Cysteine persulfidation (also called sulfhydration or sulfuration) has emerged as a potential redox mechanism to regulate protein functions and diverse biological processes in hydrogen sulfide (H2S) signaling. Due to its intrinsically unstable nature, working with this modification has proven to be challenging. Although methodological progress has expanded the inventory of persulfidated proteins, there is a continued need to develop methods that can directly and unequivocally identify persulfidated cysteine residues in complex proteomes.
Results:
A quantitative chemoproteomic method termed as low-pH quantitative thiol reactivity profiling (QTRP) was developed to enable direct site-specific mapping and reactivity profiling of proteomic persulfides and thiols in parallel. The method was first applied to cell lysates treated with NaHS, resulting in the identification of overall 1547 persulfidated sites on 994 proteins. Structural analysis uncovered unique consensus motifs that might define this distinct type of modification. Moreover, the method was extended to profile endogenous protein persulfides in cells expressing H2S-generating enzyme, mouse tissues, and human serum, which led to additional insights into mechanistic, structural, and functional features of persulfidation events, particularly on human serum albumin.
Innovation and Conclusion:
Low-pH QTRP represents the first method that enables direct and unbiased proteomic mapping of cysteine persulfidation. Our method allows to generate the most comprehensive inventory of persulfidated targets of NaHS so far and to perform the first analysis of in vivo persulfidation events, providing a valuable tool to dissect the biological functions of this important modification.
Get full access to this article
View all access options for this article.
References
1.
AkterS, FuL, JungY, ConteML, LawsonJR, LowtherWT, SunR, LiuK, YangJ, and CarrollKS. Chemical proteomics reveals new targets of cysteine sulfinic acid reductase. Nat Chem Biol, 14: 995–1004, 2018.
2.
AltaanyZ, JuY, YangG, and WangR. The coordination of S-sulfhydration, S-nitrosylation, and phosphorylation of endothelial nitric oxide synthase by hydrogen sulfide. Sci Signal, 7: ra87, 2014.
3.
ArocaA, BenitoJM, GotorC, and RomeroLC. Persulfidation proteome reveals the regulation of protein function by hydrogen sulfide in diverse biological processes in Arabidopsis. J Exp Bot, 68: 4915–4927, 2017.
4.
ArocaA, SernaA, GotorC, and RomeroLC. S-sulfhydration: a cysteine posttranslational modification in plant systems. Plant Physiol, 168: 334–342, 2015.
5.
BaeSK, HeoCH, ChoiDJ, SenD, JoeEH, ChoBR, and KimHM. A ratiometric two-photon fluorescent probe reveals reduction in mitochondrial H2S production in Parkinson's disease gene knockout astrocytes. J Am Chem Soc, 135: 9915–9923, 2013.
6.
BakDW, GaoJ, WangC, and WeerapanaE. A quantitative chemoproteomic platform to monitor selenocysteine reactivity within a complex proteome. Cell Chem Biol, 25: 1157–1167 e4, 2018.
7.
BarglowKT, KnutsonCG, WishnokJS, TannenbaumSR, and MarlettaMA. Site-specific and redox-controlled S-nitrosation of thioredoxin. Proc Natl Acad Sci U S A, 108: E600–E606, 2011.
8.
BaskarR, LiL, and MoorePK. Hydrogen sulfide-induces DNA damage and changes in apoptotic gene expression in human lung fibroblast cells. FASEB J, 21: 247–255, 2007.
BibliSI, HuJ, SigalaF, WittigI, HeidlerJ, ZukunftS, TsilimigrasDI, RandriamboavonjyV, WittigJ, KojonazarovB, SchurmannC, SiragusaM, SiudaD, LuckB, Abdel MalikR, FilisKA, ZografosG, ChenC, WangDW, PfeilschifterJ, BrandesRP, SzaboC, PapapetropoulosA, and FlemingI. Cystathionine gamma lyase sulfhydrates the RNA binding protein human antigen R to preserve endothelial cell function and delay atherogenesis. Circulation, 139: 101–114, 2019.
11.
BibliSI, SzaboC, ChatzianastasiouA, LuckB, ZukunftS, FlemingI, and PapapetropoulosA. Hydrogen sulfide preserves endothelial nitric oxide synthase function by inhibiting proline-rich kinase 2: implications for cardiomyocyte survival and cardioprotection. Mol Pharmacol, 92: 718–730, 2017.
12.
BocediA, CattaniG, StellaL, MassoudR, and RicciG. Thiol disulfide exchange reactions in human serum albumin: the apparent paradox of the redox transitions of Cys34. FEBS J, 285: 3225–3237, 2018.
13.
BogdandiV, IdaT, SuttonTR, BiancoC, DitroiT, KosterG, HenthornHA, MinnionM, ToscanoJP, van der VlietA, PluthMD, FeelischM, FukutoJM, AkaikeT, and NagyP. Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?. Br J Pharmacol, 176: 646–670, 2019.
14.
BonanataJ, TurellL, AntmannL, Ferrer-SuetaG, BotasiniS, MendezE, AlvarezB, and CoitinoEL. The thiol of human serum albumin: acidity, microenvironment and mechanistic insights on its oxidation to sulfenic acid. Free Radic Biol Med, 108: 952–962, 2017.
ChenYJ, LuCT, SuMG, HuangKY, ChingWC, YangHH, LiaoYC, ChenYJ, and LeeTY. dbSNO 2.0: a resource for exploring structural environment, functional and disease association and regulatory network of protein S-nitrosylation. Nucleic Acids Res, 43: D503–D511, 2015.
17.
CheungSH and LauJYW. Hydrogen sulfide mediates athero-protection against oxidative stress via S-sulfhydration. PLoS One, 13: e0194176, 2018.
18.
ChiH, LiuC, YangH, ZengWF, WuL, ZhouWJ, WangRM, NiuXN, DingYH, ZhangY, WangZW, ChenZL, SunRX, LiuT, TanGM, DongMQ, XuP, ZhangPH, and HeSM.Comprehensive identification of peptides in tandem mass spectra using an efficient open search engine. Nat Biotechnol, 2018 [Epub ahead of print]; DOI: 10.1038/nbt.4236.
19.
CuevasantaE, LangeM, BonanataJ, CoitinoEL, Ferrer-SuetaG, FilipovicMR, and AlvarezB. Reaction of hydrogen sulfide with disulfide and sulfenic acid to form the strongly nucleophilic persulfide. J Biol Chem, 290: 26866–26880, 2015.
20.
DengX, WeerapanaE, UlanovskayaO, SunF, LiangH, JiQ, YeY, FuY, ZhouL, LiJ, ZhangH, WangC, AlvarezS, HicksLM, LanL, WuM, CravattBF, and HeC. Proteome-wide quantification and characterization of oxidation-sensitive cysteines in pathogenic bacteria. Cell Host Microbe, 13: 358–370, 2013.
21.
DokaE, PaderI, BiroA, JohanssonK, ChengQ, BallagoK, PriggeJR, Pastor-FloresD, DickTP, SchmidtEE, ArnerES, and NagyP. A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems. Sci Adv, 2: e1500968, 2016.
22.
FilipovicMR, ZivanovicJ, AlvarezB, and BanerjeeR. Chemical Biology of H2S Signaling through Persulfidation. Chem Rev, 118: 1253–1337, 2018.
23.
FuL, LiuK, FerreiraRB, CarrollKS, and YangJ. Proteome-wide analysis of cysteine S-sulfenylation using a benzothiazine-based probe. Curr Protoc Protein Sci, 95: e76, 2019.
24.
FuL, LiuK, SunM, TianC, SunR, Morales BetanzosC, TallmanKA, PorterNA, YangY, GuoD, LieblerDC, and YangJ. Systematic and quantitative assessment of hydrogen peroxide reactivity with cysteines across human proteomes. Mol Cell Proteomics, 16: 1815–1828, 2017.
25.
GaoXH, KrokowskiD, GuanBJ, BedermanI, MajumderM, ParisienM, DiatchenkoL, KabilO, WillardB, BanerjeeR, WangB, BebekG, EvansCR, FoxPL, GersonSL, HoppelCL, LiuM, ArvanP, and HatzoglouM. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response. Elife, 4: e10067, 2015.
26.
GeigerT, VelicA, MacekB, LundbergE, KampfC, NagarajN, UhlenM, CoxJ, and MannM. Initial quantitative proteomic map of 28 mouse tissues using the SILAC mouse. Mol Cell Proteomics, 12: 1709–1722, 2013.
27.
GuoL, YangW, HuangQ, QiangJ, HartJR, WangW, HuJ, ZhuJ, LiuN, and ZhangY. Selenocysteine-specific mass spectrometry reveals tissue-distinct selenoproteomes and candidate selenoproteins. Cell Chem Biol, 25: 1380–1388 e4, 2018.
28.
GuptaV, YangJ, LieblerDC, and CarrollKS. Diverse redoxome reactivity profiles of carbon nucleophiles. J Am Chem Soc, 139: 5588–5595, 2017.
29.
GygiSP, RistB, GerberSA, TurecekF, GelbMH, and AebersoldR. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol, 17: 994–999, 1999.
30.
Huang daW, ShermanBT, and LempickiRA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc, 4: 44–57, 2009.
31.
IdaT, SawaT, IharaH, TsuchiyaY, WatanabeY, KumagaiY, SuematsuM, MotohashiH, FujiiS, MatsunagaT, YamamotoM, OnoK, Devarie-BaezNO, XianM, FukutoJM, and AkaikeT. Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling. Proc Natl Acad Sci U S A, 111: 7606–7611, 2014.
32.
ItoG, ArigaH, NakagawaY, and IwatsuboT. Roles of distinct cysteine residues in S-nitrosylation and dimerization of DJ-1. Biochem Biophys Res Commun, 339: 667–672, 2006.
33.
JaroszAP, WeiW, GauldJW, AuldJ, OzcanF, AslanM, and MutusB. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is inactivated by S-sulfuration in vitro. Free Radic Biol Med, 89: 512–521, 2015.
34.
JuY, FuM, StokesE, WuL, and YangG. H(2)S-Mediated Protein S-Sulfhydration: a prediction for its formation and regulation. Molecules, 22: pii: E1334, 2017.
35.
JuY, WuL, and YangG. Thioredoxin 1 regulation of protein S-desulfhydration. Biochem Biophys Rep, 5: 27–34, 2016.
36.
JuY, ZhangW, PeiY, and YangG. H(2)S signaling in redox regulation of cellular functions. Can J Physiol Pharmacol, 91: 8–14, 2013.
37.
KrishnanN, FuC, PappinDJ, and TonksNK. H2S-Induced sulfhydration of the phosphatase PTP1B and its role in the endoplasmic reticulum stress response. Sci Signal, 4: ra86, 2011.
38.
KumarR, JangirDK, VermaG, ShekharS, HanpudeP, KumarS, KumariR, SinghN, Sarovar BhaveshN, Ranjan JanaN, and Kanti MaitiT. S-nitrosylation of UCHL1 induces its structural instability and promotes alpha-synuclein aggregation. Sci Rep, 7: 44558, 2017.
39.
LevittMD, Abdel-RehimMS, and FurneJ. Free and acid-labile hydrogen sulfide concentrations in mouse tissues: anomalously high free hydrogen sulfide in aortic tissue. Antioxid Redox Signal, 15: 373–378, 2011.
40.
LiJ, ChenS, WangX, ShiC, LiuH, YangJ, ShiW, GuoJ, and JiaH. Hydrogen Sulfide Disturbs Actin Polymerization via S-Sulfhydration Resulting in Stunted Root Hair Growth. Plant Physiol, 178: 936–949, 2018.
41.
LiangM, JinS, WuDD, WangMJ, and ZhuYC. Hydrogen sulfide improves glucose metabolism and prevents hypertrophy in cardiomyocytes. Nitric Oxide, 46: 114–122, 2015.
42.
LiuC, SongCQ, YuanZF, FuY, ChiH, WangLH, FanSB, ZhangK, ZengWF, HeSM, DongMQ, and SunRX. pQuant improves quantitation by keeping out interfering signals and evaluating the accuracy of calculated ratios. Anal Chem, 86: 5286–5294, 2014.
43.
LongenS, RichterF, KohlerY, WittigI, BeckKF, and PfeilschifterJ. Quantitative Persulfide Site Identification (qPerS-SID) reveals protein targets of H2S releasing donors in mammalian cells. Sci Rep, 6: 29808, 2016.
44.
ManiS, CaoW, WuL, and WangR. Hydrogen sulfide and the liver. Nitric Oxide, 41: 62–71, 2014.
45.
MartellJ, SeoY, BakDW, KingsleySF, TissenbaumHA, and WeerapanaE. Global cysteine-reactivity profiling during impaired insulin/IGF-1 signaling in C. elegans identifies uncharacterized mediators of longevity. Cell Chem Biol, 23: 955–966, 2016.
46.
MengG, XiaoY, MaY, TangX, XieL, LiuJ, GuY, YuY, ParkCM, XianM, WangX, FerroA, WangR, MoorePK, ZhangZ, WangH, HanY, and JiY. Hydrogen sulfide regulates kruppel-like factor 5 transcription activity via specificity protein 1 S-sulfhydration at Cys664 to prevent myocardial hypertrophy. J Am Heart Assoc, 5: pii: e004160, 2016.
47.
MustafaAK, GadallaMM, SenN, KimS, MuW, GaziSK, BarrowRK, YangG, WangR, and SnyderSH. H2S signals through protein S-sulfhydration. Sci Signal, 2: ra72, 2009.
48.
NagumoY, KakeyaH, ShojiM, HayashiY, DohmaeN, and OsadaH. Epolactaene binds human Hsp60 Cys442 resulting in the inhibition of chaperone activity. Biochem J, 387: 835–840, 2005.
49.
NakashimaF, ShibataT, KamiyaK, YoshitakeJ, KikuchiR, MatsushitaT, IshiiI, Gimenez-BastidaJA, SchneiderC, and UchidaK. Structural and functional insights into S-thiolation of human serum albumins. Sci Rep, 8: 932, 2018.
50.
O'SheaJP, ChouMF, QuaderSA, RyanJK, ChurchGM, and SchwartzD. pLogo: a probabilistic approach to visualizing sequence motifs. Nat Methods, 10: 1211–1212, 2013.
51.
OlsonKR and GaoY. Effects of inhibiting antioxidant pathways on cellular hydrogen sulfide and polysulfide metabolism. Free Radic Biol Med, 135: 1–14, 2019.
52.
OlsonKR, GaoY, DeLeonER, ArifM, ArifF, AroraN, and StraubKD. Catalase as a sulfide-sulfur oxido-reductase: an ancient (and modern?) regulator of reactive sulfur species (RSS). Redox Biol, 12: 325–339, 2017.
53.
ParkCM, MacinkovicI, FilipovicMR, and XianM. Use of the “tag-switch” method for the detection of protein S-sulfhydration. Methods Enzymol, 555: 39–56, 2015.
54.
PaulBD and SnyderSH. H(2)S signalling through protein sulfhydration and beyond. Nat Rev Mol Cell Biol, 13: 499–507, 2012.
55.
PaulBD and SnyderSH. Modes of physiologic H2S signaling in the brain and peripheral tissues. Antioxid Redox Signal, 22: 411–423, 2015.
56.
PengH, ZhangY, PalmerLD, Kehl-FieTE, SkaarEP, TrinidadJC, and GiedrocDP. Hydrogen sulfide and reactive sulfur species impact proteome S-sulfhydration and global virulence regulation in Staphylococcus aureus. ACS Infect Dis, 3: 744–755, 2017.
57.
PetrovaB, LiuK, TianC, KitaokaM, FreinkmanE, YangJ, and Orr-WeaverTL. Dynamic redox balance directs the oocyte-to-embryo transition via developmentally controlled reactive cysteine changes. Proc Natl Acad Sci U S A, 115: E7978–E7986, 2018.
58.
PhillipsCM, ZatarainJR, NichollsME, PorterC, WidenSG, ThankiK, JohnsonP, JawadMU, MoyerMP, RandallJW, HellmichJL, MaskeyM, QiuS, WoodTG, DruzhynaN, SzczesnyB, ModisK, SzaboC, ChaoC, and HellmichMR. Upregulation of cystathionine-beta-synthase in colonic epithelia reprograms metabolism and promotes carcinogenesis. Cancer Res, 77: 5741–5754, 2017.
59.
SearcyDG, WhiteheadJP, and MaroneyMJ. Interaction of Cu,Zn superoxide dismutase with hydrogen sulfide. Arch Biochem Biophys, 318: 251–263, 1995.
60.
SenN.Functional and molecular insights of hydrogen sulfide signaling and protein sulfhydration. J Mol Biol, 429: 543–561, 2017.
61.
SenN, PaulBD, GadallaMM, MustafaAK, SenT, XuR, KimS, and SnyderSH. Hydrogen sulfide-linked sulfhydration of NF-kappaB mediates its antiapoptotic actions. Mol Cell, 45: 13–24, 2012.
62.
ShenX, PeterEA, BirS, WangR, and KevilCG. Analytical measurement of discrete hydrogen sulfide pools in biological specimens. Free Radic Biol Med, 52: 2276–2283, 2012.
63.
SunR, FuL, LiuK, TianC, YangY, TallmanKA, PorterNA, LieblerDC, and YangJ. Chemoproteomics reveals chemical diversity and dynamics of 4-Oxo-2-nonenal modifications in cells. Mol Cell Proteomics, 16: 1789–1800, 2017.
64.
SunR, ShiF, LiuK, FuL, TianC, YangY, TallmanKA, PorterNA, and YangJ. A Chemoproteomic platform to assess bioactivation potential of drugs. Chem Res Toxicol, 30: 1797–1803, 2017.
65.
ThompsonA, SchaferJ, KuhnK, KienleS, SchwarzJ, SchmidtG, NeumannT, JohnstoneR, MohammedAK, and HamonC. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Anal Chem, 75: 1895–1904, 2003.
66.
TianC, SunR, LiuK, FuL, LiuX, ZhouW, YangY, and YangJ. Multiplexed thiol reactivity profiling for target discovery of electrophilic natural products. Cell Chem Biol, 24: 1416–1427 e5, 2017.
67.
UntereinerAA, OlahG, ModisK, HellmichMR, and SzaboC. H2S-induced S-sulfhydration of lactate dehydrogenase a (LDHA) stimulates cellular bioenergetics in HCT116 colon cancer cells. Biochem Pharmacol, 136: 86–98, 2017.
68.
van der ReestJ, LillaS, ZhengL, ZanivanS, and GottliebE. Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress. Nat Commun, 9: 1581, 2018.
VasasA, DokaE, FabianI, and NagyP. Kinetic and thermodynamic studies on the disulfide-bond reducing potential of hydrogen sulfide. Nitric Oxide, 46: 93–101, 2015.
71.
WangC, WeerapanaE, BlewettMM, and CravattBF. A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles. Nat Methods, 11: 79–85, 2014.
72.
WedmannR, OnderkaC, WeiS, SzijartoIA, MiljkovicJL, MitrovicA, LangeM, SavitskyS, YadavPK, TorregrossaR, HarrerEG, HarrerT, IshiiI, GollaschM, WoodME, GalardonE, XianM, WhitemanM, BanerjeeR, and FilipovicMR. Improved tag-switch method reveals that thioredoxin acts as depersulfidase and controls the intracellular levels of protein persulfidation. Chem Sci, 7: 3414–3426, 2016.
WeichselA, GasdaskaJR, PowisG, and MontfortWR. Crystal structures of reduced, oxidized, and mutated human thioredoxins: evidence for a regulatory homodimer. Structure, 4: 735–751, 1996.
75.
WintnerEA, DeckwerthTL, LangstonW, BengtssonA, LevitenD, HillP, InskoMA, DumpitR, VandenEkartE, ToombsCF, and SzaboC. A monobromobimane-based assay to measure the pharmacokinetic profile of reactive sulphide species in blood. Br J Pharmacol, 160: 941–957, 2010.
76.
XieZZ, ShiMM, XieL, WuZY, LiG, HuaF, and BianJS. Sulfhydration of p66Shc at cysteine59 mediates the antioxidant effect of hydrogen sulfide. Antioxid Redox Signal, 21: 2531–2542, 2014.
77.
YadavPK, MartinovM, VitvitskyV, SeravalliJ, WedmannR, FilipovicMR, and BanerjeeR. Biosynthesis and reactivity of cysteine persulfides in signaling. J Am Chem Soc, 138: 289–299, 2016.
78.
YadavV, GaoXH, WillardB, HatzoglouM, BanerjeeR, and KabilO. Hydrogen sulfide modulates eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation status in the integrated stress-response pathway. J Biol Chem, 292: 13143–13153, 2017.
79.
YangJ, GuptaV, CarrollKS, and LieblerDC. Site-specific mapping and quantification of protein S-sulphenylation in cells. Nat Commun, 5: 4776, 2014.
80.
YangJ, GuptaV, TallmanKA, PorterNA, CarrollKS, and LieblerDC. Global, in situ, site-specific analysis of protein S-sulfenylation. Nat Protoc, 10: 1022–1037, 2015.
81.
YangJ, TallmanKA, PorterNA, and LieblerDC. Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells. Anal Chem, 87: 2535–2541, 2015.
82.
ZhangD, MacinkovicI, Devarie-BaezNO, PanJ, ParkCM, CarrollKS, FilipovicMR, and XianM. Detection of protein S-sulfhydration by a tag-switch technique. Angew Chem Int Ed Engl, 53: 575–581, 2014.
83.
ZhaoK, LiS, WuL, LaiC, and YangG. Hydrogen sulfide represses androgen receptor transactivation by targeting at the second zinc finger module. J Biol Chem, 289: 20824–20835, 2014.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
