type or mutant Chapl or fragments of Chapl or complements thereof are transfected and expressed in cells so as to modulate Chapl -mediated adhesion or to induce an immune response or both. Still further, in other embodiments nucleic acids encoding fragments of Stch that bind to a chaperone protein can be transfected so as to modulate the assembly of a Chapl /ubiquitin-like protein complex.

According to other aspects, the modulation of Chapl -mediated adhesion to a chaperone is achieved by using a modulator that is a protein-based embodiment. For example, Chapl is delivered to cells by liposome mediated transfer so as to raise the intracellular concentration of Chapl and thereby promote Chapl -mediated adhesion to a chaperone or, alternatively, wild-type or mutant Chapl , fragments of Chapl, and/or fragments of Stch that bind to a chaperone protein are delivered to cells by liposome- ediated transfer so as to inhibit Chapl -mediated adhesion to a chaperone. Peptidomimetics that resemble Chapl or fragments thereof or fragments of Stch that bind to a chaperone protein are also modulators of the invention and can be used to effect Chapl mediated adhesion or to induce an immune response or both. Many chemicals can also be modulators and can be identified by their ability to effect

Chapl mediated adhesion using the Chapl characterization assays and teachings herein.

Approaches in rational drug design can be employed, for example, to identify novel agents that interact with Chapl so as to modulate Chapl -mediated adhesion to a chaperone or that can be used to induce an immune response in a patient. In these embodiments, protein models of Chapl, fragments of Chapl , and agents that interact with Chapl or fragments of Chapl are constructed and approaches in combinatorial chemistry are used to develop agents that modulate Chapl -mediated adhesion to a chaperone or induce an immune response. Accordingly, novel agents that interact with Chapl are developed, screened in a Chapl characterization assay (e.g., a Chapl adhesion assay), and the identity of each agent and its performance in a Chapl characterization assay, its effect on the modulation Chapl- mediated adhesion to a chaperone or its ability to induce an immune response is recorded on software or hardware. The recorded data can be used to create a library of Chapl modulating agents. These libraries can be employed to identify more agents that modulate Chapl -mediated adhesion to a chaperone and are valuable clinical tools for manufacturing and selecting an appropriate pharmaceutical to treat a particular type of chaperone-related disease.

The nucleic acid and protein-based embodiments of the invention can also be used as biotechnological tools and probes in diagnostic assays. In some aspects, for example, the nucleic acid embodiments are employed as nucleic acid probes in hybridization assays, cloning, or as primers for Polγmerase Chain Reaction (PCR). Similarly, the protein- based embodiments can be used, for example, to characterize Chapl, identify related proteins, and study Chapl - mediated adhesion to a chaperone.

In some diagnostic embodiments, nucleic acids complementary to full-length Chapl or fragments of Chapl are used to identify Chapl nucleic acids (e.g., mRNA) present in a biological sample. Depending on the type of disease, the composition of Chapl or a nucleic acid encoding Chapl may differ. That is, it is contemplated that polymorphic variants of Chapl or nucleic acids encoding Chapl exist. One approach to identify these polymorphisms involves identifying a first population of subjects that suffers from a Chapl -related disease and a second population of subjects that do not suffer from a Chapl -related disease; obtaining a Chapl nucleic acid or protein sample from the subjects in both populations and comparing the nucleic acid and/or protein samples from the subjects in the first population with the subjects in the second population. The existence of a polymorphism can be verified by detecting a difference in the nucleic acid and/or proteins from the two populations of samples. Haplotype linkage analysis can be performed according to techniques standard in the art to draw an association between the presence of a particular disease (e.g., Alzheimer's disease or Spirocerebellar ataxia) and polymorphic variants of Chapl.

Depending on the extent of disease, the concentration or expression level of nucleic acid encoding mutant or wild type Chapl in a biological sample can also differ. That is, individuals at different stages of a Chapl -related disease can be accurately diagnosed using the methods and compositions of the invention. Additionally, healthy individuals will not express Chapl and disease-state profiles of healthy individuals can be used to provide a baseline for diagnostic determinations. For example, a Chapl -disease state profile comprising a concentration range of a nucleic acid encoding Chapl in a biological sample can be created for healthy and diseased individuals and these Chapl disease state profiles can be compared to the concentrations or expression levels of a nucleic acid encoding

Chapl detected in a tested individual so as to predict or follow the disease state of that individual. Thus, in some embodiments, the term "Chapl -disease state profile" refers to the concentration or expression level or concentration range or expression level range of a nucleic acid encoding Chapl that is detected in a biological sample. Desirably, addressable arrays comprising nucleic acid probes complementary to the full-length Chapl or fragments of Chapl are used to create such Chapl -disease state profiles. Such arrays or individual probes are also components of diagnostic kits.

In similar fashion to that discussed above, a Chapl -disease state profile comprising concentration ranges or levels of Chapl in healthy and diseased individuals can be created and can be used to predict or follow the disease state of an individual, in some embodiments, the term "Chapl -disease state profile" refers to the concentration or expression level or concentration range or expression level range of a protein corresponding to Chapl that is detected in a biological sample. Thus, by comparing a Chapl -disease state profile from healthy individuals and subjects infected with a chapl -related disease (e.g., cancer or an autoimmmune disease), a clinician can rapidly diagnose whether the tested subject has a Chapl -related disease or the effectivity of a treatment protocol designed to restore normal levels of Chapl . Desirably, addressable arrays comprising antibodies that recognize epitopes of Chapl are used to create such Chapl -disease state profiles. Such arrays or antibodies are also components of diagnostic kits.

In the therapeutic and prophylactic embodiments of the invention, Chapl, polypeptide fragments of Chapl, fragments of Stch that bind to a chaperone protein, nucleic acids encoding these molecules, and agents that interact with a Chapl -chaperone complex are incorporated into pharmaceuticals. These pharmaceuticals can be delivered by any conventional route including, but not limited to, transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar. In addition to the active ingredients mentioned above, the pharmaceutical embodiments can comprise carriers, proteins, supports, adjuvants, or components that facilitate or enhance drug delivery. These pharmaceuticals can be employed in therapeutic protocols for the treatment and prevention of a Chapl -related disease. By one approach, first a subject at risk for contracting a Chapl-related disease or a subject already afflicted with a chapl- related disease is identified by conventional techniques or the diagnostic assays described herein and then is administered an effective amount of an agent that inhibits or promotes Chapl -mediated adhesion to a chaperone protein. The discovery of the Chapl gene and Chapl protein and its characterization as a molecule that mediates adhesion to a chaperone is disclosed below.

Identification and Isolation of the Gene Encoding Chapl and Chapl Protein We obtained several HF7c His + transformants after screening a human lung yeast two-hybrid cDNA library using a pGBTΘ-Stch bait plasmid. These clones represented distinct human chaperoπe-associated proteins (Chap) that shared the property of containing a ubiquitin-domain preceded, in the case of Chapl, by an N-terminal peptide resembling a mitochondrial import signal. Three overlapping clones were isolated for the Chapl gene, one of which represented a full-length cDNA spanning 3,360 nucleotides and encoding an open reading frame of 624 residues (FIGURE 1). A Blastp search (Altschul et al., J Mol Biol, 215:403-10 (1990)) revealed that this gene represented the human homologue for the yeast DSK2 gene. (Biggins et al., J Cell Biol, 133:1331-46 (1996)). inspection of the amino acid alignment between the human and yeast homologues shows that the human gene encodes a 21 -residue leader sequence followed by a conserved 70-amino acid, ubiquitin-like domain at the amino-terminal end. Chapl /Dsk2, however, is approximately twice the size of the yeast homologue, apparently resulting from a duplication of the yeast sequence distal of the ubiquitin-like domain. In addition, both the yeast and human genes contain a ubiquitin- associated (Uba) domain at their C-termini. (Hofmann and Bucher, Trends Biochem Sci, 21 :172-3 (1996)).

Neither the N-terminal ubiquitin-like domain nor the C-terminal Uba domain of Chap1/Dsk2 were required for binding to Stch and the smallest cDNA clone isolated in our screen spanned 200 residues of one monomer DSK2 unit suggesting that the duplication of the yeast sequence was also not essential for Stch binding. Interestingly, this minimal binding domain contained a region with limited sequence similarity to the conserved C-terminal domain of chaperone-binding proteins p60/Sti1 and Hip (Chapl residues: 190-237 and 360 to 399 align with the S. cerevisiae Stil residues: 149-191 and 540 to 579) that in Stil l is implicated in Hsp90-bindiπg. (Hohfeld et al., Cell, 83:589-98 (1995); and Lassie et al., J Biol Chem, 272:1876-84 (1997)). In addition, the minimal binding domain of Chap1/Dsk2 spanned a 65-residue glycine/proiine collagen-like repeat region that resembles, but is distinct, from the GGMP repeat observed in the Hip product.

To examine the functional activity of Chap1/Dsk2, a temperature-sensitive dsk2 rad23 S. cerevisiae strain (MY5156) was tested for cell growth following transformation with either the full-length yeast DSK2 gene (yDsk2), a dominant yeast DSK2 mutation in the ubiquitin domain (yDSK2-1), the human Chap1 /Dsk2 cDNA (hDsk2), or the vector alone. All of the proteins were expressed under the control of the GAL1 galactose-inducible promoter. While single mutants for either dsk2 or rad23 do not exhibit cell growth defects at 37° C, double mutants at the restrictive temperature are arrested at G2/M with defects in duplication of the mitotic spindle pole body. We observed that high levels of yDSK2 and yDSK2-1 expression were toxic in wild type S. cerevisiae cells.

However, when expressed under its own promoter, yDSK2 could reverse the block at G2/M observed in dsk2 rad23 cells (FIGURE 2). Following galactose induction, the human Chap1/Dsk2 gene efficiently suppressed the growth arrest of the dsk2 rad23 cells, and, in contrast to yeast DSK2, high level expression of the human homologue was not toxic to wild type cells. In addition, we analyzed the cell cycle distribution of the dsk2 rad23 transf ormants following 10-hour incubation at the restrictive temperature. We observed a reduction in the frequency of 'aberrant' large-budded cells at G2/M in cells transformed with the human Chapl /Dsk2 cDNA clone, confirming the ability of the human homologue to complement the dsk2 rad23 defect (FIGURE 2). These results provide evidence that the sequence conservation between the yeast and human proteins extends to functional conservation.

To test the specificity of the binding interaction with the ATPase domain of Stch, we used the two-hybrid technique to map the binding site for Chapl /Dsk2 on the Stch protein (FIGURE 3). Using both β-galactosidase activity and growth on SC-His plates, we observed that a series of carboxy-terminal Stch truncations showed complete loss of protein binding. Since Stch contains a unique carboxy-terminal sequence that is conserved in all Stch homologues, but not shared with other members of the Hsp70 family, we designed two peptide sequences that would either include the carboxy-terminal 30 amino acid residues that are conserved in all Hsp70 members (clone 7) or would only include unique Stch sequences (clone 8). We observed that clone 7 bound Chap1/Dsk2 with the same efficiency as full-length Stch, while the smaller peptide containing exclusively Stch specific, carboxy-terminal sequences had no binding activity. Therefore, Stch binds Chapl /Dsk2 via a region that is highly conserved in Hsp70 proteins. The interaction of Chap1/Dsk2 with the ATPase domain of Stch resembles the properties of the Bag1/Rap46 protein, which was recently shown to bind to the ATPase domain of 70 kDa heat shock proteins. (Takayama et al., Embo J, 16:4887-96 (1997); and Zeiner et al., Embo J, 16:5483-90 (1997)). Bag1/Rap46 also contains a ubiquitin- like domain that is related to the N-terminal sequence of Chap1/Dsk2, and it has been shown to bind to a varied group of cellular proteins, including the anti-apoptosis factor Bcl-2. (Takayama et al.,Cell, 80:279-84 (1995)). Further, we have isolated multiple cDNA clones for a third ubiquitin-linked gene, designated Chap2, that bound efficiently to pGBT9-Stch in the two-hybrid screen. Chap2 represented the human homologue for the Bat3/Scythe gene (Banerji et al., Proc Natl Acad Sci U S A, 87:2374-8 (1990); Thress et al., Embo J, 17:6135-43 (1998)) and exhibits several similarities with Chap1/Dsk2 including the presence of an N-terminal ubiquitin domain that was again not required for Stch binding and showed an identical pattern of binding exclusively to the short conserved motif localized on Stch ATPase peptide 7 (FIGURE 3).

To confirm the Scythe-Stch interaction, recombinant GST-Stch protein linked to glutathione sepharose was incubated in Xenopus egg extracts. After pelleting and washing the GST-Stch resin, the bead-bound material was examined by SDS-PAGE and immunoblotting with anti-Scythe sera. When we used this method to compare binding of endogenous Xenopus Scythe to either GST-Stch, GST alone, or the originally reported ligand of Scythe, GST-reaper, we found that both rat and human Stch precipitated Scythe with an efficiency similar to the pro-apoptosis Drosophila reaper product, while the GST leader peptide had no Scythe-binding activity. These observations demonstrate that the ATPase domain of Hsp70-like members binds to a growing family of ubiquitin-linked proteins. Thus, we believe that Rad23 and Bag1 /Rap46, also ubiquitin-like proteins, serve as adaptors to link substrates with the proteasomal machinery or to generate specific heteromeric complexes. The best-characterized ubiquitin-related protein is Rad23p, which controls UV sensitivity (Sugasawa et al., Mol Cell Biol, 16:4852-61 (1996); and Watkins et al., Mol Cell Biol, 13:7757-65 (1993)), regulates Rad4p activity, and directly interacts with the 26S proteasome through its amino-terminal ubiquitin domain. (Schauber et al., Nature, 391:715-8 (1998)). Since Rad23p binds to the Rad4p DNA repair protein through its carboxy-terminal region (Wang et al., Mol Cell Biol, 17:635-43 (1997)), this protein represents a direct link between DNA repair and the ubiquitin/proteasome pathways. (Schauber et al., Nature, 391:715-8 (1998)). Similar links between the 26S proteasome and protein chaperone activity can be made since both pathways attack protein substrates with exposed hydrophobic residues (Laπey and Hochstrasser, Cell, 97:427-30 (1999)), proteasome mediated degradation is associated with Hsp70-like activity (Bercovich et al., J Biol Chem, 272:9002-10 (1997); and Fisher et al., J Biol Chem, 272:20427-34 (1997)), and proteasomal components and Hsp70 chaperones are enriched together in purified centrosomes isolated from human cells. (Wigley et al., J Cell Biol, 145:481-90 (1999)).

In light of our findings herein, we believe that other protein members containing an N-terminal ubiquitin domain and/or a C-terminal UBA domain, such as Dsk2p, also link substrates with the proteasomal degradation pathways, analogous to Rad23p. In addition to the presence of an amino-terminal, ubiquitin-like sequence, Dsk2p and Rad23p share several other features in common, including a functional redundancy for the ability to complement cell growth in S. cerevisiae cells containing both dsk2 and rad23 mutations and the presence of a conserved carboxy- terminal Uba domain that can participate in the regulation of substrate specificity (FIGURE 4). (Hofmaπn and Bucher, Trends Biochem Sci, 21 :172-3 (1996); and Dieckmann et al., Nat Struct Bioli, 5:1042-7 (1998). The data disclosed herein provide evidence that a common function can extend to other ubiquitin-linked genes as well. We have identified, for example, three different ubiquitin-linked proteins, Chap1/Dsk2, Bat3/Scythe, and Bag1/Rap46, that modulate the cell cycle and/or apoptotic machinery and that retain the capacity to bind to the ATPase domain of Hsp70-like molecules (FIGURE 4).

Additionally, we mapped the location of gene on the human X chromosome. The Ubiquilin-2 cDNA sequence (alias Chap1/Dsk2; GenBank accession no. AF189009) was used to design primer pairs from the 3' untranslated region. Forward primer: nucleotide no. 2244; 5' TGA TGC ATT TTA AGA TGG AGT CCC 3' (SEQ. ID. NO. 3) and reverse primer: nucleotide no. 2512; 5' CCA ACC TGT GAA GGT TGA TACCTG 3' (SEQ. ID. NO. 4) amplified the expected 268-base pair fragment from human genomic DNA at an annealing temperature of 58°C.

The GeneBridge4 radiation hybrid screening panel (Research Genetics, Huntsville, AL) was then used for mapping and the results were analyzed using the software from the Whitehead Institute at the MIT Center for Genome Research. The hybrid data vector: 00011 00100 00010 00110 00100 00111 00101 01011 0101 1 01100 00110

10001 01 110 00000 011 11 00001 00011 01100 000 was entered in the WhiteHead institute/MIT Radiation HybridMapper at http://carbon.wi.mit. edu:8000/cgi-bin/contig/rhmapper.pl. The results placed Ubiquilin2 on human chromosome X closest to NIB 1 132 with a lod score > 3 and between the markers NTB1132 (12.44cR) and WI-9877 (9.8 cR). The Genome DataBase Mapview (http://www.gdb.org/gdb-bin/genera/generaSF/hgd/Amplimer?! action = query form) localizes these two markers within the human chromosome bands Xp11.23 and Xpl 1.1, respectively. This chromosomal region has been linked to a wide range of human neuropsychiatric disorders that may involve abnormalities in protein processing and degradation. Spirocerebeliar ataxia and Alzheimer's disease, for example, may involve aberrant protein processing and/or degradation events mediated by disfunctional Chapl. Our findings establish a broader role for the Hsp70-like family in regulating brain-specific cellular events, cell cycle events in general, and, analogous to Rad23p, provide a direct link between protein chaperone activity, cell cycle regulation, and the 26S proteosome.

The disclosure below provides several software and hardware embodiments of the invention, as well as, computational methods that can be used to further characterize the Chapl nucleic acid sequence and the Chapl polypeptide sequence, as well as, identify agents that inhibit Chapl -mediated adhesion to a chaperone.

Software and Hardware Embodiments

The Chapl nucleic acid sequence and the Chapl protein sequence was entered onto a computer readable medium for recording and manipulation. It will be appreciated by those skilled in the art that a computer readable medium having the Chapl nucleic acid sequence or the Chapl protein sequence or both is useful for the determination of homologous sequences, structural and functional domains, and the construction of protein models for rational drug design. The functionality of a computer readable medium having the Chapl nucleic acid sequence or the Chapl protein sequence or both includes the ability to compare the sequence to others stored on databases, to ascertain structural and functional information, to develop protein models, and to conduct rational drug design.

The Chapl nucleic acid sequence or the Chapl protein sequence or both can be stored, recorded, and manipulated on any medium that can be read and accessed by a computer. As used herein, the words "recorded" and

"stored" refer to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide or polypeptide sequence information of this embodiment.

A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide or polypeptide sequence. The choice of the data storage structure will generally be based on the component chosen to access the stored information. Computer readable media include magnetically readable media, optically readable media, or electronically readable media. For example, the computer readable media can be a hard disc, a floppy disc, a magnetic tape, zip disk, CD-ROM, DVD-ROM, RAM, or ROM as well as other types of other media known to those skilled in the art. The computer readable media on which the sequence information is stored can be in a personal computer, a network, a server or other computer systems known to those skilled in the art.

Embodiments include systems, particularly computer-based systems that contain the sequence information described herein. The term "a computer-based system" refers to the hardware, software, and any database used to analyze the Chapl nucleic acid sequence or the Chapl protein sequence or both, or fragments of these biomolecules. The computer-based system preferably includes the storage media described above, and a processor for accessing and manipulating the sequence data. The hardware of the computer-based systems of this embodiment comprise a central processing unit (CPU) and a data database. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable.

In one particular embodiment, the computer system includes a processor connected to a bus that is connected to a main memory (preferably implemented as RAM) and a variety of secondary storage devices, such as a hard drive and removable medium storage device. The removable medium storage device may represent, for example, a floppy disk drive, a DVD drive, an optical disk drive, a compact disk drive, a magnetic tape drive, etc. A removable storage medium, such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded therein (e.g., the Chapl nucleic acid sequence or the Chapl protein sequence or both or fragments thereof) can be inserted into the removable storage device. The computer system includes appropriate software for reading the control logic and/or the data from the removable medium storage device once inserted in the removable medium storage device.

The Chapl nucleic acid sequence or the Chapl protein sequence or both can be stored in a well known manner in the main memory, any of the secondary storage devices, and/or a removable storage medium. Software for accessing and processing the Chapl nucleic acid sequence or the Chapl protein sequence or both (such as search tools, compare tools, and modeling tools etc.) reside in main memory during execution. As used herein, "a database" refers to memory that can store nucleotide or polypeptide sequence information, protein model information, information on other peptides, chemicals, peptidomimetics, and other agents that interact with proteins, and values or results from Chapl characterization assays. Additionally, a "database" refers to a memory access component that can access manufactures having recorded thereon nucleotide or polypeptide sequence information, protein model information, information on other peptides, chemicals, peptidomimetics, and other agents that interact with proteins, and values or results from Chapl characterization assays. In other embodiments, a database stores a Chapl disease-state profile comprising concentrations or expression levels or concentration ranges or expression level ranges of Chapl or Chapl or both detected in biological samples from different subjects (e.g., subjects with and without a disease related to Chapl). In more embodiments, a database stores a Chapl disease-state profile comprising concentration ranges or levels of Chapl detected in biological samples obtained from various tissue or fluid sources from diseased and healthy subjects. Many databases are known to those of skill in the art and several will be discussed below.

The sequence data on Chapl or Chapl or both can be stored and manipulated in a variety of data processor programs in a variety of formats. For example, the sequence data can be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT, an ASCII file, a html file, or a pdf file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE. A "search program" refers to one or more programs that are implemented on the computer-based system to compare a nucleotide or polypeptide sequence with other nucleotide or polypeptide sequences and agents including but not limited to peptides, peptidomimetics, and chemicals stored within a database. A search program also refers to one or more programs that compare one or more protein models to several protein models that exist in a database and one or more protein models to several peptides, peptidomimetics, and chemicals that exist in a database. A search program is used, for example, to compare regions of the Chapl nucleic acid sequence or the Chapl protein sequence or both that match sequences in nucleic acid and protein data bases so as to identify homologies and structural or functional motifs. Further, a search program is used to compare an unknown nucleic acid or protein sequence with the Chapl nucleic acid sequence or the Chapl protein sequence so as to identify homologies and related structural or functional domains. Additionally, a search program is used to compare a Chapl -disease state profile from a tested subject to Chapl -disease state profiles from diseased and healthy subjects present in a database. Still further, a search program is used to compare values or results from Chapl characterization assays.

A "retrieval program" refers to one or more programs that are implemented on the computer based system to identify a homologous nucleic acid sequence, a homologous protein sequence, or a homologous protein model. A retrieval program is also used to identify peptides, peptidomimetics, and chemicals that interact with a nucleic acid sequence, a protein sequence, or a protein model stored in a database. Further a retrieval program is used to identify a disease state of an individual by obtaining a Chapl disease-state profile from the database that matches the Chapl -disease state profile from the tested subject. Additionally, a retrieval program is used to obtain "a Chapl -agent profile" that can be composed of a nucleic acid or polypeptide sequence or model thereof or one or more symbols that represent these sequences and/or models, an identifier that represents one or more Chapl modulating agents, and a value or result from a Chapl characterization assay.

The discussion below describes embodiments of the invention having nucleic acids that encode Chapl.

Use of Nucleic Acids Encoding Chapl or Fragments of Chapl

The cDNA sequence encoding Chapl is provided in the sequence listing (SEQ. ID NO.: 1 ). Full-length Chapl and fragments of Chapl are embodiments of the invention. Further, embodiments include nucleic acids that complement full-length Chapl and nucleic acids that complement fragments of Chapl . Desired embodiments include nucleic acids having at least 9 consecutive bases of Chapl or a sequence complementary thereto, wherein the nucleic acid encodes a polypeptide that binds to a chaperone or wherein the nucleic acid complements a nucleic acid that encodes a polypeptide that binds to a chaperone. In this regard, the nucleic acid embodiments of the invention can have from 9 to 3,359 consecutive nucleotides in length that encode a fragment of Chapl or full-legth Chapl or a complementary nucleic acid, whose complement encodes a fragment of Chapl or full-length Chapl . However, one of skill in the art will appreciate that Chapl nucleic acids can be joined to an exogenous nucleic acid so as to create a nucleic acid embodiment having virtually any length. Thus, a nucleic acid having a portion (9 to 3,359 consecutive nucleotides) or full-length Chapl are embodiments of the invention. That is, a nucleic acid having less than or equal to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44,

45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,

74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125,

150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, and 3,359 nucleotides are embodied. Preferably, the nucleic acid embodiments, however, comprise at least 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleotides from Chapl or a nucleic acid that complements Chapl , as conditions dictate. More preferably, the nucleic acid embodiments comprise at least 20-30 consecutive nucleotides from Chapl or a nucleic acid that complements Chapl . In some cases, the nucleic acid embodiments comprise more than 30 nucleotides from the nucleic acids encoding Chapl or a nucleic acid that complements Chapl and in other cases, the nucleic acid embodiments comprise at least 40, at least 50, at least 75, at least 100, at least 150, or at least 200 consecutive nucleotides from the nucleic acids encoding Chapl or a nucleic acid that complements Chapl. These nucleic acid oligomers have biotechnological and diagnostic use, e.g., in nucleotide acid hybridization assays, Southern and Northern Blot analysis, etc. and the prognosis of Chapl -related diseases.

Some embodiments comprise recombinant nucleic acids having all or part of the Chapl gene or recombinant nucleic acids that complement all or part of Chapl. Desirable embodiments comprise full-length Chapl and fragments of Chapl that encode a polypeptide that binds to a chaperone and nucleic acids that complement full-length Chapl and fragments of Chapl that encode a polypeptide that binds to a chaperone. A recombinant construct can be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct can become integrated into the chromosomal DNA of the host cell. Such a recombinant polynucleotide comprises a polynucleotide of genomic or cDNA, of semi-synthetic or synthetic origin by virtue of human manipulation. Therefore, recombinant nucleic acids comprising sequences otherwise not naturally occurring are provided by embodiments of this invention. Although nucleic acids encoding Chapl or nucleic acids having sequences that complement Chapl as they appear in nature can be employed, they will often be altered, e.g., by deletion, substitution, or insertion and will be accompanied by sequence not present in humans.

The nucleic acid embodiments of this invention can be altered by mutation such as substitutions, additions, or deletions that provide for sequences encoding functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences that encode substantially the same Chapl amino acid sequence as depicted in SEQ. ID NO.: 2 can be used in some embodiments of the present invention. These include, but are not limited to, nucleic acid sequences comprising all or portions of Chapl or nucleic acids that complement all or part of Chapl that have been altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.

In addition, recombinant Chapl-encoding nucleic acid sequences and their complementary sequences can be engineered so as to modify processing or expression of Chapl . For example, and not by way of limitation, the Chapl gene can be combined with a promoter sequence and/or ribosome binding site, or a signal sequence can be inserted upstream of Chapl-encoding sequences to permit secretion of Chapl and thereby facilitate harvesting or bioavailability. Additionally, a given Chapl nucleic acid can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction sites or destroy preexisting ones, or to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis. (Hutchinson et al., J Biol Chem, 253:6551 (1978)). Further, nucleic acids encoding other proteins or domains of other proteins can be joined to nucleic acids encoding Chapl so as to create a fusion protein. The resulting fusion proteins are used as biotechnological tools or pharmaceuticals or both, as will be discussed below.

The nucleic acid embodiments can also be used as biotechnological tools for isolation procedures and diagnostic assays. By using the Chapl nucleic acid sequence disclosed in the sequence listing (SEQ ID NO.: 1), probes that complement Chapl can be designed and manufactured by oligonucleotide synthesis. These probes can be used to screen cDNA or genomic libraries so as to isolate natural sources of the nucleic acid embodiments of the present invention. Additionally, these probes can be used to isolate other nucleotide sequences capable of hybridizing to them. Further, sequences from nucleic acids complementing Chapl, or portions thereof can be used to make oligonucleotide primers by conventional oligonucleotide synthesis for use in isolation and diagnostic procedures. These oligonucleotide primers can be used, for example, to isolate the nucleic acid embodiments of this invention by amplifying the sequences resident in genomic DNA or other natural sources by using the Polymerase Chain Reaction (PCR) or other nucleic acid amplification techniques. Further, the nucleic acid embodiments of the invention can be used to modulate Chapl -mediated adhesion to a chaperone (e.g., by upregulating or dowπregulating the expression of ChapD and, therefore, have several uses in addition to biotechnological research including therapeutic and prophylactic applications, as will be discussed below. Alternatively, the nucleic acids encoding Chapl or fragments thereof are manipulated using conventional techniques in molecular biology to create recombinant constructs that express Chapl or fragments of Chapl .

The discussion that follows describes some of the expression constructs and protein embodiments of the invention.

Chapl Polypeptides and Fragments of Chapl

The Chapl polypeptides or derivatives thereof, include but are not limited to, those containing as a primary amino acid sequence all of the amino acid sequence substantially as depicted in the sequence listing (SEQ. ID NO.: 2) and fragments of SEQ. ID. NO.: 2 at least three amino acids in length that comprise amino acid sequence found in a polypeptide that binds to a chaperone, including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. Accordingly, one or more amino acid residues within the Chapl polypeptide of SEQ ID. NO.: 2 and fragments of SEQ. ID. NO.: 2 that comprise an amino acid sequence found in a peptide that binds a chaperone can be substituted by another amino acid of a similar polarity that acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence can be selected from other members of the class to which the amino acid belongs. For example, the non-polar

(hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. The aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

The Chapl fragments can be less than or equal to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 250, 300,

350, 400, 500, 600, or 623 amino acids in length. One preferred embodiment, for example, comprises a polypeptide fragment having the sequence encompassing the Stch binding domain (amino acids 319 - 520). These polypeptide fragments can be differentially modified during or after translation, e.g., by phosphorylation, glycosylation, cross- linking, acylation, proteolytic cleavage, linkage to an antibody molecule, membrane molecule, or other ligand. (Ferguson et al., Ann. Rev. Biochem. 57:285-320 (1988)).

Other embodiments include polypeptides that have homology to Chapl and bind to a chaperone. By "homology to Chapl" is meant either protein sequence homology or three-dimensional homology. As will be discussed below, several techniques exist to determine protein sequence homology and/or three-dimensional homology. These methods are routinely employed to discover related sequences and novel ligands, as well as, determine the extent of homology that one sequence, domain, or model has to a target sequence, domain, or model. Because the region of

Chapl (e.g., a region encompassing the Stch binding domain) that mediates a chaperone adhesion can be quite small embodiments of the invention can exhibit a vast degree of homology to full-length Chapl. For example, a fusion protein having a small region of Chapl can exhibit a low degree of overall homology to Chapl yet retain the ability to bind a chaperone. Thus, embodiments of the invention can have from 1 % homology to 100% homology to full-length Chapl. That is, embodiments can have 1.0%, 2.0%, 3.0%, 4.0%,. 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%,

12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21.0%, 22.0%, 23.0%, 24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%, 30.0%, 31.0%, 32.0%, 33.0%, 34.0%, 35.0%, 36.0%, 37.0%, 38.0%, 39.0%, 40.0%, 41.0%, 42.0%, 43.0%, 44.0%, 45.0%, 46.0%, 47.0%, 48.0%, 49.0%, 50.0%, 51.0%, 52.0%, 53.0%, 54.0%,. 55.0%, 56.0%, 57.0%, 58.0%, 59.0%, 60.0%, 61.0%, 62.0%, 63.0%, 64.0%, 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%, 79.0%, 80.0%, 81.0%,

82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%, 94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, and 100.0% homology to Chapl . Therefore, embodiments of the invention include polypeptides varying in size from 3 amino acids up to and including the full-length Chapl protein that have 1 % - 100% homology to Chapl and exhibit the ability to bind to a chaperone. In several embodiments, the Chapl polypeptide of SEQ ID. NO.: 2 and fragments of SEQ. ID. NO.: 2 that comprise an amino acid sequence found in a peptide that binds a chaperone are expressed in a cell line. The term "isolated" requires that the material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or protein present in a living cell is not isolated, but the same nucleic acid or protein, separated from some or all of the coexisting materials in the natural system, is isolated. In accordance with this definition, Chapl nucleic acid or Chapl protein or nucleic acid or polypeptide fragments present in a cell lysate are "isolated". The term "purified" does not require absolute purity; rather it is intended as a relative definition. For example, recombinant nucleic acids and proteins are routinely purified to electrophoretic homogeneity, as detected by ethidum bromide staining or Coomassie staining, and are suitable in several assays despite having the presence of contaminants. To express the proteins encoded by Chapl or portions thereof, nucleic acids containing the coding sequence for

Chapl or fragments of Chapl are obtained and cloned into a suitable expression vector such that the coding region is operably linked to a heterologous promoter. The nucleic acid encoding the protein or polypeptide to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology. The expression vector can be in any of the mammalian, yeast, amphibian, insect, parasite, or bacterial expression systems known in the art. Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute

(Cambridge, MA), Stratagene (La Jolla, California), Promega (Madison, Wisconsin), and Invitrogen (San Diego, California). If desired, to enhance expression and facilitate proper protein folding, the codon context and codon pairing of the sequence can be optimized for the particular expression organism in which the expression vector is introduced, as explained by Hatfield, et al., U.S. Patent No. 5,082,767. Further, a secretory leader sequence can be incorporated so as to facilitate purification of the protein.

The following is provided as one exemplary method to express the proteins encoded by the nucleic acids described above. First, the methionine initiation codon for the gene and the poly A signal of the gene are identified. If the nucleic acid encoding the polypeptide to be expressed lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques. Similarly, if the nucleic acid lacks a poly A signal, this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using Bgll and Sail restriction endonuclease enzymes and incorporating it into the mammalian expression vector pXTI (Stratagene). The vector pXT1 contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection. The vector includes the Herpes Simplex Thymidine Kinase promoter and the selectable πeomycin gene. The nucleic acid encoding the polypeptide to be expressed can be obtained by PCR from the bacterial vector using oligonucleotide primers complementary to the nucleic acid and containing restriction endonuclease sequences for Pst I incorporated into the 5 primer and Bglll at the 5 end of the corresponding cDNA 3 primer, taking care to ensure that the nucleic acid is positioned in frame with the poly A signal. The purified fragment obtained from the resulting PCR reaction is digested with Pstl, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXT1, now containing a poly A signal and digested with Bglll. The ligated product is transfected into a suitable cell line, e.g., mouse NIH 3T3 cells, using Lipofectin (Life Technologies, Inc., Grand Island, New York) under conditions outlined in the product specification. Positive transfectants are selected after growing the transfected cells in 600ug/ml G418 (Sigma, St. Louis, Missouri). Preferably the expressed protein is released into the culture medium, thereby facilitating purification.

Another embodiment utilizes the "Xpress system for expression and purification" (Invitrogen, San Diego, CA). The Xpress system is designed for high-level production and purification of recombinant proteins from bacterial, mammalian, and insect cells. The Xpress vectors produce recombinant proteins fused to a short N-terminal leader peptide that has a high affinity for divalent cations. Using a nickel-chelating resin (Invitrogen), the recombinant protein can be purified in one step and the leader can be subsequently removed by cleavage with enterokinase.

One preferred vector for the expression of Chapl and fragments of Chapl is the pBlueBacHis2 Xpress. The pBlueBacHis2 Xpress vector is a Baculovirus expression vector containing a multiple cloning site, an ampicillin resistance gene, and a lac z gene. By one approach, the Chapl nucleic acid, or portion thereof is cloned into the pBlueBacHis2 Xpress vector and SF9 cells are infected. The expression protein is then isolated or purified according to the maufacturer's instructions. Several other cultured cell lines having recombinant constructs or vectors comprising Chapl or portions thereof are embodiments of the present invention and their manufacture would be routine given the present disclosure. Proteins in the culture medium can also be separated by gel electrophoresis. The separated proteins are then detected using techniques such as Coomassie or silver staining or by using antibodies against the protein. Coomassie, silver staining, and immuπolabeling of proteins are techniques familiar to those skilled in the art. If desired, the proteins can also be ammonium sulfate precipitated or separated based on size or charge prior to electrophoresis.

The protein encoded by Chapl or portion thereof can also be purified using standard immunochromatography techniques. In such procedures, a solution containing the protein, such as the culture medium or a cell extract, is applied to a column having antibodies against the protein attached to the chromatography matrix. The protein is allowed to bind the immunochromatography column. Thereafter, the column is washed to remove non-specifically bound proteins. The specifically bound protein is then released from the column and recovered using standard techniques.

Further, Chapl or portion therof can be incorporated into expression vectors designed for use in purification schemes employing chimeric polypeptides. In such strategies, the coding sequence of Chapl or portion therof is inserted in frame with the gene encoding the other half of the chimera. The other half of the chimera may be -globin or a nickel binding polypeptide encoding sequence. A chromatography matrix having antibody to -globin or nickel attached thereto is then used to purify the chimeric protein. Protease cleavage sites can be engineered between the -globin gene or the nickel binding polypeptide and the Chapl cDNA such as enterokinase. Thus, the two polypeptides of the chimera can be separated from one another by protease digestion.

One useful expression vector for generating -globin chimerics is pSG5 (Stratagene), which encodes rabbit - globin. Intron II of the rabbit -globin gene facilitates splicing of the expressed transcript, and the polyadenylation signal incorporated into the construct increases the level of expression. These techniques as described are well known to those skilled in the art of molecular biology. Standard methods are published in methods texts such as Davis et al., (Basic Methods in Molecular Biology, LG. Davis, M.D. Dibner, and J.F. Battey, ed., Elsevier Press, NY, 1986) and many of the methods are available from Stratagene, Life Technologies, Inc., or Promega. Polypeptide may additionally be produced from the construct using in vitro translation systems, such as the In vitro Express™ Translation Kit (Stratagene).

In addition to isolating or purifying Chapl and fragments of Chapl by using recombinant DNA techniques, these molecules can be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using methods known in the art such as those set forth by Merrifield et al. (J Am Chem Soc, 85:2149 (1964), Houghten et al. (Proc Natl Acad Sci USA, 82:51:32 (1985), and Stewart and Young (solid phase peptide synthesis, Pierce Chem Co., Rockford, IL (1984)). Such polypeptides can be synthesized with or without a methionine on the amino terminus. Chemically synthesized Chapl and fragments of Chapl can be oxydized using methods set forth in these references to form disulfide bridges. Chapl and fragments of Chapl can be employed as biologically active or immunological substitutes for natural, purified Chapl and fragments of Chapl. Further, peptidomimetics that structurally and/or functionally resemble Chapl or fragments of Chapl can be made and evaluated for their ability to interact with Chapl in a Chapl characterization assay or to induce an immune response in a subject. Several approaches to make peptidomimetics that resemble polypeptides have been described. A vast number of methods, for example, can be found in U.S. Patent Nos. 5,288,707; 5,552,534; 5,811,515; 5,817,626; 5,817,879; 5,821 ,231; and 5, 874,529. Following synthesis or expression and isolation or purification of the proteins encoded by Chapl or a portion thereof, the isolated or purified proteins can be used to generate antibodies and tools for identifying agents that interact with Chapl and fragments of Chapl . Antibodies that recognize Chapl and fragments of Chapl have many uses including, but not limited to, biotechnological applications, therapeutic/prophylactic applications, and diagnostic applications. Such antibodies include, but are not limited to, polycloπal, monoclonal, chimeric, single chain. Fab fragments and fragments produced by a Fab expression library. Neutralizing antibodies, i.e., those that inhibit Chapl - mediated adhesion, are especially preferred for diagnostics and therapeutics.

For the production of antibodies, various hosts including goats, rabbits, rats, mice, etc can be immunized by injection with Chapl or any portion, fragment or oligopeptide that retains immunogenic properties. Depending on the host species, various adjuvants can be used to increase immunological response. Such adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum are potentially useful adjuvants.

Peptides used to induce specific antibodies can have an amino acid sequence consisting of at least three amino acids, preferably at least 10 or 15 amino acids. Desirably, short stretches of amino acids encoding fragments of Chapl are fused with those of another protein such as keyhole limpet hemocyanin and antibody is produced against the chimeric molecule. While antibodies capable of specifically recognizing Chapl can be generated by injecting into mice synthetic 3-mer, 10-mer, and 15-mer peptides that correspond to a protein sequence of Chapl, a more diverse set of antibodies can be generated by using recombinant or purified Chapl and fragments of Chapl.

To generate antibodies to Chapl and fragments of Chapl, substantially pure Chapl or a fragment of Chapl is isolated from a transfected or transformed cell. The concentration of the polypeptide in the final preparation is adjusted, for example, by concentration on an Amicon filter device, to the level of a few micrograms/ml. Monoclonal or polyclonal antibody to the polypeptide of interest can then be prepared as follows:

Monoclonal antibodies to Chapl or a fragment of Chapl can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497 (1975), the human B-cell hybridoma technique (Kosbor et al. Immunol Today 4:72 (1983); Cote et al Proc Natl Acad Sci 80:2026-2030 (1983), and the EBV-hybridoma technique Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc. New York N.Y., pp 77-96 (1985). In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used. (Morrison et al. Proc Natl Acad Sci 81:6851-6855 (1984); Neuberger et al. Nature

312:604-608 (1984); and Takeda et al. Nature 314:452-454 (1985). Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce Chapl -specific single chain antibodies. Antibodies can also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al., Proc Natl Acad Sci 86: 3833-3837 (1989), and Winter G. and Milstein C; Nature 349:293-299 (1991 ).

Antibody fragments that contain specific binding sites for Chapl can also be generated. For example, such fragments include, but are not limited to, the F(ab')₂ fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (Huse W. D. et al. Science 256:1275-1281 (1989).

By one approach, monoclonal antibodies to Chapl of fragments thereof are made as follows. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused in the presence of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising amiπopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (Meth Enzymol, 70:419 (1980)), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. (Basic Methods in Molecular Biology Elsevier, New York. Section 21 -2).

Polyclonal antiserum containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom described above, which can be unmodified or modified to enhance immunogeπicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogeπic than others and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple iπtradermal sites appears to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis et al. (J Clin Endocrinol Metab, 33:988-991 (1971)).

Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, 0. et al., Chap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 M). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980). Antibody preparations prepared according to either protocol are useful in quantitative immunoassays that determine concentrations of antigen-bearing substances in biological samples; they are also used semi- quantitatively or qualitatively (e.g., in diagnostic embodiments that identify the presence of Chapl in biological samples).

Additionally, Chapl and fragments of Chapl can be used to induce antibody production in humans. That is, Chapl and fragments of Chapl, whether made chemically or as detailed above, can be used as an antigen or vaccine so as to elicit an immune response in a patient. Accordingly, Chapl or fragments of Chapl can be joined to or administered with another protein, carrier, support, or adjuvant so as to generate a pharmaceutical or vaccine that will induce potent immune response. Additionally, nucleic acids encoding Chapl or fragments of Chapl can be administered by themselves or with Chapl or with fragments of Chapl and, as above, can be joined to or administered with a protein, carrier, support, or adjuvant. These nucleic acids can be administered "naked" or can be incorporated into vectors. Vaccination protocols can include, for example, identifying a subject in need of a vaccine and administering to said subject a therapeutically effective amount of either a protein or a nucleic acid-based vaccines or combinations of protein and nucleic acid vaccines.

In the following, disclosure concerning the use of Chapl characterization assays and methods to identify agents that modulate Chapl -mediated adhesion to a chaperone are provided.

Modulation of Chapl -dependent Adhesion

The data above establishes that Chapl efficiently associates with a chaperone to form a Chapl -chaperone complex. The association of Chapl to a chaperone can be measured using many techniques. By one approach, Chapl dependent adhesion to a chaperone is analyzed by contacting a support having a chaperone or a representative fragment of a chaperone with Chapl or a representative fragment of Chapl. If the Chapl or fragment thereof is detectably labeled (e.g., ¹²⁵l), the association to immobilized a chaperone (or a chaperone fragment) can be directly determined by detecting the signal (e.g., scintillation counting). Alternatively, the association of Chapl or fragment thereof with a chaperone can be determined indirectly by employing a detectably labeled antibody that has an epitope that corresponds to a region of Chapl. In these assays, the support can be a resin, plastic, a membrane, a lipid, and a cell. Additionally, the Chapl can be joined to a second support. Many Chapl characterization assays can be automated (e.g., high throughput screening employing a fluorescently labeled Chapl or fragment of Chapl ) so as to quickly identify regions of the molecule that are involved in binding to a chaperone. Values or results from these assays can be recorded on a computer readable media (e.g., in a database) and analyzed with a search program and retrieval program. Of course, embodiments of the invention include the converse of the assay described above. That is, immobilizing Chapl or fragments thereof on a support and detecting the adhesion of labeled a chaperone or fragments of a chaperone. Additional embodiments include methods of identifying agents that modulate Chapl -mediated adhesion to a chaperone. By one approach, an agent that modulates Chapl dependent adhesion (e.g., a fragment of a chaperone or protein that associates in a biological complex comprising Cfιap-1) can be identified by contacting a support having a chaperone or a representative fragment thereof with Chapl or a fragment of Chapl in the presence of the agent. Detection of Chapl dependent adhesion is accomplished, as described above, and successful agents are identified according to their ability to induce a desired modulation of the formation of the biological complex comprising Chapl and the chaperone. As above, the support can be a resin, a membrane, plastic, a lipid, or a cell and the Chapl can be joined to a second support so as to more nearly reproduce native binding conditions. In another approach, a support having Chapl or a representative fragment thereof can be used to capture directly or indirectly labeled chaperone proteins or fragments of a chaperone. In some aspects, the fragments of Chapl that are used have a polypeptide sequence that binds to a chaperone and is at least 80% homologous to Chapl . In other aspects, fragments of Stch that bind to a chaperone protein are used as agents. Desirable fragments of Stch include the polypeptide sequence of clone 7 and preferable fragments of Stch include the boxed region shown in FIGURE 3. More preferable fragments, however, include the approximately 20 amino acid residues of clone 7 that are not found in clone 8. Other embodiments include agents that comprise a cytoskeletal protein or neuronal protein or fragment thereof that interacts with Chap 1. As above, binding is conducted in the presence of the agent and Chapl dependent adhesion to a chaperone is determined by the amount of labeled a chaperone bound to the immobilized Chapl. In this method, the support can be a resin, a membrane, plastic, a lipid, and a cell and the a chaperone can also be joined to a second support. In some aspects of the invention, nucleic acids encoding Chapl, nucleic acids complementary to Chapl ,

Chapl protein, and polypeptide fragments of Chapl are agents that modulate (e.g., inhibit or enhance) the formation of the Chapl -chaperone complex. One embodiment of an Chapl modulating agent is an antisense oligonucleotide or ribozyme that hybridizes to nucleic acid encoding regions of Chapl . By "antisense oligonucleotide" is meant a nucleic acid or modified nucleic acid including, but not limited to DNA, RNA, modified DNA or RNA (including branched chain nucleic acids and 2' O-methyl RNA) and PNA (polyamide nucleic acid).

Several ribozymes known to those of skill in the art can be easily designed to hybridize to nucleic acid sequence encoding Chapl and thereby inhibit the production of functional protein. Desirably, antisense oligonucleotides or ribozymes that hybridize to the start codon of Chapl are used. In one embodiment, full length antisense Chapl is used to significantly reduced Chapl -dependent adhesion to a chaperone. Many other antisense oligonucleotides or ribozymes that interfere with the formation of a Chapl-chaperone complex can be designed and screened by the methods detailed previously.

The antisense nucleic acids should have a length and melting temperature sufficient to permit formation of an intracellular duplex having sufficient stability to inhibit the expression of the mRNA in the duplex. Strategies for designing antisense nucleic acids suitable for use in gene therapy are disclosed in Green et al. (Ann Rev Biochem, 55:569-597 (1986)) and Izant and Weintraub (Cell, 36:1007-1015 (1984)). In some strategies, antisense molecules are obtained from a nucleotide sequence encoding Chapl by reversing the orientation of the coding region with respect to a promoter so as to transcribe the opposite strand from that which is normally transcribed in the cell. Antisense molecules and ribozymes can be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Additionally, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding Chapl. Such DNA sequences can be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Further, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues. Still further, oligonucleotides that are complementary to the mRNA encoding Chapl can be synthesized in vitro. Thus, antisense nucleic acids are capable of hybridizing to Chapl mRNA to create a duplex. In some embodiments, the antisense sequences can contain modified sugar phosphate backbones to increase stability and make them less sensitive to RNase activity. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosiπe and wybutosine as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine that are not as easily recognized by endogenous endoπucleases. Further examples are described by Rossi et al., Pharmacol. Ther., 50(2):245-254, (1991).

Various types of antisense oligonucleotides complementary to the Chapl mRNA can be used. In one preferred embodiment, stable and semi-stable antisense oligonucleotides described in International Application No. PCT W094/23026 are used. In these moleucles, the 3 end or both the 3 and 5 ends are engaged in intramolecular hydrogen bonding between complementary base pairs. These molecules are better able to withstand exonuclease attacks and exhibit increased stability compared to conventional antisense oligonucleotides. In another preferred embodiment, the antisense oligodeoxyπucleotides described in International Application No. WO 95/04141 are used. In yet another preferred embodiment, the covalently cross-linked antisense oligonucleotides described in International Application No. WO 96/31523 are used. These double- or single-stranded oligonucleotides comprise one or more, respectively, inter- or intra- oligonucleotide covalent cross-linkages, wherein the linkage consists of an amide bond between a primary amine group of one strand and a carboxyl group of the other strand or of the same strand, respectively, the primary amine group being directly substituted in the 2' position of the strand nucleotide monosaccharide ring, and the carboxyl group being carried by an aliphatic spacer group substituted on a nucleotide or nucleotide analog of the other strand or the same strand, respectively.

The antisense oligodeoxynucleotides and oligonucleotides disclosed in International Application No. WO 92/18522 can also be used. These molecules are stable to degradation and contain at least one transcription control recognition sequence that binds to control proteins and are effective as decoys therefor. These molecules can contain "hairpin" structures, "dumbbell" structures, "modified dumbbell" structures, "cross-linked" decoy structures and "loop" structures. In another preferred embodiment, the cyclic double-stranded oligonucleotides described in European Patent Application No. 0 572 287 A2 are used. These ligated oligonucleotide "dumbbells" contain the binding site for a transcription factor and inhibit expression of the gene under control of the transcription factor by sequestering the factor. Use of the closed antisense oligonucleotides disclosed in International Application No. WO 92/19732 is also contemplated. Because these molecules have no free ends, they are more resistant to degradation by exonucleases than are conventional oligonucleotides. These oligonucleotides can be multifunctional, interacting with several regions that are not adjacent to the target mRNA.

The appropriate level of antisense nucleic acids required to inhibit formation of the Chapl- chaperone complex can be determined using in vitro expression analysis and the Chapl characterization assays described herein. The antisense molecule can be introduced into the cells expressing Chapl by diffusion, injection, infection or transfection using procedures known in the art. For example, the antisense nucleic acids can be introduced into the body as a bare or naked oligonucleotide, oligonucleotide encapsulated in lipid, oligonucleotide sequence encapsidated by viral protein, or as an oligonucleotide operably linked to a promoter contained in an expression vector. The expression vector can be any of a variety of expression vectors known in the art, including retroviral or viral vectors, vectors capable of extrachromosomal replication, or integrating vectors. The vectors can be DNA or RNA. The antisense molecules are introduced onto cell samples at a number of different concentrations preferably between 1x10¹⁰M to 1x10⁴M. Once the minimum concentration that can adequately control gene expression is identified, the optimized dose is translated into a dosage suitable for use in vivo. For example, an inhibiting concentration in culture of 1x10⁷ translates into a dose of approximately 0.6 mg/kg bodyweight. Levels of oligonucleotide approaching 100 mg/kg bodyweight or higher can be possible after testing the toxicity of the oligonucleotide in laboratory animals. It is additionally contemplated that cells from a vertebrate, such as a mammal or human, are removed, treated with the antisense oligonucleotide, and reintroduced into the vertebrate.

Ribozymes can also be used to reduce or eliminate Chapl expression. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Within the scope of aspects of the invention, are engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of a sequence encoding Chapl, for example. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites that include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for secondary structural features that may render the oligonucleotide inoperable. The suitability of candidate targets can also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Delivery of antisense and ribozyme agents by transfection and by liposome are quite well known in the art.

Another embodiment of an Chapl modulating agent is a polypeptide that interferes with the association of Chapl with a chaperone. Polypeptide fragments that inhibit the association of Chapl with a chaperone can be rapidly engineered and identified given the present disclosure and candidate polypeptides can contain regions of Chapl or a chaperone. Desirable fragments of Stch include the polypeptide sequence of clone 7 and preferable fragments of Stch include the boxed region shown in FIGURE 3. More preferable fragments, however, include the approximately 20 amino acid residues of clone 7 that are not found in clone 8. Additionally, fragments of other molecules that bind a biological complex comprising Chap-1 and a chaperone (e.g., cγtoskeletal proteins and/or neuronal-specific proteins) can be agents that perturb the stability of the Chap-1 /chaperone complex. The screening of polypeptide fragments and mutant proteins that modulate the association of a complex having Chapl would be routine given the present disclosure and assays described herein. For example, polypeptide Chapl modulating agents can be identified by their ability to disrupt the formation of the Chapl -chaperone complex by employing conventional affinity chromatography techniques, sandwich assays, ELISA assays, or other binding assays known to those of skill in the art and described above. A screening method, for example, wherein the polypeptide Chapl modulating agent is administered to cells expressing Chapl in culture and cell lysates are analyzed by immunoprecipitation and Western blot can rapidly evaluate the polypeptide's ability to inhibit the association of a an Chapl -chaperone complex.

In another embodiment, concentrations of Chapl or a chaperone are raised in a cell so as to enhance Chapl - mediated adhesion to a chaperone. There may be many ways to raise the concentration of Chapl or a chaperone or both in a cell. Liposome-mediated transfer, is one approach to deliver Chapl or a chaperone or both protein to a cell. Alternatively, the concentration of Chapl or a chaperone or both can be raised in a cell by transfecting constructs encoding Chapl or a chaperone or both. A construct for use in the transfection of Chapl into cells in culture was described previously and many others can be developed by those of skill in the art. Retroviral constructs for the delivery of nucleic acid encoding Chapl a chaperone or fragments thereof or complements thereof are also contemplated and many retroviral vectors can be engineered to produce Chapl or a chaperone. Other embodiments of Chapl inhibitory or enhancing agents (collectively refered to as "modulators") include antibodies, peptidomimetics, and chemicals that inhibit or enhance Chapl -dependent adhesion to a chaperone. Several other methods for identifying agents that modulate the formation of the Chapl -chaperone complex and, concomitantly, effect Chapl -dependent a chaperone adhesion can be used.

In the discussion below, we describe methods of molecular modeling, combinatorial chemistry, and rational drug design for the identification of molecules that interact with Chapl and thereby modulate the formation of a Chapl -chaperone complex.

Methods of Rational Drug Design

Combinatorial chemistry is the science of synthesizing and testing compounds for bioactivity en masse, instead of one by one, the aim being to discover drugs and materials more quickly and inexpensively than was formerly possible. In some embodiments, search programs are employed to compare regions of Chapl that modulate the formation of a

Chapl -chaperone complex with other molecules, such as peptides, peptidomimetics, and chemicals so that therapeutic interactions of the molecules can be predicted and new derivative molecules can be designed. In other embodiments, search programs are employed to compare regions of molecules that interact with Chapl and, thereby modulate the formation of a Chapl -chaperone complex, with other molecules such as peptides, peptidomimetics, and chemicals, so that therapeutic interactions of the molecules can be predicted and new derivative Chapl modulating agents can be designed. (Schneider, Genetic Engineering News, December: page 20 (1998); Tempczyk et al., Molecular Simulations Inc. Solutions, April (1997); Buteπhof, Molecular Simulations Inc. Case Notes (August 1998)). This process of directed combinatorial chemistry is referred to as "rational drug design". Libraries of molecules that resemble Chapl or interact with Chapl and, thereby inhibit or enhance the function of Chapl ("modulate" Chapl activity) can be created. In some contexts, the term "Chapl modulating agent" or "modulators" includes Chapl, polypeptide fragments corresponding to Chapl, Stch, fragments of Stch that bind to a chaperone protein, fusion proteins comprising Chapl or polypeptide fragments of Chapl, fusion proteins comprising fragments of Stch that bind to a chaperone protein, nucleic acids encoding these molecules, peptidomimetics, chemicals, and other molecules that modulate Chapl -dependent Chapl -mediated adhesion to a chaperone.

One goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, null compounds) in order to fashion drugs that are, for example, more or less potent forms of the molecule. (See, e.g., Hodgson, Bio Technology, 9:19-21

(1991 )). Rational drug design has been used to develop HIV protease inhibitors and agonists for five different somatostatin receptor subtypes. (Erickson et al., Science, 249:527-533 (1990); Berk et al.. Science, 282:737 (1998)).

By starting with the sequence or protein models of Chapl or Stch, and/or fragments thereof, polypeptides having two-dimensional and/or three-dimensional homology can be rapidly identified. In a two-dimensional approach, a percent sequence identity can be determined by standard methods that are commonly used to compare the similarity and position of the amino acid of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptides are aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences, or along a predetermined portion of one or both sequences). Such programs provide "default" opening penalty and a "default" gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in: Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978)) can be used in conjunction with the computer program. The percent identity can then be calculated as:

total number of identical matches X 100 [length of the longer sequence within the matched span + number of gaps introduced into the longer sequence in order to align the two sequences]

Accordingly, the protein sequence corresponding to Chapl or Stch is compared to known sequences on a protein basis. Protein sequences corresponding to Chapl or Stch are compared, for example, to known amino acid sequences found in Swissprot release 35, PIR release 53 and Genpept release 108 public databases using BLASTP with the parameter W=8 and allowing a maximum of 10 matches. In addition, the protein sequences encoding Chapl are compared to publicly known amino acid sequences of Swissprot using BLASTX with the parameter E = 0.001. Because the region involved in a chaperone binding can be as small as three amino acids, the embodied polypeptides can have the following degrees of homology to Chapl or Stch: 1.0%, 2.0%, 3.0%, 4.0%,. 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 1 1.0%, 12.0%, 13.0%, 14.0%, 15.0%, 16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21.0%, 22.0%, 23.0%,

24.0%, 25.0%, 26.0%, 27.0%, 28.0%, 29.0%, 30.0%, 31.0%, 32.0%, 33.0%, 34.0%, 35.0%, 36.0%, 37.0%, 38.0%, 39.0%, 40.0%, 41.0%, 42.0%, 43.0%, 44.0%, 45.0%, 46.0%, 47.0%, 48.0%, 49.0%, 50.0%, 51.0%, 52.0%, 53.0%, 54.0%,. 55.0%, 56.0%, 57.0%, 58.0%, 59.0%, 60.0%, 61.0%, 62.0%, 63.0%, 64.0%, 65.0%, 66.0%, 67.0%, 68.0%, 69.0%, 70.0%, 71.0%, 72.0%, 73.0%, 74.0%, 75.0%, 76.0%, 77.0%, 78.0%, 79.0%, 80.0%, 81.0%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 89.0%, 90.0%, 91.0%, 92.0%, 93.0%,

94.0%, 95.0%, 96.0%, 97.0%, 98.0%, 99.0%, and 100.0% The candidate polypeptides are identified and are subsequently examined using the functional assays described herein. Candidate polypeptides that interact with Chapl or Stch to modulate the formation of a Chapl- chaperone complex and thereby effect adhesion to a chaperone can be identified in this manner. Additionally, a search program can be used to compare the three-dimensional structure of Chapl, Stch, or fragments of Chapl with other known three-dimensional structures. Once candidate related structures are identified, these molecules can be made recombiπantly or by peptide or chemical synthesis. The newly generated compounds are then screened in Chapl characterization assays so as to identify modulators that interact with Chapl and thereby effect the formation of a Chapl- chaperone complex. In the past, the three-dimensional structure of proteins has been determined in a number of ways. Perhaps the best known way of determining protein structure involves the use of x-ray crystallography. A general review of this technique can be found in Van Holde, K.E. Physical Biochemistry, Prentice-Hall, N.J. pp. 221-239 (1971 ). Using this technique, it is possible to elucidate three-dimensional structure with good precision. Additionally, protein structure can be determined through the use of techniques of neutron diffraction, or by nuclear magnetic resonance (NMR). (See, e.g., Moore, W.J., Physical Chemistry, 4* Edition, Prentice-Hall, N.J. (1972)).

Alternatively, the protein model embodiments of the present invention can be constructed using computer- based protein modeling techniques. By one approach, the protein folding problem is solved by finding target sequences that are most compatible with profiles representing the structural environments of the residues in known three- dimensional protein structures. (See, e.g., Eisenberg et al., U.S. Patent No. 5,436,850 issued July 25, 1995). In another technique, the known three-dimensional structures of proteins in a given family are superimposed to define the structurally conserved regions in that family. This protein modeling technique also uses the known three-dimensional structure of a homologous protein to approximate the structure of a polypeptide of interest. (See e.g., Srinivasan, et al., U.S. Patent No. 5,557,535 issued September 17, 1996). Conventional homology modeling techniques have been used routinely to build models of proteases and antibodies. (Sowdhamini et al., Protein Engineering, 10:207, 215 (1997)). Comparative approaches can also be used to develop three-dimensional protein models when the protein of interest has poor sequence identity to template proteins. In some cases, proteins fold into similar three-dimensional structures despite having very weak sequence identities. For example, the three-dimensional structures of a number of helical cytokines fold in similar three-dimensional topology in spite of weak sequence homology.

The recent development of threading methods and "fuzzy" approaches now enables the identification of likely folding patterns and functional protein domains in a number of situations where the structural relatedness between target and template(s) is not detectable at the sequence level. By one method, fold recognition is performed using Multiple Sequence Threading (MST) and structural equivalences are deduced from the threading output using the distance geometry program DRAGON that constructs a low resolution model. A full-atom representation is then constructed using a molecular modeling package such as QUANTA. According to this 3-step approach, candidate templates are first identified by using the novel fold recognition algorithm MST, which is capable of performing simultaneous threading of multiple aligned sequences onto one or more 3-D structures. In a second step, the structural equivalences obtained from the MST output are converted into interresidue distance restraints and fed into the distance geometry program DRAGON, together with auxiliary information obtained from secondary structure predictions. The program combines the restraints in an unbiased manner and rapidly generates a large number of low resolution model confirmations. In a third step, these low resolution model confirmations are converted into full-atom models and subjected to energy minimization using the molecular modeling package QUANTA. (See e.g., Aszόdi et al., Proteins:Structure, Function, and Genetics, Supplement, 1:38-42 (1997)).

In one approach, a three-dimensional structure of a polypeptide of interest (e.g., Chapl, and/or fragments thereof or a Chapl modulating agent) is determined by x-ray crystallography, NMR, or neutron diffraction and computer modeling, as described above. Useful protein models of the polypeptide of interest can also be gained by computer modeling alone. Combinatorial chemistry can then be employed to design derivatives of the polypeptide of interest based on the three-dimensional models. The candidate Chapl modulating agents are then tested in functional assays. The assays, described herein and assays that evaluate the formation of a Chapl- chaperone complex in the presence of Chapl or fragments thereof that will be apparent to one of skill in the art given the disclosure herein

(referred to collectively as "Chapl characterization assays") are performed on the Chapl modulating agents and groups of Chapl modulating agents (wherein the grouping is based on the potency of modulation of the formation of a Chapl - chaperone complex) are identified and recorded on a computer readable media. Further cycles of modeling and Chapl characterization assays can be employed to more narrowly define the parameters needed in an optimal Chapl modulating agent.

By another approach, a Chapl modulating agent that interacts with Chapl can be manufactured and identified as follows. First, a molecular model of one or more Chapl modulating agents or portions of Chapl modulating agents that interact with Chapl (e.g., clone 7, the boxed region of Stch shown in FIGURE 3, or the approximately 20 amino acid residues of clone 7 that are not found in clone 8.) are created using one of the techniques discussed above or as known in the art. Chapl modulating agents that are known to interact with Chapl include antibodies and fragments of a chaperone. Next, chemical libraries and databases are searched for molecules similar in structure to the known Chapl modulating agents. Identified candidate Chapl modulating agents are then screened in the Chapl characterization assays, described above, and the agents that produce the desired response are used as templates for further library construction. Libraries of Chapl modulating agents are synthesized on solid support beads by split-and-pool synthesis, a multistage process for producing very large numbers of compounds. The support-bound agents are then used in Chapl characterization assays or "free mixtures" are created by cleaving the agent from the support and these free mixtures are screened in the Chapl characterization assays. Compounds that produce desirable responses are identified, recorded on a computer readable media, and the process is repeated to select optimal Chapl modulating agents. Each Chapl modulating agent and its response in a Chapl characterization assay can be recorded on a computer readable media and a database or library of Chapl modulating agents and respective responses in the Chapl characterization assay can be generated. These databases or libraries can be used by researchers to identify important differences between active and inactive molecules so that compound libraries are enriched for Chapl modulating agents that have favorable characteristics. Further, enrichment can be achieved by using approaches in dynamic combinatorial chemistry. (See e.g., Angnew, Chem Int Ed, 37:2828 (1998)). For example, a target biomolecule, such as Chapl, is joined to a support and is bound by the Chapl modulating agents from the libraries generated above. The Chapl resin bound with one or more candidate Chapl modulating agents is removed from the binding reaction, the Chapl modulating agents are eluted from the support, and are identified. Cycles of immobilized target binding assays are conducted, classes of Chapl modulating agents that exhibit desired binding characteristics are identified, and this data is recorded on a computer readable media and is used to select more Chapl modulating agents that produce a desired modulation of the formation of a Chapl- chaperone complex.

In addition, a peptide of interest (e.g., Chapl, and/or fragments thereof or a Chapl modulating agent) can be analyzed by an alanine scan (Wells, Methods in Enzymol, 202:390-411 (1991)) or other types of site-directed mutagenesis analysis. In alanine scan, for example, an amino acid residue is replaced by alanine, and its affect on the peptide's activity is measured by functional assays, such as the Chapl characterization assays described herein. Each of the amino acid residues of the peptide is analyzed in this manner and the regions important for a specific modulation of the formation of a Chapl- chaperone complex are identified. Subsequently, these functionally important regions are recorded on a computer readable medium, stored in a first database in a computer system, and a search program is employed to generate protein models of the functionally important regions. Once protein models of the functionally important regions have been generated, a second database comprising one or more libraries having peptides, chemicals, peptidomimetics and other agents is accessed by a search program and individual agents are compared to the protein models to identify agents that comprise homologous regions or domains that resemble the identified functionally important regions. Agents identified by the approach above are then tested in the Chapl characterization assays and are used to construct multimeric agents and/or are incorporated into pharmaceuticals, as detailed below. In another embodiment, computer modeling and the sequence-to-structure-to-function paradigm is exploited to identify more Chapl modulating agents that modulate the formation of a Chapl- chaperone complex. By this approach, first the structure of a Chapl modulating agent having a known response in a Chapl characterization assay (e.g., Chapl, and fragments thereof, and antibodies to Chapl, is determined from its sequence using a threading algorithm, which aligns the sequence to the best matching structure in a structural database. Next, the protein's active site (i.e., the site important for a desired response in the Chapl characterization assay) is identified and a "fuzzy functional form" (FFF) -- a three-dimensional descriptor of the active site of a protein -- is created. (See e.g., Fetrow et al., J Mol Biol, 282:703-711 (1998); Fetrow and Skolnick, J Mol Biol, 281:949-968 (1998)).

The FFFs are built by itteratively superimposing the protein geometries from a series of functionally related proteins with known structures. The FFFs are not overly specific, however, and the degree to which the descriptors can be relaxed is explored. In essence, conserved and functionally important residues for a desired response are identified and a set of geometric and conformational constraints for a specific function are defined in the form of a computer algorithm. The program then searches experimentally determined protein structures from a protein structural database for sets of residues that satisfy the specified constraints. In this manner, homologous three- dimensional structures can be compared and degrees (e.g., percentages of three-dimensional homology) can be ascertained.

By using this computational protocol, genome sequence data bases such as maintained by various organizations including: http://www.tigr.org/tdb: http://www.genetics.wisc.edu: http://genome- www.stanford.edurball; http:llhiv-web.lanl.gov; http://wwwncbi.nlm.nih.gov: http://www.ebi.ac.uk; http://pasteur.fr/other/biology; and http://www-genome.wi.mit.edu, can be rapidly screened for specific protein active sites and for identification of the residues at those active sites that resemble a desired molecule. Several other groups have developed databases of short sequence patterns or motifs designed to identify a given function or activity of a protein. These databases, notably Prosite (http://expasy.hcuge.ch/sprot/prosite.html): Blocks

(http://www.blocks.fhcrc.org); and Prints (http://www.biochem.ucl.ac.uk/bsm/dbbrowser/PRINTS/PRINTS.html). use short stretches of sequence information to identify sequence patterns that are specific for a given function; thus they avoid the problems arising from the necessity of matching entire sequences. In this manner, new Chapl modulating agents are rationally selected for further identification by Chapl characterization assays, as described above. Rounds or cycles of functional assays on the molecules and derivatives thereof and further FFF refinement and database searching allows an investigator to more narrowly define classes of Chapl modulating agents that produce a desired modulation of the formation of a Chapl-chaperone complex.

Many computer programs and databases can be used with embodiments of the invention to identify agents that modulate Chapl -mediated adhesion to a chaperone. The following list is intended not to limit the invention but to provide guidance to programs and databases that are useful with the approaches discussed above. The programs and databases that may be used include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J Mol Biol, 215: 403 (1990)), FASTA (Pearson and Lipman, Proc Natl Acad Sci USA, 85:2444 (1988)), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), CeriuslDBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology (Molecular

Simulations Inc.), Modeler (Molecular Simulations Inc.), Modeller 4 (Sali and Blundell, J Mol Biol. 234:217-241 (1997)), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), the EMBL/Swissprotein database, the MDL Available Chemicals Directory database, the MDL Drug Data Report data base, the Comprehensive Medicinal Chemistry database, Derwents's World Drug Index database, and the

BioByteMasterFile database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure.

Libraries of information on Chapl modulating agents with their corresponding response in Chapl characterization assays can be generated by performing the rational drug design approaches above in conjunction with the Chapl characterization assays. A record of the results for each Chapl modulating agent is generated and groups of Chapl modulating agents are identified and stored on a computer readable media. Databases of this information are valuable to investigators and clinicians for selecting the type of Chapl modulating agent-based pharmaceutical to treat or elicit a particular response. Preferable libraries are created by performing the assays above on Chapl and fragments thereof.

Many of the Chapl modulating agents are provided in biotechnological tools, diagnostics, and pharmaceuticals as multimeric or multimerized agents or both that can be joined to a support. In the disclosure below, we discuss the preparation of multimeric supports and multimerized Chapl modulating agents comprising Chapl or fragments of Chapl, complementary nucleic acids to Chapl, Chapl or fragments of Chapl, antibodies or antibody fragments that recognize epitopes of Chapl, and Chapl fusion proteins.

Preparation of Multimeric Supports and Multimerized Chapl Modulators

Biotechnological tools and components to prophylactic and therapeutic agents desirably provide Chapl, fragments of Chapl, complementary nucleic acids to Chapl, Chapl, fragments of Chapl, Stch 1, fragments of Stch that bind to a chaperone protein, antibodies or antibody fragments that recognize epitopes of Chapl , or Stch, and Chapl or Stch fusion proteins in such a form or in such a way that a sufficient affinity, modulation of Chapl - chaperone complex formation is achieved. While a natural monomeric agent (that is, an agent that presents a discrete molecule, thus, carrying only one binding epitope or domain) can be sufficient to achieve a desired response, a synthetic agent or a multimeric agent (that is, an agent that presents multiple molecules, thus, having several binding epitopes or domains) often times can elicit a greater response. It should be noted that the term "multimeric" refers to the presence of more than one molecule on an agent, for example, several individual molecules of an antibody joined to a support, as distinguished from the term "multimerized" that refers to an agent that has more than one molecule joined as a single discrete compound molecule on a support, for example several antibody molecules joined to form a single compound molecule that is joined to a support.

A multimeric agent (synthetic or natural) that modulates the formation of a Chapl - chaperone complex is obtained by joining Chapl, fragments of Chapl, complementary nucleic acids to Chapl, Chapl, fragments of Chapl, Stch, fragments of Stch that bind to a chaperone protein, antibodies or antibody fragments that recognize epitopes of

Chapl or Stch, and Chapl or Stch fusion proteins (collectively referred to as "Chapl modulating agents" or "modulators") to a macromolecular support. Chapl modulating agents including peptidomimetics and chemical molecules that resemble these ligands are also joined to supports so as to create the multimeric agents of the invention. A "support" can also be a carrier, a protein, a resin or any macromolecular structure used to join or immobilize a Chapl modulating agent. Solid supports include, but are not limited to, the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, Duracyte® artificial cells, and others. In several embodiments, the macromolecular support has a hydrophobic surface that interacts with a portion of the Chapl modulating agent by a hydrophobic non-covaieπt interaction. In some cases, the hydrophobic surface of the support is a polymer such as plastic or any other polymer in which hydrophobic groups have been linked such as polystyrene, polyethylene or polyvinyl. Additionally, the Chapl modulating agent is covalently bound to carriers including proteins and oligo/polysaccarides (e.g. cellulose, starch, glycogen, chitosane or aminated sepharose). In these later embodiments, a reactive group on a Chapl modulating agent, such as a hydroxy or an amino group, is used to join to a reactive group on the carrier so as to create the covalent bond. Embodiments also comprise a support with a charged surface that interacts with the Chapl modulating agent. Additional embodiments comprise a support that has other reactive groups that are chemically activated so as to attach a Chapl modulating agent, such as a peptide or chemical compound. For example, cyanogen bromide activated matrices, epoxy activated matrices, thio and thiopropyl gels, nitrophenyl chloroformate and N-hydroxy succinimide chlorformate linkages, or oxirane acrylic supports are used. (Sigma). Inorganic carriers, such as silicon oxide material (e.g. silica gel, zeolite, diatomaceous earth or aminated glass) to which the Chapl modulating agent is covalently linked through a hydroxy, carboxy or amino group and a reactive group on the carrier are also embodiments. Carriers for use in the body, (i.e. for prophylactic or therapeutic applications) are desirably physiological, non-toxic and preferably, non-immunoresponsive. Contemplated carriers for use in the body include poly-L-lysine, poly-D, L-alanine and Chromosorb' (Johns-Manville Products, Denver Co.). Conjugated Chromosorb* (Synsorb-Pk) has been tested in humans for the prevention of hemolytic-uremic syndrome and was reported as not presenting adverse reactions. (Armstrong et al., J Infectious Diseases, 171 :1042-1045 (1995)).

For some embodiments, the administration of a "naked" carrier (i.e., lacking an attached Chapl modulating agent) that has the capacity to attach a Chapl modulating agent that modulates the formation of a Chapl- chaperone complex inside the body of a subject is performed. By this approach, a "prodrug-type" therapy is administered in which the naked carrier is provided separately from the desired Chapl modulating agent and, once both are in the body, the carrier and the Chapl modulating agent assemble into a multimeric complex and modulate the formation of a Chapl- chaperone complex.

In another embodiment, linkers, such as 8 linkers, of an appropriate length are inserted between the Chapl modulating agent and the support so as to encourage greater flexibility in the Chapl modulating agent and thereby overcome any steric hindrance that is presented by the support. The determination of an appropriate length of linker that allows for optimal binding and modulation of the formation of a Chapl- chaperone complex, is made by screening the Chapl modulating agents with varying linkers in the Chapl characterization assays.

A composite support having more than one type of Chapl modulating agent is also an embodiment. A "composite support" is a carrier, a resin, or any macromolecular structure used to join or immobilize two or more different Chapl modulating agents that modulate the formation of a Chapl-chaperone complex. The composite supports are also constructed by utilizing hydrophobic interactions and covalent linkages formed through reactive groups, as detailed above. Further, linkers, such as 8 linkers, of an appropriate length between the Chapl modulating agents and the support are inserted in some embodiments so as to encourage greater flexibility in the molecule and overcome steric hindrance. The determination of an appropriate length of linker that allows for optimal binding and modulation of the formation of a Chapl-chaperone complex, is made by screening the Chapl modulating agents with varying linkers in the Chapl characterization assays detailed in the present disclosure.

In other embodiments of the present invention, the multimeric and composite supports discussed above have attached multimerized Chapl modulating agents so as to create a "multimerized-multimeric support" and a "muftimerized-composite support", respectively. An embodiment of a multimerized Chapl modulating agent, for example, is obtained by creating an expression construct having two or more nucleotide sequences encoding the

Chapl modulating agent joined together by using conventional techniques in molecular biology. The expressed fusion protein is one embodiment of a multimerized agent and is then joined to a support. A support having many such multimerized agents is termed a multimerized-multimeric support. The multimerized form of the Chapl modulating agent can be advantageous for many applications because of the ability to obtain an agent with a better ability to modulate the formation of a Chapl- chaperone complex. The incorporation of linkers or spacers, such as flexible 8 linkers, between the protein domains that make-up the multimerized agent can also be advantageous for some embodiments. The insertion of 8 linkers of an appropriate length between protein binding domains, for example, encourages greater flexibility in the molecule and overcomes steric hindrance between the several proteins. Similarly, the insertion of linkers between the multimerized Chapl modulating agent and the support encourages greater flexibility and reduces steric hindrance presented by the support. The determination of an appropriate length of linker that allows for optimal binding and modulation of the formation of a Chapl-chaperone complex can be accomplished by screening the Chapl modulating agents with varying linkers in the Chapl characterization assays detailed in this disclosure. In a similar fashion composite-multimerized-multimeric supports with and without linkers can be constructed by joining more than one different multimerized Chapl modulating agent to a support. The discussion that follows describes several diagnostic embodiments of the invention. Diagnostic Embodiments

Several diagnostic and prognostic tools that detect the concentration and expression level of nucleic acids encoding Chapl and the concentration and expression level of Chapl in various tissues and fluids are used to determine whether an individual is suffering from a Chapl-related disease or is likely to suffer from a Chapl-related disease. A "Chapl-related disease" can be a disease associated with aberrant protein degradation, cell cycle control or apoptosis and may involve neuropsychiatric disorders (e.g., Alzheimer's disease or Spirocerebellar ataxia).

Generally, the diagnostics and methods of use thereof can be classified according to whether the diagnostic detects the concentration or expression level of Chapl nucleic acid or Chapl protein in a biological sample (e.g., blood). Accordingly, the concentration and expression level of Chapl in a biological sample can be determined by monitoring the amount of RNA in the sample. The detection of an abnormal amount RNA encoding Chapl in a sample indicates the existence or predilection to a Chapl-related disease. Further, a detection of an abnormal amount of DNA encoding Chapl in a biological sample indicates the existence or predilection to a Chapl-related disease. Similarly, the concentration and expression level of Chapl in a biological sample can be determined by monitoring the amount of Chapl protein in the sample. The detection of an abnormal amount of Chapl in a sample indicates the existence or predilection to a Chapl-related disease.

To determine the presence of Chapl or Chapl in a subject, first a biological sample is obtained. Several methods known to those in the art can be employed to obtain a biological sample having red blood cells (e.g., phlebotomy). Once a biological sample from a subject in need of testing is obtained, many different techniques can be used to detect the concentration and expression level of Chapl or Chapl including, but not limited to, antibody-based detection techniques (e.g., ELISA, sandwich assays, immunoprecipitation, and immunoblots), bacteriophage display techniques, hybridization techniques (e.g., Southern and Northern), and enzymatic digestion (e.g., RNAse protection) techniques. Some of these techniques can involve disposing the proteins and/or nucleic acids present in the biological sample on a support, and contacting the support with detection reagents such as antibodies to Chapl or nucleic acid probes complementary to Chapl mRNA. Desirably, the levels of expression or concentration of Chapl or Chapl or both from diseased and healthy individuals is compared to the level detected in the subject tested.

In preferred embodiments, the nucleic acid embodiments of the present invention are attached to a support in an ordered array wherein a plurality of nucleic acid probes are attached to distinct regions of the support that do not overlap with each other. Preferably, such an ordered array is designed to be "addressable" where the distinct locations of the probe are recorded and can be accessed as part of an assay procedure.

In some embodiments, addressable nucleic acid arrays comprise a plurality of nucleic acid probes that complement Chapl . These probes are joined to a support in different known locations. The knowledge of the precise location of each nucleic acid probe makes these "addressable" arrays particularly useful in binding assays. For example, an addressable array can comprise a support having several regions to which are joined a plurality nucleic acid probes that complement Chapl. The nucleic acids from a preparation of several biological samples from a plurality of human subjects or a plurality of tissues or fluids from a single subject are labeled by conventional approaches (e.g., radioactivity or fluorescence) and the labeled samples are applied to the array under conditions that permit hybridization. If a nucleic acid in the sample hybridizes to a probe on the array, then a signal will be detected at a position on the support that corresponds to the location of the hybrid. Since the identity of each labeled sample is known and the region of the support on which the labeled sample was applied is known, an identification of the presence, concentration, and/or expression level can be rapidly determined. That is, by employing labeled standards of a known concentration of a nucleic acid encoding Chapl, (e.g., RNA), an investigator can accurately determine the concentration of a nucleic acid encoding Chapl in a sample and from this information can assess the expression level of Chapl. Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of a nucleic acid encoding Chapl . These approaches are easily automated using technology known to those of skill in the art of high throughput diagnostic analysis.

Additionally, an opposite approach to that presented above can be employed. Nucleic acids present in biological samples (e.g., tissues or fluids from one or more subjects or one or more sources in a subject's body) can be disposed on a support so as to create an addressable array. Preferably, the samples are disposed on the support at known positions that do not overlap. The presence of nucleic acids encoding Chapl in each sample is determined by applying labeled nucleic acid probes that complement nucleic acids that encode Chapl and detecting the presence of a signal at locations on the array that correspond to the positions at which the biological samples were disposed. Because the identity of the biological sample and its position on the array is known, an identification of the presence, concentration, and/or expression level of a nucleic acid encoding Chapl is rapidly determined. That is, by employing labeled standards of a known concentration of a nucleic acid encoding Chapl, (e.g., RNA), an investigator can accurately determine the concentration of a nucleic acid encoding Chapl in a sample and from this information can assess the expression level of Chapl . Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of a nucleic acid encoding Chapl . These approaches are also easily automated using technology known to those of skill in the art of high throughput diagnostic analysis. Any addressable array technology known in the art can be employed with this aspect of the invention. One particular embodiment of polynucleotide arrays is known as Geπechips™, and has been generally described in US Patent 5,143,854; PCT publications WO 90/15070 and 92/10092. These arrays are generally produced using mechanical synthesis methods or light directed synthesis methods, which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis. (Fodor et al., Science, 251:767-777, (1991 )). The immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as "Very Large Scale Immobilized Polymer Synthesis" (VLSIPS™) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSIPS™ technologies are provided in US Patents 5,143,854 and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO

95/11995, which describe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques. In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and diagnostic information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256.

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid assays. There are several ways to produce labeled nucleic acids for hybridization or PCR

(Polymerase Chain Reaction) including, but not limited to, oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, a nucleic acid encoding Chapl , or any portion of it, can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides. A number of companies such as Pharmacia Biotech (Piscataway N.J.),

Promega (Madison Wis.), and U.S. Biochemical Corp (Cleveland Ohio) supply commercial kits and protocols for these procedures. Suitable reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as, substrates, cofactors, inhibitors, magnetic particles and the like.

For diagnostic and prognostic purposes, nucleic acid probes having a sequence complementary to a nucleic acid encoding Chapl or a portion thereof can be used to detect and quantitate gene expression in biological samples.

Preferably, nucleic acid probes that are complementary to mRNA encoding Chapl are used to screen for polynucleotides present in blood. RNA-detection-based diagnostic assays, such as Northern hybridization, Northern dot blots, RNA in situ hybridization, and ELISA assays, are particularly useful to distinguish between the absence or presence of Chapl and to monitor Chapl levels during therapeutic intervention. Included in the scope of embodiments of the invention are the use of oligonucleotide sequences, antisense

RNA and DNA molecules, and PNAs that complement Chapl sequences for the determination of Chapl concentrations and expression levels in the cells of a subject by RNA-based detection techniques. The form of such qualitative and/or quantitative methods can include Northern analysis, dot blot or other membrane-based technologies; PCR technologies; dip stick, pin, chip, and ELISA technologies. All of these techniques are well known in the art and are the basis of many commercially available diagnostic kits.

In one aspect, RNA probes complementary to Chapl mRNA are used in assays that detect a Chapl-related disease. Accordingly, the nucleotide sequence encoding Chapl or a fragment thereof is used to design suitable RNA probes. The RNA probes are labeled by methods known in the art and are added to a DNAse treated fluid or tissue sample from a subject under conditions suitable for the formation of hybridization complexes. Hybridization complexes are isolated or the sample is treated with an agent that removes unhybridized nucleic acids. After an incubation period, the sample is washed with a compatible fluid that optionally contains a dye (or other label requiring a developer) if the nucleotide has been labeled with an enzyme. After the compatible fluid is rinsed off, the dye is quantitated and compared with a standard. If the amount of dye in the sample is significantly elevated over that of a comparable control sample, the nucleotide sequence has hybridized with RNA in the sample, and the presence of elevated levels of RNA encoding Chapl or a portion thereof in the sample indicates the presence of a Chapl-related disease, such as cancer.

Such assays can also be used to evaluate the efficacy of a particular therapeutic treatment regime in animal studies, in clinical trials, or in monitoring the treatment of an individual patient. In order to provide a basis for the diagnosis of disease, a normal or standard profile for Chapl expression in isolated cells, extracts, or tissue is desirably established. This is accomplished by combining body fluids or cell extracts taken from healthy subjects with RNA probes encoding Chapl, or a portion thereof, under conditions suitable for hybridization. Standard hybridization can be quantified by comparing the values obtained for healthy and diseased subjects with a dilution series of Chapl RNA run in the same experiment where a known amount of substantially purified Chapl is used. Standard values obtained from samples from healthy and diseased subjects are then compared with values obtained from samples from the tested subjects. Deviation between standards and the values obtained for the subject tested establishes the presence or predilection for a Chapl-related disease.

Additionally, PCR methods that can be used to quantitate the concentration and expression level of a particular molecule include radiolabeling (Melby et al., J Immunol Methods, 159:235-44 (1993)) or biotinylating nucleotides (Duplaa, Anal Biochem, 212:229-236 (1993)), coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated. Quantitation of multiple samples can be processed more rapidly by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation. A definitive diagnosis of this type can allow health professionals to create a disease state profile for a patient, begin aggressive treatment for the Chapl-related disease, and prevent further worsening of the condition. Similarly, further assays and reference to the changing disease state profile can help clinicians monitor the progress of a patient during treatment. That is, once a disease state is established, a therapeutic agent is administered and an initial disease state profile is generated. The assays above can be repeated on a regular basis to evaluate whether the values in the subject's disease state profile progresses toward or returns back to the initial disease state profile. Successive treatment profiles can be used to show the efficacy of treatment over a period of several days or several months.

As mentioned above, PCR technology can be used to identify and quantitate concentration and expression levels of Chapl. For amplification of mRNAs, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by PCR (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Patent No. 5,322,770, or, to use Reverse Transcriptase Asymmetric Gap Ligase Chain Reaction (RT-AGLCR), as described by Marshall et al. (PCR Methods and Applications, 4:80-84, 1994).

A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see

Molecular Cloning to Genetic Engineering White, B.A. Ed. in Methods in Molecular Biology 67: Humana Press, Totowa

(1997), and the publication entitled "PCR Methods and Applications" (1991, Cold Spring Harbor Laboratory Press). In each of these PCR procedures, PCR primers on either side of the Chapl sequence to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR has further been described in several patents including US Patents 4,683,195, 4,683,202 and 4,965,188.

The primers are selected to be substantially complementary to a portion of the sequence of Chapl mRNA and a portion of the sequence that complements the sequence of Chapl mRNA, thereby allowing the sequences between the primers to be amplified. The length of the primers for use with this aspect of the present invention be identical to most of the lengths of the nucleic acid embodiments provided previously. That is, primer length can be less than or equal to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,

34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, and 3,340 nucleotides. Preferably, however primers are 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 nucleotides in length. Shorter primers tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer primers are expensive to produce and can sometimes self-hybridize to form hairpin structures. The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer, the ionic strength of the solution and the G+C content. The higher the G + C content of the primer, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The G + C content of the amplification primers of the present invention preferably ranges between 10 and 75 %, more preferably between 35 and 60 %, and most preferably between 40 and 55 %. The appropriate length for primers under a particular set of assay conditions may be empirically determined by one of skill in the art.

The spacing of the primers determines the length of the segment to be amplified. In the context of the present invention amplified segments carrying nucleic acid sequence encoding fragments of Chapl can range in size from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers for Chapl can be of any sequence that allows the specific amplification of any DNA fragment carrying nucleic acid sequence unique Chapl . Amplification primers can be labeled or immobilized on a solid support as described above. The presence of Chapl protein can be detected by screening for the presence of the protein using conventional assays. For example, monoclonal antibodies immunoreactive with Chapl can be used to screen biological samples for the presence, concentration, and expression level of Chapl and, thereby, provide diagnostic information about Chapl-related diseases. Such immunological assays can be done in many convenient formats.

In one embodiment, antibodies are used to immunoprecipitate Chapl from solution and, in another embodiment, antibodies are used to react with Chapl on Western or Immunoblots of a polyacrylamide gel. in desirable embodiments, antibodies are used to detect Chapl in paraffin or frozen sections, using immunocytochemical techniques. Favored embodiments for detecting Chapl include enzyme-linked immunosorbant assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al., in U.S. Patent Nos. 4,376, 110 and 4,486,530.

In preferred protein-based diagnostic embodiments, antibodies of the present invention are attached to a support in an ordered array wherein a plurality of antibodies are attached to distinct regions of the support that do not overlap with each other. As with the nucleic acid-based arrays, the protein-based arrays are ordered arrays that are designed to be "addressable" such that the distinct locations are recorded and can be accessed as part of an assay procedure.

In some embodiments, addressable antibody arrays comprise a plurality of antibodies that recognize Chapl . These probes are joined to a support in different known locations. The knowledge of the precise location of each probe makes these "addressable" arrays particularly useful in binding assays. For example, an addressable array can comprise a support having several regions to which are joined a plurality antibody probes that recognize Chapl . Proteins from a preparation of several biological samples from a plurality of human subjects or a plurality of tissues or fluids from a single subject are labeled by conventional approaches (e.g., radioactivity, colorimetrically, or fluorescently) and the labeled samples are applied to the array under conditions that permit binding. If a protein in the sample binds to an antibody probe on the array, then a signal will be detected at a position on the support that corresponds to the location of the antibody-protein complex. Since the identity of each labeled sample is known and the region of the support on which the labeled sample was applied is known, an identification of the presence, concentration, and/or expression level is rapidly determined. That is, by employing labeled standards of a known concentration of Chapl, an investigator can accurately determine the protein concentration of Chapl in a sample and from this information can assess the expression level of Chapl. Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of Chapl . These approaches are easily automated using technology known to those of skill in the art of high throughput diagnostic analysis.

In another embodiment, an opposite approach to that presented above can be employed. Proteins present in biological samples (e.g., tissues or fluids from one or more subjects or one or more sources in a subject's body) can be disposed on a support so as to create an addressable array. Preferably, the protein samples are disposed on the support at known positions that do not overlap. The presence of a protein encoding Chapl in each sample is then determined by applying labeled antibody probes that recognize epitopes of Chapl and detecting a signal at locations on the array that correspond to the positions at which the biological samples were disposed. Because the identity of the biological sample and its position on the array is known, an identification of the presence, concentration, and/or expression level Chapl is rapidly determined. That is, by employing labeled standards of a known concentration of Chapl, an investigator can accurately determine the concentration of Chapl in a sample and from this information can assess the expression level of Chapl . Conventional methods in densitometry can also be used to more accurately determine the concentration or expression level of Chapl . These approaches are also easily automated using technology known to those of skill in the art of high throughput diagnostic analysis. As detailed above, any addressable array technology known in the art can be employed with this aspect of the invention and display the protein arrays on the chips in an attempt to maximize antibody binding patterns and diagnostic information. As discussed above, the presence or detection of Chapl can provide a diagnosis of a subject's disease state or predilection to disease and this information allows health professionals to create a disease state profile for a patient, begin aggressive treatment for the Chapl-related disease, and prevent further worsening of the condition. Similarly, further assays and reference to the changing disease state profile can help clinicians monitor the progress of a patient during treatment. That is, once a disease state is established, a therapeutic agent is administered and an initial disease state profile is generated. The assays above can be repeated on a regular basis to evaluate whether the values in the subject's disease state profile progresses toward or returns back to the initial disease state profile. Successive treatment profiles can be used to show the efficacy of treatment over a period of several days or several months

Additional embodiments include the preparation of diagnostic kits comprising detection components such as antibodies specific for Chapl or nucleic acid probes for detecting RNA encoding Chapl. The detection component will typically be supplied in combination with one or more of the following reagents. A support capable of absorbing or otherwise binding RNA or protein will often be supplied. Available supports for this purpose include, but are not limited to, membranes of nitrocellulose, nylon or derivatized nylon that can be characterized by bearing an array of positively charged substituents, and Genechips™ or their equivalents. One or more enzymes, such as Reverse Transcriptase and/or Taq polymerase, can be furnished in the kit, as can dNTPs, buffers, or non-human polynucleotides like calf-thymus or salmon-sperm DNA. Results from the kit assays can be interpreted by a healthcare provider or a diagnostic laboratory. Alternatively, diagnostic kits are manufactured and sold to private individuals for self -diagnosis.

In the discussion below, we describe several embodiments of the invention that have therapeutic or prophylactic application or both.

Therapeutic and Prophylactic Applications

The Chapl modulating agents of the invention are suitable for treatment of subjects either as a preventive measure to avoid a Chapl-related disease, or as a therapeutic to treat subjects already afflicted with the disease. Although anyone could be treated with the agents of the invention as a prophylactic, the most suitable subjects are people at risk for a Chapl-related disease. Such subjects include, but are not limited to, people with a family history of cancer or autoimmune disease.

The pharmacologically active compounds of this invention can be processed in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to patients, e.g., mammals including humans. The Chapl modulating agents can be incorporated into a pharmaceutical product with and without modification. Further, the manufacture of pharmaceuticals or therapeutic agents that deliver the Chapl modulating agent or a nucleic acid sequence encoding a Chapl modulating agent by several routes are aspects of the invention. For example, and not by way of limitation, DNA, RNA, and viral vectors having sequence encoding Chapl or a polypeptide fragment of Chapl, or a fragment of Stch that bind to a chaperone protein are within the scope of aspects of the present invention. Nucleic acids encoding a desired Chapl modulating agent can be administered alone or in combination with other Chapl modulating agents.

The compounds of this invention can be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application that do not deleteriously react with the Chapl modulating agents. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyetylene glycols, gelatine, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like that do not deleteriously react with the active compounds.

The effective dose and method of administration of a particular Chapl modulating agent formulation can vary based on the individual patient and the stage of the disease, as well as other factors known to those of skill in the art. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors that may be taken into account include the severity of the disease state of the patient, age, and weight of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Short acting pharmaceutical compositions are administered daily whereas long acting pharmaceutical compositions are administered every 2, 3 to 4 days, every week, or once every two weeks. Depending on half-life and clearance rate of the particular formulation, the pharmaceutical compositions of the invention are administered once, twice, three, four, five, six, seven, eight, nine, ten or more times per day. Routes of administration of the Chapl modulating agents include, but are not limited to, transdermal, parenteral, gastrointestinal, transbronchial, and transalveolar. Transdermal administration is accomplished by application of a cream, rinse, gel, etc. capable of allowing the Chapl modulating agent to penetrate the skin and enter the blood stream. Parenteral routes of administration include, but are not limited to, electrical or direct injection such as direct injection into a central venous line, intravenous, intramuscular, intraperitoneal or subcutaneous injection.

Gastrointestinal routes of administration include, but are not limited to, ingestion and rectal. Transbronchial and transalveolar routes of administration include, but are not limited to, inhalation, either via the mouth or intranasally.

Compositions of the Chapl modulating agents suitable for transdermal administration include, but are not limited to, pharmaceutically acceptable suspensions, oils, creams, and ointments applied directly to the skin or incorporated into a protective carrier such as a transdermal device ("transdermal patch"). Examples of suitable creams, ointments, etc. can be found, for instance, in the Physician's Desk Reference. Examples of suitable transdermal devices are described, for instance, in U.S. Patent No. 4,818,540 issued April 4, 1989 to Chinen, et al..

Compositions of the Chapl modulating agents suitable for parenteral administration include, but are not limited to, pharmaceutically acceptable sterile isotonic solutions. Such solutions include, but are not limited to, saline and phosphate buffered saline for injection into a central venous line, intravenous, intramuscular, intraperitoneal, or subcutaneous injection of the Chapl modulating agents.

Compositions of the Chapl modulating agents suitable for transbronchial and transalveolar administration include, but not limited to, various types of aerosols for inhalation. Devices suitable for transbronchial and transalveolar administration of the Chapl inhibiting agents are also embodiments. Such devices include, but are not limited to, atomizers and vaporizers. Many forms of currently available atomizers and vaporizers can be readily adapted to deliver Chapl modulating agents.

Compositions of the Chapl modulating agents suitable for gastrointestinal administration include, but not limited to, pharmaceutically acceptable powders, pills or liquids for ingestion and suppositories for rectal administration. Due to the ease of use, gastrointestinal administration, particularly oral, is the preferred embodiment of the present invention.

Several methods of treatment and prevention of a Chapl -related disease, which involve administration of the pharmaceutical embodiments of the invention are provided. In these aspects of the invention, Chapl, polypeptide fragments of Chapl, nucleic acids encoding these molecules, and agents that interact with a Chapl - chaperone complex are incorporated into pharmaceuticals and are administered to patients in need. By one approach, a subject at risk for contracting a Chapl-related disease or a subject afflicted with a Chapl-related disease is identified by conventional techniques or the diagnostic assays described above and then a therapeutically or prophylactically beneficial amount of Chapl or fragment of Chapl is administered.

In the description below, several of the materials and methods of the invention are provided.

EXAMPLE 1 Isolation of Stch binding proteins.

Human Stch cDNA (codons 2-467) was subcloned in-frame into the pGBT9 Gal4p DNA binding domain plasmid, (Clontech, Palo Alto, CA). The HF7c yeast strain was transformed with the pGBT9-Stch plasmid followed by sequential transformation of a human lung cD A library fused to the pGadl O Gal4p activation domain plasmid. Plasmids were isolated from yeast transformaπts on -L/W/H plates and subjected to nucleotide sequencing. Yeast strains were re-transformed with purified plasmids and multiple independent transformants were tested to confirm protein binding by β-galactosidase enzyme activity and by growth on SC-His media in all cases.

Functional analyses of Chap1/Dsk2. Yeast media and general techniques were as described previously (Rose et al, Methods in yeast genetics.

Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1990)). A wildtype S. cerevisiae strain, MY3492, and the dsk2 rad23 mutant strain, MY5156, were transformed with the following plasmids: pMR3429, pGAL vector alone; pMR2757 DSK2 CEN (Biggins et al., J Cell Biol, 133:1331-46 (1996)); pMR4647 (pGAL-human Chap1/Dsk2); pMR2905, pGAL-DSK2; and pMR2906, pGAL-DSK2-1. Serial dilutions of the transformants were incubated at the permissive or restrictive temperature and in the presence or absence of galactose and scored for growth. Cultures of yeast transformants were also grown in SC-ura galactose medium until early logarithmic phase as 30°C and were shifted to 37°C for 10 hours. Cells were fixed with methaπokacetone (3:1 ratio) on ice for 30 minutes and stained with DAPI on ice for 30 minutes. Greater than 100 cells were counted for each culture.

GST-Stch pull-down analysis with endogenous Bat3/Scythe

Giutathione-S-transferase (GST) fusion proteins were purified on sepharose beads as depicted and incubated with cellular extracts as previously reported (Thress et al., Embo J, 17:6135-43 (1998)). Recombinant GST protein alone or the indicated GST-fusion proteins were immobilized on glutathione sepharose beads and incubated in the presence of Xenopus egg extract for 1 hour at 4 °C. The beads were pelleted, washed three times with egg lysis buffer (50mM sucrose, 2.5mM MgCI2, 1.0mM DTT, 50mM KCI, 10mM Hepes pH 7.4), resuspended in SDS sample buffer and processed for immunoblotting using anti-peptide sera targeted against the C-terminal 16 aa of the Xenopus Scythe protein.

Protein sequence analysis The non-redundant database of protein sequences at the National Center for Biotechnology Information

(NCBI, NIH, Bethesda) was tested using the gapped BLASTP program and the Position-Specific Iterating (PSI) BLAST program (Altschul et al., Nucleic Acids Res, 25:3389-402 (1997)). Conserved signaling and interaction domains in protein sequences were identified using the SMART searching engine (Ponting et al., Nucleic Acids Res, 27:229-32

(1999)). Multiple sequence alignments were constructed using the ClustalW program (Thompson et al., Nucleic Acids Res, 22:4673-80 (1994)). The accession number for the amino acid sequences used: S. cerevisiae DSK2 (P48510), S. cerevisiae RAD23 (P32628), S. cerevisiae SSA4 (P22202), human Stch (U04735), and human BiP (P11021).

Although the invention has been described with reference to embodiments and examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All references cited herein are hereby expressly incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting the concentration or expression level of Chapl or Chapl in a biological sample from a tested subject comprising the step of comparing the concentration or expression level of a first sequence selected from the group consisting of Chapl gene, Chapl RNA, Chapl cDNA, and Chapl polypeptide from the biological sample with the concentration or expression level of a second sequence selected from the group consisting of Chapl gene, Chapl RNA, Chapl cDNA, and Chapl polypeptide from a healthy subject or Chapl gene, Chapl RNA, Chapl cDNA, and Chapl polypeptide from a subject afflicted with a Chapl-related disease.

2. A purified or isolated nucleic acid comprising the sequence of SEQ ID NO: 1 or a sequence complementary thereto.

3. A purified or isolated nucleic acid comprising at least 9 consecutive bases of the sequence of SEQ

ID NO.: 1 or a sequence complementary thereto, wherein the nucleic acid encodes a polypeptide that binds to a chaperone protein or wherein the nucleic acid complements a nucleic acid that encodes a polypeptide that binds a chaperone and, optionally, wherein said polypeptide is a Stil-like repeat sequence.

4. A purified or isolated nucleic acid encoding a polypeptide having the sequence of SEQ ID NO.: 2.

5. A recombinant construct comprising the coding region of SEQ ID NO.: 1 operably linked to a heterologous promoter.

6. A vector comprising the isolated DNA of Claim 2 or 3.

7. A vector comprising the isolated DNA of Claim 4.

8. An isolated nucleic acid molecule that hybridizes to SEQ. ID. NO. 1 at 37°C in the presence of 0.5M NaP04 (pH 7) and 7% SDS and under wash conditions of 37°C, in 6X SSC and 0.2% SDS, wherein the nucleic acid molecule has a sequence complementary to a sequence found in a gene that encodes a protein that binds to a chaperone that is at least 80% homologous to Chapl .

9. A purified or isolated protein comprising the sequence of SEQ ID NO.: 2.

10. The purified or isolated protein of Claim 9, wherein at least one acidic amino acid contained therein is replaced with a different acidic amino acid.

11. The purified or isolated protein of Claim 9, wherein at least one basic amino acid contained therein is replaced with a different basic amino acid

12. The purified or isolated protein of Claim 9, wherein at least one nonpolar amino acid contained therein is replaced with a different nonpolar amino acid.

13. The purified or isolated protein of Claim 9, wherein at least one uncharged amino acid contained therein is replaced with a different uncharged amino acid.

14. The purified or isolated protein of Claim 9, wherein at least one aromatic amino acid contained therein is replaced with a different aromatic amino acid.

15. A purified or isolated polypeptide comprising at least 3 consecutive amino acids of the sequence of SEQ ID NO.: 2, wherein the polypeptide binds to a chaperone and, optionally, wherein said polypeptide is a Stil-like repeat sequence.

16. A purified or isolated polypeptide comprising at least 3 consecutive amino acids of the sequence of SEQ ID NO.: 2, wherein the amino acid sequence of the polypeptide is found in molecule that binds to a chaperone that is at least 80% homologous to Chapl .

17. An isolated polypeptide, wherein the polypeptide is at least 80% identical to the polypeptide having the amino acid sequence of SEQ. ID. NO. 2 as determined by FASTA or BLAST using default opening and gap penalties and a PAM 250 scoring matrix.

18. A method of making a protein having the sequence of SEQ ID NO.: 2 comprising: obtaining a cDNA comprising the sequence in SEQ ID NO.: 1; inserting said cDNA in an expression vector such that said cDNA is operably linked to a promoter; and introducing said expression vector into a host cell whereby said host cell produces the protein encoded by said cDNA.

19. The method of Claim 18, further comprising isolating the protein.

20. An isolated Chapl polypeptide that promotes adhesion to a chaperone wherein the polypeptide is selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of SEQ. ID NO. 2 (b) a polypeptide encoded by the nucleic acid of Claim 2, 3, or 4; and

(c) a polypeptide that is at least 70% identical to the polypeptide of (a) or (b) as determined by FASTA or BLAST using default opening and gap penalties and a PAM 250 scoring matrix.

21. A method for constructing a transformed host cell that expresses SEQ ID NO.: 2 comprising transforming the host cell with a recombinant DNA vector comprising the sequence of SEQ ID NO.: 1.

22. A cultured cell line comprising the vector of Claim 6.

23. A cultured cell line comprising the vector of Claim 7.

24. A purified or isolated antibody capable of specifically binding Chapl.

25. The antibody of Claim 26, wherein the antibody is a monoclonal antibody.

26. An isolated or purified biological complex comprising Chapl and a chaperone.

27. A method of preparing a therapeutic agent comprising: providing an agent that modulates Chapl -dependent adhesion to a chaperone; and mixing with the agent a pharmaceutically acceptable carrier.

28. A method of treatment and prevention of a Chapl -related disease comprising: identifying a patient at risk for contracting a Chapl -related disease or a patient afflicted with a Chapl-related disease; and administering a therapeutically effective amount of the therapeutic agent of Claim 27.

29. An isolated or purified polypeptide fragment of a chapperone that binds to a ubiquitin-like protein.

30. The polypeptide of Claim 29, wherein the chapperone is Stch.

31. The polypeptide of Claim 29, wherein the fragment is the ATPase domain or portion thereof.

32. The polypeptide of Claim 30, wherein the chapperone is Stch.

33. A method of identifying a polymorphism that is linked to a Chapl -related disease comprising: identifying a first population of subjects that have a Chapl -related disease or a predilection to contract a Chapl-related disease- identifying a second population of subjects that do not have a Chapl-related disease or do not have a predilection to contract a Chapl -related disease; isolating a sample from said first and said second populations, wherein said sample comprises a nucleic acid encoding Chapl or a protein corresponding to Chapl; comparing said nucleic acid encoding Chapl or said protein corresponding to Chapl in said sample from said first population of subjects with said nucleic acid encoding Chapl or said protein corresponding to Chapl in said sample from said second population of subjects; and detecting a difference between said nucleic acid encoding Chapl or protein corresponding to

Chapl in said sample from said first population of subjects and said nucleic acid encoding Chapl or protein corresponding to Chapl in said sample from said second population of subjects, whereby said polymorphism that is linked to a Chapl-related disease is identified.

34. The method of Claim 31 , wherein said Chapl-related disease is a neurodegenerative disease.

35. The method of Claim 31 , wherein said Chapl-related disease is Alzheimer's disease or Spirocerebellar ataxia.

36. An isolated or purified biological complex comprising Chap2 or a fragment thereof and a chaperone.