Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-05-07
2002-01-01
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007100, C435S007200, C435S007400, C435S007940, C435S069100, C250S281000
Reexamination Certificate
active
06335157
ABSTRACT:
1. FIELD OF THE INVENTION
The present invention is directed to methods for screening for fragments and derivatives of Hsp90 that are altered in their subcellular localization, and thus, altered in their activity as compared to wild type Hsp90. The present invention is also directed to screening for modulators of Hsp90 activity as determined by assaying for altered subcellular localization of Hsp90. The present invention is further directed to diagnostic, prognostic and screening uses of Hsp90/centrosome co-purification.
2. BACKGROUND OF THE INVENTION
The Hsp90 family of proteins is a group of highly conserved stress proteins that are expressed in all eukaryotic cells, and are essential in yeast and Drosophila. For a general review of heat shock proteins, see Parsell and Lindquist, 1993, Ann. Rev. Genet. 27:437-496. Hsp90 is one of the most abundant proteins in the eukaryotic cell, constituting up to about 1-2% of the cellular protein under normal physiologic conditions, and its expression is increased several-fold in response to stress. Up to now, members of the Hsp90 family were found in the cytosol, the endoplasmic reticulum, and chloroplasts; however, evidence for the actual subcellular localization in the cell or possible association with cellular organelles has so far been cryptic and at best contradictory. Scheibel and Buchner, 1997, The Hsp90 Family—An Overview, In:
Guidebook to Molecular Chaperones and Protein Catalysts
, Oxford University Press, pp. 147-151. Although family members of Hsp90 have interchangeable functions, the respective genes are differentially regulated in eukaryotes. Borkovich et al., 1989, Mol. Cell Biol. 9:3919-3930. In most eukaryotic cells, one of the two Hsp90 family members is expressed constitutively at a high level at physiological temperature and is induced only 2-3 times by heat shock. A second family member is expressed at a low basal level at normal temperatures, but its expression is enhanced strongly under restrictive growth conditions, like heat treatment. See, Borkovich et al., 1989, Mol. Cell Biol. 9:3919-3930; Krone and Sass, 1994, Biochem. Biophys. Res. Commun. 204:746-752.
The two genes that encode Hsp90 in humans, Hsp90&agr; and Hsp90&bgr;, are 86% homologous. Further, there is extensive homology with lower species. The 63 kDa Hsp90 homolog in
Escherichia coli
is 42% identical in amino acid sequence to human Hsp90. The 83 kDa Hsp90 protein homolog of Drosophila (Hsp83) is 78% identical/similar to human Hsp90. See, e.g., Alique et al., 1994, EMBO J. 13:6099-6106; Rebbe et al., 1987, Gene 53:235-245; Blackman et al., 1986, J. Mol. Biol. 188:499-515.
The Hsp90 family has been implicated as an important component of intracellular signaling pathways as well as in assisting protein folding. Dimeric Hsp90 proteins bind molecules such as steroid hormone receptors and the receptor kinases v-src, Raf, and casein kinase II (Catelli et al., 1985, EMBO J. 4:3131-3135; Miyata and Yahara, 1992, J. Biol. Chem. 267:7042-7047; Stancato et al., 1993, J. Biol. Chem. 268:21711-21716; Xu and Lindquist, 1993, Proc. Natl. Acad. Sci, USA 90:7074-7078; Wartmann and Davis, 1994, J. Biol. Chem. 269:6695-6701; van der Straten et al., 1997, EMBO J. 16:1961-1969). In the case of steroid receptors, this interaction is required for efficient ligand binding and transcriptional regulation (Bohen and Yamamoto, 1994, Modulation of Steroid Receptor Signal Transduction by Heat Shock Proteins, In:
The Biology of Heat Shock Proteins and Molecular Chaperones
, Cold Spring Harbor Laboratory Press, pp. 313-334).
Although the molecular structure of Hsp90 is known in detail and a long list of interacting partners is known, very little is known about the subcellular localization of Hsp90 except that Hsp90 is observed in the cytoplasm and nucleus.
Citation or identification of any reference in Section 2 or in any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present inventors have discovered that at least a significant portion of the Hsp90 molecules in a cell are localized to, and are a core component of (tightly associated with) the centrosome. Further, the localization of Hsp90 to the centrosome, and thus, its function and role in mitosis and fidelity of chromosome segregation, is conserved over evolution. Hsp90 is known to play important roles in the control of cell signaling and the cell cycle, as well as in transcription, therefore the detection of fragments and derivatives of Hsp90 that have altered localization identifies those Hsp90 molecules that have different activities, and allows for the screening of molecules that alter Hsp90 activity by altering its location.
The present invention is directed to methods of identifying a molecule that alters the centrosomal location of Hsp90 in a cell comprising the following steps in the order stated: (a) contacting the cell with one or more candidate molecules; and (b) detecting localization of Hsp90 molecules in the cell, wherein an increase or decrease in the amount of Hsp90 localized to the centrosome relative to said amount in a cell not so contacted with the one or more candidate molecules indicates that the candidate molecules alter the centrosomal localization of Hsp90. In another embodiment, the present invention is directed to methods of identifying a molecule that alters the centrosomal location of Hsp90 in a cell comprising the following steps in the order stated: (a) recombinantly expressing within the cell one or more candidate molecules; and (b) detecting localization of Hsp90 molecules in the cell, wherein an increase or decrease in the amount of Hsp90 localized to the centrosome relative to said amount in a cell in which the one or more candidate molecules were not so expressed indicates that the candidate molecules alter the centrosomal localization of Hsp90. In a specific aspect of either embodiment, step (b) comprises contacting the cell with an antibody to Hsp90 or a binding region of said antibody, and a fluorescently labeled binding partner of said antibody under conditions conducive to immunospecific binding. In an alternative aspect, the method of detecting comprises contacting the cell with a fluorescently labeled antibody to Hsp90 or a binding region of said antibody under conditions conducive to immunospecific binding. In yet another embodiment, the method of detecting comprises sequencing by mass spectroscopy centrosomal proteins isolated from the cell.
In another embodiment of the present invention, methods of identifying a molecule that affects Hsp90 function in a cell are provided which comprise the following steps in the order stated: (a) contacting the cell with one or more candidate molecules; and (b) detecting or measuring altered centrosomal or chromosomal structure or function, wherein an alteration of centrosomal or chromosomal structure or function relative to said structure or function in a cell not so contacted with the one or more candidate molecules indicates that the candidate molecules affects Hsp90 function. In an alternative embodiment, the method of identifying a molecule that affects Hsp90 function in a cell comprises the following steps in the order stated: (a) recombinantly expressing within the cell one or more candidate molecules; and (b) detecting or measuring altered centrosomal or chromosomal structure or function, wherein an alteration of centrosomal or chromosomal structure or function relative to said structure or function in a cell in which the one or more candidate molecules were not so expressed indicates that the candidate molecules affects Hsp90 function. In a specific aspect of either embodiment, the altered centrosomal or chromosomal structure or function is evidenced by an aberrant mitotic figure, which mitotic figure includes but is not limited to monopolar spindles, aneuploydies, chromosomal missegregation, or chromosome non-joinder.
In yet another embodiment, the present invention is directed to methods of identifying a deri
Gonzalez Cayetano
Lange Bodo
Caputa Anthony C.
Nickol Gary B.
Pennie & Edmonds LLP
The European Molecular Biology Laboratory
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