Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has diverse biological functions including its nuclear translocation in response to oxidative stress. We show that GAPDH physically associates with APE1, an essential enzyme involved in the repair of abasic sites in damaged DNA, as well as in the redox regulation of several transcription factors. This interaction allows GAPDH to convert the oxidized species of APE1 to the reduced form, thereby reactivating its endonuclease activity to cleave abasic sites. The GAPDH variants C152G and C156G retain the ability to interact with but are unable to reactivate APE1, implicating these cysteines in catalyzing the reduction of APE1. Interestingly, GAPDH-small interfering RNA knockdown sensitized the cells to methyl methane sulfonate and bleomycin, which generate lesions that are repaired by APE1, but showed normal sensitivity to 254-nm UV. Moreover, the GAPDH knockdown cells exhibited an increased level of spontaneous abasic sites in the genomic DNA as a result of diminished APE1 endonuclease activity. Thus, the nuclear translocation of GAPDH during oxidative stress constitutes a protective mechanism to safeguard the genome by preventing structural inactivation of APE1.The evolutionary conserved enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 3 exists as a tetramer that catalyzes a critical reaction in the second stage of the glycolytic pathway (1). It uses the oxidized form of nicotinamide adenine dinucleotide (NAD ϩ ) and converts glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with the concomitant release of NADH in an oxido-reduction reaction (1). GAPDH is also a key redox-sensitive protein that possesses an active site cysteine sulfhydryl that is susceptible to oxidation (2). Under oxidative stress, GAPDH rapidly undergoes disulfide bond formation leading to reduction in its enzymatic activity (2, 3). GAPDH has the propensity to interact with several proteins that are vulnerable to aggregation and are associated with neurodegenerative disorders such as in the case of the pro-oxidant amyloid ␤ peptide involved in Alzheimer disease (4). Recent studies have documented that GAPDH is also involved in several other nuclear processes that include histone H2B gene expression, nuclear RNA export, apoptosis, and cellular response to DNA damage (5-8).Several lines of evidence support a role for GAPDH in DNA damage and repair (5, 9). For example, GAPDH can translocate from the cytoplasm to the nucleus when cells are challenged with the potent chemical oxidant and DNA-damaging agent H 2 O 2 , although it is not clear what is the function executed by GAPDH under this stress condition (10). However, a more recent study documented that nitric oxide can also induce nuclear localization of GAPDH where it is acetylated by the acetyltransferase p300/CBP via direct protein interaction, which in turn causes stimulation of the catalytic activity of p300/CBP, resulting in the activation of downstream targets such as p53 (11). Other studies have shown that GAPDH is associated w...

The mitochondrial genome is continuously subject to attack by reactive oxygen species generated through aerobic metabolism. This leads to the formation of a variety of highly genotoxic DNA lesions, including abasic sites. Yeast Apn1p is localized to the nucleus, where it functions to cleave abasic sites, and apn1 ⌬ mutants are hypersensitive to agents such as methyl methanesulfonate (MMS) that induce abasic sites. Here we demonstrate for the first time that yeast Apn1p is also localized to the mitochondria. We found that Pir1p, initially isolated as a cell wall constituent of unknown function, interacts with the C-terminal end of Apn1p, which bears a bipartite nuclear localization signal. Further analysis revealed that Pir1p is required to cause Apn1p mitochondrial localization, presumably by competing with the nuclear transport machinery. pir1⌬ mutants displayed a striking (ϳ3-fold) increase of Apn1p in the nucleus, which coincided with drastically reduced levels in the mitochondria. To explore the functional consequences of the Apn1p-Pir1p interaction, we measured the rate of mitochondrial mutations in the wild type and pir1⌬ and apn1⌬ mutants. pir1⌬ and apn1⌬ mutants exposed to MMS exhibited 3.6-and 5.8-fold increases, respectively, in the rate of mitochondrial mutations, underscoring the importance of Apn1p in repair of the mitochondrial genome. We conclude that Pir1p interacts with Apn1p, at the level of either the cytoplasm or nucleus, and facilitates Apn1p transport into the mitochondria to repair damaged DNA.

The ubiquitin-specific protease (USP) structural class represents the largest and most diverse family of deubiquitinating enzymes (DUBs). Many USPs assume important biological roles and emerge as potential targets for therapeutic intervention. A clear understanding of USP catalytic mechanism requires a functional evaluation of the proposed key active site residues. Crystallographic data of ubiquitin aldehyde adducts of USP catalytic cores provided structural details on the catalytic triad residues, namely the conserved Cys and His, and a variable putative third residue, and inferred indirect structural roles for two other conserved residues (Asn and Asp), in stabilizing via a bridging water molecule the oxyanion of the tetrahedral intermediate (TI). We have expressed the catalytic domain of USP2 and probed by site-directed mutagenesis the role of these active site residues in the hydrolysis of peptide and isopeptide substrates, including a synthetic K48-linked diubiquitin substrate for which a label-free, mass spectrometry based assay has been developed to monitor cleavage. Hydrolysis of ubiquitin-AMC, a model substrate, was not affected by the mutations. Molecular dynamics simulations of USP2, free and complexed with the TI of a bona fide isopeptide substrate, were carried out. We found that Asn271 is structurally poised to directly stabilize the oxyanion developed in the acylation step, while being structurally supported by the adjacent absolutely conserved Asp575. Mutagenesis data functionally confirmed this structural role independent of the nature (isopeptide vs peptide) of the bond being cleaved. We also found that Asn574, structurally located as the third member of the catalytic triad, does not fulfill this role functionally. A dual supporting role is inferred from double-point mutation and structural data for the absolutely conserved residue Asp575, in oxyanion hole formation, and in maintaining the correct alignment and protonation of His557 for catalytic competency.

The members of the Endo IV family of DNA repair enzymes, including Saccharomyces cerevisiae Apn1 and Escherichia coli endonuclease IV, possess the capacity to cleave abasic sites and to remove 3'-blocking groups at single-strand breaks via apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase activities, respectively. In addition, Endo IV family members are able to recognize and incise oxidative base damages on the 5'-side of such lesions. We previously identified eight amino acid substitutions that prevent E. coli endonuclease IV from repairing damaged DNA in vivo. Two of these substitutions were glycine replacements of Glu145 and Asp179. Both Glu145 and Asp179 are among nine amino acid residues within the active site pocket of endonuclease IV that coordinate the position of a trinuclear Zn cluster required for efficient phosphodiester bond cleavage. We now report the first structure-function analysis of the eukaryotic counterpart of endonuclease IV, yeast Apn1. We show that glycine substitutions at the corresponding conserved amino acid residues of yeast Apn1, i.e., Glu158 and Asp192, abolish the biological function of this enzyme. However, these Apn1 variants do not exhibit the same characteristics as the corresponding E. coli mutants. Indeed, the Apn1 Glu158Gly mutant, but not the E. coli endonuclease IV Glu145Gly mutant, is able to bind DNA. Moreover, Apn1 Asp192Gly completely lacks enzymatic activity, while the activity of the E. coli counterpart Asp179Gly is reduced by approximately 40-fold. The data suggest that although yeast Apn1 and E. coli endonuclease IV exhibit a high degree of structural and functional similarity, differences exist within the active site pockets of these two enzymes.

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