P21 Peptide acetate

We have employed computer-based molecular modeling approaches to design peptides from the ras-p21 and p53 proteins that either induce tumor cell reversion to the untransformed phenotype or induce tumor cell necrosis without affecting normal cells. For rasp21, we have computed and superimposed the average low energy structures for the wild-type protein and oncogenic forms of this protein and found that specific domains change conformation in the oncogenic proteins. We have synthesized peptides corresponding to these and found that ras peptides, 35-47 (PNC-7) and 96-110 (PNC-2), block oncogenic ras-p21-induced oocyte maturation but have no effect on insulin-induced oocyte maturation that requires activation of endogenous wild-type ras-p21. These results show signal transduction pathway differences between oncogenic and activated wild-type ras-p21. Both peptides, attached to a membrane-penetrating peptide (membrane residency peptide or MRP), either induce phenotypic reversion to the untransformed phenotype or tumor cell necrosis of several ras-transformed cell lines, but have no effect on the growth of normal cells. Using other computational methods, we have designed two peptides, PNC-27 and 28, containing HDM-2-protein-binding domain sequences from p53 linked on their C-termini to the MRP that induce pore formation in the membranes of a wide range of cancer cells but not any normal cells tested. This is due to the expression of HDM-2 in the cancer cell membrane that does not occur in normal cells. These peptides eradicate a highly malignant tumor in nude mice with no apparent side effects. Both ras and p53 peptides show promise as anti-tumor agents in humans.

Oxidative stress in the retinal pigment epithelium (RPE) can cause mitochondrial dysfunction and is likely a causative factor in the pathogenesis of age-related macular degeneration (AMD). Under oxidative stress conditions, some of the RPE cells become senescent and a contributory role for RPE senescence in AMD pathology has been proposed. The purpose of this study is to 1) characterize senescence in human RPE; 2) investigate the effect of an αB Crystallin chaperone peptide (mini Cry) in controlling senescence, in particular by regulating mitochondrial function and senescence-associated secretory phenotype (SASP) production and 3) develop mouse models for studying the role of RPE senescence in dry and nAMD. Senescence was induced in human RPE cells in two ways. First, subconfluent cells were treated with 0.2 μg/ml doxorubicin (DOX); second, subconfluent cells were treated with 500 μM H2O2. Senescence biomarkers (senescence-associated beta-galactosidase (SA-βgal), p21, p16) and mitochondrial proteins (Fis1, DRP1, MFN2, PGC1-α, mtTFA) were analyzed in control and experimental groups. The effect of mini Cry on mitochondrial bioenergetics, glycolysis and SASP was determined. In vivo, retinal degeneration was induced by intravenous injection of NaIO3 (20 mg/kg) and subretinal fibrosis by laser-induced choroidal neovascularization. Increased SA-βgal staining and p16 and p21 expression was observed after DOX- or H2O2-induced senescence and mini Cry significantly decreased senescence-positive cells. The expression of mitochondrial biogenesis proteins PGC-1 and mTFA increased with senescence, and mini Cry reduced expression significantly. Senescent RPE cells were metabolically active, as evidenced by significantly enhanced oxidative phosphorylation and anaerobic glycolysis, mini Cry markedly reduced rates of respiration and glycolysis. Senescent RPE cells maintain a proinflammatory phenotype characterized by significantly increased production of cytokines (IFN-ˠ, TNF-α, IL1-α IL1-β, IL-6, IL-8, IL-10), and VEGF-A; mini Cry significantly inhibited their secretion. We identified and localized senescent RPE cells for the first time in NaIO3-induced retinal degeneration and laser-induced subretinal fibrosis mouse models. We conclude that mini Cry significantly impairs stress-induced senescence by modulating mitochondrial biogenesis and fission proteins in RPE cells. Characterization of senescence could provide further understanding of the metabolic changes that accompany the senescent phenotype in ocular disease. Future studies in vivo may better define the role of senescence in AMD and the therapeutic potential of mini Cry as a senotherapeutic.

Purpose: We have previously found that a synthetic peptide corresponding to ras-p21 residues 96 110 (PNC2) selectively blocks oncogenic (Val 12-containing) ras-p21 protein-induced oocyte maturation. With a view to introducing this peptide into ras-transformed human cells to inhibit their proliferation, we synthesized an inducible plasmid that expressed this peptide sequence. Our purpose was to test this expression system in oocytes to determine if it was capable of causing selective inhibition of oncogenic ras-p21. Methods: We injected this plasmid and a plasmid expressing a control peptide into oocytes either together with oncogenic p21 or in the presence of insulin (that induces maturation that is dependent on normal cellular ras-p21) in the presence and absence of the inducer isopropylthioglucose (IPTG). Results: Microinjection of this plasmid into oocytes together with Val 12-p21 resulted in complete inhibition of maturation in the presence of inducer. Another plasmid encoding the sequence for the unrelated control peptide, X13, was unable to inhibit Val 12-p21-induced maturation. In contrast, PNC2 plasmid had no effect on the ability of insulin-activated normal cellular or wild-type ras-p21 to induce oocyte maturation, suggesting that it is selective for blocking the mitogenic effects of oncogenic (Val 12) ras p21. Conclusion: We conclude that the PNC2 plasmid selectively inhibits oncogenic ras-p21 and may therefore be highly effective in blocking proliferation of ras-induced cancer cells. Also, from the patterns of inhibition, by PNC2 and other ras- and raf-related peptides, of raf- and constitutively activated MEK-induced maturation, we conclude that PNC2 peptide inhibits oncogenic ras p21 downstream of raf.