Risheng Xu

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.

Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington’s disease

Huntington’s disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes. Huntington’s disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington’s disease may reflect the striatally selective small Gu2009protein Rhes binding to mHtt and enhancing its neurotoxicity. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington’s disease tissues, which may mediate Huntington’s disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington’s disease tissues and in intact mouse models of Huntington’s disease, suggesting therapeutic potential.

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.

Inositol polyphosphate multikinase is a physiologic PI3-kinase that activates Akt/PKB

The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP3 as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP3 generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.

Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2.

The apoptotic actions of p53 require its phosphorylation by a family of phosphoinositide-3-kinase-related-kinases (PIKKs), which include DNA-PKcs and ATM. These kinases are stabilized by the TTT (Tel2, Tti1, Tti2) cochaperone family, whose actions are mediated by CK2 phosphorylation. The inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks), of which IP6K2 has been implicated in p53-associated cell death. In the present study we report an apoptotic signaling cascade linking CK2, TTT, the PIKKs, and p53. We demonstrate that IP7, formed by IP6K2, binds CK2 to enhance its phosphorylation of the TTT complex, thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine 15 to activate the cell death program in human cancer cells and in murine B cells.

Inositol pyrophosphates promote tumor growth and metastasis by antagonizing liver kinase B1

Significance Inositol pyrophosphates are messenger molecules incorporating the energetic pyrophosphate bond. Although they have been implicated in diverse biologic processes, their physiologic functions remain enigmatic. We show that the catalytic activity of inositol hexakisphosphate kinase 2 (IP6K2), one of the principal enzymes generating the inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), mediates cancer cell migration and tumor metastasis both in cell culture and intact mice. In this process, IP6K2 diminishes cell–cell adhesion, enabling cells to invade the intercellular matrix. Drugs that inhibit IP6K2 may be beneficial in cancer therapy. The inositol pyrophosphates, molecular messengers containing an energetic pyrophosphate bond, impact a wide range of biologic processes. They are generated primarily by a family of three inositol hexakisphosphate kinases (IP6Ks), the principal product of which is diphosphoinositol pentakisphosphate (IP7). We report that IP6K2, via IP7 synthesis, is a major mediator of cancer cell migration and tumor metastasis in cell culture and in intact mice. IP6K2 acts by enhancing cell-matrix adhesion and decreasing cell–cell adhesion. This action is mediated by IP7-elicited nuclear sequestration and inactivation of the tumor suppressor liver kinase B1 (LKB1). Accordingly, inhibitors of IP6K2 offer promise in cancer therapy.

Inositol Polyphosphate Multikinase Is a Coactivator of p53-Mediated Transcription and Cell Death

By promoting p53 acetylation and activation, IPMK contributes to DNA damage–induced cell death. Activating p53 with IMPK The transcription factor p53 mediates cell death in response to cellular stress. Xu et al. found that the transcriptional activity of p53 was enhanced by inositol polyphosphate multikinase (IPMK). IPMK bound to p53 directly and stimulated its acetylation by the acetyltransferase p300. Loss of IPMK or disruption of the IPMK-p53 interaction decreased acetylation of both p53 and the promoters of its target genes, reduced the transcription of p53 target genes, and decreased p53-mediated apoptosis. Thus, IPMK functions as a transcriptional coactivator of p53. The tumor suppressor protein p53 is a critical stress response transcription factor that induces the expression of genes leading to cell cycle arrest, apoptosis, and tumor suppression. We found that mammalian inositol polyphosphate multikinase (IPMK) stimulated p53-mediated transcription by binding to p53 and enhancing its acetylation by the acetyltransferase p300 independently of its inositol phosphate and lipid kinase activities. Genetic or RNA interference (RNAi)–mediated knockdown of IPMK resulted in decreased activation of p53, decreased recruitment of p53 and p300 to target gene promoters, abrogated transcription of p53 target genes, and enhanced cell viability. Additionally, blocking the IPMK-p53 interaction decreased the extent of p53-mediated transcription. These results suggest that IPMK acts as a transcriptional coactivator for p53 and that it is an integral part of the p53 transcriptional complex facilitating cell death.

Inositol polyphosphate multikinase is a transcriptional coactivator required for immediate early gene induction

Significance The induction of immediate early genes (IEGs) by neural stimuli underlies much of the plasticity of brain function, but regulatory mechanisms have been obscure. Inositol polyphosphate multikinase (IPMK) is a notably pleiotropic enzyme that displays inositol phosphate kinase activity and phosphatidylinositol kinase activity and exhibits physiologically noncatalytic actions such as stabilizing the mammalian target of rapamycin complex 1 complex. We report that IPMK is required for IEG induction by neural activation and neurotrophic stimuli. We have elucidated the molecular mechanisms responsible for IPMK influences; namely, that it enhances the transcriptional coactivation ability of Creb-binding protein (CBP). This epigenetic regulation of IEGs may have both neural and nonneural implications, as IPMK and CBP are broadly expressed in a variety of tissues. Profound induction of immediate early genes (IEGs) by neural activation is a critical determinant for plasticity in the brain, but intervening molecular signals are not well characterized. We demonstrate that inositol polyphosphate multikinase (IPMK) acts noncatalytically as a transcriptional coactivator to mediate induction of numerous IEGs. IEG induction by electroconvulsive stimulation is virtually abolished in the brains of IPMK-deleted mice, which also display deficits in spatial memory. Neural activity stimulates binding of IPMK to the histone acetyltransferase CBP and enhances its recruitment to IEG promoters. Interestingly, IPMK regulation of CBP recruitment and IEG induction does not require its catalytic activities. Dominant-negative constructs, which prevent IPMK-CBP binding, substantially decrease IEG induction. As IPMK is ubiquitously expressed, its epigenetic regulation of IEGs may influence diverse nonneural and neural biologic processes.

Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

Significance Attachment of the small protein ubiquitin to other proteins, a process called “ubiquitylation,” triggers protein degradation. The Cullin and signalosome protein families cooperatively mediate this process, but how they are regulated has been obscure. We show that an inositol polyphosphate with seven phosphates, hence designated IP7, is critical. Under basal conditions, the enzyme that generates IP7 is bound to the Cullin/signalosome complex, which is thereby maintained in an inactive state. Stressful stimuli, such as UV radiation, stimulate the enzyme to form IP7, which dissociates the complex, leading to activation of the Cullins with attendant ubiquitylation and degradation of target proteins. This process may play a key role in how cells respond to environmental stressors. Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1–3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN–CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4–CSN complex, thereby regulating nucleotide excision repair and cell death.

Behavioral Effects of Cocaine Mediated by Nitric Oxide-GAPDH Transcriptional Signaling

Cocaines behavioral-stimulant effects derive from potentiation of synaptic signaling by dopamine and serotonin leading to transcriptional alterations in postsynaptic cells. We report that a signaling cascade involving nitric oxide (NO) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mediates cocaines transcriptional and behavioral actions. Lower, behavioral-stimulant doses enhance the cAMP response element-binding (CREB) signaling system, while higher, neurotoxic doses stimulate the p53 cytotoxic system. The drug CGP3466B, which potently and selectively blocks GAPDH nitrosylation and GAPDH-Siah binding, prevents these actions as well as behavioral effects of cocaine providing a strategy for anticocaine therapy.