Characterization of surface-exposed reactive cysteine residues in Saccharomyces cerevisiae.
Numerous cellular processes are subject to redox regulation, and thiol-dependent redox control, acting through reactive cysteine (Cys) residues, is among the major mechanisms of redox regulation. However, information on the sets of proteins that provide thiol-based redox regulation or are affected by it is limited. Here, we describe proteomic approaches to characterize proteins that contain reactive thiols and methods to identify redox Cys in these proteins. Using Saccharomyces cerevisiae as a eukaryotic model organism, we identified 284 proteins with exposed reactive Cys and determined the identities of 185 of these residues. We then characterized subsets of these proteins as in vitro targets of major cellular thiol oxidoreductases, thioredoxin and glutaredoxin, and found that these enzymes can control the redox state of a significant number of thiols in target proteins. We further examined common features of exposed reactive Cys and compared them with an unbiased control set of Cys using computational approaches. This analysis (i) validated the efficacy of targeting exposed Cys in proteins in their native, folded state, (ii) quantified the proportion of targets that can be redox regulated via thiol oxidoreductase systems, and (iii) revealed the theoretical range of the experimental approach with regard to protein abundance and physicochemical properties of reactive Cys. From these analyses, we estimate that approximately one-fourth of exposed Cys in the yeast proteome can be regarded as functional sites, either subject to regulation by thiol oxidoreductases or involved in structural disulfides and metal binding.
Marino
Stefano M
SM
Department of Biochemistry and Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588, USA.
Li
Yehua
Y
Fomenko
Dmitri E
DE
Agisheva
Natalia
N
Cerny
Ronald L
RL
Gladyshev
Vadim N
VN
D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D054477:Glutaredoxins / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
D054477:Glutaredoxins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D013438:Sulfhydryl Compounds; D013879:Thioredoxins; D010088:Oxidoreductases; D003545:Cysteine