Metalloproteinases

Details Metalloproteinases Metalloproteinases

1999-08-11

2001-11-06

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S212000, C435S226000

Reexamination Certificate

active

06312937

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to novel metalloproteinases. More specifically, isolated nucleic acid molecules are provided encoding: (1) a human TNF-alpha converting enzyme (“TACE”)-like protein, and (2) a matrilysin-like protein. TACE-like and matrilysin-like polypeptides are also provided, as are vectors, host cells, and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of the activity of these two enzymes. Also provided are diagnostic and therapeutic methods for detecting and treating diseases or disorders that involve these enzymes.
2. Related Art
Tumor necrosis factor-alpha (TNF-) is a potent cytokine, secreted primarily by activated monocytes and macrophages, that contributes to a variety of inflammatory disease states and is broadly involved in immunomodulation. TNF-alpha is processed from an immature, membrane-bound form to a mature, secreted form by a metalloproteinase called TNF-alpha converting enzyme, or “TACE.” See, R. A. Black et al.,
Nature
385:729-733 (February 1997); M. L. Moss et al.,
Nature
385:733-736 (February 1997); M. L. Moss et al.,
J. Neuroimmunol
. 72:127-129 (February 1997). Inhibitors of the enzyme TACE block secretion of TNF-alpha.
TACE is a new member of a protein family called “ADAMs” (proteins which contain A Disintegrin And Metalloprotease domain; also called adamalysins). See, Wolfsberg et al.,
Dev. Bio
. 169:378-383 (1995).
The TACE/ADAM family is composed of membrane proteins with structural homology to the snake venom metalloproteases and disintegrins. Snake venom disintegrins are a family of anticoagulant peptides with a high cysteine content. A new member of TACEIADAM in Drosophila, called the kuzbanian gene (“KUZ”), was found to be involved in Drosophila neurogenesis (Rooke, J. et al.,
Science
273:1227-1231 (August 1996)).
Approximately 11-13 ADAM genes have now been identified, including fertilin alpha and beta (involved in the integrin mediated binding and fusion of egg and sperm; previously known as PH-30 alpha and beta), epididymal apical protein I, cyritestin, MDC (a candidate for tumor suppressor in human breast cancer), meltrin- (mediates fusion of myoblast fusion in the process of myotube formation), MS2 (a macrophage surface antigen), and metargidin. Typical ADAMs are cell surface proteins which consist of pro-, metalloprotease-like, disintegrin-like, cysteine-rich, epidermal growth factor-like repeat, transmembrane and cytoplasmic domains.
A new ADAM family gene, named ADAMTS-1, containing a disintegrin and metalloproteinase domain with thrombospondin (TSP) motifs, has now been shown to be closely associated with various inflammatory processes, as well as development of cancer cachexia. Kuno, K. et al.,
J. Biol. Chem
. 272:556-562 (1997).
The disintegrin domain of ADAM family proteins functions in the prevention of integrin-mediated cell to cell and cell to matrix interactions, such as platelet aggregation, adhesion, and migration of tumor cells or neutrophils, and angiogenesis. Previously described disintegrins, such as contortrostatin (Trikha et al.,
Cancer Research
54:4993-4998 (1994) have been used to inhibit human metastatic melanoma (M24 cells) cell adhesion to type I collagen, vitronectin, and fibronection, but not laminin. Further, contortrostatin inhibits lung colonization of M24 cells in a murine metastasis model.
The matrix metalloproteinases (MMPs) compose a family of structurally similar zinc-dependent enzymes that degrade all of the major components of the extracellular matrix. MMPs include the collagenases, gelatinases A and B, the stromelysins, matrilysin, metalloelastase, and the membrane-type matrix metalloproteinases. Over-expression and activation of MMPs have been linked with a range of diseases, such as arthritis, cancer, and multiple sclerosis. Regarding cancer, although MMPs classically have been implicated in basement membrane destruction associated with late-stage tumor cell invasion and metastasis, one MMP member, matrilysin, has recently been shown to be expressed in early stage human colorectal tumors. (C. L. Wilson et al.,
Proc. Natl. Acad. Sci
. USA 94:1402-1407 (February 1997)).
Clearly, members of the TACE/ADAM family of proteins have a high potential for becoming valuable therapeutically and diagnostically. ADAM proteins, peptides derived from the sequence of ADAM proteins, and ADAM protein antagonists may become desirable components of molecular methods of assisting or preventing fertilization. Furthermore, specific TACE/ADAM proteins or derivatives may be useful in the detection and prevention of muscle disorders. TACE-like proteins also have an exciting potential potential in the treatment of inflammation, thrombosis, cancer, and cancer metastasis. TACE-like factors, or antagonists thereof, may also become usefull agents in promoting macrophage or T-cell adhesion to matrices or cells' access to bound cytokines and other regulatory molecules.
Inhibitors of members of the matrix metalloproteinase (MMP) family (such as matrilysin) have been studied in the treatment or prophylaxis of cancer, cancer metastasis, as well as in the treatment of arthritis, corneal ulcers, pleural effusion, arid multiple sclerosis. For a review on recent advances in matrix metalloproteinase research, see, Beckett, R. P. et al., DDT 1:16-26 (January 1996).
Clearly, there is a need in the art for novel TACE-like and matrilysin-like molecules, exhibiting sequence relatedness to known metalloproteinases with recognized therapeutic and diagnostic usefulness.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the TACE-like polypeptide having the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 209042 on May 15, 1997. The present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the matrilysin-like polypeptide having the amino acid sequence shown in SEQ ID NO:5 or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 209055 on May 16, 1997.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of TACE-like or matrilysin-like polypeptides or peptides by recombinant techniques.
The invention fer provides isolated TACE-like or matrilysin-like polypeptides having an amino acid sequence encoded by a polynucleotide described herein.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by the TACE-like or matrilysin-like protein, which involves contacting cells which express the TACE-like or matrilysin-like protein with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
The invention provides a diagnostic method useful during diagnosis of cancer.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of TACE-like activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated TACE-like polypeptide of the invention or an agonist thereof.
An additional aspect of the invention is related to a method for treating an individual in need of a decreased level of TACE-like activity in the body comprising administering to such an individual a composition comprising

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