The cysteine proteome

Abstract

The cysteine (Cys) proteome is a major component of the adaptive interface between the genome and the exposome. The thiol moiety of Cys undergoes a range of biologic modifications enabling biological switching of structure and reactivity. These biological modifications include sulfenylation and disulfide formation, formation of higher oxidation states, S-nitrosylation, persulfidation, metalation, and other modifications. Extensive knowledge about these systems and their compartmentalization now provides a foundation to develop advanced integrative models of Cys proteome regulation. In particular, detailed understanding of redox signaling pathways and sensing networks is becoming available to allow the discrimination of network structures. This research focuses attention on the need for atlases of Cys modifications to develop systems biology models. Such atlases will be especially useful for integrative studies linking the Cys proteome to imaging and other omics platforms, providing a basis for improved redox-based therapeutics. Thus, a framework is emerging to place the Cys proteome as a complement to the quantitative proteome in the omics continuum connecting the genome to the exposome.

Keywords: Cysteine proteome; Free radicals; Functional network; Redox proteome; Redox signaling; Thiol.

Fig. 1

The Cys proteome exists as an adaptive interface between the exposome and the functional genome. Research on integrated omics has emphasized the role of the metabolome and the proteome as intermediates between environmental exposures and the genome, epigenome and transcriptome. The Cys proteome has a more direct role to support sensing and signaling of nutritional and environmental conditions and also to provide an array of reactive nucleophiles to neutralize toxic electrophilic chemicals. Thus, the Cys proteome is an important functional component of the translated proteome, directly interacting with the metabolome to utilize environmental resources and defend against environmental threats.

Fig. 2

Multiple lines of evidence support function of redox-sensitive Cys measured by mass spectrometry. A. Targeted searches show that Cys detected in redox proteomic analyses are highly conserved across metazoans compared to other amino acids in the same proteins. B. Examination of x-ray crystal structures shows that redox-sensitive Cys are commonly present in functional motifs of proteins. Examples shown include specific Cys adjacent to GTP-binding site in RhoQ, Cys in RNA binding site of heteronuclear ribonucleoprotein (HNRNP) A/B and DNA interaction site of PCNA. Lower panels show oxidation in response to challenge with cadmium or inhibition of thioredoxin with auranofin (Aur) or GSH depletion with buthionine sulfoximine (BSO).

Fig. 3

Protein Cys map to functional pathways based upon percent oxidation. Ingenuity Pathway Analysis of most reduced protein Cys in colon carcinoma HT29 cells under steady-state non-challenged conditions showed associations with mitochondrial pathways for energy metabolism, cell growth and proliferation; cytoplasmic cytoskeletal pathways and nuclear ran-import mechanisms and RNA processing. Most oxidized protein Cys were associated with mitochondrial intermediary metabolism, cytoplasmic cell survival, Myc-mediated apoptosis and 14-3-3 signaling, and nuclear granzyme B signaling. Data from [6, 8, 24, 49].

Fig. 4

Functional modifications of the Cys proteome. Protein Cys (RSH) undergoes a range of enzymatic and non-enzymatic modifications, often mediated by pH-dependent ionization to Cys thiolate (RS⁻; large yellow sphere). Interaction of RS⁻ with metal ions (blue) is common as a structural element, as a binding motif for nucleic acids and as a component of catalytic sites. RS⁻ is subject to oxidation ([O]) to Cys sulfenate (RSO⁻, yellow) by H₂O₂ and other 2-electron oxidants. Thiol (RSH) also undergoes exchange with disulfide (R₁S-SR₁) to generate different thiol (R₁SH) and disulfide (RS-SR₁) in a process termed thiol-disulfide exchange. Non-equilibrium steady-state oxidation of specific RSH occurs due to the presence of low nanomolar concentrations of H₂O₂ in cells. RSO⁻ reacts with RSH or GSH to produce the respective disulfides (RSSR, RSSG; dark yellow, top). Many RSO⁻ can undergo hyperoxidation to sulfinate (RSO₂⁻; orange, bottom) and sulfonate (RSO₃⁻; red) in the presence of excess oxidant. RS⁻ also reacts with hypothiocyanous acid (HOSCN) to produce sulfenyl thiocyanate (RSSCN; dark yellow, upper left), which rapidly hydrolyzes, and with nitric oxide through multiple means of nitronium ion transfer (NO⁺) to produce a corresponding nitrosothiol (RSNO; orange, bottom left). RSO⁻ can also react with hydrogen sulfide (HS⁻) to form a cysteinyl persulfide (RSS⁻, green), which can be oxidized to a cysteinyl thiosulfate (RSSO₃⁻). Alternatively, RSO⁻ can react with primary amines of neighboring amino acids or separate biomolecules to form sulfenamide (RSNHR) which can be further oxidized to sulfinamide (RS(O)NHR) and sulfonamide (RS(O)₂NHR), frequently resulting in cyclic intramolecular structures.

Fig. 5

Cys proteome networks regulated by Trx and GSH systems. Accumulating evidence shows that redox networks exist in different subcellular compartments supported by NADPH as reductant functioning in opposition to oxidation by H₂O₂ and other oxidants. Trx is the most common intermediary reductant, with a smaller number of proteins supported by GSH. Cysteine and cystine constitute the most abundant extracellular low molecular weight thiol/disulfide couple, apparently functioning in regulation of protein Cys on the extracellular surface. References provide detailed information on interactions of Cys proteins and functional Cys residues illustrated in this figure. TrxR-actin [110]; Trx-dependent regulation of proteins [139]; Ref1-AP1 [258]; TrxR/AP1 [259]; PDGFR [260]; xCT [261]; PTEN-Trx [262, 263]; PTEN-GSH [264]; ASK1-Trx2 [264]; GSTp1-Prx6 [265]; GAPDH-GSH [266, 267]; ND-GSH [268]; p53-Trx [269]; Trx2-Prx3 [270]; Srx-Prx1 and Srx-Prx3 [271, 272]; Grx2-Prx3 [273]; Grx2-Prx3-Trx2 [274]; PDI-UPR sensor [275]; Era-PDI [276]; PDI-TF [277]; Ero-α-PDI and GSH [278]; GPx7-ER [279]; Nrf2 regulatory Cys residues [280]; Srx functional Cys residues [281].