AIMS: 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. INNOVATION AND CONCLUSION: 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.
AIMS: 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 mPTPopening in a cyclophilin D-dependent manner. INNOVATION AND CONCLUSION: 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: Tsu-Kung Lin; Gillian Hughes; Aleksandra Muratovska; Frances H Blaikie; Paul S Brookes; Victor Darley-Usmar; Robin A J Smith; Michael P Murphy Journal: J Biol Chem Date: 2002-02-22 Impact factor: 5.157
Authors: Reiko Matsui; Beatriz Ferran; Albin Oh; Dominique Croteau; Di Shao; Jingyan Han; David Richard Pimentel; Markus Michael Bachschmid Journal: Antioxid Redox Signal Date: 2020-01-23 Impact factor: 8.401