Thiol/disulfide redox states in signaling and sensing

Abstract

Rapid advances in redox systems biology are creating new opportunities to understand complexities of human disease and contributions of environmental exposures. New understanding of thiol-disulfide systems have occurred during the past decade as a consequence of the discoveries that thiol and disulfide systems are maintained in kinetically controlled steady states displaced from thermodynamic equilibrium, that a widely distributed family of NADPH oxidases produces oxidants that function in cell signaling and that a family of peroxiredoxins utilize thioredoxin as a reductant to complement the well-studied glutathione antioxidant system for peroxide elimination and redox regulation. This review focuses on thiol/disulfide redox state in biologic systems and the knowledge base available to support development of integrated redox systems biology models to better understand the function and dysfunction of thiol-disulfide redox systems. In particular, central principles have emerged concerning redox compartmentalization and utility of thiol/disulfide redox measures as indicators of physiologic function. Advances in redox proteomics show that, in addition to functioning in protein active sites and cell signaling, cysteine residues also serve as redox sensors to integrate biologic functions. These advances provide a framework for translation of redox systems biology concepts to practical use in understanding and treating human disease. Biological responses to cadmium, a widespread environmental agent, are used to illustrate the utility of these advances to the understanding of complex pleiotropic toxicities.

Figure 1

Genealogy of thiol redox systems biology. Thousands of scientific studies have examined thiol systems in detail, yet integrated knowledge of the complex thiol interactions is only beginning to emerge. Systems biology provides approaches and tools to address this challenging problem. Mass spectrometry-based redox proteomics methods, in particular, provide the ability to simultaneously study redox effects on specific cysteine residues in hundreds of proteins. A search of “redox proteomics” resulted in 995 papers, suggesting a growing influence of this approach. Improved analytic throughput with such methods is expected to support rapid progress in thiol redox systems biology development.

Figure 2

The cysteine proteome co-evolved with structural and functional complexity of higher organisms. A, Modified figure from Miseta and Csutora’s study (Fig1 of (Miseta and Csutora, 2000)) The occurrences of the 20 coded amino acids were counted in representative samples of humans, bovines, mice, fruit flies, C. elegans, maize, rice, tomatoes, yeast, Cyanobacteria, E. coli, R. sphaeroides, P. aeruginosa,H. marismortui, T. aquaticus, and Sulpho-archea. The total of all 20 coded amino acids was considered 100%. B, The percentage of conserved/evolved Cys content in cytoplasmic and mitochondrial ribosomal proteins for small subunit (top) and large subunit (bottom) are increased with complexity of the organism, supporting Miseta and Csutora’s finding. Abbreviations for species’ names; J, Methanococcus jannaschii (cytoplasmic); E, Escherichia coli (mitochondrial); S, Saccharomyces cerevisiae; C, Caenorhabditis elegans; A, Anopheles gambiae; D, Drosophila melanogaster; Z, Danio rerio; X, Xenopus laevis; M, Mus musculus; R, Rattus norvegicus; B, Bos taurus; H, Homo sapiens. C, Modified figure from Go et al. (Fig 8 of (Go et al., 2011)). Evolutionary conservation among vertebrates of mitochondrial peptidyl Cys are associated with functional networks. The UniProt database was searched for protein amino acid sequences of 10 vertebrate species for the peptidyl Cys associated with functional pathways in Fig. 7 of the study by Go et al. (Go et al., 2011). The species included human (Homo sapiens), Sumatran orangutan (Pongo abelii), bovine (Bos taurus), mouse (Mus musculus), rat (Rattus norvegicus), chicken (Gallus gallus), African clawed frog (Xenopus laevis), Western clawed frog (Xenopus tropicalis), zebrafsh (Danio rerio) and Atlantic salmon (Salmo salar). The percentage of identity relative to humans was calculated for each of the Cys (filled circles), and the percentage of amino acid identity relative to humans was obtained from a BLAST search in Uniprot (filled triangles), showing that Cys are much more highly conserved.

Figure 3

The Cys proteome exists in a multidimensional functional space. While many Cys are contained within internal protein structures and not accessible to reversible modifications or interactions, Cys is a polar amino acid commonly found on the surface of proteins. In the vicinity of cationic amino acids, Cys can undergo ionization to a thiolate, which readily binds transition metals and is reactive with oxidants and electrophiles. These characteristics are associated with a large number of covalent modifications and ionic interactions, which are used in control of protein activity, structure, subcellular distribution, cell signaling, macromolecular interactions and trafficking. As shown on the top left, thiols are present in proteins as monothiols and vicinal dithiols; in some proteins larger clusters are also present. Modifications of monothiols are presented on the right, and functional responses to modification of monothiols and vicinal dithiols are shown on the lower left.

Figure 4

Hierarchical redox structure for control of the redox proteome. Protein Cys residues are maintained in kinetically controlled redox states dependent upon opposing reductive and oxidative systems. An overriding level of control occurs at the physical level of subcellular compartmentation because of restricted redox communication between compartments. Within compartments, reductive systems are conceptualized as occurring in redox regulons, in which primary reductant systems such as NADPH/thioredoxin reductase or thioredoxin function as hubs controlling multiple redox nodes. Oxidative processes are depicted as occurring through opposing redox regulons, such as a H₂O₂ redox regulon. Many Cys are directly controlled by secondary reductant or oxidant systems, and these are presented as additional levels of organization within the hierarchical structure. Individual Cys residues are conceptualized as occurring in redox modules in which Cys in different proteins have evolved to share common redox properties, providing coordination of functional systems. Based upon known reaction types and specific examples, one can extrapolate that such a structure is sufficient to provide specific control of all 214,000 Cys encoded in mammalian genomes.

Figure 5

Functional systems and representative proteins with redox-sensitive thiols in cell nuclei. Multiple Cys residues are redox-sensitive in some proteins. Mass spectrometry-based redox proteomic analysis enables the measurement of redox state of multiple proteins in different subcellular compartments in response to diverse extracellular signals. This figure is derived from data reported in (Go et al., 2011) and illustrates redox-sensitive nuclear proteins that appear to be organized into redox modules. In such modules, multiple protein Cys have similar redox dependences with a character suggesting that redox mechanisms coordinate function. Redox-sensitive nuclear proteins are; DNMT1, DNA methyltransferase; HNRNP, heterogeneous nuclear ribonucleoproteins; NCBP, Nuclear cap-binding protein; NOP56, Nucleolar protein 56; PCNA, Proliferating cell nuclear antigen; PRKDC, DNA-dependent protein kinase catalytic subunit; RNU2, U2 small nuclear ribonucleoprotein.