| Plant pr-5 proteins as mammalian therapeutic agents -> Monitor Keywords |
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Plant pr-5 proteins as mammalian therapeutic agentsRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 25 Or More Peptide Repeating Units In Known Peptide Chain StructurePlant pr-5 proteins as mammalian therapeutic agents description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060069024, Plant pr-5 proteins as mammalian therapeutic agents. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is related to U.S. provisional patent applications Ser. No. 60/613,647, filed Sep. 27, 2004 and Ser. No. 60/657,335, filed Feb. 28, 2005. TECHNICAL FIELD [0002] The present invention relates to the treatment of mammalian disorders, specifically disorders of glucose and lipid metabolism, to therapeutic agents for the treatment of such conditions, and to the design and/or identification of such therapeutic agents. BACKGROUND OF THE INVENTION [0003] Among families of antimicrobial proteins associated with plant defense are the pathogenesis related proteins of family 5 (PR-5) that are structurally related to the sweet tasting protein thaumatin (Veronese et al., 2003). PR-5 proteins are distinguished by having (a) a three domain structure consisting of a .beta. barrel (Domain I) that forms the compact core of the molecule (this structure is commonly found in lectins and is hereafter referred to as a "lectin-like .beta. barrel"); a domain (Domain II) that extends from Domain I and consists of several loops stabilized by four disulfide bonds; and Domain III that also extends from domain I and consists of a small loop stabilized by two disulfide bonds, (b) an alanine commonly located at the cleavage site of the N-terminal leader sequence, (c) up to 16 cysteine residues that have a conserved spatial distribution throughout the protein and linked by disulfide bridges (Min, et al., 2004), and (d) a cleft formed by Domains I & II that could be associated with biological activity. [0004] Osmotin is an antifungal tobacco PR-5 protein. It induces programmed cell death in S. cerevisiae by signaling suppression of cellular stress responses via RAS2/cAMP (Narasimhan et at., 2001). Most of the PR-5 proteins, including osmotin, have specific wide-spectrum antifungal activities, suggesting that target recognition may be determined by their interaction with pathogen cell surface components. In the case of osmotin, specific fungal cell wall components enhance or suppress osmotin antifungal activity (Narasimhan et al., 2003; Veronese et al., 2003). In S. cerevisiae, the PIR family of cell wall glycoproteins are osmotin-resistance determinants (Yun et al., 1997). Phosphomannans of yeast cell wall glycoproteins have been reported to increase osmotin toxicity, probably by serving as docking structures for osmotin, thereby increasing its local concentration and diffusion across the cell wall (Ibeas et al., 2000). Genetic analyses have revealed that SSD1, a protein that affects cell wall morphogenesis and deposition of PIR proteins, is a determinant of resistance to osmotin (Ibeas et al., 2001). Unidentified changes in the yeast cell wall that enhance toxicity are induced by osmotin via activation of a mitogen-activated protein kinase cascade (Yun et al., 1998). Specific interactions at the plasma membrane also appear to be required for osmotin antifungal activity because yeast spheroplasts that are susceptible to tobacco osmotin can be resistant to PR-5 proteins from other plant species (Yun et al., 1997). Although the best studied role of PR-5 proteins is their antifungal activity, a signaling or recognition role has been suggested. Veronese et al., 2003 [0005] Adiponectin (also called 30-kDa adipocyte complement-related protein--Acrp30) is an antidiabetic and antiatherosclerotic protein hormone in mammals that conditions sensing of energy status, fatty acid oxidation and glucose transport upon interaction with adiponectin receptors. Known adiponectin receptors include the human AdipoR1 and AdipoR2 (Diez and Iglesias, 2003; Yamauchi et al., 2003a,b), and the pig adiponectin receptor genebank NM.sub.--001007193) (Ding et al. 2004). Additional adiponectin receptor sequences can be found in GeneBank. Serum adiponectin levels are decreased under conditions of obesity, insulin resistance, and type II diabetes (Yamauchi, et al., 2003a), while administration of adiponectin lowers serum glucose levels and ameliorates insulin resistance in mice (Yamauchi, et al. 2003a). The mammalian adiponectin receptors AdipoR1 and AdipoR2 are predicted to have seven transmembrane domains, a characteristic feature of G protein-coupled receptors (GPCRs). SUMMARY OF THE INVENTION [0006] Proteins of the PR-5 family control apoptosis in yeast through receptor comprising seven transmembrane domains, a characteristic of GPCRs. Receptors which specifically bind to PR-5 proteins have been found to be homologous to mammalian adiponectin receptors, and PR-5 proteins can act as functional homologues of adiponectin and control adiponectin response in mammals. [0007] A plasma membrane determinant of osmotin sensitivity in yeast cells was isolated, after being identified by its ability to confer an osmotin supersensitive phenotype upon gene overexpression. The gene, ORE20/PHO36, encodes a seven transmembrane domain receptor-like protein (PHO36), having structural homology to GPCRs, and significant sequence identity (29%) to mammalian adiponectin receptor R1. PHO36 regulates yeast lipid and phosphate metabolism, and is required for full sensitivity to osmotin in yeast. PHO36 functions upstream of RAS2 in the osmotin-induced apoptotic pathway. A mammalian homolog of PHO36 is a receptor for the hormone adiponectin, which regulates cellular lipid and sugar metabolism. While the osmotin receptor PHO36 and adiponectin receptors share significant sequence homology, osmotin and adiponectin, the corresponding receptor-binding proteins, do not share sequence similarity (.ltoreq.10% homology). However, the tertiary structures of a lectin-like .beta. barrel domain of both proteins have been found to be very similar and can be overlapped. It has been found that osmotin can bind selectively to mammalian adiponectin receptors, and acts as an adiponectin agonist in mammalian systems. [0008] Thus, osmotin, and related proteins in the PR-5 family having a structurally homologous lectin-like .beta. barrel domain, suitably formulated in a pharmaceutically acceptable carrier, can be used as therapeutic agents for the treatment of a wide variety of mammalian disorders in which adiponectin receptor-mediated pathways are implicated. Such disorders include diabetes, arteriosclerosis, and heart disease. Osmotin, and related PR-5 proteins having a structurally homologous lectin-like .beta. barrel domain, can also be used as surrogates for adiponectin in the development of therapeutic methods for treatment of mammalian disorders in which adiponectin receptor-mediated pathways are implicated. Osmotin, and related PR-5 proteins having a structurally homologous lectin-like .beta. barrel domain, can also be used as a basis for the rational design of new therapeutic agents for the treatment of mammalian disorders in which adiponectin receptor-mediated pathways are implicated. [0009] Receptors to which osmotin, and related PR-5 proteins having a structurally homologous lectin-like .beta. barrel domain, can specifically bind (for example the yeast receptor PHO36) can be used as screening systems for new therapeutic agents useful in the treatment of mammalian disorders in which adiponectin receptor-mediated pathways are implicated. Such screening methods can be carried out using a preparation of the isolated receptor (e.g., in solution, or bound for use in a column packing, thin-layer plate, or micro-array), or using a cell line or tissue culture in which the receptor is expressed. [0010] The yeast strain BWG7a expresses the osmotin receptor PHO36 and is sensitive to osmotin. When caused to over-express PHO36, this yeast strain was found to be supersensitive to the apoptosis-inducing effects of osmotin. A cell, such as the yeast strain BWG7a or any other suitable cell line expressing or over-expressing PHO36 or similarly sensitive PR-5 protein receptors, may thus be used as a primary screen to identify chemical and protein/peptide agonists or antagonists of adiponectin receptor function in mammals. This screen allows identification of potential adiponectin receptor-mediated therapeutic agents. In a preferred embodiment, the cell is one that over-expresses PHO36 or similarly sensitive PR-5 receptor. [0011] A nucleic acid having the osmotin genomic sequence [SEQ ID NO. 1] or cDNA sequence [SEQ ID NO. 2], or their protein products and therapeutic products derived therefrom, or derivatives and functional homologues thereof, can be used in structure/function analyses of adiponectin receptor binding, in order to improve the efficacy of adiponectin, PR-5 proteins, or derivatives or functional homologues thereof, as therapeutic agents. Such structure/function analyses can include comparative analyses using in vitro molecular evolution approaches, such as DNA shuffling, or enhanced selective mutagenesis and phage display or other selective methods for improved peptide function, and rational design approaches to develop improved therapeutic agents, including drugs that are agonists or antagonists of all or part of the adiponectin targets in human cells. [0012] One embodiment of the invention is a pharmaceutical composition comprising a PR-5 protein having a lectin-like .beta. barrel domain. In a preferred embodiment, the PR-5 protein is osmotin [SEQ ID NO. 3], or a homolog thereof having a lectin-like .beta. barrel domain. [0013] Another embodiment of the invention is a method for treating a mammal suffering from a disorder that is the result of activation or inhibition of a metabolic pathway mediated by adiponectin or an adiponectin-like protein, comprising administration of a PR-5 protein having a lectin-like .beta. barrel domain. In preferred embodiments the disorder is selected from the group consisting of type II diabetes, insulin resistance, hyperlipidemia, arteriosclerosis, and heart disease. In more preferred embodiments, the method comprises administration of osmotin [SEQ ID NO. 3], or a homolog of osmotin having a lectin-like .beta. barrel domain. [0014] Another embodiment of the invention is the use of a PR-5 protein in the rational design of a therapeutic agent that is an agonist or an antagonist of all or part of the adiponectin targets in a mammalian cell. In a preferred embodiment the therapeutic agent is an agonist. In another preferred embodiment, the therapeutic agent is an antagonist. In a more preferred embodiment, the rational design includes structure/function analysis. In a most preferred embodiment, the PR-5 protein is osmotin [SEQ ID NO: 3]. [0015] Another embodiment of the invention is the use of a nucleic acid sequence that encodes a PR-5 protein having a lectin-like .beta. barrel domain in the rational design of a therapeutic agent that is an agonist or antagonist of all or part of the adiponectin targets in a mammalian cell. In a preferred embodiment, the therapeutic agent is an agonist. In another preferred embodiment, the therapeutic agent is an antagonist. In a more preferred embodiment, the rational design includes structure/function analysis, preferably structure/function analysis includes one or more technique selected from the group consisting of DNA shuffling, enhanced selective mutagenesis and phage display. In the most preferred embodiments, the nucleic acid sequence is SEQ ID NO. 1 or SEQ ID NO. 2. [0016] Another embodiment of the invention is the use of a receptor protein, having specific binding affinity for a PR-5 protein having a lectin-like .beta. barrel domain, as a primary screen to identify a therapeutic agent that is an agonist or an antagonist of all or part of the adiponectin targets in a mammalian cell. In one preferred embodiment the screen is carried out using a receptor that is in isolated form. In another preferred embodiment, the screen is carried out using a cell line or tissue culture expressing the receptor, more preferably overexpressing the receptor. A preferred cell line for use in this embodiment is a yeast cell line, most preferably a yeast cell line overexpressing the receptor. The most preferred PR-5 receptor for use in this embodiment is PHO36. BRIEF DESCRIPTION OF THE DRAWINGS [0017] FIG. 1: PHO36 Mediates Sensitivity to Osmotin in Yeast. A. Aliquots (2.5 .mu.l) of 10-fold serial dilutions of log phase cultures (A.sub.600nm 0.4) of the yeast strain BWG7a transformed with the centromeric plasmid pRS316 without (vector) or with PHO36t insert (pPHO36t) were spotted on selective SC-galactose media without (none) or with osmotin supplement (1.6 .mu.M) and allowed to grow for 3 days at 28.degree. C. B. Spheroplasts (10.sup.6 ml.sup.-1) derived from the wild-type strain BWG7a (.quadrature.) or the isogenic .DELTA.pho36 mutant (.box-solid.) were diluted and plated on YPD agar containing 0.8 M sorbitol and the indicated concentrations of osmotin. Viable spheroplasts were counted after 3 days incubation at 28.degree. C. Viable counts are normalized to the value without added osmotin. Values are the average of three different experiments .+-.SE (inset). The results of a similar experiment with spheroplasts of BWG7a strain carrying the plasmid pRS316 without (.smallcircle.) or with PHO36t insert (pPHO36t;.circle-solid.), performed in selective SC-galactose media supplemented with 0.8 M sorbitol, are shown. [0018] FIG. 2: PHO36 Functions via RAS2 in Osmotin-Induced Apoptosis. A. Cells (10.sup.8 ml.sup.-1) of the wild-type strain (wt), and isogenic .DELTA.pho36, .DELTA.ras2, and .DELTA.pho36.DELTA.ras2 mutant strains were incubated with 5 .mu.M osmotin in YPD at 30.degree. C. under apoptosis-inducing conditions. Cells were washed after 3 hr osmotin treatment and the percent of total cells that were able to survive and form colonies was measured. Values represent the mean of four determinations .+-.SE. B. Cells (10.sup.8 ml.sup.-1) of the wild type (wt), and .DELTA.ras2 mutant strains transformed with p426GPD without (vec) or with PHO36 insert (pPHO36) were incubated with 2 .mu.M osmotin in selective SC-glucose medium at 30.degree. C. under apoptosis-inducing conditions. The percent of total cells able to survive and form colonies was measured after 1 hr osmotin treatment as above. Values represent the mean of two determinations .+-.SE. C. Osmotin sensitivity was assayed by spotting aliquots (2.5 .mu.l) of serial 10-fold dilutions of A.sub.600nm 0.4 cultures of strains BWG7a (wt) and the .DELTA.pho36 mutant transformed with pAD4M (vec) or pAD.sub.4M-RAS.sub.2.sup.G19V (pRAS.sup.G19V) on selective SC-glucose medium without (none) or with the indicated osmotin supplements. Plates were photographed after 5 days incubation at room temperature. D. Cells (10.sup.8 ml.sup.-1) of strain BWG7a (wt) and .DELTA.pho36, .DELTA.pho36, .DELTA.ras2, and .DELTA.ras2 mutants, transformed with pSTRE-lacZ(LEU2), were treated without (minus) or with (plus) 8 .mu.M osmotin in YPD for 45 min at 30.degree. C. under apoptosis-inducing conditions. Shown are the ratios of .beta.-galactosidase activities measured in cell-free extracts. Each bar represents the mean .+-.SD (n=3). Statistical comparison of the groups is reported: *p<0.01; no asterisk, no difference. .beta.-galactosidase activities in absence of osmotin were, respectively, respectively, 152.+-.4, 133.+-.3, 705.+-.23, and 139.+-.6 units. The experiment was repeated once with similar results. E. A model for osmotin-mediated cell death pathways is shown: Osmotin activates the RAS2/cAMP pathway and induces suppression of cellular stress responses (STRE-lacZ reporter) followed by accumulation of reactive oxygen species and cell death. Interaction of osmotin with PHO36 activities cell death via RAS2. There may be unidentified upstream components that stimulate the RAS2 cell death pathway in response to osmotin. Pathways controlling stress responses are even more complex, as evidenced by osmotin-independent effects on STRE-lacZ activity in a .DELTA.pho36.DELTA.ras2 genetic background, and are not shown. Symbols: ?, unknown components; .dwnarw., activation; .perp., inhibition; .fwdarw., cell wall weakening; CW, cell wall; PM, plasma membrane. F. Aliquots (2.5 .mu.l) of 10-fold serial dilutions of A.sub.600 nm 0.4 cultures of strain BWG7a (wt) and the indicated mutant strains were spotted on YPD agar without (none) or with the indicated osmotin supplements. Plates were photographed after incubation for 2 days at 28.degree. C. [0019] FIG. 3: PHO36 is Localized on the Plasma Membrane. A. The PHO36MH construct sequence is shown. The EcoR1-Xhol fragment contained 46 nt of the PHO36 gene preceding the start codon, and the natural PHO36 ORF (thin) fused in-frame near its C terminus to a c-myc-tag (thick) and a His tag (dotted underline). B-C. Immunogold localization of tagged PHO36MH protein. Ultrathin sections of spheroplasts of strain BWG7a carrying the single copy plasmid p416GPD without (B) or with the PHO36MH insert (pPHO36MHS[C]) are shown. The 20 nm gold particles, which appear as small black dots, indicate the location of PHO36MH protein. D-F. Distribution of PHO36MH protein in cellular membranes. Extracts of cells carrying pPHO36MHS were fractionated, and distribution of proteins in the membrane-free supernatant fraction (lane 1), total membrane fraction (lane 2), and in sucrose density gradient membrane fractions, from lighter to denser fractions (lanes 3-7, respectively) was analyzed. PHO36MH (D) and plasma membrane H.sup.+-ATPase marker (E) were detected on blots of proteins separated by SDS-PAGE with mycl-9E10 monoclonal antibodies and PMA1 antibodies, respectively. The activity of the vacuolar .alpha.-mannosidase marker was measured in these fractions (F) and is given in arbitrary units as increments of A.sub.400nm min.sup.-1 mg.sup.-1 protein. Continue reading about Plant pr-5 proteins as mammalian therapeutic agents... 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