A multi-iron enzyme installs copper-binding oxazolone/thioamide pairs on a nontypeable Haemophilus influenzae virulence factor.

The multinuclear nonheme iron-dependent oxidases (MNIOs) are a rapidly growing family of enzymes involved in the biosynthesis of ribosomally synthesized, posttranslationally modified peptide natural products (RiPPs). Recently, a secreted virulence factor from nontypeable Haemophilus influenzae (NTHi) was found to be expressed from an operon, which we designate the hvf operon, that also encodes an MNIO. Here, we show by Mössbauer spectroscopy that the MNIO HvfB contains a triiron cofactor. We demonstrate that HvfB works together with HvfC [a RiPP recognition element (RRE)-containing partner protein] to perform six posttranslational modifications of cysteine residues on the virulence factor precursor peptide HvfA. Structural characterization by tandem mass spectrometry and NMR shows that these six cysteine residues are converted to oxazolone and thioamide pairs, similar to those found in the RiPP methanobactin. Like methanobactin, the mature virulence factor, which we name oxazolin, uses these modified residues to coordinate Cu(I) ions. Considering the necessity of oxazolin for host cell invasion by NTHi, these findings point to a key role for copper during NTHi infection. Furthermore, oxazolin and its biosynthetic pathway represent a potential therapeutic target for NTHi.

Cell-Free Translation Quantification via a Fluorescent Minihelix.

Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems.

Engineered reversible inhibition of SpyCatcher reactivity enables rapid generation of bispecific antibodies.

The precise regulation of protein function is essential in biological systems and a key goal in chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we perform a cysteine scan of the structured region of SpyCatcher. Except for two known reactive and catalytic residues, none of these mutations abolish reactivity. In a second screening step, we modify the cysteines with disulfide bond-forming small molecules. Here we identify 8 positions at which modifications strongly inhibit reactivity. This inhibition can be reversed by reducing agents. We call such a reversibly inhibitable SpyCatcher "SpyLock". Using "BiLockCatcher", a genetic fusion of wild-type SpyCatcher and SpyLock, and SpyTagged antibody fragments, we generate bispecific antibodies in a single, scalable format, facilitating the screening of a large number of antibody combinations. We demonstrate this approach by screening anti-PD-1/anti-PD-L1 bispecific antibodies using a cellular reporter assay.

Sulfur dioxide inhibits mast cell degranulation by sulphenylation of galectin-9 at cysteine 74.

Objectives: Mast cell (MC) degranulation is a key process in allergic reactions and inflammatory responses. Aspartate aminotransferase 1 (AAT1)-derived endogenous sulfur dioxide (SO2) is an important regulator of MC function. However, the mechanism underlying its role in MC degranulation remains unclear. This study aimed to investigate the mechanism by which endogenous SO2 controlled MC degranulation. Methods: HMC-1 and Rat basophilic leukemia cell MC line (RBL-2H3) were used in the cell experiments. SO2 content was detected by in situ fluorescent probe. MC degranulation represented by the release rate of MC ß-hexosaminidase was determined using a colorimetric assay. Sulfenylation of galectin-9 (Gal-9) in MCs and purified protein was detected using a biotin switch assay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the exact sulfenylation sites of Gal-9 by SO2. Animal models of passive cutaneous anaphylaxis (PCA) and hypoxia-driven pulmonary vascular remodeling were used to investigate the effect of SO2 on mast cell activation in vivo. Site-directed mutation of Gal-9 was conducted to confirm the exact site of SO2 and support the significance of SO2/Gal-9 signal axis in the regulation of MC degranulation. Results: Degranulation was increased in AAT1-knockdowned MCs, and SO2 supplementation reversed the increase in MC degranulation. Furthermore, deficiency of endogenous SO2 contributed to IgE-mediated degranulation in vitro. Besides, SO2 inhibited IgE-mediated and hypoxia-driven MC degranulation in vivo. Mechanistically, LC-MS/MS analysis and site-directed mutation results showed that SO2 sulfenylated Gal-9 at cysteine 74. Sulfenylation of the 74th cysteine of Gal-9 protein was required in the SO2-inhibited MC degranulation under both physiological and pathophysiological conditions. Conclusion: These findings elucidated that SO2 inhibited MC degranulation via sulfenylating Gal-9 under both physiological and pathophysiological conditions, which might provide a novel treatment approach for MC activation-related diseases.

Amyloid formation and depolymerization of tumor suppressor p16INK4a are regulated by a thiol-dependent redox mechanism.

The conversion of a soluble protein into polymeric amyloid structures is a process that is poorly understood. Here, we describe a fully redox-regulated amyloid system in which cysteine oxidation of the tumor suppressor protein p16INK4a leads to rapid amyloid formation. We identify a partially-structured disulfide-bonded dimeric intermediate species that subsequently assembles into fibrils. The stable amyloid structures disassemble when the disulfide bond is reduced. p16INK4a is frequently mutated in cancers and is considered highly vulnerable to single-point mutations. We find that multiple cancer-related mutations show increased amyloid formation propensity whereas mutations stabilizing the fold prevent transition into amyloid. The complex transition into amyloids and their structural stability is therefore strictly governed by redox reactions and a single regulatory disulfide bond.

The cysteine-rich virulence factor NipA of Arthrobotrys flagrans interferes with cuticle integrity of Caenorhabditis elegans.

Animals protect themself from microbial attacks by robust skins or a cuticle as in Caenorhabditis elegans. Nematode-trapping fungi, like Arthrobotrys flagrans, overcome the cuticle barrier and colonize the nematode body. While lytic enzymes are important for infection, small-secreted proteins (SSPs) without enzymatic activity, emerge as crucial virulence factors. Here, we characterized NipA (nematode induced protein) which A. flagrans secretes at the penetration site. In the absence of NipA, A. flagrans required more time to penetrate C. elegans. Heterologous expression of the fungal protein in the epidermis of C. elegans led to blister formation. NipA contains 13 cysteines, 12 of which are likely to form disulfide bridges, and the remaining cysteine was crucial for blister formation. We hypothesize that NipA interferes with cuticle integrity to facilitate fungal entry. Genome-wide expression analyses of C. elegans expressing NipA revealed mis-regulation of genes associated with extracellular matrix (ECM) maintenance and innate immunity.

Evidence of Gas Phase Glucosyl Transfer and Glycation in the CID/HCD-Spectra of S-Glucosylated Peptides.

Protein cysteine S-glycosylation is a relatively rare and less well characterized post-translational modification (PTM). Creating reliable model proteins that carry this modification is challenging. The lack of available models or natural S-glycosylated proteins significantly hampers the development of mass-spectrometry-based (MS-based) methodologies for detecting protein cysteine S-glycosylation in real-world proteomic studies. There is also limited MS-sequencing data describing it as easier to create synthetic S-glycopeptides. Here, we present the results of an in-depth manual analysis of automatically annotated CID/HCD spectra for model S-glucopeptides. The CID spectra show a long series of y/b-fragment ions with retained S-glucosylation, regardless of the dominant m/z signals corresponding to neutral loss of 1,2-anhydroglucose from the precursor ions. In addition, the spectra show signals manifesting glucosyl transfer from the cysteine position onto lysine, arginine (Lys, Arg) side chains, and a peptide N-terminus. Other spectral evidence indicates that the N-glucosylated initial products of transfer are converted into N-fructosylated (i.e., glycated) structures due to Amadori rearrangement. We discuss the peculiar transfer of the glucose oxocarbenium ion (Glc+) to positively charged guanidinium residue (ArgH+) and propose a mechanism for the gas-phase Amadori rearrangement involving a 1,2-hydride ion shift.

Cysteine Oxidation in Human Galectin-1 Occurs Sequentially via a Folded Intermediate to a Fully Oxidized Unfolded Form.

Galectins are multifunctional effectors in cellular homeostasis and dysregulation. Oxidation of human galectin-1 (Gal-1) with its six sulfhydryls produces a disulfide-bridged oxidized form that lacks normal lectin activity yet gains new glycan-independent functionality. Nevertheless, the mechanistic details as to how Gal-1 oxidation occurs remain unclear. Here, we used 15N and 13C HSQC NMR spectroscopy to gain structural insight into the CuSO4-mediated path of Gal-1 oxidation and identified a minimum two-stage conversion process. During the first phase, disulfide bridges form slowly between C16-C88 and/or C42-C66 to produce a partially oxidized, conformationally flexible intermediate that retains the ability to bind lactose. Site-directed mutagenesis of C16 to S16 impedes the onset of this overall slow process. During the second phase, increased motional dynamics of the intermediate enable the relatively distant C2 and C130 residues to form the third and final disulfide bond, leading to an unfolded state and consequent dimer dissociation. This fully oxidized end state loses the ability to bind lactose, as shown by the hemagglutination assay. Consistent with this model, we observed that the Gal-1 C2S mutant maintains intermediate-state structural features with a free sulfhydryl group at C130. Incubation with dithiothreitol reduces all disulfide bonds and allows the lectin to revert to its native state. Thus, the sequential, non-random formation of three disulfide bridges in Gal-1 in an oxidative environment acts as a molecular switch for fundamental changes to its functionality. These data inspire detailed bioactivity analysis of the structurally defined oxidized intermediate in, e.g., acute and chronic inflammation.

Reverse Thiol Trapping Approach to Assess the Thiol Status of Metal-Binding Mitochondrial Proteins.

Thiol-disulfide interconversions are pivotal in the intricate chemistry of biological systems. They play a vital role in governing cellular redox potential and shielding against oxidative harm. These interconversions can also act as molecular switches within an expanding array of redox-regulated proteins, facilitating dynamic and responsive processes. Furthermore, metal-binding proteins often use thiols for coordination. Reverse thiol trapping is a valuable analytical tool to study the redox state of cysteines in biological systems. By selectively capturing and stabilizing free thiol species with an alkylating agent, reverse thiol trapping allows for their subsequent identification and quantification. Various methods can be employed to analyze the trapped thiol adducts, including electrophoresis-based methods, mass spectrometry, nuclear magnetic resonance spectroscopy, and chromatographic techniques. In this chapter, we will focus on describing a simple and sensitive method to sequentially block thiols in their cellular state with a cell-permeant agent (iodoacetamide), and following reduction and denaturation of the samples, trap the native disulfides with a second blocker that shifts the apparent molecular weight of the protein. The oxidation status of proteins for which suitable antibodies are available can then be analyzed by immunoblotting. We present examples of mitochondrial proteins that use cysteine thiols to coordinate metal factors such as iron-sulfur clusters, zinc, and copper.

Advancements in inhibitors of crucial enzymes in the cysteine biosynthetic pathway: Serine acetyltransferase and O-acetylserine sulfhydrylase.

Infectious diseases have been jeopardized problem that threaten public health over a long period of time. The growing prevalence of drug-resistant pathogens and infectious cases have led to a decrease in the number of effective antibiotics, which highlights the urgent need for the development of new antibacterial agents. Serine acetyltransferase (SAT), also known as CysE in certain bacterial species, and O-acetylserine sulfhydrylase (OASS), also known as CysK in select bacteria, are indispensable enzymes within the cysteine biosynthesis pathway of various pathogenic microorganisms. These enzymes play a crucial role in the survival of these pathogens, making SAT and OASS promising targets for the development of novel anti-infective agents. In this comprehensive review, we present an introduction to the structure and function of SAT and OASS, along with an overview of existing inhibitors for SAT and OASS as potential antibacterial agents. Our primary focus is on elucidating the inhibitory activities, structure-activity relationships, and mechanisms of action of these inhibitors. Through this exploration, we aim to provide insights into promising strategies and prospects in the development of antibacterial agents that target these essential enzymes.

Palmitoylation at a conserved cysteine residue facilitates gasdermin D-mediated pyroptosis and cytokine release.

Gasdermin D (GSDMD)-mediated pyroptotic cell death drives inflammatory cytokine release and downstream immune responses upon inflammasome activation, which play important roles in host defense and inflammatory disorders. Upon activation by proteases, the GSDMD N-terminal domain (NTD) undergoes oligomerization and membrane translocation in the presence of lipids to assemble pores. Despite intensive studies, the molecular events underlying the transition of GSDMD from an autoinhibited soluble form to an oligomeric pore form inserted into the membrane remain incompletely understood. Previous work characterized S-palmitoylation for gasdermins from bacteria, fungi, invertebrates, as well as mammalian gasdermin E (GSDME). Here, we report that a conserved residue Cys191 in human GSDMD was S-palmitoylated, which promoted GSDMD-mediated pyroptosis and cytokine release. Mutation of Cys191 or treatment with palmitoyltransferase inhibitors cyano-myracrylamide (CMA) or 2-bromopalmitate (2BP) suppressed GSDMD palmitoylation, its localization to the membrane and dampened pyroptosis or IL-1ß secretion. Furthermore, Gsdmd-dependent inflammatory responses were alleviated by inhibition of palmitoylation in vivo. By contrast, coexpression of GSDMD with palmitoyltransferases enhanced pyroptotic cell death, while introduction of exogenous palmitoylation sequences fully restored pyroptotic activities to the C191A mutant, suggesting that palmitoylation-mediated membrane localization may be distinct from other molecular events such as GSDMD conformational change during pore assembly. Collectively, our study suggests that S-palmitoylation may be a shared regulatory mechanism for GSDMD and other gasdermins, which points to potential avenues for therapeutically targeting S-palmitoylation of gasdermins in inflammatory disorders.

Rule-based omics mining reveals antimicrobial macrocyclic peptides against drug-resistant clinical isolates.

Antimicrobial resistance remains a significant global threat, driving up mortality rates worldwide. Ribosomally synthesized and post-translationally modified peptides have emerged as a promising source of novel peptide antibiotics due to their diverse chemical structures. Here, we report the discovery of new aminovinyl-(methyl)cysteine (Avi(Me)Cys)-containing peptide antibiotics through a synergistic approach combining biosynthetic rule-based omics mining and heterologous expression. We first bioinformatically identify 1172 RiPP biosynthetic gene clusters (BGCs) responsible for Avi(Me)Cys-containing peptides formation from a vast pool of over 50,000 bacterial genomes. Subsequently, we successfully establish the connection between three identified BGCs and the biosynthesis of five peptide antibiotics via biosynthetic rule-guided metabolic analysis. Notably, we discover a class V lanthipeptide, massatide A, which displays excellent activity against gram-positive pathogens, including drug-resistant clinical isolates like linezolid-resistant S. aureus and methicillin-resistant S. aureus, with a minimum inhibitory concentration of 0.25 µg/mL. The remarkable performance of massatide A in an animal infection model, coupled with a relatively low risk of resistance and favorable safety profile, positions it as a promising candidate for antibiotic development. Our study highlights the potential of Avi(Me)Cys-containing peptides in expanding the arsenal of antibiotics against multi-drug-resistant bacteria, offering promising drug leads in the ongoing battle against infectious diseases.

Cysteine in the R3 Tau Peptide Modulates Hemin Binding and Reactivity.

Tau is a neuronal protein involved in axonal stabilization; however under pathological conditions, it triggers the deposition of insoluble neurofibrillary tangles, which are one of the biomarkers for Alzheimer's disease. The factors that might influence the fibrillation process are i) two cysteine residues in two pseudorepetitive regions, called R2 and R3, which can modulate protein-protein interaction via disulfide cross-linking; ii) an increase of reactive oxygen species affecting the post-translational modification of tau; and iii) cytotoxic levels of metals, especially ferric-heme (hemin), in hemolytic processes. Herein, we investigated how the cysteine-containing R3 peptide (R3C) and its Cys→Ala mutant (R3A) interact with hemin and how their binding affects the oxidative damage of the protein. The calculated binding constants are remarkably higher for the hemin-R3C complex (LogK1 = 5.90; LogK2 = 5.80) with respect to R3A (LogK1 = 4.44; LogK2 < 2), although NMR and CD investigations excluded the direct binding of cysteine as an iron axial ligand. Both peptides increase the peroxidase-like activity of hemin toward catecholamines and phenols, with a double catalytic efficiency detected for hemin-R3C systems. Moreover, the presence of cysteine significantly alters the susceptibility of R3 toward oxidative modifications, easily resulting in peptide dopamination and formation of cross-linked S-S derivatives.

Zn2+-dependent association of cysteine-rich protein with virion orchestrates morphogenesis of rod-shaped viruses.

The majority of rod-shaped and some filamentous plant viruses encode a cysteine-rich protein (CRP) that functions in viral virulence; however, the roles of these CRPs in viral infection remain largely unknown. Here, we used barley stripe mosaic virus (BSMV) as a model to investigate the essential role of its CRP in virus morphogenesis. The CRP protein γb directly interacts with BSMV coat protein (CP), the mutations either on the His-85 site in γb predicted to generate a potential CCCH motif or on the His-13 site in CP exposed to the surface of the virions abolish the zinc-binding activity and their interaction. Immunogold-labeling assays show that γb binds to the surface of rod-shaped BSMV virions in a Zn2+-dependent manner, which enhances the RNA binding activity of CP and facilitates virion assembly and stability, suggesting that the Zn2+-dependent physical association of γb with the virion is crucial for BSMV morphogenesis. Intriguingly, the tightly binding of diverse CRPs to their rod-shaped virions is a general feature employed by the members in the families Virgaviridae (excluding the genus Tobamovirus) and Benyviridae. Together, these results reveal a hitherto unknown role of CRPs in the assembly and stability of virus particles, and expand our understanding of the molecular mechanism underlying virus morphogenesis.

Construction of a mitochondria-targeted probe to monitor cysteine levels in cancer cells and zebrafish.

Cysteine (Cys) plays an indispensable role as an antioxidant in the maintenance of bioredox homeostasis. We have constructed an efficient fluorescent probe Mito-Cys based on the binding of indole and naphthol. The acrylic ester group serves as a recognition switch for specific detection of Cys, which undergoes Michael addition and intramolecular cyclization reactions, thereby ensuring the chemical kinetics priority of Cys compared to other biothiols. The probe has good water solubility, large Stokes shift (137 nm), with a detection limit of 21.81 nM. In addition, cell imaging experiments have shown that the probe has excellent mitochondrial targeting ability (R = 0.902). The probe can distinguish between Cys, homocysteine (Hcy) and glutathione (GSH), and can detect Cys specifically and quickly (100 s) to ensure accurate quantitative analysis of Cys changes in cells. More importantly, the probe confirms that ferroptosis inducing factors trigger thiol starvation in mitochondria, which helps to gain a deeper understanding of the physiological and pathological functions related to Cys and ferroptosis.

Kinetic and Spectroscopic Investigation of the Y157F and C93G/Y157F Variants of Cysteine Dioxygenase: Dissecting the Roles of the Second-Sphere Residues C93 and Y157.

In mammals, l-cysteine (Cys) homeostasis is maintained by the mononuclear nonheme iron enzyme cysteine dioxygenase (CDO), which oxidizes Cys to cysteine sulfinic acid. CDO contains a rare post-translational modification, involving the formation of a thioether cross-link between a Cys residue at position 93 (Mus musculus CDO numbering) and a nearby tyrosine at position 157 (Cys-Tyr cross-link). As-isolated CDO contains both the cross-linked and non-cross-linked isoforms, and formation of the Cys-Tyr cross-link during repeated enzyme turnover increases CDO's catalytic efficiency by ∼10-fold. Interestingly, while the C93G CDO variant lacks the Cys-Tyr cross-link, it is similarly active as cross-linked wild-type (WT) CDO. Alternatively, the Y157F CDO variant, which also lacks the cross-link but maintains the free thiolate at position 93, exhibits a drastically reduced catalytic efficiency. These observations suggest that the untethered thiolate moiety of C93 is detrimental to CDO activity and/or that Y157 is essential for catalysis. To further assess the roles of residues C93 and Y157, we performed a spectroscopic and kinetic characterization of Y157F CDO and the newly designed C93G/Y157F CDO variant. Our results provide evidence that the non-cross-linked C93 thiolate stabilizes a water at the sixth coordination site of Cys-bound Y157F Fe(II)CDO. A water is also present, though more weakly coordinated, in Cys-bound C93G/Y157F Fe(II)CDO. The presence of a water molecule, which must be displaced by cosubstrate O2, likely makes a significant contribution to the ∼15-fold and ∼7-fold reduced catalytic efficiencies of the Y157F and C93G/Y157F CDO variants, respectively, relative to cross-linked WT CDO.

Cysteine Homeostasis Disturbance in Escherichia coli Caused by Exposure to Ciprofloxacin.

E. coli exposure to ciprofloxacin disturbs cysteine homeostasis; an increase in the intracellular concentration of cysteine is dangerous due to its ability to enhance ROS generation. Unlike wild-type bacteria, in which the cysteine content did not exceed the control level, cells of the gshA mutant lacking glutathione are characterized by increased concentration of intracellular cysteine in proportion to the concentrations of the antibiotic, despite the intensive export of cysteine into the medium. At low concentrations of ciprofloxacin, the mutant strain formed half as many colonies as the parent strain in the survival test. These findings attest to the important role of the incorporation of excess cysteine into glutathione as one of the mechanisms of cysteine homeostasis during the stress response to antibiotic.

N-terminal cysteine acetylation and oxidation patterns may define protein stability.

Oxygen homeostasis is maintained in plants and animals by O2-sensing enzymes initiating adaptive responses to low O2 (hypoxia). Recently, the O2-sensitive enzyme ADO was shown to initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O2-dependent protein stability and the hypoxic response.

Metabolic engineering of CHO cells towards cysteine prototrophy and systems analysis of the ensuing phenotype.

Chinese hamster ovary (CHO) cells require cysteine for growth and productivity in fed-batch cultures. In intensified processes, supplementation of cysteine at high concentrations is a challenge due to its limited solubility and instability in solution. Methionine can be converted to cysteine (CYS) but key enzymes, cystathionine beta-synthase (Cbs) and cystathionine gamma-lyase (Cth), are not active in CHO cells resulting in accumulation of an intermediate, homocysteine (HCY), in cell culture milieu. In this study, Cbs and Cth were overexpressed in CHO cells to confer cysteine prototrophy, i.e., the ability to grow in a cysteine free environment. These pools (CbCt) needed homocysteine and beta-mercaptoethanol (ßME) to grow in CYS-free medium. To increase intracellular homocysteine levels, Gnmt was overexpressed in CbCt pools. The resultant cell pools (GnCbCt), post adaptation in CYS-free medium with decreasing residual HCY and ßME levels, were able to proliferate in the HCY-free, ßME-free and CYS-free environment. Interestingly, CbCt pools were also able to be adapted to grow in HCY-free and CYS-free conditions, albeit at significantly higher doubling times than GnCbCt cells, but couldn't completely adapt to ßME-free conditions. Further, single cell clones derived from the GnCbCt cell pool had a wide range in expression levels of Cbs, Cth and Gnmt and, when cultivated in CYS-free fed-batch conditions, performed similarly to the wild type (WT) cell line cultivated in CYS supplemented fed-batch culture. Intracellular metabolomic analysis showed that HCY and glutathione (GSH) levels were lower in the CbCt pool in CYS-free conditions but were restored closer to WT levels in the GnCbCt cells cultivated in CYS-free conditions. Transcriptomic analysis showed that GnCbCt cells upregulated several genes encoding transporters as well as methionine catabolism and transsulfuration pathway enzymes that support these cells to biosynthesize cysteine effectively. Further, 'omics analysis suggested CbCt pool was under ferroptotic stress in CYS-free conditions, which, when inhibited, enhanced the growth and viability of these cells in CYS-free conditions.

Thiol Redox Proteomics for Identifying Redox-Sensitive Cysteine Residues Within the Protein of Interest During Stress.

Redox modulation is a common posttranslational modification to regulate protein activity. The targets of oxidizing agents are cysteine residues (Cys), which have to be exposed at the surface of the proteins and are characterized by an environment that favors redox modulation. This includes their protonation state and the neighboring amino acids. The Cys redox state can be assessed experimentally by redox titrations to determine the midpoint redox potential in the protein. Exposed cysteine residues and putative intramolecular disulfide bonds can be predicted by alignments with structural data using dedicated software tools and information on conserved cysteine residues. Labeling with light and heavy reagents, such as N-ethylmaleimide (NEM), followed by mass spectrometric analysis, allows for the experimental determination of redox-responsive cysteine residues. This type of thiol redox proteomics is a powerful approach to assessing the redox state of the cell, e.g., in dependence on environmental conditions and, in particular, under abiotic stress.