Regulation of apoptosis by protein S-nitrosylation.

S-nitrosylation/denitrosylation of critical cysteine residues on proteins serves as a redox switch that regulates the function of a wide array of proteins. A key signaling pathway that is regulated by S-nitrosylation is apoptotic cell death. Here we will review the proteins in apoptotic pathways that are known to be S-nitrosylated by endogenous NO production. The targets and functional consequences of S-nitrosylation during apoptosis are multifaceted, allowing cells to fine tune their response to apoptotic signals.

S-Nitrosylation of mitochondrial caspases.

Caspase-3 is a cysteine protease located in both the cytoplasm and mitochondrial intermembrane space that is a central effector of many apoptotic pathways. In resting cells, a subset of caspase-3 zymogens is S-nitrosylated at the active site cysteine, inhibiting enzyme activity. During Fas-induced apoptosis, caspases are denitrosylated, allowing the catalytic site to function. In the current studies, we sought to identify the subpopulation of caspases that is regulated by S-nitrosylation. We report that the majority of mitochondrial, but not cytoplasmic, caspase-3 zymogens contain this inhibitory modification. In addition, the majority of mitochondrial caspase-9 is S-nitrosylated. These studies suggest that S-nitrosylation plays an important role in regulating mitochondrial caspase function and that the S-nitrosylation state of a given protein depends on its subcellular localization.

Accelerated s-nitrosothiol breakdown by amyotrophic lateral sclerosis mutant copper,zinc-superoxide dismutase.

Mutations in copper,zinc-superoxide dismutase (SOD) have been implicated in familial amyotrophic lateral sclerosis (FALS). We have investigated the breakdown of S-nitrosothiols by wild-type (WT) SOD and two common FALS mutants, alanine-4 valine (A4V) SOD and glycine-37 arginine (G37R) SOD. In the presence of glutathione, A4V SOD and G37R SOD catalyzed S-nitrosoglutathione breakdown three times more efficiently than WT SOD. Indeed, A4V SOD catabolized GSNO more efficiently than WT SOD throughout the physiological range of GSH concentrations. Moreover, a variety of additional S-nitrosothiols were catabolized more readily by A4V SOD than by WT SOD. Initial rate data for fully reduced WT SOD and A4V SOD, and data using ascorbic acid as the reductant, suggest that FALS mutations in SOD may influence the efficiency of reduction of the copper center by glutathione. We have identified a potentially toxic gain of function of two common FALS mutations that may contribute to neurodegeneration in FALS.

Nitric oxide modulates HIV-1 replication.

Although nitric oxide (NO) production is increased in HIV-1-infected patients, and NO is known to inhibit the replication of several viruses, very little is known about the effects of NO on HIV-1 replication. In the present studies, we find that S-nitrosothiols (RSNOs), a class of NO donor compounds present in the human circulatory system, inhibit HIV-1 replication in acutely infected human peripheral blood mononuclear cells (PBMCs) and have an additive inhibitory effect on HIV-1 replication in combination with 3'-azido-3'-deoxythymidylate (AZT). RSNOs inhibit HIV-1 replication in acutely infected PBMCs at a step in the viral replicative cycle after reverse transcription, but before or during viral protein expression through a cGMP-independent mechanism. In the latently infected U1 cell line, NO donor compounds and intracellular NO production stimulate HIV-1 reactivation. These studies suggest that NO both inhibits HIV-1 replication in acutely infected cells and stimulates HIV-1 reactivation in chronically infected cells. Thus, NO may have a physiologic role in HIV-1 replication, and NO donor compounds, which have been used for decades in the treatment of coronary artery disease with limited toxicity, might be useful in the treatment of HIV-1 disease by inhibiting acute infection, reactivating latent virus, or both.

Fas-induced caspase denitrosylation.

Only a few intracellular S-nitrosylated proteins have been identified, and it is unknown if protein S-nitrosylation/denitrosylation is a component of signal transduction cascades. Caspase-3 zymogens were found to be S-nitrosylated on their catalytic-site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway. Decreased caspase-3 S-nitrosylation was associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active-site thiol. Protein S-nitrosylation/denitrosylation can thus serve as a regulatory process in signal transduction pathways.

Nitric oxide inhibits Fas-induced apoptosis.

The Fas antigen (CD95, APO-1) is a transmembrane cell surface receptor that mediates apoptosis of many cell types when bound by Fas ligand or cross-linked by agonist antibody. The cellular factors regulating Fas-induced apoptosis have not been well defined. Here we show that basal nitric-oxide synthase (NOS) activity in human leukocytes inhibits Fas-induced apoptosis via a cGMP-independent mechanism. Further, NOS inhibits Fas-induced cleavage of poly(ADP-ribose) polymerase by members of the caspase family of cysteine proteases. These data suggest that Fas activity is under the control of the NO signaling pathway. NOS regulating the function of this member of the tumor necrosis factor receptor family suggests a new role for nitric oxide (or related molecules) in the human immune response.

The Epstein-Barr virus nuclear antigen leader protein associates with hsp72/hsc73.

Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) is important for primary B-lymphocyte growth transformation. We now demonstrate that the W repeat-encoded domain of EBNA-LP significantly associates with proteins of the heat shock protein 70 family (hsp72/hsc73). hsp72/hsc73 may mediate the previously observed interaction between EBNA-LP and the retinoblastoma protein or p53.

Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation.

Nitric oxide (NO) produced by murine macrophages is important in murine resistance to ectromelia virus, herpes simplex virus, and vaccinia virus infection. In contrast, NO production by human mononuclear cells has been difficult to demonstrate, and a role for NO in human responses to infection is uncertain. We report constitutive, low level, macrophage-type NO synthase (iNOS) expression in Epstein-Barr virus (EBV)-transformed human B lymphocytes and Burkitt's lymphoma cell lines. Immune NOS activity is involved in maintaining EBV latency through down-regulation of the expression of the immediate-early EBV transactivator Zta. NO also inhibits apoptosis in B lymphocyte cell lines. The effects of NO are largely independent of cGMP and influential on signaling pathways regulated by (sulfhydryl) redox status. These results suggest that NO plays a physiological role in human B cell biology by inhibiting programmed cell death and maintaining viral latency.

The Epstein-Barr virus nuclear protein encoded by the leader of the EBNA RNAs is important in B-lymphocyte transformation.

These experiments evaluate the role of the Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) in B-lymphocyte growth transformation by using a recombinant EBV molecular genetic approach. Recombinant viruses encoding for a mutant EBNA-LP lacking the carboxy-terminal 45 amino acids were markedly impaired in their ability to transform primary B lymphocytes compared with EBNA-LP wild-type but otherwise isogenic recombinant viruses. This impairment was particularly evident when primary B lymphocytes were infected under conditions of limiting virus dilution. The impairment could be partially corrected by growth of the infected lymphocytes with fibroblast feeder layers or by cocultivation of primary B lymphocytes with relatively highly permissive mutant virus-infected cells. One of the five mutant recombinants recovered by growth of infected cells on fibroblast feeder cultures was a partial revertant which had a normal transforming phenotype. Several lymphoblastoid cell lines infected with the EBNA-LP mutant recombinant viruses had a high percentage of cells with bright cytoplasmic immunoglobulin staining, as is characteristic of cells undergoing plasmacytoid differentiation. Expression of the other EBV latent or lytic proteins and viral replication were not affected by the EBNA-LP mutations. Thus, the EBNA-LP mutant phenotype is not mediated by an effect on expression of another EBV gene. These data are most compatible with the hypothesis that EBNA-LP affects expression of a B-lymphocyte gene which is a mediator of cell growth or differentiation.