We found that Ku70, a known DNA repair factor, has a novel function to bind and inhibit Bax (Bcl-2-associated X protein), a key mediator of apoptosis. Pentapeptides derived from the Bax-binding domain of Ku70 were cell-permeable and protected cells from Bax-mediated apoptosis. These pentapeptides were called BIPs (Bax-inhibiting peptides). BIPs may become a useful therapeutic tool to reduce cellular damage. We also generated BIP mutant pentapeptides that do not inhibit Bax, but retain their cell-penetrating activity. Since both BIPs and BIP mutants are cell-permeable, these peptides were designated CPP5s (cell-penetrating pentapeptides). Among the CPP5s discovered, VPTLK (BIP) and KLPVM (BIP mutant) were confirmed to possess protein transduction activity by examination of the delivery of GFP (green fluorescent protein) into cells by these peptides. The mechanism of cell penetration by CPP5s is not known. CPP5s enter the cell at 0 and 4 degrees C. In preliminary studies, various inhibitors of endocytosis and pinocytosis did not show any significant suppression of CPP5 cell entry. CPP5s have very low toxicity in vitro and in vivo and so may be useful tools in order to develop non-toxic drug-delivery technologies.

Polyglutamine (polyQ) diseases, such as Huntington's disease and Machado-Joseph disease (MJD), are caused by gain of toxic function of abnormally expanded polyQ tracts. Here, we show that expanded polyQ of ataxin-3 (Q79C), a gene that causes MJD, stimulates Ku70 acetylation, which in turn dissociates the proapoptotic protein Bax from Ku70, thereby promoting Bax activation and subsequent cell death. The Q79C-induced cell death was significantly blocked by Ku70 or Bax-inhibiting peptides (BIPs) designed from Ku70. Furthermore, expression of SIRT1 deacetylase and the addition of a SIRT1 agonist, resveratrol, reduced Q79C toxicity. In contrast, mimicking acetylation of Ku70 abolished the ability of Ku70 to suppress Q79C toxicity. These results indicate that Bax and Ku70 acetylation play important roles in Q79C-induced cell death, and that BIP may be useful in the development of therapeutics for polyQ diseases. Nine inherited neurodegenerative disorders with expanded polyglutamine (polyQ) are caused by mutations in different genes, but they likely share the common pathology in which expanded polyQ gains toxic functions. 1 The molecular mechanism of neuronal toxicity of polyQ remains enigmatic. Recent findings suggest that the intracellular aggregation of polyQ causes cellular stress responses that trigger neuronal cell death. 1 It is hypothesized that polyQ aggregation suppresses neuronal transcriptional activity by sequestering histone acetyltransferases (HAT) from chromosomes and thus polyQ causes neuronal cell death. [2][3][4] Bax is a proapoptotic member of Bcl-2 family proteins that plays a key role in programmed cell death in neurons. 5,6 Recently, mutant huntingtin with expanded polyQ was shown to activate p53 and increase the expression level of Bax. 7 Based on these previous findings, we became interested in examining the role of Bax in polyQ-induced cell death. Recently, we developed a series of cytoprotective membrane-permeable pentapeptides that rescue cells from Baxmediated cell death. These peptides are named Bax-inhibiting peptides (BIPs) and were designed from the Bax binding domain of Ku70. [8][9][10] Ku70 is a multifunctional protein playing roles in DNA repair and cell survival. 11 Ku70 has been shown to inhibit Bax-mediated cell death by binding Bax in the cytosol. 12-14 The present study demonstrates that BIP can rescue cells from polyQ toxicity, and that polyQ promotes Bax-mediated cell death by inducing Ku70 acetylation that activates Bax. ResultsBIP suppresses Q79C-induced cell death. BIPs consisting of five amino acids (e.g. VPMLK and VPTLK) were used in this study. A mutated peptide (i.e. IPMIK) that does not bind Bax but retains cell permeability was also used in this study as a negative control (NC). For the investigation of polyQ toxicity, we used the C-terminal, truncated fragment of the Machado-Joseph disease 1 (MJD1) gene product, ataxin-3, which includes an expanded polyQ stretch (79 glutamine repeats, Q79C). 15,16 As a negative control, ataxin-3 C-terminus with 22 or 35 glutamine rep...

Previously, we developed cell-penetrating penta-peptides (CPP5s). In the present study, VPTLK and KLPVM, two representative CPP5s, were used to characterize the cell-penetration and protein-transduction activities of these small molecules. Various inhibitors of endocytosis and pinocytosis (chlorpromazine, cytochalasin D, Filipin III, amiloride, methyl-β-cyclodextrin, and nocodazole) were tested. Only cytochalasin D showed suppression of CPP5 entry, though the effect was partial. In addition, CPP5s were able to enter a proteoglycan-deficient CHO cell line. These results suggest that pinocytosis and endocytosis may play only a minor role in the cell entry of CPP5s. By mass spectrometry, we determined that the intracellular concentration of VPTLK ranged from 20 nM to 6.0 μM when the cells were cultured in medium containing 1 μM – 1.6 mM VPTLK. To determine the protein-transduction activity of CPP5s, the Tex-LoxP EG cell line, which has a Cre-inducible green fluorescent protein (GFP) gene, was used. VPTLK and KLPVM were added to the N-terminus of Cre, and these fusion proteins were added to the culture medium of Tex-LoxP EG cells. Both VPTLK-Cre and KLPVM-Cre were able to turn on GFP expression in these cells, suggesting that CPP5s have protein-transduction activity. Since CPP5s have very low cytotoxic activity, even at a concentration of 1.6 mM in the medium, CPP5s could be utilized as a new tool for drug delivery into cells.

Previously, we reported that Ku70 binds and inhibits Bax activity in the cytosol, and that ubiquitin-dependent proteolysis of cytosolic Ku70 facilitates Bax-mediated apoptosis. We found that Hdm2 (Human Homologue of Murine Double Minute) has an ability to ubiquitinate Ku70 and that Hdm2 overexpression in cultured cells causes a decrease in Ku70 expression levels. An interaction between Ku70 and Hdm2 was demonstrated by means of immunoprecipitation, while none could be shown between Ku80 and Hdm2. Vascular endothelial growth factor (VEGF) is known to inhibit endothelial cell (EC) apoptosis through an Akt-mediated survival kinase signal, however, the mechanism underlying this inhibition of apoptosis has not been fully elucidated. We found that VEGF inhibited cytosolic Ku70 degradation induced by apoptotic stress. It is known that Akt-dependent phosphorylation of Hdm2 causes nuclear translocation of Hdm2 followed by Hdm2-mediated inactivation of p53. We found that VEGF stimulated nuclear translocation of Hdm2 in EC, and efficiently inhibited Ku70 degradation. We also found that constitutively active Akt, but not kinase-dead Akt, inhibited Ku70 degradation in the cytosol. Furthermore, Ku70 knock-down diminished anti-apoptotic activity of Akt. Taken together, we propose that Hdm2 is a Ku70 ubiquitin ligase and that Akt inhibits Bax-mediated apoptosis, at least in part, by maintaining Ku70 levels through the promotion of Hdm2 nuclear translocation.

Expression of the clusterin (CLU) gene results in the synthesis of a conventional secretory isoform set (pre-and mature secretory clusterin proteins, psCLU/sCLU), as well as another set of intracellular isoforms, appearing in the cytoplasm (pre-nuclear CLU, pnCLU) and in the nucleus as an ϳ55-kDa mature nuclear clusterin (nCLU) form. These two isoform sets have opposing cell functions: pro-survival and pro-death, respectively. Although much is known about the regulation and function of sCLU as a pro-survival factor, the regulation and function of endogenous nCLU in cell death are relatively unexplored. Here, we show that depletion of endogenous nCLU protein using siRNA specific to its truncated mRNA increased clonogenic survival of ionizing radiation (IR)-exposed cells. nCLU-mediated apoptosis was Bax-dependent, and lethality correlated with accumulation of mature nCLU protein.