Supramolecular complex formation between Rad6 and proteins of the p53 pathway during DNA damage-induced response

The HR6A and -B genes, homologues of the yeast Rad6 gene, encode ubiquitin-conjugating enzymes that are required for postreplication repair of DNA and damage-induced mutagenesis. Using surface plasmon resonance, we show here that HR6 protein (referred as Rad6) physically interacts with p53. Analysis of proteins coimmunoprecipitated with Rad6 antibody from metabolically labeled normal MCF10A human breast epithelial cells not only confirmed Rad6-p53 interactions in vivo but also demonstrated for the first time that exposure of MCF10A cells to cisplatin or adriamycin (ADR) induces recruitment of p14ARF into Rad6-p53 complexes. Further analysis of ADR-induced p53 response showed that stable Rad6-p53-p14ARF complex formation is associated with a parallel increase and decrease in monoubiquitinated and polyubiquitinated p53, respectively, and arrest in G(2)/M phase of the cell cycle. Interestingly, the ADR-induced suppression of p53 polyubiquitination correlated with a corresponding decline in intact Hdm2 protein levels. Treatment of MCF10A cells with MG132, a 26S proteasome inhibitor, effectively stabilized monoubiquitinated p53 and rescued ADR-induced downregulation of Hdm2. These data suggest that ADR-induced degradation of Hdm2 occurs via the ubiquitin-proteasome pathway. Rad6 is present in both the cytoplasmic and nuclear compartments of normal MCF10A cells, although in response to DNA damage it is predominantly found in the nucleus colocalizing with ubiquitinated p53, whereas Hdm2 is undetectable. Consistent with in vivo data, results from in vitro ubiquitination assays show that Rad6 mediates addition of one (mono-) to two (multimono-) ubiquitin molecules on p53 and that inclusion of Mdm2 is essential for its polyubiquitination. The data presented in the present study suggest that Rad6-p53-p14ARF complex formation and p53 ubiquitin modification are important damage-induced responses that perhaps determine the fidelity of DNA postreplication repair.     

Nitric oxide inhibits ornithine decarboxylase via S-nitrosylation of cysteine 360 in the active site of the enzyme.

Ornithine decarboxylase is the initial and rate-limiting enzyme in the polyamine biosynthetic pathway. Polyamines are found in all mammalian cells and are required for cell growth. We previously demonstrated that N-hydroxyarginine and nitric oxide inhibit tumor cell proliferation by inhibiting arginase and ornithine decarboxylase, respectively, and, therefore, polyamine synthesis. In addition, we showed that nitric oxide inhibits purified ornithine decarboxylase by S-nitrosylation. Herein we provide evidence for the chemical mechanism by which nitric oxide and S-nitrosothiols react with cysteine residues in ornithine decarboxylase to form an S-nitrosothiol(s) on the protein. The diazeniumdiolate nitric oxide donor agent 1-diethyl-2-hydroxy-2-nitroso-hydrazine acts through an oxygen-dependent mechanism leading to formation of the nitrosating agents N(2)O(3) and/or N(2)O(4). S-Nitrosoglutathione inhibits ornithine decarboxylase by an oxygen-independent mechanism likely by S-transnitrosation. In addition, we provide evidence for the S-nitrosylation of 4 cysteine residues per ornithine decarboxylase monomer including cysteine 360, which is critical for enzyme activity. Finally S-nitrosylated ornithine decarboxylase was isolated from intact cells treated with nitric oxide, suggesting that nitric oxide may regulate ornithine decarboxylase activity by S-nitrosylation in vivo.     

S-nitrosylation of FLICE inhibitory protein determines its interaction with RIP1 and activation of NF-κB

Death receptor (DR) ligation can lead to divergent signaling pathways causing either caspase-mediated cell death or cell proliferation and inflammation. These variations in cellular fate are determined by adaptor proteins that are recruited to the DR signaling complex. FLICE inhibitory protein (FLIP) is an established inhibitor of caspase-8-mediated apoptosis, and it is also involved in NF-κB activation. However, the molecular mechanism that regulates FLIP within this complex is unknown. In this study, we provide new evidence for the regulation of NF-κB by FLIP through S-nitrosylation, which involves covalent modification of the protein’s cysteine thiol by nitric oxide to form S-nitrosothiol. Point mutations of FLIP at cysteine residues 254 and 259 prevent FLIP S-nitrosylation and its ability to activate NF-κB. The mechanism by which FLIP nitrosylation regulates NF-κB activity involves RIP1 binding and redistribution, whereas TRAF2 binding and distribution are unaffected. We further show that FLIP processing and cleavage is dependent on its nitrosylation status. Collectively, our study reveals a novel pathway for FLIP regulation of NF-κB through protein S-nitrosylation, which is a key posttranslational mechanism controlling DR-mediated cell death and survival. Since increased expression of FLIP and nitric oxide are frequently observed in chemotherapy-resistant tumors, S-nitrosylation of FLIP could be a key mechanism of chemoresistance and tumor growth.     

Methionine adenosyltransferase S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target thiol.

S-Adenosylmethionine serves as the methyl donor for many biological methylation reactions and provides the propylamine group for the synthesis of polyamines. S-Adenosylmethionine is synthesized from methionine and ATP by the enzyme methionine adenosyltransferase. The cellular factors regulating S-adenosylmethionine synthesis have not been well defined. Here we show that in rat hepatocytes S-nitrosoglutathione monoethyl ester, a cell-permeable nitric oxide donor, markedly reduces cellular S-adenosylmethionine content via inactivation of methionine adenosyltransferase by S-nitrosylation. Removal of the nitric oxide donor from the incubation medium leads to the denitrosylation and reactivation of methionine adenosyltransferase and to the rapid recovery of cellular S-adenosylmethionine levels. Nitric oxide inactivates methionine adenosyltransferase via S-nitrosylation of cysteine 121. Replacement of the acidic (aspartate 355) or basic (arginine 357 and arginine 363) amino acids located in the vicinity of cysteine 121 by serine leads to a marked reduction in the ability of nitric oxide to S-nitrosylate and inactivate hepatic methionine adenosyltransferase. These results indicate that protein S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target cysteine.     

Studying the S-nitrosylation of model peptides and eNOS protein by mass spectrometry.

Oxidative addition of a nitric oxide (NO) molecule to the thiol group of cysteine residues is a physiologically important post-translational modification that has been implicated in several metabolic and pathophysiological events. Our previous studies have indicated that S-nitrosylation can result in the disruption of the endothelial NO synthase (eNOS) dimer. It has been suggested that for S-nitrosylation to occur, the cysteine residue must be flanked by hydrophilic residues either in the primary structure or in the spatial proximity through appropriate conformation. However, this hypothesis has not been confirmed. Thus, the objective of this study was to determine if the nature of the amino acid residues that flank the cysteine in the primary structure has a significant effect on the rate and/or specificity of S-nitrosylation. To accomplish this, we utilized several model peptides based on the eNOS protein sequence. Some of these peptides contained point mutations to allow for different combinations of amino acid properties (acidic, basic, and hydrophobic) around the cysteine residue. To ensure that the results obtained were not dependent on the nitrosylation procedure, several common S-nitrosylation techniques were used and S-nitrosylation followed by mass spectrometric detection. Our data indicated that all peptides independent of the amino acids surrounding the cysteine residue underwent rapid S-nitrosylation. Thus, there does not appear to be a profound effect of the primary sequence of adjacent amino acid residues on the rate of cysteine S-nitrosylation at least at the peptide levels. Finally, our studies using recombinant human eNOS confirm that Cys98 undergoes S-nitrosylation. Thus, our data validate the importance of Cys98 in regulating eNOS dimerization and activity, and the utility of mass spectroscopy to identify cysteine residues susceptible to S-nitrosoylation.     

Defining the molecular basis of Arf and Hdm2 interactions.

Understanding the interaction of Arf and Hdm2 has recently become a central issue in cancer biology. In response to hyperproliferative signals, p14(Arf) stabilizes p53 by binding to Hdm2 and inhibits the ubiquitination and subsequent proteosome-dependent degradation of p53. The medical importance of the Arf-Hdm2-p53 regulatory system is highlighted by the finding that either p53 or p14(Arf) are lost or modified in virtually all human cancers. Isolated Arf and Hdm2 domains are dynamically disordered in solution, yet they retain the ability to interact in vitro and in cellular assays. Upon binding, domains of both Arf and Hdm2 undergo a dramatic transition from disordered conformations to extended structures comprised of beta-strands. The presence of domains from both proteins are necessary and sufficient for the formation of the highly stable extended beta structures. We have mapped sites within Arf and Hdm2 that interact at a resolution of five amino acid residues using surface plasmon resonance. Surface plasmon resonance and circular dichroism spectropolarimetry confirm the presence of multiple interaction domains within each protein. Both p14(Arf) (human) and p19(Arf) (mouse) interact with Hdm2 through two short motifs present in their N termini. The Arf interacting region of Hdm2 is also composed of two short sequences located in the central acidic domain, between residues 235-264 and 270-289. The binding-induced structural transition is also induced by short peptides, 15 amino acids in length, that contain the binding motifs. Micro-injection and live cell imaging of proteins tagged with fluorescent labels was used to confirm the in vivo function of the interaction domains. Arf and Hdm2 thus appear to interact through a novel mechanism that exerts control over the cell division cycle. The novel molecular mechanism of interaction and the limited size of the protein domains involved provide opportunities for the development of anticancer therapeutics.     

Recognition of the tumor suppressor protein p53 and other protein targets by the calcium-binding protein S100B

S100B is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effect by binding and affecting various target proteins. A consensus sequence for S100B target proteins was published as (K/R)(L/I)xWxxIL and matches a region in the actin capping protein CapZ (V.V. Ivanenkov, G.A. Jamieson, Jr., E. Gruenstein, R.V. Dimlich, Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ, J. Biol. Chem. 270 (1995) 14651-14658). Several additional S100B targets are known including p53, a nuclear Dbf2 related (NDR) kinase, the RAGE receptor, neuromodulin, protein kinase C, and others. Examining the binding sites of such targets and new protein sequence searches provided additional potential target proteins for S100B including Hdm2 and Hdm4, which were both found to bind S100B in a calcium-dependent manner. The interaction between S100B and the Hdm2 and/or the Hdm4 proteins may be important physiologically in light of evidence that like Hdm2, S100B also contributes to lowering protein levels of the tumor suppressor protein, p53. For the S100B-p53 interaction, it was found that phosphorylation of specific serine and/or threonine residues reduces the affinity of the S100B-p53 interaction by as much as an order of magnitude, and is important for protecting p53 from S100B-dependent down-regulation, a scenario that is similar to what is found for the Hdm2-p53 complex.     

S-nitrosylation-dependent inactivation of Akt/protein kinase B in insulin resistance

Inducible nitric-oxide synthase (iNOS) has been implicated in many human diseases including insulin resistance. However, how iNOS causes or exacerbates insulin resistance remains largely unknown. Protein S-nitrosylation is now recognized as a prototype of a redox-dependent, cGMP-independent signaling component that mediates a variety of actions of nitric oxide (NO). Here we describe the mechanism of inactivation of Akt/protein kinase B (PKB) in NO donor-treated cells and diabetic (db/db) mice. NO donors induced S-nitrosylation and inactivation of Akt/PKB in vitro and in intact cells. The inhibitory effects of NO donor were independent of phosphatidylinositol 3-kinase and cGMP. In contrast, the concomitant presence of oxidative stress accelerated S-nitrosylation and inactivation of Akt/PKB. In vitro denitrosylation with reducing agent reactivated recombinant and cellular Akt/PKB from NO donor-treated cells. Mutated Akt1/PKBalpha (C224S), in which cysteine 224 was substituted by serine, was resistant to NO donor-induced S-nitrosylation and inactivation, indicating that cysteine 224 is a major S-nitrosylation acceptor site. In addition, S-nitrosylation of Akt/PKB was increased in skeletal muscle of diabetic (db/db) mice compared with wild-type mice. These data suggest that S-nitrosylation-mediated inactivation may contribute to the pathogenesis of iNOS- and/or oxidative stress-involved insulin resistance.     

Biological selectivity and functional aspects of protein tyrosine nitration

The formation of nitric oxide in biological systems has led to the discovery of a number of post-translational protein modifications that could regulate protein function or potentially be utilized as transducers of nitric oxide signaling. Principal among the nitric oxide-mediated protein modifications are: the nitric oxide-iron heme binding, the S-nitrosylation of reduced cysteine residues, and the C-nitration of tyrosine and tryptophan residues. With the exception of the nitric oxide binding to heme iron proteins, the other two modifications appear to require secondary reactions of nitric oxide and the formation of nitrogen oxides. The rapid development of analytical and immunological methodologies has allowed for the quantification of S-nitrosylated and C-nitrated proteins in vivo revealing an apparent selectivity and specificity of the proteins modified. This review is primarily focused upon the nitration of tyrosine residues discussing parameters that may govern the in vivo selectivity of protein nitration, and the potential biological significance and clinical relevance of this nitric oxide-mediated protein modification.     

Nitric oxide-mediated inhibition of taurocholate uptake involves S-nitrosylation of NTCP

The sodium-taurocholate (TC) cotransporting polypeptide (NTCP) facilitates bile formation by mediating sinusoidal Na(+)-TC cotransport. During sepsis-induced cholestasis, there is a decrease in NTCP-dependent uptake of bile acids and an increase in nitric oxide (NO) levels in hepatocytes. In rat hepatocytes NO inhibits Na(+)-dependent uptake of taurocholate. The aim of this study was to extend these findings to human NTCP and to further investigate the mechanism by which NO inhibits TC uptake. Using a human hepatoma cell line stably expressing NTCP (HuH-NTCP), we performed experiments with the NO donors sodium nitroprusside and S-nitrosocysteine and demonstrated that NO inhibits TC uptake in these cells. Kinetic analyses revealed that NO significantly decreased the V(max) but not the K(m) of TC uptake by NTCP, indicating noncompetitive inhibition. NO decreased the amount of NTCP in the plasma membrane, providing a molecular mechanism for the noncompetitive inhibition of TC uptake. One way that NO can modify protein function is through a posttranslational modification known as S-nitrosylation: the binding of NO to cysteine thiols. Using a biotin switch technique we observed that NTCP is S-nitrosylated under conditions in which NO inhibits TC uptake. Moreover, dithiothreitol reversed NO-mediated inhibition of TC uptake and S-nitrosylation of NTCP, indicating that NO inhibits TC uptake via modification of cysteine thiols. In addition, NO treatment led to a decrease in Ntcp phosphorylation. Taken together these results indicate that the inhibition of TC uptake by NO involves S-nitrosylation of NTCP.     

Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO

Background

Endothelial nitric oxide synthase (eNOS) is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.

Methods

Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.

Results

Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER)-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3) was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.

Conclusion

Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.     

BH3 activation blocks Hdmx suppression of apoptosis and cooperates with Nutlin to induce cell death

The Hdmx protein restricts p53 activity in vivo and is overexpressed in a significant fraction of human tumors that retain the wild type p53 allele. An understanding of how Hdmx limits p53 activation and blocks apoptosis could therefore lead to development of novel therapeutic agents. We previously showed that Hdmx modulates tumor cell sensitivity to Nutlin-3a, a potent antagonist of the p53/Hdm2 interaction. In this report, we demonstrate that this also applies to MI-219, another Hdm2 antagonist. Thus, the inability to disrupt Hdmx/p53 complexes is a potential barrier to the efficacy of these compounds as single agents. We show that sensitivity to apoptosis in cells with high Hdmx levels is restored by combined treatment with Hdm2 and a Bcl-2 family member antagonist to activate Bax. The data are consistent with a model in which Hdmx attenuates p53-dependent activation of the intrinsic apoptotic pathway, and that this occurs upstream of Bax activation. Thus, selectively inhibiting Hdm2 and activating Bax is one effective strategy to induce apoptosis in tumors with high Hdmx levels. Our findings also indicate that preferential induction of apoptosis in tumor versus normal cells occurs using appropriate drug doses.     

Development and application of site-specific proteomic approach for study protein S-nitrosylation

Protein S-nitrosylation is the covalent redox-related modification of cysteine sulfhydryl groups with nitric oxide, creating a regulatory impact similar to phosphorylation. Recent studies have reported a growing number of proteins to be S-nitrosylated in vivo resulting in altered functions. These studies support S-nitrosylation as a critical regulatory mechanism, fine-tuning protein activities within diverse cellular processes and biochemical pathways. In addition, S-nitrosylation appears to have key roles in the etiology of a broad range of human diseases. In this review, we discuss recent advances in proteomic approaches for the enrichment, identification, and quantitation of cysteine S-nitrosylated proteins and peptides. These advances have provided analytical tools with the power to interpret the impact of S-nitrosylation at the system level, providing a new platform for drug discovery and the identification of diagnostic markers for human diseases.