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Mitochondrial copper metabolism and delivery to cytochrome c oxidase and mitochondrially localized CuZn-superoxide dismutase (Sod1) requires a growing number of intermembrane space proteins containing a twin Cx9C motif. Among them, Cmc1 was recently identified by our group. Here we describe another conserved mitochondrial metallochaperone-like protein, Cmc2, a close homologue of Cmc1, whose function affects both cytochrome c oxidase and Sod1. In the yeast Saccharomyces cerevisiae, Cmc2 localizes to the mitochondrial inner membrane facing the intermembrane space. In the absence of Cmc2, cytochrome c oxidase activity measured spectrophoto-metrically and cellular respiration measured polarographically are undetectable. Additionally, mutant cmc2 cells display 2-fold increased mitochondrial Sod1 activity, whereas CMC2 overexpression results in Sod1 activity decreased to 60% of wild-type levels. CMC1 overexpression does not rescue the respiratory defect of cmc2 mutants or vice versa. However, Cmc2 physically interacts with Cmc1 and the absence of Cmc2 induces a 5-fold increase in Cmc1 accumulation in the mitochondrial membranes. Cmc2 function is conserved from yeast to humans. Human CMC2 localizes to the mitochondria and CMC2 expression knockdown produces cytochrome c oxidase deficiency in Caenorhabditis elegans. We conclude that Cmc1 and Cmc2 have cooperative but nonoverlapping functions in cytochrome c oxidase biogenesis. [ABSTRACT FROM AUTHOR]

Most agents that damage DNA act through posttranslational modifications of p53 and activate its downstream targets. However, whether cellular responses to nucleoside analogue-induced DNA damage also operate through p53 posttranslational modification has not been reported. In this study, the relationship between p53 activation and its posttranslational modifications was investigated in the human cancer cell lines A549 and HCT116 in response to 5-aza-2′-deoxycytidine (5-aza-CdR) or cytarabine treatment. 5-Aza-CdR induces p53 posttranslational modifications through activation of an ATM- and Rad3-related (AIR) signaling pathway, and 5-aza-CdR-induced association of replication protein A with chromatin is required for the binding of AIR to chromatin. Upon treatment with 5-aza-CdR, AIR activation is clearly associated with p53 phosphorylation at Ser15, but not at Thr18, Ser20, or Ser37. This specific p53 phosphorylation at Ser15 in turn results in acetylation of p53 at Lys320 and Lys373/Lys382 through transcriptional cofactors p300/CBP-associated factor and p300, respectively. These p53 posttranslational modifications are directly responsible for 5-aza-CdR induced p21Waf1/Cip1 expression because the binding activity of acetylated p53 at Lys320/Lys373/Lys382 to the p21Waf1/Cip1 promoter, as well as p21Waf1/Cip1 expression itself are significantly increased after 5-aza-CdR treatment. It is of interest that p53 phosphorylation at Ser15 and acetylations at Lys320/ Lys373/Lys382 mutually interact in the 5-aza-CdR induced p21Waf1/Cip1 expression shown by transfection of artificially mutated p53 expression vectors including S15A, K320R, and K373R/K382R into p53-null H1299 cells. These data taken together show for the first time that 5-aza-CdR activates the ATR signaling pathway, which elicits a specific p53 phosphorylation-acetylation cascade to induce p21Waf1/Cip1 expression. [ABSTRACT FROM AUTHOR]