Two Critical Cysteine Residues Implicated in Disulfide Bond Formation and Proper Folding of Kir2.1

Biochemistry ◽  
2000 ◽  
Vol 39 (16) ◽  
pp. 4649-4657 ◽  
Author(s):  
Hee Cheol Cho ◽  
Robert G. Tsushima ◽  
The-Tin T. Nguyen ◽  
H. Robert Guy ◽  
Peter H. Backx
Keyword(s):  
Disulfide Bond ◽  
Bond Formation ◽  
Cysteine Residues ◽  
Disulfide Bond Formation

scholarly journals Regulation of the Yeast Yap1p Nuclear Export Signal Is Mediated by Redox Signal-Induced Reversible Disulfide Bond Formation

2001 ◽  
Vol 21 (18) ◽  
pp. 6139-6150 ◽  
Author(s):  
Shusuke Kuge ◽  
Minetaro Arita ◽  
Asako Murayama ◽  
Kazuhiro Maeta ◽  
Shingo Izawa ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nuclear Localization ◽  
Disulfide Bond ◽  
Nuclear Export ◽  
Bond Formation ◽  
Nuclear Export Signal ◽  
Cysteine Residues ◽  
Disulfide Bond Formation ◽  
Disulfide Linkage ◽  
Export Signal

ABSTRACT Yap1p, a crucial transcription factor in the oxidative stress response of Saccharomyces cerevisiae, is transported in and out of the nucleus under nonstress conditions. The nuclear export step is specifically inhibited by H2O2 or the thiol oxidant diamide, resulting in Yap1p nuclear accumulation and induction of transcription of its target genes. Here we provide evidence for sensing of H2O2 and diamide mediated by disulfide bond formation in the C-terminal cysteine-rich region (c-CRD), which contains 3 conserved cysteines and the nuclear export signal (NES). The H2O2 or diamide-induced oxidation of the c-CRD in vivo correlates with induced Yap1p nuclear localization. Both were initiated within 1 min of application of oxidative stress, before the intracellular redox status of thioredoxin and glutathione was affected. The cysteine residues in the middle region of Yap1p (n-CRD) are required for prolonged nuclear localization of Yap1p in response to H2O2 and are thus also required for maximum transcriptional activity. Using mass spectrometry analysis, the H2O2-induced oxidation of the c-CRD in vitro was detected as an intramolecular disulfide linkage between the first (Cys598) and second (Cys620) cysteine residues; this linkage could be reduced by thioredoxin. In contrast, diamide induced each pair of disulfide linkage in the c-CRD, but in this case the cysteine residues in the n-CRD appeared to be dispensable for the response. Our data provide evidence for molecular mechanisms of redox signal sensing through the thiol-disulfide redox cycle coupled with the thioredoxin system in the Yap1p NES.

scholarly journals Identification of a Thiol-Disulfide Oxidoreductase (SdbA) catalyzing Disulfide Bond Formation in the Superantigen SpeA in Streptococcus pyogenes

2021 ◽  
Author(s):  
Song F. Lee ◽  
Lydia Li ◽  
Naif Jalal ◽  
Scott A. Halperin
Keyword(s):  
Streptococcus Pyogenes ◽  
Disulfide Bond ◽  
Biological Activities ◽  
Bond Formation ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Initial Reaction ◽  
Disulfide Bond Formation ◽  
Cxxc Motif ◽  
Disulfide Oxidoreductase

Mechanisms of disulfide bond formation in the human pathogen Streptococcus pyogenes is currently unknown. To date, no disulfide bond forming thiol-disulfide oxidoreductase (TDOR) has been described and at least one disulfide bonded protein is known in S. pyogenes . This protein is the superantigen SpeA, which contains 3 cysteine residues (Cys 87, Cys90, and Cys98), and has a disulfide bond is formed between Cys87 and Cys98. In this study, candidate TDORs were identified from the genome seuence of S. pyogenes MGAS8232. Using mutational and biochemical approaches, one of the candidate proteins, SpyM18_2037 (named here SdbA), was shown to be the catalyst that introduces the disulfide bond in SpeA. SpeA in the culture supernatant remained reduced when sdbA was inactivated and restored to the oxidized state when a functional copy of sdbA was returned to the sdbA -knockout mutant. SdbA has a typical C 46 XXC 49 active site motif commonly found in TDORs. Site-directed mutagenesis experiments showed that the cysteines in the CXXC motif were required the disulfide bond in SpeA to form. Interactions between SdbA and SpeA were examined using cysteine variant proteins. The results showed that SdbA C49A formed a mixed disulfide with SpeA C87A , suggesting that the N-terminal Cys46 of SdbA and the C-terminal Cys98 of SpeA participated in the initial reaction. SpeA oxidized by SdbA displayed biological activities suggesting that SpeA was properly folded following oxidation by SdbA. In conclusion, formation of the disulfide bond in SpeA is catalyzed by SdbA and the findings represent the first report of disulfide bond formation in S. pyogenes . IMPORTANCE Here, we reported the first example of disulfide bond formation in Streptococcus pyogenes . The results showed that a thiol-disulfide oxidoreductase, named SdbA, is responsible for introducing the disulfide bond in the superantigen SpeA. The cysteine residues in the CXXC motif of SdbA are needed for catalyzing the disulfide bond in SpeA. The disulfide bond in SpeA and neighboring amino acids form a disulfide loop that is conserved among many superantigens, including those from Staphylococcus aureus . SpeA and staphylococcal enterotoxins lacking the disulfide bond are biologically inactive. Thus, the discovery of the enzyme that catalyzes the disulfide bond in SpeA is important to understanding the biochemistry of SpeA production and presents a target for mitigating the virulence of S. pyogenes .

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