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• W2140307910 abstract "•S-Nitrosylation of MEF2 at conserved cysteine residues occurs in plants and animal•S-Nitrosylation of MEF2 inhibits its DNA binding and transcriptional activity•S-Nitrosylation of MEF2 inhibits both neurogenesis and neuronal survival Redox-mediated posttranslational modifications represent a molecular switch that controls major mechanisms of cell function. Nitric oxide (NO) can mediate redox reactions via S-nitrosylation, representing transfer of an NO group to a critical protein thiol. NO is known to modulate neurogenesis and neuronal survival in various brain regions in disparate neurodegenerative conditions. However, a unifying molecular mechanism linking these phenomena remains unknown. Here, we report that S-nitrosylation of myocyte enhancer factor 2 (MEF2) transcription factors acts as a redox switch to inhibit both neurogenesis and neuronal survival. Structure-based analysis reveals that MEF2 dimerization creates a pocket, facilitating S-nitrosylation at an evolutionally conserved cysteine residue in the DNA binding domain. S-Nitrosylation disrupts MEF2-DNA binding and transcriptional activity, leading to impaired neurogenesis and survival in vitro and in vivo. Our data define a molecular switch whereby redox-mediated posttranslational modification controls both neurogenesis and neurodegeneration via a single transcriptional signaling cascade. Redox-mediated posttranslational modifications represent a molecular switch that controls major mechanisms of cell function. Nitric oxide (NO) can mediate redox reactions via S-nitrosylation, representing transfer of an NO group to a critical protein thiol. NO is known to modulate neurogenesis and neuronal survival in various brain regions in disparate neurodegenerative conditions. However, a unifying molecular mechanism linking these phenomena remains unknown. Here, we report that S-nitrosylation of myocyte enhancer factor 2 (MEF2) transcription factors acts as a redox switch to inhibit both neurogenesis and neuronal survival. Structure-based analysis reveals that MEF2 dimerization creates a pocket, facilitating S-nitrosylation at an evolutionally conserved cysteine residue in the DNA binding domain. S-Nitrosylation disrupts MEF2-DNA binding and transcriptional activity, leading to impaired neurogenesis and survival in vitro and in vivo. Our data define a molecular switch whereby redox-mediated posttranslational modification controls both neurogenesis and neurodegeneration via a single transcriptional signaling cascade. IntroductionRedox modification of cysteine, reminiscent of phosphorylation and ubiquitination of critical amino acids, regulates protein activity (Hess et al., 2005Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Protein S-nitrosylation: purview and parameters.Nat. Rev. Mol. Cell Biol. 2005; 6: 150-166Crossref PubMed Scopus (1704) Google Scholar). One type of redox modification involves reaction of nitric oxide (NO) with cysteine thiol. In this manner, NO functions as a signaling molecule influencing multiple targets. In brain, NO regulates physiologic functions including neural development, neurotransmitter release, and synaptic plasticity, but excess NO promotes neuronal damage in a variety of neurodegenerative disorders (Nakamura et al., 2013Nakamura T. Tu S. Akhtar M.W. Sunico C.R. Okamoto S. Lipton S.A. Aberrant protein s-nitrosylation in neurodegenerative diseases.Neuron. 2013; 78: 596-614Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Previously, we and our colleagues demonstrated that NO reacts with critical cysteine thiols, termed S-nitrosylation, on various proteins to modulate their activity (Hess et al., 2005Hess D.T. Matsumoto A. Kim S.O. Marshall H.E. Stamler J.S. Protein S-nitrosylation: purview and parameters.Nat. Rev. Mol. Cell Biol. 2005; 6: 150-166Crossref PubMed Scopus (1704) Google Scholar). Recently, we found that S-nitrosylation of the transcription factor myocyte enhancer factor 2C (MEF2C) occurs in cell-based Parkinson’s disease (PD) models, resulting in inhibition of transcriptional activity and resultant cell death via a PGC1α-mediated pathway (Ryan et al., 2013Ryan S.D. Dolatabadi N. Chan S.F. Zhang X. Akhtar M.W. Parker J. Soldner F. Sunico C.R. Nagar S. Talantova M. et al.Isogenic human iPSC Parkinson’s model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription.Cell. 2013; 155: 1351-1364Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). Heretofore, however, it remained unknown if S-nitrosylation of a common target could molecularly “switch off” both neurogenesis and neuronal survival. Here, we demonstrate that S-nitrosylation of MEF2A or MEF2C at an inhibitory “cysteine switch” abrogates DNA binding. This redox reaction shuts off two distinct transcriptional cascades involved in neuronal survival and neurogenesis.MEF2 integrates diverse signals and plays roles in the immune, muscular, and nervous systems. In brain, we and others have shown that various MEF2 isoforms modulate neurogenesis and neuronal survival (Flavell et al., 2006Flavell S.W. Cowan C.W. Kim T.K. Greer P.L. Lin Y. Paradis S. Griffith E.C. Hu L.S. Chen C. Greenberg M.E. Activity-dependent regulation of MEF2 transcription factors suppresses excitatory synapse number.Science. 2006; 311: 1008-1012Crossref PubMed Scopus (441) Google Scholar, Li et al., 2008Li H. Radford J.C. Ragusa M.J. Shea K.L. McKercher S.R. Zaremba J.D. Soussou W. Nie Z. Kang Y.J. Nakanishi N. et al.Transcription factor MEF2C influences neural stem/progenitor cell differentiation and maturation in vivo.Proc. Natl. Acad. Sci. USA. 2008; 105: 9397-9402Crossref PubMed Scopus (171) Google Scholar, Mao et al., 1999Mao Z. Bonni A. Xia F. Nadal-Vicens M. Greenberg M.E. Neuronal activity-dependent cell survival mediated by transcription factor MEF2.Science. 1999; 286: 785-790Crossref PubMed Scopus (441) Google Scholar, Okamoto et al., 2000Okamoto S. Krainc D. Sherman K. Lipton S.A. Antiapoptotic role of the p38 mitogen-activated protein kinase-myocyte enhancer factor 2 transcription factor pathway during neuronal differentiation.Proc. Natl. Acad. Sci. USA. 2000; 97: 7561-7566Crossref PubMed Scopus (170) Google Scholar, Okamoto et al., 2002Okamoto S. Li Z. Ju C. Schölzke M.N. Mathews E. Cui J. Salvesen G.S. Bossy-Wetzel E. Lipton S.A. Dominant-interfering forms of MEF2 generated by caspase cleavage contribute to NMDA-induced neuronal apoptosis.Proc. Natl. Acad. Sci. USA. 2002; 99: 3974-3979Crossref PubMed Scopus (115) Google Scholar, Shalizi et al., 2006Shalizi A. Gaudillière B. Yuan Z. Stegmüller J. Shirogane T. Ge Q. Tan Y. Schulman B. Harper J.W. Bonni A. A calcium-regulated MEF2 sumoylation switch controls postsynaptic differentiation.Science. 2006; 311: 1012-1017Crossref PubMed Scopus (373) Google Scholar). Here, we used cerebral ischemia models to study the influence of NO on MEF2 in neuronal cell death, because both NO and MEF2 have been previously linked to such damage (Huang et al., 1994Huang Z. Huang P.L. Panahian N. Dalkara T. Fishman M.C. Moskowitz M.A. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase.Science. 1994; 265: 1883-1885Crossref PubMed Scopus (1449) Google Scholar, Okamoto et al., 2002Okamoto S. Li Z. Ju C. Schölzke M.N. Mathews E. Cui J. Salvesen G.S. Bossy-Wetzel E. Lipton S.A. Dominant-interfering forms of MEF2 generated by caspase cleavage contribute to NMDA-induced neuronal apoptosis.Proc. Natl. Acad. Sci. USA. 2002; 99: 3974-3979Crossref PubMed Scopus (115) Google Scholar). Additionally, as a tractable paradigm to study dysfunctional neurogenesis in the adult nervous system, we used Alzheimer’s disease (AD) models because NO and MEF2 have both been implicated in the generation of new neurons (Packer et al., 2003Packer M.A. Stasiv Y. Benraiss A. Chmielnicki E. Grinberg A. Westphal H. Goldman S.A. Enikolopov G. Nitric oxide negatively regulates mammalian adult neurogenesis.Proc. Natl. Acad. Sci. USA. 2003; 100: 9566-9571Crossref PubMed Scopus (286) Google Scholar, Li et al., 2008Li H. Radford J.C. Ragusa M.J. Shea K.L. McKercher S.R. Zaremba J.D. Soussou W. Nie Z. Kang Y.J. Nakanishi N. et al.Transcription factor MEF2C influences neural stem/progenitor cell differentiation and maturation in vivo.Proc. Natl. Acad. Sci. USA. 2008; 105: 9397-9402Crossref PubMed Scopus (171) Google Scholar).ResultsS-Nitrosylation of MEF2 Transcription FactorsTo confirm that the consensus motif for nitrosylation on MEF2 (Stamler et al., 1997Stamler J.S. Toone E.J. Lipton S.A. Sucher N.J. (S)NO signals: translocation, regulation, and a consensus motif.Neuron. 1997; 18: 691-696Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar) can indeed be S-nitrosylated (to form SNO-MEF2), we initially expressed Myc-tagged MEF2C in human embryonic kidney (HEK) 293 cells and exposed the transfected cells to the physiological NO donors, S-nitrosocysteine (SNOC) or nitroso-S-glutathione (GSNO). Using the biotin switch assay in which biotin is selectively substituted for the NO moiety of SNO-cysteine residues (Jaffrey et al., 2001Jaffrey S.R. Erdjument-Bromage H. Ferris C.D. Tempst P. Snyder S.H. Protein S-nitrosylation: a physiological signal for neuronal nitric oxide.Nat. Cell Biol. 2001; 3: 193-197Crossref PubMed Scopus (1217) Google Scholar), we found that these NO donors induced S-nitrosylation of MEF2C (Figure S1A).Next, we asked if endogenous SNO-MEF2 is increased in neurodegenerative conditions. We primarily focused on MEF2C because this isoform is known to predominate in cerebrocortical neurons, although our findings pertain to all isoforms (Leifer et al., 1993Leifer D. Krainc D. Yu Y.T. McDermott J. Breitbart R.E. Heng J. Neve R.L. Kosofsky B. Nadal-Ginard B. Lipton S.A. MEF2C, a MADS/MEF2-family transcription factor expressed in a laminar distribution in cerebral cortex.Proc. Natl. Acad. Sci. USA. 1993; 90: 1546-1550Crossref PubMed Scopus (183) Google Scholar, Li et al., 2008Li H. Radford J.C. Ragusa M.J. Shea K.L. McKercher S.R. Zaremba J.D. Soussou W. Nie Z. Kang Y.J. Nakanishi N. et al.Transcription factor MEF2C influences neural stem/progenitor cell differentiation and maturation in vivo.Proc. Natl. Acad. Sci. USA. 2008; 105: 9397-9402Crossref PubMed Scopus (171) Google Scholar). Because neuronal cell death triggered by excessive activation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor is known to be mediated, at least in part, by generation of endogenous NO (Dawson et al., 1991Dawson V.L. Dawson T.M. London E.D. Bredt D.S. Snyder S.H. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures.Proc. Natl. Acad. Sci. USA. 1991; 88: 6368-6371Crossref PubMed Scopus (2098) Google Scholar), and MEF2C activity has been shown to ameliorate such damage (Okamoto et al., 2002Okamoto S. Li Z. Ju C. Schölzke M.N. Mathews E. Cui J. Salvesen G.S. Bossy-Wetzel E. Lipton S.A. Dominant-interfering forms of MEF2 generated by caspase cleavage contribute to NMDA-induced neuronal apoptosis.Proc. Natl. Acad. Sci. USA. 2002; 99: 3974-3979Crossref PubMed Scopus (115) Google Scholar), we exposed cultured rat cerebrocortical neurons to 50 μM NMDA and monitored SNO-MEF2C formation by biotin switch assay. We observed SNO-MEF2C within 30 min of NMDA application, an effect blocked by cotreatment with the NO synthase (NOS) inhibitor L-NG-nitroarginine methyl ester (L-NAME; Figures 1A and S1B). Because apoptosis does not occur in this paradigm until the next day, these results imply that MEF2C is S-nitrosylated at an early stage of cell-death signaling (Bonfoco et al., 1995Bonfoco E. Krainc D. Ankarcrona M. Nicotera P. Lipton S.A. Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures.Proc. Natl. Acad. Sci. USA. 1995; 92: 7162-7166Crossref PubMed Scopus (1855) Google Scholar, Cho et al., 2009Cho D.H. Nakamura T. Fang J. Cieplak P. Godzik A. Gu Z. Lipton S.A. S-nitrosylation of Drp1 mediates beta-amyloid-related mitochondrial fission and neuronal injury.Science. 2009; 324: 102-105Crossref PubMed Scopus (823) Google Scholar). NO-induced neurotoxicity has also been implicated in in vivo studies, including focal cerebral ischemia (stroke) (Hara and Snyder, 2007Hara M.R. Snyder S.H. Cell signaling and neuronal death.Annu. Rev. Pharmacol. Toxicol. 2007; 47: 117-141Crossref PubMed Scopus (190) Google Scholar, Huang et al., 1994Huang Z. Huang P.L. Panahian N. Dalkara T. Fishman M.C. Moskowitz M.A. Effects of cerebral ischemia in mice deficient in neuronal nitric oxide synthase.Science. 1994; 265: 1883-1885Crossref PubMed Scopus (1449) Google Scholar). Robust NO production has been observed during the first few hours of cerebral ischemia in rodent middle cerebral artery (MCA) occlusion models (Kader et al., 1993Kader A. Frazzini V.I. Solomon R.A. Trifiletti R.R. Nitric oxide production during focal cerebral ischemia in rats.Stroke. 1993; 24: 1709-1716Crossref PubMed Scopus (251) Google Scholar, Zhang et al., 1995Zhang Z.G. Chopp M. Bailey F. Malinski T. Nitric oxide changes in the rat brain after transient middle cerebral artery occlusion.J. Neurol. Sci. 1995; 128: 22-27Abstract Full Text PDF PubMed Scopus (119) Google Scholar). Accordingly, we occluded the right MCA of mice for 1 hr and observed S-nitrosylation of MEF2C in the ischemic but not the contralateral cerebral cortex (Figures 1B and S1C), showing that MEF2C is S-nitrosylated in vivo during ischemia.In addition to promoting acute neuronal injury in conditions such as stroke, excessive NO generation has been observed in chronic neurodegenerative diseases, including human AD brains (Fernández-Vizarra et al., 2004Fernández-Vizarra P. Fernández A.P. Castro-Blanco S. Encinas J.M. Serrano J. Bentura M.L. Muñoz P. Martínez-Murillo R. Rodrigo J. Expression of nitric oxide system in clinically evaluated cases of Alzheimer’s disease.Neurobiol. Dis. 2004; 15: 287-305Crossref PubMed Scopus (92) Google Scholar, Vodovotz et al., 1996Vodovotz Y. Lucia M.S. Flanders K.C. Chesler L. Xie Q.W. Smith T.W. Weidner J. Mumford R. Webber R. Nathan C. et al.Inducible nitric oxide synthase in tangle-bearing neurons of patients with Alzheimer’s disease.J. Exp. Med. 1996; 184: 1425-1433Crossref PubMed Scopus (262) Google Scholar). By biotin switch assay, we examined brains of Tg2576 mice, an AD model overexpressing amyloid precursor protein (APP) with the Swedish mutation, and in postmortem human AD brains. We found increased SNO-MEF2C levels in both mouse (Figures 1C and S1D) and human AD brains but not in controls (Figures 1D and S1E). Taken together, these findings suggest that MEF2C is S-nitrosylated in vivo both in acute neurodegenerative conditions such as stroke and in chronic neurodegenerative conditions such as AD.To determine which cysteine residue(s) is S-nitrosylated on MEF2C and other MEF2 isoforms, we first performed a top-down analysis of the protein using an linear trap quadrupole (LTQ)-Orbitrap-XL mass spectrometry with electron transfer dissociation (ETD). Exposure to the physiological NO donor SNOC shifted the MEF2C spectral cluster reflecting S-nitrosothiol formation on a single cysteine residue (739.9426 m/z), whereas expected clusters of MEF2C with two S-nitrosothiols (741.4986 m/z) or more were not observed. These results indicate the presence of one S-nitrosylation site on MEF2C (Figure 2A). To identify that site, we mutated each Cys residue in MEF2C to Ala and then transfected expression vectors encoding these mutated forms into HEK293 cells. We exposed the cells to SNOC and analyzed S-nitrosylated MEF2C by the biotin switch method. Mutation of Cys39 (C39A) abrogated formation of SNO-MEF2C, whereas other mutations such as MEF2C(C41A) did not (Figures 2B and S2A, densitometric quantification). To confirm S-nitrosylation of Cys39, we employed a direct biochemical analysis for S-nitrosothiols, as described previously, by monitoring conversion of 2,3-diaminonaphthalene (DAN) to 2,3-naphthotriazole (NAT) from immunoprecipitates of HEK293 cells transfected with wild-type (WT) or mutant MEF2C(C39A) (Wink et al., 1999Wink D.A. Vodovotz Y. Grisham M.B. DeGraff W. Cook J.C. Pacelli R. Krishna M. Mitchell J.B. Antioxidant effects of nitric oxide.Methods Enzymol. 1999; 301: 413-424Crossref PubMed Scopus (92) Google Scholar). In this assay, the C39A mutation abolished SNO-MEF2C formation (Figure 2C), supporting the notion that Cys39 is the primary site of S-nitrosylation.Figure 2S-Nitrosylation of MEF2 at Cys39 in the MADS-Box DNA Binding DomainShow full caption(A) Recombinant MEF2C protein was infused into a mass spectrometer (LTQ-Orbitrap-XL with ETD) to obtain molecular ion spectra in the presence or absence of NO donor, 50 μM SNOC (n = 2 independent experiments).(B) HEK293T cells transfected with WT myc-tagged MEF2C (WT), MEF2C(C39A), or MEF2C(C41A) were incubated with SNOC. SNO-MEF2C was determined by biotin switch. SNO proteins were precipitated, and SNO-MEF2C was detected with anti-myc (n = 3 independent experiments; quantification shown in Figure S2A).(C) HEK293 cells stably expressing nNOS were transfected with V5-tagged MEF2C (WT) or V5-tagged MEF2C(C39A), and nNOS activated with Ca2+ ionophore A23187. V5-tagged MEF2C proteins were immunoprecipitated with anti-V5 and subjected to DAN assay. Values are mean ± SEM (n = 3 independent experiments, ∗p < 0.05 by t test).(D) Sequence alignment of MADS-box DNA binding domains of MEF2 (human isoforms MEF2A-D; Drosophila D-MEF2). α Helix (bars) and β strands (arrows). Cys39 S-nitrosylation site (yellow). Residues involved in pocket formation (red).(E) Cys39 (yellow) is symmetrically located at the hinge between α1 helix and β1 strands in the MEF2 (monomer A in blue, monomer B in green) -DNA (pink) complex (PDB ID: 1EGW). Right: viewed at 90°.(F) Electrostatic view of MEF2 (negative charge in red, positive charge in blue). Cys39 is present at the bottom of the pocket created by Glu14, Arg17, Ser50, Ser51, Asn49, Phe21, and Gln18.See also Figure S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)S-Nitrosylation of MEF2 Inhibits DNA Binding and Transcriptional ActivityNext, we investigated the effect of SNO-MEF2 on transcriptional activity. We noted that Cys39 is conserved in the N-terminal MADS domain among all MEF2 family members, and other MEF2 isoforms can also be S-nitrosylated at this position (Figures 2D, S2B, and S2C). Because MEF2 dimerizes and binds to DNA through the MADS domain, we studied the effect that SNO-MEF2 might exert on dimerization and DNA binding. The structure of the MEF2/DNA complex has been analyzed by NMR spectroscopy and X-ray crystallography (Han et al., 2003Han A. Pan F. Stroud J.C. Youn H.D. Liu J.O. Chen L. Sequence-specific recruitment of transcriptional co-repressor Cabin1 by myocyte enhancer factor-2.Nature. 2003; 422: 730-734Crossref PubMed Scopus (88) Google Scholar, Han et al., 2005Han A. He J. Wu Y. Liu J.O. Chen L. Mechanism of recruitment of class II histone deacetylases by myocyte enhancer factor-2.J. Mol. Biol. 2005; 345: 91-102Crossref PubMed Scopus (87) Google Scholar, Santelli and Richmond, 2000Santelli E. Richmond T.J. Crystal structure of MEF2A core bound to DNA at 1.5 A resolution.J. Mol. Biol. 2000; 297: 437-449Crossref PubMed Scopus (96) Google Scholar). Our analysis of these structures revealed that Cys39 is located at a hinge region where the α-helical stretch (α1) turns into a β sheet (β1) (Figures 2D and 2E). Intriguingly, the electrostatic view demonstrates that Cys39 is at the bottom of a pocket created by seven amino acids: Glu14, Arg17, Gln18, Phe21, Asn49, Ser50, and Ser51 (Figure 2F). These residues are highly conserved throughout the entire MEF2 family (red in Figure 2D). Additionally, these same residues are often part of an amino-acid motif facilitating S-nitrosylation of a critical Cys residue (Greco et al., 2006Greco T.M. Hodara R. Parastatidis I. Heijnen H.F. Dennehy M.K. Liebler D.C. Ischiropoulos H. Identification of S-nitrosylation motifs by site-specific mapping of the S-nitrosocysteine proteome in human vascular smooth muscle cells.Proc. Natl. Acad. Sci. USA. 2006; 103: 7420-7425Crossref PubMed Scopus (232) Google Scholar, Hao et al., 2006Hao G. Derakhshan B. Shi L. Campagne F. Gross S.S. SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures.Proc. Natl. Acad. Sci. USA. 2006; 103: 1012-1017Crossref PubMed Scopus (293) Google Scholar), suggesting that the pocket may form such a nitrosylation motif.To determine whether S-nitrosylation alters MEF2 transcriptional activity, we first used a molecular dynamics approach to analyze the interaction patterns of Cys39 and SNO-Cys39 residues. Using the crystal structure of the solved MEF2A/DNA complex (Santelli and Richmond, 2000Santelli E. Richmond T.J. Crystal structure of MEF2A core bound to DNA at 1.5 A resolution.J. Mol. Biol. 2000; 297: 437-449Crossref PubMed Scopus (96) Google Scholar), we modified the Cys39 residues to SNO-Cys in both polypeptide chains of the dimer in silico. In order to determine the geometry and electrical properties of the NO-substituted side chain of Cys39, we then performed quantum mechanical calculations. The SNO-Cys dipeptide (CH3CO-NH-CH[CH2SNO]CO-NH-CH3) was used as a model molecule for deriving essential force-field parameters necessary for molecular mechanics modeling. The geometry of the SNO-Cys dipeptide was optimized using the quantum mechanical GAMESS program (Schmidt et al., 1993Schmidt M.W. Baldridge K.K. Boatz J.A. Elbert S.T. Gordon M.S. Jensen J.H. Koseki S. Matsunaga N. Nguyen K.A. Su S. et al.General atomic and molecular electronic structure system.J. Comput. Chem. 1993; 14: 1347-1363Crossref Scopus (19025) Google Scholar), and the charges on the dipeptide atoms were determined to reproduce the quantum mechanically derived electrostatic potential around the entire dipeptide using the RESP method (Bayly et al., 1993Bayly C.I. Cieplak P. Cornell W. Kollman P.A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model.J. Phys. Chem. 1993; 97: 10269-10280Crossref Scopus (5465) Google Scholar) and R.E.D.III program (Vanquelef et al., 2011Vanquelef E. Simon S. Marquant G. Garcia E. Klimerak G. Delepine J.C. Cieplak P. Dupradeau F.Y. R.E.D. Server: a web service for deriving RESP and ESP charges and building force field libraries for new molecules and molecular fragments.Nucleic Acids Res. 2011; 39: W511-W517Crossref PubMed Scopus (523) Google Scholar). We compared the resultant atomic charges of the SNO-Cys dipeptide side chain with the charge distribution of the unmodified Cys residue, for which molecular mechanical parameters are available in the AMBER force field (Cornell et al., 1995Cornell W.D. Cieplak P. Bayly C.I. Gould I. Merz K.M. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. A second generation force field for simulation proteins, nucleic acids and related organic molecules.J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11443) Google Scholar). Substitution of the H atom with the NO group in Cys39 replaced the positively charged single atom with two negatively charged atoms, making the entire side chain electronegative (Figure 3A). This modification should, in theory, substantially affect the interaction of Cys39, located at the hinge region of the DNA binding domain (Figures 2D and 2E), with the surrounding residues essential for MEF2/DNA interaction.Figure 3S-Nitrosylation of MEF2 Impairs Its DNA Binding, Transcriptional, and Prosurvival ActivitiesShow full caption(A) Computational structure analysis of S-nitrosylated Cys39. Atomic charge distribution for normal Cys and Cys-NO residues obtained from quantum mechanical calculations (see text). Electrostatic potentials: positive charge (blue) and negative charge (red). Values show excess atomic charge.(B) Optimization of molecular mechanical geometry to show possible orientation of the Cys-NO side chain in MEF2. Key residues of dimer interface are highlighted.(C) EMSA of MEF2/DNA binding activity. Nuclear extracts from cortical neurons were exposed to 50 μM SNOC (n = 3 independent experiments). MEF2/DNA binding complex (arrow).(D) EMSA of mutant MEF2/DNA binding activity. In vitro translated MEF2C(C39A) was exposed to SNOC (n = 3 independent experiments).(E) Effect of NO on MEF2 reporter gene activity. Cortical neurons were transfected with empty vector (pcDNA 3.1) or expression vectors for MEF2C or MEF2C(C39A), MEF2 luciferase reporter construct, and renilla luciferase vector to control for transfection efficiency. Luciferase activity was measured 4 hr after 50 μM SNOC exposure. Bars represent mean ± SEM (n = 12 cultures derived from three independent platings, ∗p < 0.0001 by ANOVA with post hoc Scheffé’s test).(F) Effect of iNOS transfection on endogenous MEF2 reporter gene activity. Cortical neurons were transfected with empty vector (pcDNA 3.1) or expression vectors for iNOS with or without MEF2C(C39A), and MEF2 luciferase reporter construct plus renilla luciferase; 24 hr after transfection, luciferase activity was measured. Bars represent mean ± SEM (n = 12 cultures derived from three independent platings, ∗p < 0.0001 by ANOVA with post hoc Scheffé’s test).(G) Effect of MEF2 S-nitrosylation on neuronal apoptosis. Cortical neurons were transfected with control vector, MEF2C, or MEF2C(C39A) plus enhanced green fluorescent protein (EGFP) vector to identify transfected neurons. Cultures were then exposed to 50 μM SNOC and analyzed 8 hr later. Hoechst DNA dye was used to assess condensed, apoptotic nuclei. Bars represent mean ± SEM (n = 9 cultures derived from three independent platings, ∗p < 0.01, ∗∗p < 0.05 by ANOVA with post hoc Scheffé’s test).(H) Stereotactic injection of S-nitrosylation-resistant MEF2C(C39A) mutant (LV-C39A) but not WT MEF2C into striatum improved neuronal survival in stroke penumbra. Values are mean ± SEM of survival index, representing the inverse of the apoptotic ratio for infected versus uninfected neurons for each construct (n ≥ 4 animals for each condition, ∗p < 0.05 by ANOVA with post hoc Scheffé’s test).See also Figure S3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We found that the solved atomic structure shows the Cys39 region has a network of interacting residues that controls orientation of the N-terminal polypeptide arm (composed of residues 1–17), which is involved in DNA binding (Figure 3B). One important interaction is a salt bridge between the Arg10 and Asp40 residues located in different chains of the MEF2 dimer. Another important interaction is between Arg10 and Arg17 within a single polypeptide chain. The importance of this second interaction is experimentally supported because mutation of Arg17 to Val has been shown to decrease MEF2-DNA binding activity (Molkentin et al., 1996Molkentin J.D. Black B.L. Martin J.F. Olson E.N. Mutational analysis of the DNA binding, dimerization, and transcriptional activation domains of MEF2C.Mol. Cell. Biol. 1996; 16: 2627-2636Crossref PubMed Scopus (165) Google Scholar). We thus obtained a plausible conformation of SNO-Cys39 by molecular mechanical optimization using the AMBER program and its parm99 force field (Figure 3B) (Case et al., 2005Case D.A. Cheatham 3rd, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. The Amber biomolecular simulation programs.J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6331) Google Scholar, Cornell et al., 1995Cornell W.D. Cieplak P. Bayly C.I. Gould I. Merz K.M. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. A second generation force field for simulation proteins, nucleic acids and related organic molecules.J. Am. Chem. Soc. 1995; 117: 5179-5197Crossref Scopus (11443) Google Scholar). This type of -SNO orientation could potentially perturb interactions between Arg17 and Arg10, thus affecting the strength of the salt bridge between Arg10 and Asp40. This perturbation, in turn, could dislocate the position of the MEF2 N-terminal arm from the vicinity of DNA, thus reducing DNA binding.To test empirically whether S-nitrosylation of MEF2 impairs DNA binding, as predicted by the atomic structure and quantum mechanical considerations, we exposed nuclear extracts of cortical neurons to NO donor and analyzed DNA binding by electrophoretic mobility shift assay (EMSA) (Krainc et al., 1998Krainc D. Bai G. Okamoto S. Carles M. Kusiak J.W. Brent R.N. Lipton S.A. Synergistic activation of the N-methyl-D-aspartate receptor subunit 1 promoter by myocyte enhancer factor 2C and Sp1.J. Biol. Chem. 1998; 273: 26218-26224Crossref PubMed Scopus (78) Google Scholar, Leifer et al., 1993Leifer D. Krainc D. Yu Y.T. McDermott J. Breitbart R.E. Heng J. Neve R.L. Kosofsky B. Nadal-Ginard B. Lipton S.A. MEF2C, a MADS/MEF2-family transcription factor expressed in a laminar distribution in cerebral cortex.Proc. Natl. Acad. Sci. USA. 1993; 90: 1546-1550Crossref PubMed Scopus (183) Google Scholar). We found that exposure to SNOC decreased MEF2 binding to DNA, with binding specificity documented by supershift (Figures 3C and S3A). To determine the contribution of SNO-Cys39 to this effect, we mutated Cys39 to Ala, translated the mutant protein MEF2C(C39A) in vitro, and assayed DNA binding by EMSA (Huang et al., 2000Huang K. Louis J.M. Donaldson L. Lim F.L. Sharrocks A.D. Clore G.M. Solution structure of the MEF2A-DNA complex: s" @default.
• W2140307910 created "2016-06-24" @default.
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