Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-12-01
2001-10-23
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S184000, C514S025000, C514S026000, C514S027000, C514S028000, C514S029000, C514S030000, C514S031000, C514S443000, C530S351000, C530S396000, C530S399000
Reexamination Certificate
active
06306613
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to leaderless protein export pathway and to modulators of leaderless protein export, and more specifically, to methods of identifying components of the leaderless protein export pathway and compounds that increase or decrease export of leaderless proteins into the extracellular environment.
BACKGROUND OF THE INVENTION
Many proteins exert an effect on cell growth, differentiation, and inflammation through signal transduction, mediated by binding to a cell surface receptor. Yet other proteins, such as factors that initiate or are necessary for blood clot formation, act enzymatically in blood. While these actions are generally part of normal processes, tinder certain circumstances, it may be desirable to limit or inhibit the action of certain proteins and the effects of subsequent signaling. For example, tumor growth that is promoted by a growth factor, such as bFGF acting on melanoma cells, is deleterious and often leads to fatalities.
Approaches to inhibit specific proteins have concentrated primarily on interfering with protein-substrate or protein-receptor interactions. Typically, this involves using an antibody or other molecule that competitively binds the protein, by administration of competitors for receptor binding, or by protease digestion of the protein. An alternative approach, not generally pursued, is to reduce the level of the protein by inhibiting its expression at a transcriptional or translational level. Methods of reducing protein levels by inhibiting the transcription or translation of the protein have been difficult to achieve because of inherent problems of inhibiting the specific expression of one or a few proteins.
The discovery that certain proteins, such as growth factors, mediators of inflammation, and mediators of blood clotting, are exported through a nonclassical secretory pathway allows the development of specific inhibitors for these proteins. These proteins are identified by their lack of a hydrophobic leader sequence that mediates secretion by the classical ER/Golgi pathway.
This invention provides modulators of the export of these leaderless proteins, allowing control of undesired proliferation and inflammation, as well as other related advantages.
SUMMARY OF THE INVENTION
The present invention generally provides methods of modulating the export of a leaderless protein from a cell and of identifying one or more components of a cell transport pathway. In one aspect, methods are provided for decreasing export of a leaderless protein from a cell, by contacting a cell with an effective amount of a modulator, wherein the modulator directly or indirectly prevents formation of or alters the stability of a complex or indirectly alters export, where the complex comprises a leaderless protein and a transport molecule, thereby decreasing export of the leaderless protein from the cell. In certain preferred embodiments, the transport molecule may comprise a gastrin binding protein/alpha subunit of mitochondrial fatty acid &bgr;-oxidation multienzyme complex (p70, GenBank Accession Nos. U04627/D16480), a phosphotyrosine-independent ligand of the SH2 domain of p56lck (p62, GenBank Accession No. U46751), mitochondrial fatty acid &bgr;-oxidation trifunctional protein &bgr; subunit (TP-&bgr;) (p48, GenBank Accession No. D16481), actin related protein 3 (Arp3) (p48, GenBank Accession No. U29610), K-glypican (GenBank Accession No. X83577), tubulin (p50, GenBank Accession No. AF081484) and related polypeptides that are functionally equivalent in their role as leaderless protein trafficking components. In certain other embodiments, the cell may be bacterial, yeast, plant, COS-1, BHK, CHO, HeLa, 293, NS-1, HepG2, J744, HEC-1-A, HEC-1-B, 3T3, D10.G4.1, P388D
1
, 5637, SK-HEP-1, THP-1, Caco-2, MDCK, Jurkat, U87, LnCap, primary tumor biopsies, and tumor derived cell lines. In yet other embodiments, the leaderless protein may be FGF-1, FGF-2, IL-1&agr;, IL-1&bgr;, aldose reductase, PD-ECGF, CNTF, prothymosin ax, parathymosin, galectin-1, Factor XIIIa, ATL-derived factor, annexin-1, transglutaminase, mammary-derived growth inhibitor, macrophage migration inhibitory factor (MIF), HIV tat, ATP synthase, aminoacyl-tRNA synthetase, EMAP, rhodanase, and thioredoxin-like protein.
In another aspect, modulators that decrease export of a leaderless protein from a cell are provided. The modulator should decrease export of a leaderless protein; should not inhibit secretion of a leader sequence-containing protein; and should alter the stability of a complex, the complex comprising a leaderless protein and a transport molecule. In certain preferred embodiments, the transport molecule may include, for example, a gastrin binding protein/alpha subunit of mitochondrial fatty acid &bgr;-oxidation multienzyme complex (p70, GenBank Accession Nos. U04627/D16480), a phosphotyrosine-independent ligand of the SH2 domain of p56lck (p62, GenBank Accession No. U46751), mitochondrial fatty acid &bgr;-oxidation trifunctional protein &bgr; subunit (TP-&bgr;) (p48, GenBank Accession No. D16481), actin related protein 3 (Arp3) (p48, GenBank Accession No. U29610), K-glypican (GenBank Accession No. X83577), tubulin (p50, GenBank Accession No. AF081484) and related polypeptides that are functionally equivalent in their role as leaderless protein trafficking components.
In yet another aspect, methods are provided for detecting one or more components of a cell transport pathway, by contacting cell extracts or cell membranes containing components of a cell transport pathway with a fusion protein of a transport molecule and a tag, to form a complex of the fusion protein with one or more components of the cell transport pathway; isolating the complex; and detecting one or more components of the cell transport pathway in the complex. In certain embodiments, one or more components of the cell transport pathway may include a leaderless protein and/or a transport molecule. In certain preferred embodiments, the transport molecule may include, for example, a gastrin binding protein/alpha subunit of mitochondrial fatty acid &bgr;-oxidation multienzyme complex (p70, GenBank Accession Nos. U04627/D16480), a phosphotyrosine-independent ligand of the SH2 domain of p56lck (p62, GenBank Accession No. U46751), mitochondrial fatty acid &bgr;-oxidation trifunctional protein &bgr; subunit (TP-&bgr;) (p48, GenBank Accession No. D16481), actin related protein 3 (Arp3) (p48, GenBank Accession No. U29610), K-glypican (GenBank Accession No. X83577), tubulin (p50, GenBank Accession No. AF081484) and related polypeptides that are functionally equivalent in their role as leaderless protein trafficking components. In certain other embodiments, the tag may be glutathione-S-transferase or a fragment thereof that binds glutathione.
In addition, methods for decreasing export of a leaderless protein are provided, comprising contacting a cell with an effective amount of a modulator, the modulator comprising a nucleic acid molecule capable of binding and reducing translation of RNA encoding a transport molecule, wherein the transport molecule may be a gastrin binding protein/alpha subunit of mitochondrial fatty acid &bgr;-oxidation multienzyme complex (p70, GenBank Accession Nos. U040271D16480), a phosphotyrosine-independent ligand of the SH2 domain of p56lck (p62, GenBank Accession No. U46751), mitochondrial fatty acid &bgr;-oxidation trifunctional protein &bgr; subunit (TP-&bgr;) (p48, GenBank Accession No. D16481), actin related protein 3 (Arp3) (p48, GenBank Accession No. U29610), K-glypican (GenBank Accession No. X83577), tubulin (p50, GenBank Accession No. AF081484) and related polypeptides that are functionally equivalent in their role as leaderless protein trafficking components. In one embodiment, the nucleic acid molecule may be DNA or RNA encoding at least 10 nucleotides (e.g., antisense molecules) of a transport molecule RNA.
Methods are also provided for identifying a compound that modulates leaderless protein export, which comprises contacting a cell with a candidate compoun
Baird Andrew
Florkiewicz Robert Z.
Warnock Dale E.
Baskar Padma
Ciblex Corporation
Seed Intellectual Property Law Group PLLC
Smith Lynette R. F.
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