Abstract
Hydrogen sulfide (H2S) is thought to signal through protein S-sulfuration (persulfidation; S-sulfhydration) in both mammalian systems and bacteria. We previously profiled proteome S-sulfuration in Staphylococcus aureus (S. aureus) and identified two thioredoxin-like proteins, designated TrxP and TrxQ, that were capable of reducing protein persulfides as a potential regulatory mechanism. In this study, we further characterize TrxP, TrxQ and the canonical thioredoxin, TrxA, by identifying candidate protein substrates in S. aureus cells using a mechanism-based profiling assay where we trap mixed disulfides that exist between the attacking cysteine of a FLAG-tagged Trx and a persulfidated cysteine on the candidate substrate protein in cells. Largely non-overlapping sets of four, 32 and three candidate cellular substrates were detected for TrxA, TrxP, and TrxQ, respectively, many of which were previously identified as global proteome S-sulfuration targets including for example, pyruvate kinase, PykA. Both TrxA (kcat = 0.13 s-1) and TrxP (kcat = 0.088 s-1) are capable of reducing protein persulfides on PykA, a model substrate detected as a candidate substrate of TrxP; in contrast, TrxQ shows lower activity (kcat = 0.015 s-1). This work reveals that protein S-sulfuration, central to H2S and reactive sulfur species (RSS) signaling, may impact cellular activities and appears to be regulated in S. aureus largely by TrxP under conditions of sulfide stress.
Highlights
Hydrogen sulfide (H2S) has long been studied as an environmental toxin, but is generally recognized as the third “gasotransmitter,” alongside nitric oxide (NO), and carbon monoxide (CO), which signal at physiologically relevant concentrations in the vascular and nervous systems (Wang, 2002)
All S. aureus (Sau) Trx-like proteins are expected to adopt a canonical Trx fold characterized by a core of five β-sheets surrounded by four α-helices, and this is known for both Sau TrxA (Roos et al, 2007) and TrxP (Peng et al, 2017b)
We investigate the functions of three of the four Trx-like proteins in S. aureus strain Newman using an in vivo mechanism-based profiling strategy to trap candidate cellular substrates of TrxA, TrxP, and TrxQ
Summary
Hydrogen sulfide (H2S) has long been studied as an environmental toxin, but is generally recognized as the third “gasotransmitter,” alongside nitric oxide (NO), and carbon monoxide (CO), which signal at physiologically relevant concentrations in the vascular and nervous systems (Wang, 2002). The N-terminal, more solvent exposed, cysteine is referred to as the “attacking” cysteine, which initiates disulfide reduction via disulfide exchange, forming a mixed disulfide intermediate between the target protein and the Trx. Thioredoxins are ≈12 kDa disulfide oxidoreductases that utilize a pair of cysteines in the active site, Cys-X-XCys (CXXC), where X is any amino acid, to reduce disulfide bonds. The N-terminal, more solvent exposed, cysteine is referred to as the “attacking” cysteine, which initiates disulfide reduction via disulfide exchange, forming a mixed disulfide intermediate between the target protein and the Trx This is followed by attack of the C-terminal “resolving” cysteine on the N-terminal Trx Cys, with the release of the dithiol reduced target protein, and oxidized Trx. The oxidized Trx is turned over (re-reduced) by thioredoxin reductase [TrxB in Staphylococcus aureus (S. aureus)] using a similar disulfide exchange mechanism with the reduced flavin adenine dinucleotide (FADH2) used to re-reduce TrxB disulfide and reduced nicotinamide adenine dinucleotide phosphate (NADPH) used as the final electron donor to reduce oxidized FAD (Figure 1A)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
