Cysteine (Cys) exhibits unique character. It contains sulfur in the form of sulfhydryl thiol form which is ionizable, forming a negatively charged thiolate group after deprotonation that further increases its reactivity. This thiol/thiolate group is subject to alkylation by electrophiles and oxidation by reactive oxygen, nitrogen, and sulfur species, resulting in posttranslationally modified forms that may exhibit significantly altered functions (1).

Cys is considered one of the most conserved amino acids in proteins across all species. Strong selection pressure retained Cys residues at functionally important sites and removed them from others (2). The physical and chemical properties of Cys predestine it to be a polar residue (like serine, where the sulfur atom is replaced by oxygen), however, it is considered hydrophobic because it is buried to an increased extent (protection from the solvent), and the number of unpaired Cys residues on the protein surface is minimal (2). Another unique property is its tendency to form clusters with other Cys, as in metal-binding or redox-sensitive proteins, leading to the formation of disulfide bonds. Exposed and isolated Cys are less conserved (1).

The chemical properties of Cys allow it to be both redox active and strongly nucleophilic due to the large atomic radius of the sulfur atom, the presence of lone pairs of electrons, and the low dissociation energy of the thiol S-H bond.

The thiol groups (R- SH) undergo deprotonation (loss of H⁺), giving the thiolate form R-S^-. The readiness to provide the proton is given by the pKa, the local pH and electrostatic environment. In proteins, the specific hydrogen bond donors and an electropositive local environment cause a decrease in the pKa value due to the stabilization of the negative thiolate anion, while a hydrophobic environment or an electronegative local environment has the opposite effect (1).

Another important feature is the reductive potential of the thiol group, i.e., the ability to accept or donate electrons. This ability allows the formation of so-called redox pairs, and their interconversion is defined as a reductive or oxidative half-reaction. In biological systems, electron transfer is very dynamic and involves many players. Therefore, many reactions occur even under thermodynamically unfavorable conditions and would never occur in isolation, favoring the kinetic pathways and rates associated with certain reactions (3, 4).

Thiols can produce disulfides in one or more thiol-disulfide exchange processes in intricate biological systems (1). Long assumed to merely serve to stabilize proteins structurally, it is now known that these processes also give rise to many enzymes’ diverse and dynamic functional characteristics (5). The rate-determining stage in the folding process of proteins creating structural disulfide linkages is direct thiol-disulfide exchange. Although in vivo enzyme catalysis speeds up the events, spontaneous thiol-disulfide exchange is slow (kinetically inadequate on the folding timescale). Through a sequence of intra- and intermolecular thiol-disulfide exchange processes carried out by oxidoreductases, the folding polypeptide gains a new disulfide bond. Molecular oxygen serves as the oxidizing equivalent in these reactions. A different mechanism for thiol-disulfide exchange involves the oxidative conversion of protein thiols to disulfides and their subsequent reduction. Protein thiols are also significant targets of reactive oxygen species in vivo. These processes are crucial for both antioxidant defense and redox regulation of cell signaling, and it is believed that the thiol-disulfide pool is principally responsible for maintaining intracellular redox equilibrium (6). It is now widely acknowledged that thiol-disulfide exchange and thiol oxidation/reduction reactions are dynamic, non-equilibrium processes that are kinetically rather than thermodynamically controlled in cellular systems (7–9). In other words, the partitioning of particular routes depends on relative rates, whereas redox potentials and equilibrium constants just tell whether a reaction is favorable. By fine-tuning the activation energies of reactions that control the outcome of oxidative stimuli or the location of structural disulfides in native proteins, enzymes play a crucial role in these processes.

The availability of different oxidation states of thiol-sulfur allows the formation of a variety of oxidative posttranslational modifications (PTMs) on cysteines, including S-nitrosylation (or S-nitrosation, SNO), sulfhydration (SSH), disulfide bond formation (RS-SR), sulfenylation (SOH), sulfinic acid (SO₂H), and sulfonic acid (SO₃H) (10) ( Figure 1A ). Most Cys oxidative PTMs are initiated by reactive oxygen or nitrogen species (ROS/RNS) or sulfane (H₂S) reacting with the free thiol on a Cys side chain ( Figure 1A ). Two-electron oxidation of thiol(ate) groups, e.g., by H₂O₂, peroxynitrite, and other hydroperoxides, produces the simplest oxyacid of sulfur, sulfenic acid. The rate of this reaction can vary from negligibly slow to 10⁸ M^-1 s^-1 at active sites of peroxidase (4). Sulfenic acid readily reacts with proximal thiol groups to form disulfides or is further oxidized to irreversible sulfinic or sulfonic acids in the absence of such groups (11). Sulfonic acids can also react with each other to form thiosulfinates or with amine or amide groups to form sulfenylamides (12) ( Figure 1A ). S-nitrosothiols and persulfides are additional oxidative reaction byproducts that are unquestionably significant as signaling intermediates (13, 14). The thiyl radical, another reactive species that can produce a variety of products once generated, can be produced by the one-electron oxidation of thiol groups in the presence of radicals (15).

The reactivity of cysteine to oxidative changes is determined by the proximity of oxidants (in addition to its chemical properties). In β-cells, these are mainly superoxide (O₂ ^·−) and other oxidizing byproducts generated in mitochondria, reduced flavoprotein oxidases or monooxygenases and NADPH oxidase (NOX) family; and NO produced by NOS and combining with O₂ ^·− also peroxinitrite (16). Endogenous antioxidant system such as superoxide dismutase (SOD), peroxiredoxins (Prdx), glutathione peroxidases (GPX) and compounds such as, glutathione (GSH) and thioredoxin (Trx) are well compartmentalized (17). Together they play a critical role in maintaining redox equilibrium.

For many years, β-cells were attributed as having weak antioxidant defense. However, this was based on comparison with liver or kidney, which are highly specialized detoxifying organs (36). Recently, it was shown that β-cells express Prdxs, Trxs, and SOD1/2. Thus, superoxide formed can be rapidly dismutated to H₂O₂, which can then serve as a signaling molecule due to its stability, thiol selectivity, and ability to diffuse through membranes. As mentioned earlier, thiol-containing molecules, Prxs/Trxs exhibit a high affinity for oxidants and are therefore efficient sensors for a prooxidant redox environment. Pancreatic β-cells are glucose sensors responding to high glucose by insulin secretion. Glucose stimulation induces oxidative metabolism, which increases the prooxidant redox status. We and others have shown that short-term glucose stimulation, which increases prooxidant status via cytoplasmic NOX4, is necessary for efficient insulin secretion, whereas long-term stimulation, which tends to induce oxidative stress, can lead to the development of T2D (37, 38). A possible mechanism for the role of prooxidant metabolism in pancreatic β-cells has been proposed (39–46).

The most important, but still technically elusive problem in redox signaling is the compartmentalization of redox status. Cellular localization then determines the source of ROS/RNS/RSS and their proximity to potential cysteine modifications, type of antioxidant defense, and the redox potential of cysteine residues present. Subcellular organelles maintain different pH values and redox potentials as well as concentrations of reactive metabolites that trigger specific redox signals (47, 48). The most important compartments for redox signaling in pancreatic β-cells are the mitochondria, the cytoplasm with the plasma membrane rafts, while oxidation in ER with the Golgi apparatus has more structural functions. Whereas the cytoplasm and mitochondria of quiescent cells have a more reduced environment (-200 and -300mV for the GSH/GSSG redox pair), which gives them a broad probability for redox signaling, e.g. upon induction of proliferation or some other stimulus, ER and Golgi secretion machinery exhibit a more oxidized potential (-150 and -140mV for the GSH/GSSG redox pair) (9, 49). This oxidized status is rather essential for the formation and maintenance of the structural disulfide bonds of the secretory proteins and counteracts redox signaling. It is difficult to determine the reduction potential of individual cellular compartments because different redox pairs are present (GSH/GSSG, Trx, Cys/Cys, etc.). They have individually different midpoint potentials that vary in different organelles. For example, Trx redox pairs are generally more reducing than GSH/GSSG, and while Trx1 shows a redox potential of -280 and -300 mV in the cytosol and nucleus, Trx2 shows an, even more, reducing redox pair of -340 and -360 mV in mitochondria (9, 48). The ability of cysteine to be modified by redox depends on many factors as stated above, but also depends on the presence of molecules with greater reactivity toward thiols such as peroxymonocarbonate. CO₂ is in equilibrium with bicarbonate, which forms the biological buffer within cells. Bicarbonate can react with peroxide to form peroxymonocarbonate. The rate of its formation increases with decreasing pH (50). Thus, cellular metabolism and its compartmentalization are the main trigger for redox signaling.

It is obvious that not all Cys residues are the same and that the Cys residues that confer signaling properties to proteins are unique because of their position in the protein, the surrounding amino acids, protein cellular localization, and its redox environment. Although we have extensive knowledge of the location of cysteine modifications in individual proteins, we lack a functional understanding and interplay of the various cysteine modifications in a single protein and their consequences on the global scale. It is well known that β-cells require redox signaling through cysteine modifications for efficient glucose-stimulated insulin secretion and insulin signaling because of their sensitive redox homeostasis. However, metabolic overload and imbalanced redox homeostasis may lead to the development of T2D. This increases the need for research in this area as a potential intervention in the treatment of diabetes development.

Conceptualization: BH and LP-H; Writing the original manuscript: BH and LP-H; Manuscript review and editing: BH and LP-H; Funding acquisition: LP-H. LP-H is the guarantor of this work. All authors contributed to the article and approved the submitted version.