Nilkantha Sen

H2S Signals Through Protein S-Sulfhydration

The gaseous messenger hydrogen sulfide regulates target proteins through S-sulfhydration of cysteine residues. Battling for the Same Cysteine? Recent evidence suggests that hydrogen sulfide (H2S)—a gas perhaps best known for its scent of rotten eggs—has joined nitric oxide (NO) and carbon monoxide in the select ranks of gases that act as physiologic messenger molecules. Although H2S is enzymatically generated in vivo and mediates various physiologic functions, including acting as a vasorelaxant and eliciting hibernation states, the mechanisms through which it affects its targets have been unclear. Here, Mustafa et al. show that endogenous H2S modifies cysteine residues in many proteins, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin, converting cysteine -SH groups to -SSH groups in a process the authors call S-sulfhydration. Intriguingly, H2S enhanced GAPDH activity through sulfhydration of a cysteine residue that is also a target of nitrosylation by NO, which inhibits GAPDH activity, suggesting that some targets might be subject to regulation through competitive nitrosylation and sulfhydration of the same cysteine residues. Hydrogen sulfide (H2S), a messenger molecule generated by cystathionine γ-lyase, acts as a physiologic vasorelaxant. Mechanisms whereby H2S signals have been elusive. We now show that H2S physiologically modifies cysteines in a large number of proteins by S-sulfhydration. About 10 to 25% of many liver proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are sulfhydrated under physiological conditions. Sulfhydration augments GAPDH activity and enhances actin polymerization. Sulfhydration thus appears to be a physiologic posttranslational modification for proteins.

Hydrogen sulfide-linked sulfhydration of NF-κB mediates its antiapoptotic actions.

Nuclear factor κB (NF-κB) is an antiapoptotic transcription factor. We show that the antiapoptotic actions of NF-κB are mediated by hydrogen sulfide (H(2)S) synthesized by cystathionine gamma-lyase (CSE). TNF-α treatment triples H(2)S generation by stimulating binding of SP1 to the CSE promoter. H(2)S generated by CSE stimulates DNA binding and gene activation of NF-κB, processes that are abolished in CSE-deleted mice. As CSE deletion leads to decreased glutathione levels, resultant oxidative stress may contribute to alterations in CSE mutant mice. H(2)S acts by sulfhydrating the p65 subunit of NF-κB at cysteine-38, which promotes its binding to the coactivator ribosomal protein S3 (RPS3). Sulfhydration of p65 predominates early after TNF-α treatment, then declines and is succeeded by a reciprocal enhancement of p65 nitrosylation. In CSE mutant mice, antiapoptotic influences of NF-κB are markedly diminished. Thus, sulfhydration of NF-κB appears to be a physiologic determinant of its antiapoptotic transcriptional activity.

Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis.

Besides its role in glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) initiates a cell death cascade. Diverse apoptotic stimuli activate inducible nitric oxide synthase (iNOS) or neuronal NOS (nNOS), with the generated nitric oxide (NO) S-nitrosylating GAPDH, abolishing its catalytic activity and conferring on it the ability to bind to Siah1, an E3-ubiquitin-ligase with a nuclear localization signal (NLS). The GAPDH–Siah1 protein complex, in turn, translocates to the nucleus and mediates cell death; these processes are blocked by procedures that interfere with GAPDH–Siah1 binding. Nuclear events induced by GAPDH to kill cells have been obscure. Here we show that nuclear GAPDH is acetylated at Lys 160 by the acetyltransferase p300/CREB binding protein (CBP) through direct protein interaction, which in turn stimulates the acetylation and catalytic activity of p300/CBP. Consequently, downstream targets of p300/CBP, such as p53 (Refs 10,11,12,13,14,15), are activated and cause cell death. A dominant-negative mutant GAPDH with the substitution of Lys 160 to Arg (GAPDH-K160R) prevents activation of p300/CBP, blocks induction of apoptotic genes and decreases cell death. Our findings reveal a pathway in which NO-induced nuclear GAPDH mediates cell death through p300/CBP.

GAPDH Mediates Nitrosylation of Nuclear Proteins

S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS—neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) —are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO–GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO–GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein–protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.

Camptothecin induced mitochondrial dysfunction leading to programmed cell death in unicellular hemoflagellate Leishmania donovani

AbstractThe parasites of the order kinetoplastidae including Leishmania spp. emerge from most ancient phylogenic branches of unicellular eukaryotic lineages. In their life cycle, topoisomerase I plays a significant role in carrying out vital cellular processes. Camptothecin (CPT), an inhibitor of DNA topoisomerase I, induces programmed cell death (PCD) both in the amastigotes and promastigotes form of L. donovani parasites. CPT-induced cellular dysfunction in L. donovani promastigotes is characterized by several cytoplasmic and nuclear features of apoptosis. CPT inhibits cellular respiration that results in mitochondrial hyperpolarization taking place by oligomycin-sensitive F0-F1 ATPase-like protein in leishmanial cells. During the early phase of activation, there is an increase in reactive oxygen species (ROS) inside cells, which causes subsequent elevation in the level of lipid peroxidation and decrease in reducing equivalents like GSH. Endogenous ROS formation and lipid peroxidation cause eventual loss of mitochondrial membrane potential. Furthermore, cytochrome c is released into the cytosol in a manner independent of involvement of CED3/CPP32 group of proteases and unlike mammalian cells it is insensitive to cyclosporin A. These events are followed by activation of both CED3/CPP32 and ICE group of proteases in PCD of Leishmania. Taken together, our study indicates that different biochemical events leading to apoptosis in leishmanial cells provide information that could be exploited to develop newer potential therapeutic targets.

Sulfhydration mediates neuroprotective actions of parkin

Increases in S-nitrosylation and inactivation of the neuroprotective ubiquitin E3 ligase, parkin, in the brains of patients with Parkinson’s Disease (PD) are thought to be pathogenic and suggest a possible mechanism linking parkin to sporadic PD. Here we demonstrate that physiologic modification of parkin by hydrogen sulfide (H2S), termed sulfhydration, enhances its catalytic activity. Sulfhydration sites are identified by mass spectrometry analysis and investigated by site directed mutagenesis. Parkin sulfhydration is markedly depleted in the brains of patients with PD, suggesting that this loss may be pathologic. This implies that H2S donors may be therapeutic.

GOSPEL: A Neuroprotective Protein that Binds to GAPDH upon S-Nitrosylation

We recently reported a cell death cascade whereby cellular stressors activate nitric oxide formation leading to S-nitrosylation of GAPDH that binds to Siah and translocates to the nucleus. The nuclear GAPDH/Siah complex augments p300/CBP-associated acetylation of nuclear proteins, including p53, which mediate cell death. We report a 52 kDa cytosolic protein, GOSPEL, which physiologically binds GAPDH, in competition with Siah, retaining GAPDH in the cytosol and preventing its nuclear translocation. GOSPEL is neuroprotective, as its overexpression prevents NMDA-glutamate excitotoxicity while its depletion enhances death in primary neuron cultures. S-nitrosylation of GOSPEL at cysteine 47 enhances GAPDH-GOSPEL binding and the neuroprotective actions of GOSPEL. In intact mice, virally delivered GOSPEL selectively diminishes NMDA neurotoxicity. Thus, GOSPEL may physiologically regulate the viability of neurons and other cells.

Apoptosis is induced in leishmanial cells by a novel protein kinase inhibitor withaferin A and is facilitated by apoptotic topoisomerase I-DNA complex

Protein kinase C (PKC) is an important constituent of the signaling pathways involved in apoptosis. We report here that like staurosporine, withaferin A is a potent inhibitor of PKC. In Leishmania donovani, the inhibition of PKC by withaferin A causes depolarization of ΔΨm and generates ROS inside cells. Loss of ΔΨm leads to the release of cytochrome c into the cytosol and subsequently activates caspase-like proteases and oligonucleosomal DNA cleavage. Moreover, in treated cells, oxidative DNA lesions facilitate the stabilization of topoisomerase I-mediated cleavable complexes, which also contribute to DNA fragmentation. However, withaferin A and staurosporine cannot induce cleavable complex formation in vitro with recombinant topoisomerase I nor with nuclear extracts from control cells. Taken together, our results indicate that inhibition of PKC by withaferin A is a central event for the induction of apoptosis and that the stabilization of topoisomerase I–DNA complex is necessary to amplify apoptotic process.

Betulinic Acid, a Catalytic Inhibitor of Topoisomerase I, Inhibits Reactive Oxygen Species–Mediated Apoptotic Topoisomerase I–DNA Cleavable Complex Formation in Prostate Cancer Cells but Does Not Affect the Process of Cell Death

The ubiquitious enzyme topoisomerase I can be targeted by drugs which turn these enzymes into cellular poisons and subsequently induce cell death. Drugs like staurosporine, which do not target topoisomerase I directly, can also lead to stabilization of topoisomerase I-DNA cleavable complexes by an indirect process of reactive oxygen species (ROS) generation and subsequent oxidative DNA damage. In this study, we show that betulinic acid, a catalytic inhibitor of topoisomerases, inhibits the formation of apoptotic topoisomerase I-DNA cleavable complexes in prostate cancer cells induced by drugs like camptothecin, staurosporine, and etoposide. Although events like ROS generation, oxidative DNA damage, and DNA fragmentation were observed after betulinic acid treatment, there is no topoisomerase I-DNA cleavable complex formation, which is a key step in ROS-induced apoptotic processes. We have shown that betulinic acid interacts with cellular topoisomerase I and prohibits its interaction with the oxidatively damaged DNA. Using oligonucleotide containing 8-oxoguanosine modification, we have shown that betulinic acid inhibits its cleavage by topoisomerase I in vitro. Whereas silencing of topoisomerase I gene by small interfering RNA reduces cell death in the case of staurosporine and camptothecin, it cannot substantially reduce betulinic acid-induced cell death. Thus, our study provides evidence that betulinic acid inhibits formation of apoptotic topoisomerase I-DNA complexes and prevents the cellular topoisomerase I from directly participating in the apoptotic process.

Neurotrophin-mediated degradation of histone methyltransferase by S-nitrosylation cascade regulates neuronal differentiation.

Epigenetic regulation of histones mediates neurotrophin actions with histone acetylation enhancing cAMP response element-binding (CREB)-associated transcription elicited by brain-derived neurotrophic factor (BDNF) and nerve-growth factor (NGF). Roles for histone methylation in CREBs transcriptional activity have not been well characterized. We show that depletion of the histone methyltransferase suppressor of variegation 3–9 homolog 1 (SUV39H1) selectively augments BDNF- and NGF-mediated neurite outgrowth. SUV39H1 is the principal enzyme responsible for trimethylation of histone H3 at lysine 9, a molecular mark associated with transcriptional silencing. BDNF and NGF act via a signaling cascade wherein degradation of SUV39H1 down-regulates trimethylation of H3K9 in a nitric oxide-dependent pathway. BDNF activates neuronal NOS with the nitrosylated GAPDH/seven in absentia (Siah) homolog complex translocating to the nucleus. Degradation of SUV39H1 by Siah facilitates histone H3 on lysine 9 acetylation, CREB binding to DNA, enhanced expression of CREB-regulated genes and neurite outgrowth.