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Pyridine dinucleotides (NAD and NADP) are ubiquitous cofactors involved in hundreds of redox reactions essential for the energy transduction and metabolism in all living cells. In addition, NAD also serves as a substrate for ADP-ribosylation of a number of nuclear proteins, for silent information regulator 2 (Sir2)-like histone deacetylase that is involved in gene silencing regulation, and for cyclic ADP ribose (cADPR)-dependent Ca 2؉ signaling. Pyridine nucleotide adenylyltransferase (PNAT) is an indispensable central enzyme in the NAD biosynthesis pathways catalyzing the condensation of pyridine mononucleotide (NMN or NaMN) with the AMP moiety of ATP to form NAD (or NaAD). Here we report the identification and structural characterization of a novel human PNAT (hsPNAT-3) that is located in the cytoplasm and mitochondria. Its subcellular localization and tissue distribution are distinct from the previously identified human nuclear PNAT-1 and PNAT-2. Detailed structural analysis of PNAT-3 in its apo form and in complex with its substrate(s) or product revealed the catalytic mechanism of the enzyme. The characterization of the cytosolic human PNAT-3 provided compelling evidence that the final steps of NAD biosynthesis pathways may exist in mammalian cytoplasm and mitochondria, potentially contributing to their NAD/NADP pool. The coenzymes NADϩ (H) 1 and NADP ϩ (H) have been known for many decades as the major hydrogen donor or acceptor in hundreds of metabolic redox reactions throughout the cell. Together these nucleotides have a direct impact on virtually every cellular metabolic pathway. Additionally, NAD serves as a substrate for the covalent modification of nuclear proteins by ADP-ribosylation, a process involved in DNA repair and the regulation of genomic instability (1-3). Recently, many new exciting functions have been discovered for this long-known molecule. These include its role as co-substrate in Sir2-mediated histone deacetylation involved in gene silencing regulation and in increasing the lifespan of species ranging from yeast, to worm, to certain mammals (4, 5). Moreover, several derivatives of NAD and NADP were found to be potent intracellular calcium-mobilizing agents and are involved in a variety of Ca 2ϩ -signaling pathways (6 -8). These recent developments brought a significant amount of additional interest to the investigation of cellular NAD biosynthesis and regulation.NMN and/or NaMN adenylyltransferase (NMNAT and/or NaMNAT, collectively named pyridine nucleotide adenylyltransferase, or PNAT) is an indispensable enzyme catalyzing the central step of all NAD biosynthesis pathways (9, 10). It links the AMP moiety of ATP with the nicotinamide mononucleotide (NMN, or its deamidated form NaMN) to form the dinucleotide product NAD (or deamido-NAD, NaAD). A practical aspect of human PNAT function is that it catalyzes the rate-limiting step in the metabolic conversion of the anticancer agent tiazofurin to its active form TAD (tiazofurin adenine dinucleotide, an NAD analog) (11). The development of ti...

The PDZ1 domain of the Na ؉ /H؉ exchanger regulatory factor (NHERF) binds with nanomolar affinity to the carboxyl-terminal sequence QDTRL of the cystic fibrosis transmembrane conductance regulator (CFTR) and plays a central role in the cellular localization and physiological regulation of this chloride channel. The crystal structure of human NHERF PDZ1 bound to the carboxyl-terminal peptide QDTRL has been determined at 1.7-Å resolution. The structure reveals the specificity and affinity determinants of the PDZ1-CFTR interaction and provides insights into carboxyl-terminal leucine recognition by class I PDZ domains. The peptide ligand inserts into the PDZ1 binding pocket forming an additional antiparallel ␤-strand to the PDZ1 ␤-sheet, and an extensive network of hydrogen bonds and hydrophobic interactions stabilize the complex. Remarkably, the guanido group of arginine at position ؊1 of the CFTR peptide forms two salt bridges and two hydrogen bonds with PDZ1 residues Glu 43 and Asn 22 , respectively, providing the structural basis for the contribution of the penultimate amino acid of the peptide ligand to the affinity of the interaction.

Objective To assess neurocognitive impairment after the off-pump and on-pump techniques for coronary artery bypass graft surgery in patients with triple vessel disease. Design Randomised controlled trial. Setting University Hospital of Wales, Cardiff. Participants 60 patients undergoing coronary artery bypass graft surgery for triple vessel disease prospectively randomised to the off-pump or on-pump technique. Main outcome measures Change in scores in nine standard neuropsychometric tests administered preoperatively and at 1 and 10 weeks postoperatively. Results The on-pump group showed a significantly greater deterioration in scores for two and three tests at 1 week and 10 weeks postoperatively, respectively, than the off-pump group. The on-pump group also showed a significantly higher incidence of major deterioration in one of the tests both 1 week and 10 weeks postoperatively. The incidence of neurocognitive impairment at 1 week postoperatively was 27% (8 out of 30) in the off-pump group and 63% (19 out of 30) in the on-pump group (P=0.004); and at 10 weeks postoperatively was 10% (3 out of 30) in the off-pump group and 40% (12 out of 30) in the on-pump group (P=0.017). Conclusion Off-pump coronary artery bypass graft surgery results in less neurocognitive impairment than the on-pump technique.