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Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for 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 residues to form S-nitrosocysteine, is one of the fication. Attempts to investigate this very important biological molecular mechanisms by which influences diverse question have not been possible largely because datasets of in vivo biological functions. In this study, unique MS-based proteomic modified 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 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 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 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 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 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. This false positive rate compares fa- same number of unique protein identifications (Table S1). As the vorably with the greater than 90% specificity reported for immo- MRC capture method allows for higher input, site-specific peptide bilized metal affinity chromatography used for the enrichment of capture by this method was also performed with the use of 30 mg of phosphopeptides from mouse liver (14). Therefore, pretreatment liver extract originating from three different mice. The inclusion of with UV served as negative control throughout based on previous these additional biological replicates reduced biological variance studies that documented the elimination of S-nitrosocysteine and improved the depth of the analysis while maintaining less than without affecting other cysteine modifications (15, 16). Collec- 3% false identification rate. Overall by matching sulfonic acid- tively, these experiments showed that the reaction of protein S- containing peptides with corresponding protein identifications, we

Pre-treated FIR (%) Non-cysteinyl A B peptides (%) E 2 mPEGb 3 ± 1 3 ± 4

MRC 1 ± 1 2 ± 1 Unbound Bound UV Asc Untreated DTT HgCl 1.2E+06

1E+06 UV + - + -

8E+05

6E+05 Hsp71

4E+05 Intensity (AU)

2E+05 VLCADH MEDICAL SCIENCES

0E+00 0 10 20 30 40 50 60 Time (min) GAPDH

YLLGTSLARPC IAR 97 b9 12.00 100% 1,580.84 AMU, +2 H (Parent Error: 0.59 ppm) C m/z= 791.4245 GT S L A R IA R D YLL PC+48 RAIC+48PRALSTGLL Y 10.00 75% Protein SNO Xc= 3.15 8.00 y5 (pmoles/mg protein) MRC mPEGb 6.00 50% WT 50 ± 10 + + - - + + - - 4.00 - / - - - + + - - + + 25% y6 y7 eNOS 5 ± 5 2.00 y2 y11 b12 (AU) b7 y8 y9 - 4 y3 b4 y4b5 b6 b8 y10 b11 b13 UV photolysis N.D. + - + - + - + - Relative Intensity b3 0.00 0% 790 791 792 793 794 795 0 250 500 750 1000 1250 1500 m/z

8.00 m/z= 780.8843 FELTC132YSLAPQIK b9 100% 1,559.75 AMU, +2 H (Parent Error: 0.13 ppm) F E LTC+48Y SLAP Q I K 6.00 K I Q P A L S Y C+48 T L E F 75% Abundance x 10 Xc= 2.40 y4 4.00 50% b6 y5 b8 2.00 b8-H2O 25% y8 b12 0.00 b3 y7 b7 779780781782783784 b2 b4 b5 y12 Relative Intensity 0% m/z 0 250 500 750 1000 1250 1500 m/z

Fig. 1. Site-specific identification and UV photolysis confirmation of S-nitrosocysteine. (A) Representative short (approximately 2 cm) colloidal blue-stained protein gel of mPEGb protein capture from WT mouse liver that was processed by GeLC-MS/MS analysis. Colloidal blue protein staining of bound fractions from mPEGb protein capture of untreated liver homogenates showed protein enrichment. Pretreatment with UV (for 30 min), DTT (10 mM), Cu-Asc (5 mM

Asc/copper 0.1 mM), or HgCl2 (20 mM) prevented this capture, demonstrating specificity for protein S-nitrosocysteine. (B) Representative base peak chro- matograms from mPEGb peptide capture demonstrates reduction in ion intensity after UV treatment (red) compared with the untreated sample (black). For clarity, the chromatogram corresponding to UV treatment was plotted with a y-axis offset of 1E4. Inset: Average false-positive identification rate (FIR ± SD) from mPEGb and MRC represented the percentage of peptides that were identified in both the UV-treated and untreated samples across three independent biological replicates. Also reported is the percent of noncysteinyl peptides identified (SI Materials and Methods). (C) Representative MS spectra of doubly

charged sulfonic acid-containing tryptic peptides, YLLGTSLARPC97IAR (monoisotopic m/z, 791.4256; Top, Left) and FELTC132YSLAPQIK (monoisotopic m/z, 780.8851; Bottom, Left), from argininosuccinate synthase. MS/MS spectra confirmed the sequence and site of sulfonic acid-containing peptides (C+48) from argininosuccinate synthase identified in mouse liver (SI Materials and Methods). MS/MS spectra passed automatic and manual filter criteria (Fig. S2), and were

identified with high SEQUEST cross correlation (Xc) scores at ppm mass error. Cys132 has been previously reported as a target of S-nitrosylation in human argininosuccinate synthase (20), whereas the identification of Cys97 corresponded to a previously unidentified site of S-nitrosocysteine formation. (D) Col- loidal blue protein staining of bound fractions from MRC and mPEGb enrichment of S-nitrosylated proteins from WT and eNOS−/− mouse livers. UV photolysis demonstrated specificity of the enrichment. The levels of protein S-nitrosocysteine were quantified by reductive chemistries coupled to chemiluminescent- − − based detection (41). The amount of S-nitrosylated protein captured by mPEGb from eNOS / livers (lane 8) was below the limit of detection by colloidal blue staining. (E) Western blot analysis of bound and unbound fractions from WT mouse liver that were processed by MRC protein capture.

Doulias et al. PNAS | September 28, 2010 | vol. 107 | no. 39 | 16959 Downloaded by guest on September 26, 2021 precisely pinpointed 328 S-nitrosocysteine–containing peptides in networks (Ingenuity Systems). The analysis was restricted to in- 192 proteins in untreated livers with a high level of confidence vestigate liver-related pathways. Sixteen S-nitrosylated proteins (complete list is provided in Table S1 and all of the MS/MS spectra were significantly clustered in a network that encompassed liver from peptide capture can be viewed at http://www.research.chop. responses to the hormone (Fig. S3C). Leptin is an adipocyte- edu/tools/msms/spectra.pdf). The depth of this analysis represents secreted hormone that primarily acts on the central nervous sys- asignificant advancement versus present methodologies (17). The tem to regulate energy homeostasis. Leptin also regulates liver majority of the proteins identified in the current study, 186, cor- , evident by the significant accumulation of (fatty responded to previously unidentified endogenous targets of liver) in mice deficient in leptin (ob/ob) (25, 26) or leptin long-form S-nitrosylation in the mouse liver, whereas six proteins (GAPDH, receptor (db/db) (27). The regulation of liver metabolism is at- hemoglobin, β-tubulin, argininosuccinate synthase, de- tributed to the leptin-dependent repression of liver stearoyl-CoA hydrogenase, and catalase) have been previously identified as en- desaturase-1, the rate limiting step in monosaturated fat bio- dogenously S-nitrosylated in hepatocytes and other organ systems synthesis (28). In addition, recent data indicate that leptin also (18–21). Proteins were also distributed across a wide range of regulates liver mitochondrial respiratory chain protein expression, molecular weights (13–272 kDa) and cellular localization including mitochondrial function and structure (29), remarkably similar to membrane-associated proteins, demonstrating the efficacy of the the previously recognized regulation of mitochondrial function by method to identify S-nitrosocysteine independent of protein size NO (30, 31). Interestingly, delivery of S-nitroso-N- by and location. We selected very long chain specificacyl-CoAde- gavage to ob/ob mice prevented the development of fatty liver (32). hydrogenase, heat shock cognate 71 protein, for which endogenous Mice deficient in eNOS also experience abnormal fat deposition in S-nitrosylation was not previously described, and GAPDH, which the liver (33), which was attributed in part to regulation of mito- has been known to be S-nitrosylated and independently confirmed chondrial fatty acid synthesis and activation of AMP-activated their selective enrichment by Western blot analysis after protein (33). Moreover, 14 of the 16 proteins in the leptin − − capture (Fig. 1E). network (Fig. S3C) were absent in eNOS / liver S-nitrosocysteine To further probe the biological specificity of our method proteome analysis, suggesting a potential relationship between while demonstrating its utility for comparison of endogenous eNOS-derived S-nitrosylation and leptin regulation of fatty acid S-nitrosoproteomes, we analyzed livers from mice lacking endo- metabolism. Although it requires further experimentation, the − − thelial NO synthase (eNOS / ). Using chemiluminescence-based data indicate that S-nitrosylation may be a molecular link be- quantification, a 10-fold decrease in protein S-nitrosocysteine lev- tween the actions of leptin and NO in liver fatty acid − − els of eNOS / livers was measured as compared with WT livers. and mitochondrial metabolism. Overall, cellular localization and Concomitantly, a reduced reactivity with phenylmercury com- functional analyses revealed that the S-nitrosylated proteins iden- − − pounds was observed (Fig. 1D). From the eNOS / livers, 36 sul- tified in the liver were largely cytosolic and mitochondrial fonic acid-containing peptides in 26 proteins were identified (Table that function as oxidoreductases and , which are critical S2), of which 24 were also identified in the WT liver. The data in- for regulating , energy, and biosynthesis, and may dicate that the majority of the endogenous liver S-nitrosoproteome coordinate the regulation of metabolic pathways by leptin and NO. is dependent on eNOS-generated NO. Biochemical, Biophysical, and Structural Properties of the Modified S-Nitrosylation Is Implicated in Multiple Metabolic Pathways. Pro- Cysteine Residues. This enriched S-nitrosoproteome was in- teomic experiments generate rich, diverse datasets that benefitfrom terrogated for the structural properties of the modified cysteine computational analysis to extract biologically relevant and poten- residues by using various bioinformatic tools and available crystal tially novel information. Consequently, functional and ontological structures of proteins. Table 1 provides the basic biochemical and analyses were conducted to assist in identifying cellular, molecular, biophysical properties of the modified cysteine residues using re- and biological functions in which S-nitrosylation may play a role in duced unmodified cysteine residues in the same proteins as a com- the liver. Sixty-five percent of S-nitrosylated proteins were localized parison group. Kyte-Doolittle hydropathy indices in 13-residue to the cytoplasm and mitochondrion, representing a significant windows were calculated to determine the influence of primary − enrichment compared with the mouse genome (P = 4.9e 34 and P = structure of the protein on modified cysteine residues (Fig. 2A). The − 7.2e 22,respectively;Fig. S3A). A subset of S-nitrosylated proteins average hydropathy index value was calculated to be −0.03 ± 0.69 were found to be present in cell membranes, whereas the remaining (n = 309) which did not differ significantly when compared with the proteins were distributed across nearly all cellular compartments mean value of the unmodified within the same proteins (Fig. S3A). Gene ontology analysis revealed that 99 S-nitrosylated (0.10 ± 0.77, n = 1,382). proteins had catalytic activity largely composed of oxidoreductases Using crystal structures (Protein Data Bank) and the Propka (39%) and transferases (17%; Fig. S3B). These functions were also 2.0 algorithm (34), the average predicted pKa of 142 cysteine res- − − found to be significantly overrepresented (P = 1.28e 20 and 3.2e 3, idues S-nitrosylated in vivo was 10.0, which was not significantly respectively) in the liver S-nitrosoproteome compared with the different from the average pKa of reduced unmodified cysteine mouse genome (22). This is not surprising, as the molecular func- residues in the same proteins (pKa of 9.88). Only 15 modified cys- tions of S-nitrosylated proteins were assigned to diverse metabolic teine residues had predicted pKa values lower than 7.4 (5.66 ± processes (i.e., , energy synthesis, lipid me- 1.24), indicating that these particular residues may be deproto- tabolism) that take place within the liver. nated at physiological pH (Fig. 2B). Secondary structure analysis Analysis of the data has also confirmed the presence of multiple revealed that S-nitrosocysteine residues were present in β-sheets S-nitrosylated cysteine residues in nearly 45% (86 of 192) of the (28%), helices (40%), and coils (32%). Unmodified cysteine resi- liver S-nitrosoproteome. Poly-S-nitrosylation is present in all top- dues within the same proteins were localized primarily in coils ranking molecular functions, suggesting that multiple sites of (39%) and equally distributed across helices and β-sheets (Table 1). S-nitrosylation in vivo may regulate protein activity. This is in ac- Statistical analysis revealed an overrepresentation and under- cordance with other known posttranslational modifications such representation of modified cysteines in helices and coils, respect- as and in which polyphos- ively (P < 0.02). phorylation and polyacetylation are considered regulators of pro- None of the cysteine residues found to be S-nitrosylated partic- tein function and signaling (23, 24). ipate in disulfide bonding in their known structures and none has To place these functional assignments into the context of bio- been reported to date to be modified by glutathiolation and alky- chemical and molecular signaling pathways, S-nitrosylated proteins lating agents (www.uniprot.org). Furthermore, by using predictive were assembled into biological protein interaction and signaling algorithms and literature searches, it was found that less than 20%

16960 | www.pnas.org/cgi/doi/10.1073/pnas.1008036107 Doulias et al. Downloaded by guest on September 26, 2021 Table 1. Biochemical and biophysical properties of S-nitrosylated and unmodified cysteine residues within the same proteins Variable S-nitrosocysteine residues Unmodified cysteine residues

Hydropathy index 0.03 ± 0.69 (n =309) 0.1± 0.77 (n = 382)

Predicted pKa 10.0 ± 2.10 (n = 142) 9.88 ± 2.20 (n = 559) Helices, % 40* 29 β-Sheets, % 28 32 † Coils, % 32 39 ‡ RSA 71% buried (n = 99) 77% buried (n = 561) 29% exposed (n = 40) 23% exposed (n = 171)

† *P < 0.02, P < 0.01 using unmodified residues as control group. ‡ Residue surface accessibility (RSA) for cysteine residues was calculated by the accessible surface area normalized by the accessible surface area of cysteine in the extended tripeptide Ala-Cys-Ala. A value of ≤10% was used as cutoff to denote a buried cysteine.

of the cysteine residues were predicted to be sites of oxidation (35). hibited a roughly normal distribution (Fig. 2A, Left), indicating that These data and analysis indicate that the majority of the in vivo sites they corresponded to a single population of cysteine residues. of S-nitrosylation represent a unique population of cysteine resi- Unmodified cysteine residues within the same proteins also dues not chemically modified through other biological processes. showed a roughly normal distribution as well (Fig. 2A, Right), in- dicating that there is no distinction between S-nitrosylated and S-Nitrosocysteine Residues Are Equally Distributed in Hydrophobic unmodified cysteine residues within the same proteins to localize in and Hydrophilic Areas of the Proteins. The overall slightly nega- hydrophobic or hydrophilic areas of proteins. Previous studies have tive hydropathy index (Table 1) is not indicative of a trend for indicated that S-nitrosylation is favored in hydrophobic regions of S-nitrosylated cysteine residues to localize in hydrophilic regions of the proteins (36), presumably because of the increased localized the protein. Further inspection of the hydropathy indices of concentration of NO-derived oxides, which may provide a suitable modified cysteine residues revealed that 139 of the 309 cysteine microenvironment for the S-nitrosylation of these cysteine resi- residues reside nearly in hydrophobic regions, whereas 170 of the dues. The present data imply that, for a subset of proteins, hy- 309 are located in hydrophilic regions. This observation led us to drophobicity may serve as a determinant for selective targeting of further examine whether S-nitrosylated cysteines belong to two cysteine residues for S-nitrosylation. distinct populations regarding their hydrophobicity/hydrophilicity. S-Nitrosylation Occurs on Cysteine Residues Adjacent to Flexible Kernel density estimation revealed that hydropathy indices ex- Regions Within the Protein. To further explore the location of S-nitrosylated cysteine residues in protein secondary structure, the frequency of secondary structures for flanking residues (positions A −10 to +10) was calculated and compared with the respective MEDICAL SCIENCES

5

.

6

04. .05. frequencies of the unmodified cysteine residues using the χ2 test. A

03.02 0 shift in secondary structure mainly from coils to helices was ob-

4 ytisneD . − < 03. served over the range of positions from 6to0(P 0.05), con-

0 sistent with the presence of the majority of S-nitrosocysteine

.0

2.01 residues in α-helices (Fig. 3A). Moreover, a 10% increase in the

1

.

. 0 < 00 frequency of coils from positions 0 to +3 (P 0.001) concomitant

N = 170 N = 139 0. N = 635 N = 747 .0 β < 0 with a reduction in -sheet frequency (P 0.001) indicative of -2 -1 0 1 2 -2 -1 0 1 2 3 a change in secondary structure was observed C-terminal from the Hydropathy Index modified cysteine residues. Unmodified cysteine residues within 30 the same proteins were localized primarily in coils and with lower B frequency in β-sheets and helices (Fig. S4A). Moreover, the fre-

s 25 quency of coils did not change significantly across all flanking

eniet residues (−10 to +10), indicating that shifts in secondary structure

s 20 yc were only between helices and β-sheets. Collectively, these data

detalysort demonstrate that modified cysteine residues are predominantly 15 present in secondary structures of proteins which may facilitate site-directed S-nitrosylation by protein-protein interactions.

in 10

-S 5 Surface Accessibility of S-Nitrosocysteine Residues. The relative residue surface accessibility (RSA) for all cysteines (modified and 0 unmodified) within the S-nitrosoproteome was calculated with ≥0 ≥1 ≥2 ≥3 ≥4 ≥5 ≥6 ≥7 ≥8 ≥9 ≥10 ≥11 ≥12 ≥13 Naccess 2.1.1 using the radius of a water molecule (1.4 Å2)as Predicted pKa value a probe (37). Ninety-nine S-nitrosylated residues (71%) were cal- culated with an RSA of 10% or lower, indicating that those resi- Fig. 2. Hydropathy index and pKa values of S-nitrosylated residues. (A) dues were not accessible to the solvent, whereas the remaining 40 Kernel density plot of hydropathy indices for S-nitrosocysteine (Left) and modified cysteines (29%) had a relative RSA greater than 10%, fi unmodi ed cysteine residues (Right). Hydropathy index was calculated for all meaning they were solvent-accessible. Unmodified cysteines within S-nitrosylated (n = 170 hydrophilic, n = 139 hydrophobic) and unmodified (n = 635 hydrophilic, n = 747 hydrophobic) cysteine residues within a 13 amino acid the same proteins exhibited similar distribution between buried (77%) and solvent-accessible residues (23%). A quantile–quantile window, with a negative value indicating hydrophilicity. (B) Predicted pKa value for each S-nitrosylated cysteine was calculated from the experimental probability plot was used to determine if there was enrichment for structures and the distribution was represented as a histogram. exposed or buried cysteines within the modified versus the un-

Doulias et al. PNAS | September 28, 2010 | vol. 107 | no. 39 | 16961 Downloaded by guest on September 26, 2021 A B

C D

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 E

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

-6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6

Fig. 3. Analysis of primary sequence, distribution in secondary structures, and surface accessibility of S-nitrosylated cysteine residues. (A) The frequency of S-nitrosocysteine (n = 142; black bars) and unmodified cysteine residues (n = 473; white bars) within the same proteins in different secondary structures was calculated using the available crystal structures. (B) Distribution of residues flanking S-nitrosylated cysteines in secondary structures. SNO-cysteines are located at position 0 and the frequency for 10 residues upstream (−10) and 10 residues downstream (+10) was calculated as described earlier. (C) Quantile–quantile plot of relative RSA for S-nitrosylated (x axis) versus unmodified (y axis) cysteine residues. RSA values were calculated from the biological assemblies defined by PISA (42) for available crystal structures. (D) Top three scoring sequence motifs for residues flanking S-nitrosylated cysteine residue. Note the presence of charged as well as aliphatic amino acids able to “accommodate” protein and small-molecule binding. (E) Top two sequence motifs assigned to the residues sensitive to Trx system.

modified group of cysteine residues (Fig. 3C). To produce this plot, loproteins were identified as endogenously S-nitrosylated, sug- the RSA values of modified and unmodified cysteine residues were gesting a self-catalyzed mechanism of S-nitrosylation similar to sorted and values of RSA for each percentile were plotted against the proposed mechanism for the selective S-nitrosylation of he- each other (i.e., RSA of smallest 1% of modified cysteine residues moglobin (39). The possibility also exists that these metallo- vs. RSA of smallest 1% of unmodified cysteine residues, smallest proteins can catalyze S-nitrosylation of interacting proteins, as 2% vs. smallest 2%). The plot demonstrates that S-nitrosocysteine has been indicated previously (39). This will require protein– residues have a distribution skewed toward larger surface-accessible protein association and, as discussed later, the majority of the S- areas than unmodified cysteine residues within the same proteins. nitrosylated proteins, either in primary sequence or within the In addition, 70% of S-nitrosylated cysteine residues within 6 Å tertiary structure, contain charged amino acids that can provide were surrounded by negatively or positively charged amino acids interactive interfaces for specific transfer of a NO equivalent from that had their side chains pointed away from cysteine thiol groups. a to cysteine residues. Although the presence of charged residues in the vicinity of the modified residues did not impact their predicted pKa, it may facil- Linear Motifs. To further interrogate structural elements that may itate site specificmodification by accommodating protein or S- distinguish modified cysteines from unmodified within the same nitrosoglutathione association. This is in agreement with findings of proteins, flanking amino acids were examined for the presence of Mitchell et al., who demonstrated that charged residues near cys- linear motifs using the program Motif-X (40). The top scoring teine 73 were required for interaction and transnitrosylation of motif for modified cysteines (n = 37, 12%) had exclusively procaspase-3 (38). Accordingly, the presence of cysteine residues in at position −1(P ≤ 0.001) and consisted mostly of hydrophobic highly exposed areas of proteins and in proximity to charged amino residues (Fig. 3D). Remarkably, the second (n = 31, 10%) and acids suggests a protein or small molecule transnitrosation assisted third (n = 25, 8%) top scoring motifs had negatively charged amino mechanism of S-nitrosylation. acids exclusively at positions +3 and −1, respectively (P ≤ 0.001; Fig. 3D). The same analysis for unmodified cysteines within the Metal Catalyzed S-Nitrosylation. Studies exploring the S-nitro- same proteins revealed top sequence motifs lacking negatively sylation of proteins in cells indicated that more than 50% of the charged amino acids flanking cysteine residues (Fig. S4B), in- cellular formation of protein S-nitrosocysteine is derived by dini- dicating that specific elements of the primary structure are re- trosyliron complexes (7). Within the liver S-nitrosoproteome, 13 S- quired for a cysteine to be S-nitrosylated in vivo. These motifs nitrosylated cysteine residues, which are directly involved in the could serve as scaffolds for protein and small molecule binding chelation of metal ions, were identified (Table S3). Metal ligation such as thioredoxin (Trx) or S-nitrosoglutathione. may provide site-directed modification of these residues. Alter- To further explore this possibility we used the Trx ex vivo natively, dinitrosyliron complexes could be stabilized near cysteine denitrosylation assay (17) to identify sites of S-nitrosylation that residues by interactions with neighboring acidic residues. As stated, can interact with Trx. Freshly isolated liver homogenates were more than 70% of the modified cysteine residues are in close treated with a Trx system (Trx/Trx-reductase/NADPH). This proximity (<6 Å) to acidic residues, which could serve as inter- treatment resulted in a 72% reduction in protein S-nitrosocysteine acting sites for dinitrosyliron complexes. Moreover, 15 metal- levels as quantified by chemiluminescence. In comparison with the

16962 | www.pnas.org/cgi/doi/10.1073/pnas.1008036107 Doulias et al. Downloaded by guest on September 26, 2021 untreated liver S-nitrosoproteome (Table S1), 72 proteins were no Materials and Methods longer identified after Trx treatment (Table S4). Linear motif Chemicals and Reagents. All HPLC solvents were purchased from Burdick and analysis revealed two motifs in this subset of S-nitrosylated pro- Jackson, and unless stated all other reagents were purchased from Sigma- teins. The highest-scoring motif had exclusively Aldrich. Mercury/PEG/biotin compound was designed by the authors and at position −1, whereas the second motif had exclusively thre- synthesized by SoluLink. onine at position +5 (Fig. 3E). Notably, both motifs contained charged amino acids at positions before and after the cysteine Capture of S-Nitrosylated Proteins Using MRC or mPEGb. Mercury resin syn- thesis, MRC solid-phase, and mPEGb compound-based protein and pep- residue, consistent with the idea that charged amino acids in tide enrichment are described in detail in the SI Materials and Methods and S-nitrosocysteine–containing motifs may facilitate interaction Scheme S1. with Trx. In conclusion, by using unique, highly specific MS-based proteomic Protein Sequence and Structural Analysis. A detailed description of protein methods, we identified an expanded endogenous S-nitrosoproteome sequence and structural analysis exists in SI Materials and Methods. from WT mouse liver. Despite that S-nitrosylated cysteine residues had, in general, similar hydropathy distribution and predicted pK Statistical Analyses. Graphs were constructed and statistical analyses were a fi values as nonmodified cysteine residues in the same proteins, closer performed using GraphPad Prism 5 software (GraphPad). Statistical signi - cance was determined by paired or unpaired nonparametric two-tailed t tests interrogation of the surrounding primary and secondary structures using either the Mann-Whitney (unpaired) or Wilcoxon matched-pairs test. revealed distinctions that direct site-specific S-nitrosylation of certain cysteine residues. The structural analysis of these proteins also un- ACKNOWLEDGMENTS. We thank the Protein Core at the Children’s Hospital covered structural features that can accommodate multiple mecha- of Philadelphia Research Institute for their assistance with mass spectrome- nisms for S-nitrosylation in vivo. In addition, the data also revealed try, Dr. Santosh S. Venkatesh for assistance with the χ2 test, Dr. David Schwartz (Solulink Biosciences, San Diego, CA) for the synthesis of organo- a putative link among leptin, eNOS, and protein S-nitrosylation in the mercury-polyethyleneglycol-biotin, and Dr. Qi Fang for support with struc- regulation of liver fatty acid metabolism. Overall, the use of global tural analysis. This work was supported by National Institutes of Health proteomic methods enabled structural and functional characteriza- Grants AG13966 and HL054926, National Institute of Environmental Health tion of the in vivo S-nitrosocysteine proteome and the formulation of Sciences Center of Excellence in Environmental Toxicology Grant ES013508 (to H.I), and National Institute of General Medical Sciences Award testable new hypotheses that can be explored in the future using F31GM085903 (to J.L.G.). H.I. is the Gisela and Dennis Alter Research Pro- targeted approaches. fessor of Pediatrics.

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