title: S-guanylation proteomics for redox-based mitochondrial signaling.
abstract: OBJECTIVE
  8-nitroguanosine 3',5'-cyclic monophosphate (8-Nitro-cGMP) is a nitrated derivative of cGMP that is formed via cross-talk of reactive oxygen species formed by NADPH oxidase 2 and mitochondria. This nitrated nucleotide can function as a unique electrophilic second messenger in regulation of redox signaling by inducing a post-translational modification of protein thiols via cGMP adduction (protein S-guanylation). With S-guanylation proteomics, we investigated endogenous mitochondrial protein S-guanylation.
  
  
  RESULTS
  We developed a new mass spectrometry (MS)-based proteomic method-S-guanylation proteomics-which comprised two approaches: (i) direct protein digestion followed by immunoaffinity capture of S-guanylated peptides that were subjected to liquid chromatography-tandem MS (LC-MS/MS); and (ii) two-dimensional (2D)-gel electrophoretic separation of S-guanylated proteins that were subjected to in-gel digestion, followed by LC-MS/MS. We thereby identified certain mitochondrial proteins that are S-guanylated endogenously during immunological stimulation, including mortalin and 60-kDa heat-shock protein (HSP60). Mortalin and HSP60 were recently reported to regulate mitochondrial permeability-transition pore (mPTP) opening, at least partly, by interacting with cyclophilin D, an mPTP component. Our data revealed that immunological stimulation and 8-nitro-cGMP treatment induced mPTP opening in a cyclophilin D-dependent manner.
  
  
  CONCLUSIONS
  Our S-guanylation proteomic method determined that mitochondrial HSPs may be novel targets for redox modification via protein S-guanylation that participates in mPTP regulation and mitochondrial redox signaling.
authors.affiliation: 1 Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University , Kumamoto, Japan .
authors.forename: Md Mizanur
authors.initials: MM
authors.lastname: Rahaman
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Sawa
authors.forename: Ahmed Khandaker
authors.initials: AK
authors.lastname: Ahtesham
authors.forename: Shahzada
authors.initials: S
authors.lastname: Khan
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Inoue
authors.forename: Atsuhi
authors.initials: A
authors.lastname: Irie
authors.forename: Shigemoto
authors.initials: S
authors.lastname: Fujii
authors.forename: Takaaki
authors.initials: T
authors.lastname: Akaike
mesh_terms: D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods; D051381:Rats; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry
chemical_list: C525106:8-nitroguanosine 3',5'-cyclic monophosphate; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D024101:Mitochondrial Proteins; D006152:Cyclic GMP