Quantification of the immunometabolite protein modifications S-2-succinocysteine and 2,3-dicarboxypropylcysteine.

J Hunter Cox, Richard S McCain, Emery Tran, Shoba Swaminathan, Holland H Smith, Gerardo G Piroli, Michael Shtutman, Michael D Walla, William E Cotham, Norma Frizzell
Author Information
  1. J Hunter Cox: Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina, United States. ORCID
  2. Richard S McCain: Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina, United States.
  3. Emery Tran: Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina, United States.
  4. Shoba Swaminathan: Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina, United States.
  5. Holland H Smith: Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina, United States.
  6. Gerardo G Piroli: Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina, United States.
  7. Michael Shtutman: Department of Drug Discovery & Biomedical Sciences, College of Pharmacy, University of South Carolina, Columbia, South Carolina, United States.
  8. Michael D Walla: Mass Spectrometry Center, Department of Chemistry & Biochemistry, University of South Carolina, Columbia, South Carolina, United States.
  9. William E Cotham: Mass Spectrometry Center, Department of Chemistry & Biochemistry, University of South Carolina, Columbia, South Carolina, United States.
  10. Norma Frizzell: Department of Pharmacology, Physiology & Neuroscience, School of Medicine, University of South Carolina, Columbia, South Carolina, United States. ORCID

Abstract

The tricarboxylic acid (TCA) cycle metabolite fumarate nonenzymatically reacts with the amino acid cysteine to form S-(2-succino)cysteine (2SC), referred to as protein succination. The immunometabolite Itaconate accumulates during lipopolysaccharide (LPS) stimulation of macrophages and microglia. Itaconate nonenzymatically reacts with cysteine residues to generate 2,3-dicarboxypropylcysteine (2,3-DCP), referred to as protein dicarboxypropylation. Since fumarate and Itaconate levels dynamically change in activated immune cells, the levels of both 2SC and 2,3-DCP reflect the abundance of these metabolites and their capacity to modify protein thiols. We generated ethyl esters of 2SC and 2,3-DCP from protein hydrolysates and used stable isotope dilution mass spectrometry to determine the abundance of these in LPS-stimulated Highly Aggressively Proliferating Immortalized (HAPI) microglia. To quantify the stoichiometry of the succination and dicarboxypropylation, reduced cysteines were alkylated with iodoacetic acid to form S-carboxymethylcysteine (CMC), which was then esterified. Itaconate-derived 2,3-DCP, but not fumarate-derived 2SC, increased in LPS-treated HAPI microglia. Stoichiometric measurements demonstrated that 2,3-DCP increased from 1.57% to 9.07% of total cysteines upon LPS stimulation. This methodology to simultaneously distinguish and quantify both 2SC and 2,3-DCP will have broad applications in the physiology of metabolic diseases. In addition, we find that available anti-2SC antibodies also detect the structurally similar 2,3-DCP, therefore "succinate moiety" may better describe the antigen recognized. Itaconate and fumarate have roles as immunometabolites modulating the macrophage response to inflammation. Both immunometabolites chemically modify protein cysteine residues to modulate the immune response. Itaconate and fumarate levels change dynamically, whereas their stable protein modifications can be quantified by mass spectrometry. This method distinguishes Itaconate and fumarate-derived protein modifications and will allow researchers to quantify their contributions in isolated cell types and tissues across a range of metabolic diseases.

Keywords

References

  1. Mol Cancer. 2017 Jun 7;16(1):101 [PMID: 28592321]
  2. Cell Metab. 2020 Sep 1;32(3):468-478.e7 [PMID: 32791101]
  3. Nature. 2013 Apr 11;496(7444):238-42 [PMID: 23535595]
  4. Redox Biol. 2023 Nov;67:102932 [PMID: 37883842]
  5. J Biol Chem. 2009 Sep 18;284(38):25772-81 [PMID: 19592500]
  6. Nature. 2018 Apr 5;556(7699):113-117 [PMID: 29590092]
  7. J Biol Chem. 2016 Jul 1;291(27):14274-14284 [PMID: 27189937]
  8. Br J Cancer. 2016 Jun 14;114(12):1405-11 [PMID: 27187686]
  9. Nature. 2016 Aug 31;537(7621):544-547 [PMID: 27580029]
  10. Anal Chem. 2020 Aug 4;92(15):10627-10634 [PMID: 32634308]
  11. Anal Chem. 2023 Jan 10;: [PMID: 36625376]
  12. Proc Natl Acad Sci U S A. 2013 May 7;110(19):7820-5 [PMID: 23610393]
  13. J Neuroinflammation. 2012 May 31;9:115 [PMID: 22651808]
  14. Cell Rep. 2021 Mar 9;34(10):108756 [PMID: 33691097]
  15. J Neurochem. 2008 Oct;107(2):557-69 [PMID: 18717813]
  16. Hum Pathol. 2023 Apr;134:102-113 [PMID: 36581128]
  17. J Pathol. 2011 Sep;225(1):4-11 [PMID: 21630274]
  18. Glia. 2001 Jul;35(1):53-62 [PMID: 11424192]
  19. Am J Surg Pathol. 2016 Jul;40(7):865-75 [PMID: 26900816]
  20. Mol Cell Proteomics. 2019 Mar;18(3):504-519 [PMID: 30587509]
  21. Arch Biochem Biophys. 2004 May 15;425(2):200-6 [PMID: 15111128]
  22. Cell Metab. 2016 Dec 13;24(6):807-819 [PMID: 27866838]
  23. ACS Chem Biol. 2020 Apr 17;15(4):856-861 [PMID: 32250583]
  24. J Biol Chem. 2020 Sep 25;295(39):13410-13418 [PMID: 32820045]
  25. Cell Rep. 2023 Feb 28;42(2):112104 [PMID: 36787220]
  26. Nat Metab. 2022 May;4(5):534-546 [PMID: 35655026]
  27. Nat Commun. 2023 Oct 9;14(1):6322 [PMID: 37813836]
  28. Am J Surg Pathol. 2014 May;38(5):627-37 [PMID: 24441663]
  29. J Biol Chem. 2007 Nov 23;282(47):34219-28 [PMID: 17726021]
  30. Cell Metab. 2016 Jul 12;24(1):158-66 [PMID: 27374498]
  31. J Clin Invest. 2023 Jun 1;133(11): [PMID: 37053010]
  32. Nature. 2018 Apr;556(7702):501-504 [PMID: 29670287]
  33. Am J Surg Pathol. 2022 Apr 1;46(4):537-546 [PMID: 34678832]
  34. Amino Acids. 2003 Dec;25(3-4):275-81 [PMID: 14661090]
  35. Biochem J. 2012 Jul 15;445(2):247-54 [PMID: 22524437]
  36. Nat Chem Biol. 2019 Apr;15(4):391-400 [PMID: 30718813]
  37. J Biol Chem. 2002 Aug 9;277(32):29018-27 [PMID: 12023292]
  38. Anal Biochem. 1990 May 15;187(1):136-40 [PMID: 2372110]
  39. Arch Biochem Biophys. 2006 Jun 1;450(1):1-8 [PMID: 16624247]
  40. Mol Cell Proteomics. 2016 Feb;15(2):445-61 [PMID: 26450614]
  41. Biochem J. 2014 Sep 1;462(2):231-45 [PMID: 24909641]
  42. Nature. 2023 Mar;615(7952):490-498 [PMID: 36890227]
  43. J Biol Chem. 1951 Nov;193(1):265-75 [PMID: 14907713]
  44. Cell Metab. 2023 Jun 6;35(6):961-978.e10 [PMID: 37178684]
  45. Nature. 2010 Dec 9;468(7325):790-5 [PMID: 21085121]

Grants

  1. R56 NS116174/NINDS NIH HHS
  2. R01 NS092938/NINDS NIH HHS
  3. R21 DA058586/NIDA NIH HHS
  4. R01 NS126851/NINDS NIH HHS

MeSH Term

Humans
Cysteine
Lipopolysaccharides
Proteins
Fumarates
Metabolic Diseases
Allyl Compounds
Hydrocarbons, Chlorinated
Succinates

Chemicals

itaconic acid
S-(2-succinyl)cysteine
Cysteine
2,3-dichloro-1-propene
Lipopolysaccharides
Proteins
Fumarates
Allyl Compounds
Hydrocarbons, Chlorinated
Succinates

Word Cloud

Created with Highcharts 10.0.0protein23-DCPfumarate2SCcysteineitaconatemicrogliaacidItaconatelevelsquantifymodificationsnonenzymaticallyreactsformreferredsuccinationimmunometaboliteLPSstimulationresidues3-dicarboxypropylcysteinedicarboxypropylationdynamicallychangeimmuneabundancemodifystablemassspectrometryHAPIcysteinesfumarate-derivedincreasedwillmetabolicdiseasesimmunometabolitesresponsetricarboxylicTCAcyclemetaboliteaminoS-2-succinoaccumulateslipopolysaccharidemacrophagesgenerateSinceactivatedcellsreflectmetabolitescapacitythiolsgeneratedethylestershydrolysatesusedisotopedilutiondetermineLPS-stimulatedHighlyAggressivelyProliferatingImmortalizedstoichiometryreducedalkylatediodoaceticS-carboxymethylcysteineCMCesterifiedItaconate-derivedLPS-treatedStoichiometricmeasurementsdemonstrated157%907%totaluponmethodologysimultaneouslydistinguishbroadapplicationsphysiologyadditionfindavailableanti-2SCantibodiesalsodetectstructurallysimilartherefore"succinatemoiety"maybetterdescribeantigenrecognizedrolesmodulatingmacrophageinflammationchemicallymodulatewhereascanquantifiedmethoddistinguishesallowresearcherscontributionsisolatedcelltypestissuesacrossrangeQuantificationS-2-succinocysteineimmunometabolismmodification

Similar Articles

Cited By

No available data.