| Method for pr-39 peptide mediated selective inhibition of ikappabalpha degradation -> Monitor Keywords |
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Method for pr-39 peptide mediated selective inhibition of ikappabalpha degradationRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test StripMethod for pr-39 peptide mediated selective inhibition of ikappabalpha degradation description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070065804, Method for pr-39 peptide mediated selective inhibition of ikappabalpha degradation. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE [0001] The present application is a Continuation-In-Part of prior pending U.S. patent application Ser. No. 09/276,868 filed Mar. 26, 1999 and 09/426,011 filed Oct. 25, 1999. FIELD OF THE INVENTION [0003] The present invention is concerned generally with the Rel/NF.kappa.B family of transcription factors within viable cells comprising living tissues and organs; and is particularly directed to proteasome mechanisms regulated by PR-39 peptides which result in a selective inhibition of I.kappa.B.alpha. degradation on-demand and may be used for controlled suppression of NF.kappa.B transcription factor activity and NF.kappa.B-dependent gene expression. BACKGROUND OF THE INVENTION [0004] Degradation of proteins in mammalian cells proceeds via two distinct pathways, the lysosome-dependent and proteasome-dependent systems. The proteasome-dependent system catalyzes the hydrolysis of proteins marked for degradation typically by conjugation to ubiquitin, but also can degrade certain non-ubiquitinated proteins as well. [0005] Proteasome-mediated degradation is also a principal means for controlling the intracellular levels of most cell proteins, including the recognized major regulators of gene expression such as NF.kappa.B transcription factor; the inhibitor protein I.kappa.B.alpha.; hypoxia-inducing factor (HIF)-1a; protooncogenes c-Fos, c-Jun and c-Mos; and the various cyclins. See for example: Whiteside, S. T. and A. Israel, Semin. Cancer Biol. 8: 75-82 (1997); Srinivas et al., J. Biol. Chem. 273: 18019-18022 (1998); Salceda, S. and J. Caro, J. Biol. Chem. 272: 22642-22647 (1997); Huang et al., Proc. Natl. Acad. Sci. USA 95: 7987-7992 (1998); He et al., J. Biol. Chem. 273: 25015-25019 (1998); and Pahl, H. L. and P. A. Baeverle, Curr. Opin. Cell Biol. 8: 340-347 (1996). Furthermore, the smaller-sized peptides generated by the proteasome during the course of protein breakdown are often biologically active; for example, some peptides are presented as antigens on the class I major histocompatibility complex (MHC) [Rock, K. L. and A. L. Goldberg, Annu. Rev. Immunol. 17: 739-779 (1999)]. These degradation products thus can cause different effects and major consequences in a variety of cellular processes, many of which have substantive clinical value. [0006] In particular, NF.kappa.B-dependent gene expression is recognized as playing an important role in a number of biological processes of major medical importance including immune, inflammatory and anti-apoptotic responses [Baeuerle, P. A. and D. Baltimore, Cell 87: 13-20 (1996); Beg, A. A. and D. Baltimore, Science 274: 782-784 (1996); and Antwerp et al., Science 274: 787-789 (1996)]. NF.kappa.B is a dimer molecule composed of the p50 and p65 (RelA) monomer subunits; and binding of this dimer complex to I.kappa.B inhibitor protein in a cytosol is believed to be the main cellular mechanism preventing NF.kappa.B-dependent transcription of genes under normal conditions. A number of different extracellular stimuli (including TNF.alpha., Il-1 and lipopolysaccharide) can trigger NF.kappa.B transcription factor activation, most notably by causing a rapid degradation of I.kappa.B inhibitor protein by the ubiquitin (Ub)-proteasome degradation pathway. [0007] Several steps necessary for proteasome-mediated I.kappa.B.alpha. degradation to occur have been identified. These include: phosphorylation of I.kappa.B.alpha. at two sites by a specific I.kappa.B.alpha. kinase of the SCF1 family [Whiteside, S. T. and A. Ismael, Semin. Cancer Biol. 8: 75-82 (1997); Chen et al., Cell 84: 853-862 (1996)]; the ubiquitination of the phosphorylated I.kappa.B.alpha. by a specific E3 enzyme complex [Suzuki et al., Biochem. Biophys. Res. Comm. 256: 121-126 (1996); Spencer et al., Genes Dev. 13: 284-294 (1999); Kroll et al., J. Biol. Chem. 274: 7941-7945 (1999); Yaron et al., Nature 396: 590-594 (1998); and Gonen et al., J. Biol. Chem. 274: 14923-14830 (1999)]; and the subsequent binding to VCP (valosin-containing protein) that results in a physical link between the ubiquitinated I.kappa.B.alpha. and the proteasome. [0008] Separate and distinct from these events is the PR-39 protein. PR-39 is a highly basic arginine/proline-rich peptide originally isolated from porcine intestine on the basis of its anti-bacterial activity [Agerbeth et al., Eur. J. Biochem. 202: 849-854 (1991)]. The PR-39 peptide is secreted in a prepro-protein form that includes a canonical leader sequence and rapidly undergoes cleavage of the N-terminal portion to generate the mature form composed of the 39 C-terminal amino acids [Gudmundsson et al., Proc. Natl. Acad. Sci. USA 92: 7085-7095 (1995)]. While the sequence of the N-terminal part of the prepro-protein is highly homologous to the cathelin gene family members, the sequence of the 39 C-terminal amino acids that make up the mature peptide, has no homology to any other known protein. [0009] Research investigations have shown that PR-39 protein can rapidly cross cell membranes; and, by virtue of its proline-rich composition, may interact with SH3 domains of p47.sup.phox and p130.sup.Cas [Ross et al., Proc. Natl. Acad. Sci. USA 93: 6014-6018 (1996); and Chan, Y. R. and R. L. Gallo, J. Biol. Chem. 273: 28978-28985 (1998)]. The PR-39 peptide (predominantly produced by blood-derived macrophages) is found at the sites of active inflammation including skin wounds and myocardial infarction and is seen as playing an important role by inducing expression of heparan sulfate-carrying core proteins, syndecan 1 and 4 [Li et al., Circ. Res. 81: 785-796 (1997); and Gallo et al., Proc. Natl. Acad. Sci. USA 91: 11035-11039 (1994)] and inhibiting degradation of the hypoxia-inducible factor (HIF)-1.alpha. protein. However, the molecular events and mechanism of action involved in this peptide's actions remain largely unknown. [0010] Accordingly, although there have been many investigations, publications, and developments of these various entities, there remains a general ignorance and failure of understanding by research investigators and clinicians alike regarding useful and effective specific means and methods for suppressing NF.kappa.B-dependent gene expression on-demand within living cells, tissues, and organs. Thus, while the value and desirability of selectively controlling NF.kappa.B transcription factor activity--especially within cells at localized tissue areas on an as-needed basis for individual subjects--is well recognized, these aims have remained a long-sought goal yet to be achieved to date in a practical manner. SUMMARY OF THE INVENTION [0011] The present invention has multiple aspects and uses. A first aspect provides a method for selectively inhibiting degradation of I.kappa.B.alpha. within a targeted collection of viable cells in-situ, said method comprising the steps of: [0012] identifying a collection of cells comprising viable cells in-situ as a target for inhibiting I.kappa.B.alpha. degradation; [0013] providing means for effecting an introduction of at least one member selected from the group consisting of the PR-39 oligopeptide collective to the cytoplasm of said targeted collection of cells; [0014] introducing at least one member of the PR-39 oligopeptide collective to the cytoplasm of said targeted collection of cells using said effecting means; [0015] allowing said introduced PR-39 oligopeptide collective member to interact with such I.kappa.B.alpha. and proteasomes as are present within the cytoplasm of said targeted collection of cells whereby [0016] (a) at least some of the proteasomes interact with said PR-39 oligopeptide collective member, [0017] (b) at least a part of the proteolytic activity mediated by said interacting proteasomes becomes selectively altered, and [0018] (c) the selectively altered proteolytic activity of said proteasomes results in a marked inhibition of I.kappa.B.alpha. degradation in-situ within the cytoplasm of said targeted collection of viable cells. [0019] A second aspect of the invention provides a method for decreasing the activity of NF.kappa.B transcription factor in-situ within a collection of viable cells, said method comprising the steps of: [0020] identifying a collection of cells comprising viable cells in-situ as a target for decreased NF.kappa.B activity; [0021] providing means for effecting an introduction of at least one member selected from the group consisting of the PR-39 oligopeptide collective to the cytoplasm of said targeted collection of cells; [0022] introducing at least one member of the PR-39 oligopeptide collective to the cytoplasm of said targeted collection of cells using said effecting means; [0023] allowing said introduced PR-39 oligopeptide collective member to interact with such I.kappa.B.alpha. and proteasomes as are present within the cytoplasm of said targeted collection of cells whereby [0024] (a) at least some of the proteasomes interact with the PR-39 oligopeptide collective member, [0025] (b) at least a part of the proteolytic activity mediated by said proteasomes becomes selectively altered by said interaction, [0026] (c) the selectively altered proteolytic activity of said proteasomes results in a marked reduction of I.kappa.B.alpha. degradation in-situ within the cytoplasm of said targeted collection of cells; and [0027] (d) said reduction of I.kappa.B.alpha. degradation results in a decrease in activity for such NF.kappa.B transcription factor as is present intracellularly. 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