Human actin regulatory proteins and methods for detection,...

Details Human actin regulatory proteins and methods for detection,... Human actin regulatory proteins and methods for detection,...

1999-10-15

2002-06-11

Huff, Sheela (Department: 1642)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C530S380000

Reexamination Certificate

active

06403766

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the field of control of cytoskeletal structure and changes in the cytoskeletal structure, especially as it relates to the regulation of cell motility, transformation and tumorigenesis. Specifically the invention relates to human actin-binding regulatory proteins, nucleic acids encoding these proteins, epitopes of these proteins and antibodies specific for these epitopes. The invention also relates to screening methods for the identification of potential drug candidate molecules and the use of such molecules in cancer therapy and the treatment of other disorders of cell motility, cell proliferation, wound healing, growth and division.
BACKGROUND
The conversion of epithelial cells from sessile, non-dividing cells in monolayers to motile, proliferating cells of invasive carcinomas must be tightly coupled to highly regulated rearrangements of the actin cytoskeleton. In malignant carcinoma tumors, invasion of transformed epithelial cells into the underlying connective tissue occurs by cell migration (References 1-3). Metastasis of carcinoma tumors also involves cell migration from the primary tumor site into blood vessels by diapedesis through the vessel endothelium (Ref. 2).
Migration of metastatic tumor cells was clearly described by Waldeyer in 1872 as amoeboid movement (Ref. 4), a form of cell motility that requires coordinated mobilization and remodeling of the actin cytoskeleton by actin-binding proteins (Refs. 5-10). An initial step in cortical actin cytoskeleton rearrangement includes site-specific actin polymerization onto actin filament ends that have been generated by severing or uncapping of existing filaments (Ref. 11). Two families of actin filament fragmenting/capping proteins are presently recognized, the severin/fragmin/gelsolin family containing shared 125 amino acid repeat domains (Refs. 12-16), and the actin depolymerization factor family of ADF (Ref. 17), depactin (Ref. 18), destrin (Ref. 19), and actophorin (Ref. 20). Severin from Dictyostelium amoebae (Refs. 21,22) and fragmin in Physarum slime molds (Ref. 23) are the earliest phylogenetic examples of actin filament fragmenting proteins. The parallel actin severing protein in mammalian cells is gelsolin, an 80 kDa protein derived from duplication of the ancestral severin gene (Ref 24). A cytoplasmic gelsolin is expressed in epithelial cells, fibroblasts and leucocytes, and secreted plasma gelsolin is present in blood (Refs. 5,6,25). In gelsolin, it is the conservation of severin amino acid sequences that accounts for the actin filament severing activity (Refs. 13,26,27).
Gelsolin is implicated in mammalian cell motility by the demonstration that increased expression of gelsolin in fibroblasts by gene transfection proportionally enhances the rate of migration (Ref. 28). Actin binding protein, ABP 120, has also been implicated in cell motility by functional phenotype analysis (Refs. 29,30). Paradoxically, despite the heightened migratory behavior of invasive tumor cells, gelsolin is extensively down-regulated during transformation of mammary epithelium and fibroblasts (Refs. 31,32).
The following patents and scientific publications may be useful in practicing the full scope of the invention. These patents are incorporated herein by reference in their entirety. The scientific literature is cited to give an indication of the available art known to the skilled artisan in the field. These patents and publications are provided for illustrative purposes.
U.S. Pat. No. 5,374,544 is entitled “Mutated skeletal actin promoter.”U.S. Pat. No. 5,464,817 is entitled “Methods for reducing the viscosity of pathological mucoid airway contents in the respiratory tract comprising addministering actin-binding compounds with or without DNAse I.”
U.S. Pat. No. 5,508,265 entitled “Therapeutic uses of actin-binding compounds” discloses the use of actin-binding compounds, including gelsolin and biologically active fragments thereof in the treatment of actin-related disorders.
U.S. Pat. No. 5,593,964 is entitled “Methods of treating septic shock by preventing actin polymerization.”
U.S. Pat. No. 5,656,589 is entitled “Method for the reduction of viscous purulent airway contents in the respiratory tract comprising administering actin-binding compounds with or without DNAse I.”
U.S. Pat. No. 5,851,786 is entitled “Product and process to regulate actin polymerization.”
U.S. Pat. No. 5,071,773 entitled “Hormone receptor-related bioassays” discloses assay methods using transcriptional reporter genes generally useful for high throughput screening. Such screens may be adapted for use of assays employing genes encoding actin-binding and regulatory proteins in addition to the steroid hormone receptors which act as transcription factors.
U.S. Pat. No. 5,401,629 discloses further screening methods using readouts based on detecting changes in the transcription of reporter genes engineered to express a detectable signal in response to activation by intracellular signaling pathways.
U.S. Pat. No. 5,482,835 entitled “Methods of Testing in Yeast Cells for Agonists and Antagonists of Mammal G protein-Coupled Receptors” discloses methods for screening;
U.S. Pat. No. 5,747,267 also discloses yeast screens and is entitled “Method for Identifying a G Protein-Coupled Glutamate Receptor Agonist and Antagonist”;
U.S. Pat. No. 5,750,353 entitled “Assay for Non-peptide Agonists to Peptide Hormone Receptors” discloses further screening methods; as does U.S. Pat. No. 5,925,529 entitled “Method for Discovery of Peptide Agonists”;
U.S. Pat. No. 5,744,303 is entitled “Functional Assays for Transcriptional Regulator genes”; and U.S. Pat. No. 5,569,588 discloses “Methods for Drug Screening”.
Andre, E. A., M. Brink, G. Gerisch, G. Isenberg, A. Noegel, M. Schleicher, J. E. Segall, and E. Wallraff. 1989.
J. Cell Biol.
108: 985-995. Is entitled: “A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis”.
Yin, H. L. et al. 1990. FEBS LETT. 264(1): 78-80 is entitled “Severin is a gelsolin phenotype”.
Jones, J. G., J. Segal and J. Condeelis. 1991.
Experientia
-
Suppl.
59: 1-16 is entitled “Molecular analysis of aioeboid chemotaxis: parallel observations in amoeboid phagacytes and metastatic tumor cells.”
Eichinger et al. 1991. J. Cell. Biol. 112(4): 665-76 is entitled “Domain structure in actin-binding proteins: expression and functional characterization of truncated severin.”
Prendergast, G. C. and E. B. Ziff 1991. EMBO J. 10(4): 757-66 is entitled “Mbh1: a novel gelsolin/severin-related protein which binds actin in vitro and exibits nuclear localization in vivo.”
Finidori et al. 1992. J. Cell. Biol. 116(5): 1145-55 is entitled “In vivo analysis of functional domains from villin and gelsolin.”
Eichinger, L. and M. Schleicher. 1992. Biochemistry 31(20) 4779-87 is entitled “Characterization of actin- and lipid-binding domains in severin, a Ca(2+)-dependent F-actin fragmenting protein.”
Schnuchel et al. 1995. J. Mol. Biol. 247(1): 21-7 is entitled “Structure of severin domain 2 in solution.”
Folger, P. A. 1996. Ph.D. thesis, Cornell University, entitled “Identification, isolation and expression of M-severin, a novel actin filament severing preotein in murine carcinoma tumors.”
Markus et al. 1997. Protein Sci. 6(6): 1197-1209 is entitled “Refined structure of villin 14T and a detailed comparison with other actin-severing domains.”
Eichinger, L. et al. 1998. J. Biol. Chem. 273(21): 12952-9 is entitled “Characterization and cloning of a Dictyostelium Ste20-like protein kinase that phosphorylates the actin-binding protein severin.”
Weber, I., Niewohner, J., and Faix, J. 1999. Biochem. Soc. Symp. 65:245-65 is entitled “Cytoskeletal protein mutations and cell motility in Dictyostelium.”
Despite a longfelt need to isolate the human actin regulatory proteins corresponding to M-severin and M-30 these these proteins have not been provided until the disclosure of the present invention.
Nowhere in these references is there any disclosure, suggestion or even hint of the use of

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