title: Proteome-wide light/dark modulation of thiol oxidation in cyanobacteria revealed by quantitative site-specific redox proteomics.
abstract: Reversible protein thiol oxidation is an essential regulatory mechanism of photosynthesis, metabolism, and gene expression in photosynthetic organisms. Herein, we present proteome-wide quantitative and site-specific profiling of in vivo thiol oxidation modulated by light/dark in the cyanobacterium Synechocystis sp. PCC 6803, an oxygenic photosynthetic prokaryote, using a resin-assisted thiol enrichment approach. Our proteomic approach integrates resin-assisted enrichment with isobaric tandem mass tag labeling to enable site-specific and quantitative measurements of reversibly oxidized thiols. The redox dynamics of ∼2,100 Cys-sites from 1,060 proteins under light, dark, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (a photosystem II inhibitor) conditions were quantified. In addition to relative quantification, the stoichiometry or percentage of oxidation (reversibly oxidized/total thiols) for ∼1,350 Cys-sites was also quantified. The overall results revealed broad changes in thiol oxidation in many key biological processes, including photosynthetic electron transport, carbon fixation, and glycolysis. Moreover, the redox sensitivity along with the stoichiometric data enabled prediction of potential functional Cys-sites for proteins of interest. The functional significance of redox-sensitive Cys-sites in NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxin (AhpC/TSA family protein Sll1621), and glucose 6-phosphate dehydrogenase was further confirmed with site-specific mutagenesis and biochemical studies. Together, our findings provide significant insights into the broad redox regulation of photosynthetic organisms.
authors.affiliation: From the ‡Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, 99352;
authors.forename: Jia
authors.initials: J
authors.lastname: Guo
authors.affiliation: ¶Department of Biology, Washington University, St. Louis, Missouri, 63130;
authors.forename: Amelia Y
authors.initials: AY
authors.lastname: Nguyen
authors.affiliation: ‖Energy and Efficiency Division, Pacific Northwest National Laboratory, Richland, Washington, 99352;
authors.forename: Ziyu
authors.initials: Z
authors.lastname: Dai
authors.affiliation: From the ‡Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, 99352;
authors.forename: Dian
authors.initials: D
authors.lastname: Su
authors.affiliation: From the ‡Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, 99352;
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Gaffrey
authors.affiliation: From the ‡Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, 99352;
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Moore
authors.affiliation: From the ‡Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, 99352;
authors.forename: Jon M
authors.initials: JM
authors.lastname: Jacobs
authors.affiliation: From the ‡Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, 99352;
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Monroe
authors.affiliation: From the ‡Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, 99352; ‡‡Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington, 99352.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.affiliation: ‡‡Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington, 99352.
authors.forename: David W
authors.initials: DW
authors.lastname: Koppenaal
authors.affiliation: ¶Department of Biology, Washington University, St. Louis, Missouri, 63130;
authors.forename: Himadri B
authors.initials: HB
authors.lastname: Pakrasi
authors.affiliation: From the ‡Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, 99352; weijun.qian@pnnl.gov.
authors.forename: Wei-Jun
authors.initials: WJ
authors.lastname: Qian
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D057486:Carbon Cycle / Q000187:drug effects / Q000235:genetics; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004237:Diuron / Q000633:toxicity; D004579:Electron Transport / Q000187:drug effects / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006019:Glycolysis / Q000187:drug effects / Q000235:genetics; D006540:Herbicides / Q000633:toxicity; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D054464:Peroxiredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017440:Photoperiod; D010788:Photosynthesis / Q000187:drug effects / Q000235:genetics; D020543:Proteome / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013237:Stereoisomerism; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D046939:Synechocystis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006540:Herbicides; D020543:Proteome; D013438:Sulfhydryl Compounds; D004237:Diuron; D005954:Glucosephosphate Dehydrogenase; D054464:Peroxiredoxins; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+); D003545:Cysteine