Human embryonic stem (hES) cells activate a rapid apoptotic response after DNA damage but the underlying mechanisms are unknown. A critical mediator of apoptosis is Bax, which is reported to become active and translocate to the mitochondria only after apoptotic stimuli. Here we show that undifferentiated hES cells constitutively maintain Bax in its active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having pre-activated Bax. After DNA damage, active Bax rapidly translocated to the mitochondria by a p53-dependent mechanism. Interestingly, upon differentiation, Bax was no longer active and cells were not acutely sensitive to DNA damage. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development.

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...

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