Structural Profiling of Endogenous S-Nitrosocysteine Residues Reveals
Total Page：16
File Type：pdf, Size：1020Kb
Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation Paschalis-Thomas Douliasa,1, Jennifer L. Greenea,1, Todd M. Grecoa, Margarita Tenopouloua, Steve H. Seeholzera, Roland L. Dunbrackb, and Harry Ischiropoulosa,2 aChildren’s Hospital of Philadelphia Research Institute and Department of Pharmacology, Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA 19104; and bProgram in Molecular Medicine, Fox Chase Cancer Center, Philadelphia, PA 19111 Edited by Michael A. Marletta, University of California, Berkeley, CA, and approved August 19, 2010 (received for review June 9, 2010) S-nitrosylation, the selective posttranslational modification of pro- appreciate the biological selectivity of this posttranslational modi- tein cysteine residues to form S-nitrosocysteine, is one of the fication. Attempts to investigate this very important biological molecular mechanisms by which nitric oxide influences diverse question have not been possible largely because datasets of in vivo biological functions. In this study, unique MS-based proteomic modified proteins are not available (1, 11). Previous structural approaches precisely pinpointed the site of S-nitrosylation in 328 analyses have been attempted using limited data sets or by including peptides in 192 proteins endogenously modified in WT mouse liver. all sites of modification identified after exposing tissues or cells to Structural analyses revealed that S-nitrosylated cysteine residues S-nitrosylating agents (11). However, as the authors of these articles were equally distributed in hydrophobic and hydrophilic areas of have indicated, these sites of modification represent putative sites ± proteins with an average predicted pKa of 10.01 2.1. S-nitrosylation but not necessarily those modified in vivo (11). We used organo- sites were over-represented in α-helices and under-represented in fi fi fi mercury reagents that react directly, ef ciently, and speci cally coils as compared with unmodi ed cysteine residues in the same pro- with S-nitrosocysteine and thus enable the precise identification teins (χ2 test, P < 0.02). A quantile–quantile probability plot indicated of S-nitrosocysteine–containing peptides and independently S- that the distribution of S-nitrosocysteine residues was skewed to- nitrosylated proteins to assemble the in vivo S-nitrosocysteine ward larger surface accessible areas compared with the unmod- ified cysteine residues in the same proteins. Seventy percent of the proteome of the mouse liver. By using bioinformatic tools, we then S-nitrosylated cysteine residues were surrounded by negatively or interrogated this enriched endogenous S-nitrosocysteine proteome fi positively charged amino acids within a 6-Å distance. The location of to de ne the biochemical, biophysical, and structural environment fi cysteine residues in α-helices and coils in highly accessible surfaces of the cysteine residues modi ed by S-nitrosylation, elements that bordered by charged amino acids implies site directed S-nitrosylation might inform on how specificity of S-nitrosylation is achieved. mediated by protein–protein or small molecule interactions. Moreover, 13 modified cysteine residues were coordinated with metals and Results and Discussion 15 metalloproteins were endogenously modified supporting metal- Complementary MS-Based Proteomics Identify the Endogenous Liver catalyzed S-nitrosylation mechanisms. Collectively, the endogenous S- S-Nitrosocysteine Proteome. The reaction of phenylmercury com- nitrosoproteome in the liver has structural features that accommodate pounds with S-nitrosocysteine results in the formation of a relatively multiple mechanisms for selective site-directed S-nitrosylation. stable thiol–mercury bond (12). Therefore, we used an organomercury resin (MRC) synthesized by conjugation of ρ-amino-phenylmercuric cysteine modification | nitric oxide | S-nitrosation | posttranslational acetate to N-hydroxysuccinimide–activated Affi-Gel 10 agarose modification | proteomics beads and a phenylmercury-polyethyleneglycol-biotin (mPEGb) compound to capture S-nitrosylated proteins and peptides (Fig. ysteine S-nitrosylation is a reversible and apparently selective S1). The method consists of three steps: (i) blocking of reduced Cposttranslational protein modification that regulates protein cysteine residues with methyl methanethiosulfonate (MMTS), (ii) activity, localization, and stability within a variety of organs and capture and release of S-nitrosylated proteins or peptides, and (iii) cellular systems (1–6). Despite the considerable biological impor- liquid chromatography/tandem MS analysis. β-Mercaptoethanol or tance of this posttranslational modification, significant gaps exist performic acid was used to release captured proteins or pep- regarding its in vivo specificity and origin. The identification of tides, respectively. Mild performic acid was used to selectively and in vivo S-nitrosylated proteins has indicated that not all reduced quantitatively release the bound peptides and more importantly cysteine residues and not all proteins with reduced cysteine residues oxidize cysteine thiols to sulfonic acid, thereby creating a unique are modified, implying a biased selection. Several biological MS signature that permits site-specific identification of the modi- chemistries have been proposed to account for the S-nitrosylation fied cysteine residues (13). Under the workflow used (Fig. S1), of proteins in vivo (1, 7, 8). Broadly, these include (i) oxidative 100% of cysteine-containing peptides were detected with the sul- S-nitrosation by higher oxides of NO, (ii) transnitrosylation by small fonic acid modification. molecular weight NO carriers such as S-nitrosoglutathione or dinitrosyliron complexes, (iii) catalysis by metalloproteins, and (iv) protein-assisted transnitrosation, as elegantly documented for the Author contributions: H.I. designed research; P.-T.D., J.L.G., M.T., and S.H.S. performed S-nitrosylation of caspase-3 by S-nitrosothioredoxin (9, 10). With research; P.-T.D., T.M.G., and H.I. contributed new reagents/analytic tools; P.-T.D., J.L.G., the exception of the protein-assisted transnitrosylation and metal- T.M.G., S.H.S., R.L.D., and H.I. analyzed data; and P.-T.D., J.L.G., T.M.G., and H.I. wrote the loprotein catalyzed S-nitrosylation, which we presume necessitates paper. protein–protein interaction, the other proposed mechanisms are The authors declare no conflict of interest. rather nondiscriminatory unless the microenvironment of selective This article is a PNAS Direct Submission. cysteine residues in proteins can specifically accommodate these 1P.-T.D. and J.L.G. contributed equally to this work. chemical modifications. Therefore, structural interrogation of en- 2To whom correspondence should be addressed. E-mail: [email protected]. fi fi dogenous S-nitrosylated proteins with site speci cidenti cation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the modified cysteine residues could provide valuable insights to 1073/pnas.1008036107/-/DCSupplemental. 16958–16963 | PNAS | September 28, 2010 | vol. 107 | no. 39 www.pnas.org/cgi/doi/10.1073/pnas.1008036107 Downloaded by guest on September 26, 2021 To control for the inclusion of false-positive protein S- nitrosocysteine with the phenylmercury compounds is specificand nitrosocysteine that may result from incomplete blocking of re- efficient and achieved selective identification of the modified res- duced cysteine residues or nonspecific interactions with the resins idue, the ultimate qualifier for the unambiguous assignment of S- during enrichment, pretreatment with UV, DTT, and copper- nitrosylated proteins through the inclusion of negative controls. ascorbate (Cu-Asc) reduction or reaction with mercury chloride The ability of the method to reveal endogenous S- were used to displace NO before the reaction with the phenyl- nitrosoproteomes was assessed in WT mouse liver homogenates. mercury compounds (Fig. 1A and Fig. S2A). For these experi- Initially, three different mouse livers were analyzed independently ments, we used mouse liver homogenates with 50 ± 10 pmol for protein and peptide capture by using both the MRC and protein S-nitrosocysteine per milligram of protein as quantified by mPEGb approach with 3 mg of starting material. From the three reductive chemistries coupled to ozone-based chemiluminescence. biological replicates, sulfonic acid-containing peptides identified Displacement of NO from S-nitrosocysteine residues by pre- by SEQUEST database searches were pooled and those also treatment with UV, DTT, Cu-Asc, or mercury chloride decreased present in the UV-pretreated samples were removed. Similarly, all protein S-nitrosocysteine levels by greater than 99% as quantified proteins identified by protein capture were pooled (those also by chemiluminescence (n = 3) and also eliminated reaction with identified in the UV-pretreated samples were removed). The use the phenylmercury compounds (Fig. 1A and Fig. S2A). On average, of solid-phase and in-solution–based enrichment approaches was only 3% of peptides (Fig. 1B) and 5% of proteins (Fig. S2B) largely complementary, as the number of shared protein identi- were identified as false positives (present in untreated and UV- fications between protein capture methods was approximately pretreated samples), demonstrating that the method maintains 50%, whereas each method individually contributed about the high specificity at each step.