The transient receptor potential melastatin-related channel 2 (TRPM2) is a nonselective cation channel, whose prolonged activation by oxidative and nitrative agents leads to cell death. Here, we show that the drug puromycin selectively targets TRPM2-expressing cells, leading to cell death. Our data suggest that the silent information regulator 2 (Sir2 or sirtuin) family of enzymes mediates this susceptibility to cell death. Sirtuins (2). This influx is thought to occur as a consequence of TRPM2 channel activation via ADP-ribose (ADPr) binding to the cytoplasmic, C-terminal domain of the channel. This domain, termed NudT9-H (NudT9 homology), contains an apparent Nudix enzymatic motif and shares significant homology to the mitochondrial NudT9 enzyme (4, 5), a member of the Nudix ADPr hydrolase family. Indeed, electrophysiology experiments have shown that low micromolar levels of ADPr activate the TRPM2 channel (4, 6), and that this activation is mediated through the NudT9-H domain (7, 8), implicating a direct ADPr and NudT9-H interaction.The NudT9-H domain has been reported to have hydrolytic activity toward ADPr, albeit at a ϳ100-fold lower rate than NudT9 (4, 9). Although it was initially thought that the ADPr hydrolase activity of TRPM2 might have a role in regulating TRPM2 gating via hydrolysis of bound ADPr, recent data have demonstrated that mutations in putative catalytic residues of NudT9-H do not affect TRPM2 channel gating, leaving the function of this activity unclear (8). Some studies suggest that hydrogen peroxide directly activates the TRPM2 channel (3, 10); however, it has been reported recently that this oxidative agent acts indirectly by triggering the release of ADPr from an intracellular compartment (8, 11). The mechanism of TRPM2 activation by hydrogen peroxide may prove to be more complex, as hydrogen peroxide and ADPr have also been suggested to act cooperatively in TRPM2 channel activation (12).In this article, we have investigated the ability of TRPM2 to sensitize cells to cellular insults other than those inducing oxidative stress. Here, we demonstrate that puromycin, a well known pleiotropic cell stress agent, selectively targets TRPM2-expressing cells, leading to cell death. Our studies suggest that Sir2 enzymes play an important role in mediating this response. We found that TRPM2 has the capacity to sense OAADPr, the unique metabolite of the Sir2 protein deacetylase reaction, and induce cell death in response to puromycin. Furthermore, we report the first direct evidence that OAADPr binds to the NudT9-H domain and modulates TRPM2 channel activity. Our data have important implications for the potential physiological roles of ADPr-related molecules as secondary messengers. This report links Sir2 enzymes (sirtuins) with the TRPM2 channel through the Sir2 metabolite OAADPr. This newly discovered association provides a plausible mechanism for previously described sirtuin functions, which include control of stress response pathways and metabolic regulation. EXPERIMENTAL PROCEDURESCell Cul...

NEMO (NF-B essential modulator) is a regulatory protein essential to the canonical NF-B signaling pathway, notably involved in immune and inflammatory responses, apoptosis, and oncogenesis. Here, we report that the zinc finger (ZF) motif, located in the regulatory C-terminal half of NEMO, forms a specific complex with ubiquitin. We have investigated the NEMO ZF-ubiquitin interaction and proposed a structural model of the complex based on NMR, fluorescence, and mutagenesis data and on the sequence homology with the polymerase ubiquitinbinding zinc finger involved in DNA repair. Functional complementation assays and in vivo pull-down experiments further show that ZF residues involved in ubiquitin binding are functionally important and required for NF-B signaling in response to tumor necrosis factor-␣. Thus, our findings indicate that NEMO ZF is a bona fide ubiquitin-binding domain of the ubiquitin-binding zinc finger type.The NF-B transcription factors regulate the expression of genes involved in inflammation, immunity, cell proliferation, apoptosis, and oncogenesis (1). The classical NF-B signaling pathway is induced in response to proinflammatory cytokines (interleukin-1, TNF-␣), 5 antigens, and endotoxins. Although different receptors and downstream molecules are engaged depending on the stimulus, they all transduce signals through the IKK complex. These active complexes comprise two catalytic kinase subunits, IKK␣ and/or IKK␤, and a dimer of regulatory subunits termed NEMO, which is required for IKK complex activity and subsequent NF-B activation (2, 3).The N-terminal part of NEMO interacts with the IKK kinases, whereas the regulatory C-terminal half is involved in signal recognition (4, 5). The latter comprises the CC2-LZ domain, including a coiled-coil (CC2) and a leucine zipper (LZ) motif and a CCHC-type zinc finger (ZF) domain (Fig. 1). The CC2-LZ domain is required for NEMO oligomerization (6, 7) and contains a ubiquitin-binding domain (UBD) that preferentially interacts with Lys-63-polyubiquitin (Lys-63-polyUb) chains (8, 9). Lys-63-polyUb binding through the CC2-LZ domain of NEMO is required for NF-B signaling from several different receptors. In the case of TNF-␣ receptor 1 (TNFR1), the IKK complex seems to be recruited to the TNFR1 receptor via interaction with Lys-63-polyubiquitinated RIP1 protein (8, 9). In addition to binding Lys-63-polyUb, a minor fraction of NEMO undergoes ubiquitination (post-translational modification via the covalent attachment of ubiquitin) within the regulatory portion of NEMO. Several monoubiquitination and Lys-63-polyubiquitination sites on NEMO have been identified, including a Lys-399 residue in the ZF domain (10), and the site of modification and its impact on IKK activity appear to be signal-dependent (11-13).Although the CC2-LZ domain of NEMO is required for a wide range of NF-B-inducing signals, the role of the NEMO ZF domain remains controversial (14, 15) and appears to depend on the stimulus. Several studies reported that NEMO ZF is required for interleukin-1 and T...

The Sir2 family of histone/protein deacetylases (sirtuins) is comprised of homologues found across all kingdoms of life. These enzymes catalyse a unique reaction in which NAD+ and acetylated substrate are converted into deacetylated product, nicotinamide, and a novel metabolite O‐acetyl ADP‐ribose. Although the catalytic mechanism is well conserved across Sir2 family members, sirtuins display differential specificity toward acetylated substrates, which translates into an expanding range of physiological functions. These roles include control of gene expression, cell cycle regulation, apoptosis, metabolism and ageing. The dependence of sirtuin activity on NAD+ has spearheaded investigations into how these enzymes respond to metabolic signals, such as caloric restriction. In addition, NAD+ metabolites and NAD+ salvage pathway enzymes regulate sirtuin activity, supporting a link between deacetylation of target proteins and metabolic pathways. Apart from physiological regulators, forward chemical genetics and high‐throughput activity screening has been used to identify sirtuin inhibitors and activators. This review focuses on small molecule regulators that control the activity and functions of this unusual family of protein deacetylases.

). To complement this biochemical analysis, we undertook a genetic approach to the analysis of the structure and function of the A20 protein. Here we report the application of clustered charge-to-alanine mutagenesis of the A20 gene. Eight mutant viruses containing altered A20 alleles were isolated using this approach; two of these, tsA20-6 and tsA20-ER5, have tight temperature-sensitive phenotypes. At the nonpermissive temperature, neither virus forms macroscopic plaques and the yield of infectious virus is <1% of that obtained at the permissive temperature. Both viruses show a profound defect in the accumulation of viral DNA at the nonpermissive temperature, although both the A20 protein and DNA polymerase accumulate to wild-type levels. Cytoplasmic extracts prepared from cells infected with the tsA20 viruses show a defect in processive polymerase activity; they are unable to direct the formation of RFII product using a singly primed M13 template. In sum, these data indicate that the A20 protein plays an essential role in the viral life cycle and that viruses with A20 lesions exhibit a DNA ؊ phenotype that is correlated with a loss in processive polymerase activity as assayed in vitro. The vaccinia virus A20 protein can, therefore, be considered a new member of the family of proteins (E9, B1, D4, and D5) with essential roles in vaccinia virus DNA replication.Vaccinia virus, the prototypic member of the poxvirus family, displays a great deal of genetic and physical autonomy from the host. The virus replicates solely within the cytoplasm of the host, and the 192-kb genome is thought to encode most if not all of the functions required for genome replication, gene expression, and virion morphogenesis. The centerpiece of the replication apparatus is the E9 DNA polymerase, which displays significant homology to the ␣ and ␦ families of eucaryotic replicative polymerases as well as the polymerases encoded by herpesviruses. We and others have characterized the polymerase both genetically and biochemically (4, 5,9, 10, 12, 29-31, 36, 38, 39, 41). Temperature-sensitive (ts) alleles, mutator and anti-mutator alleles, and mutants conferring resistance to aphidicolin, phosphonoacetic acid, and cytosine arabinoside have been isolated and studied. The polymerase has been overexpressed and purified and shown to have both polymerase and proofreading exonuclease activities. We have also shown that the enzyme is inherently distributive in vitro, being able to catalyze the addition of Ͻ10 nucleotides (nt) per binding event when moderate levels of salt (40 mM NaCl) or divalent cations (8 mM MgCl 2 ) are present (31). In sharp contrast, the cytoplasmic lysates of infected cells are able to catalyze the addition of as many as 7,000 nt in a single binding event under the same reaction conditions (29). We demonstrated that the protein(s) responsible for conferring processivity on the viral polymerase was present in extracts prepared from infected cells in which only early proteins were present but not in extracts prepared from uninfect...