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Cancer cells require nutrients to grow and proliferate. The availability of the amino acid cysteine impacts whether cancer cells die by an iron-dependent, non-apoptotic form of cell death executed by lipid peroxidation called ferroptosis. Ferroptosis can be induced if the availability of the amino acid cysteine is limiting or by inhibiting the phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4). Extracellular metabolites such as monounsaturated fatty acids (MUFAs) and cysteine can protect from ferroptosis. However, it is unclear how cancer cells acquire cysteine to evade ferroptosis or what MUFAs they can use to evade ferroptosis. Using human-derived cancer cell lines in both adherent (2D) and suspension (3D) culture, this study demonstrates that the cysteine-rich extracellular protein albumin protects from ferroptosis caused by cysteine deprivation. Albumin in combination with inhibition of the nutrient and energy sensing kinase, mammalian target of rapamycin complex 1 (mTORC1), is required to robustly protect from cysteine deprivation-induced ferroptosis in 2D. This combination treatment protects from ferroptosis by increasing intracellular levels of cysteine. Furthermore, this process is dependent on the catabolism of albumin into its amino acid constituents. Additionally, albumin protects from cystine deprivation-induced ferroptosis in 3D culture. This study reveals that a variety of exogenous MUFAs that have not previously been characterized as ferroptosis-protective metabolites protect HT-1080 cells in monolayer and HT-1080 and Panc-1 spheroids from GPX4 inhibition-induced ferroptosis. These MUFAs exhibit different protective abilities in adherent compared to spheroid culture and this suggests growth-context dependent differences in MUFA protection. These results describe novel metabolite-mediated strategies of ferroptosis evasion in 2D and 3D culture. Understanding how cells utilize exogenous protective metabolites such as albumin and MUFAs in death-inducing conditions can reveal novel ways to induce more complete cancer cell death by curtailing alternative avenues of protection. Targeting these mechanisms to induce more effective cell death in cancer cells reveals potential therapeutic relevance of this work.

Caspases are a family of cysteine-dependent proteases with important cellular functions in inflammation and apoptosis, while also implicated in human diseases. Classical chemical tools to study caspase functions lack selectivity for specific caspase family members due to highly conserved active sites and catalytic machinery. To overcome this limitation, we targeted a non-catalytic cysteine residue (C264) unique to caspase-6 (C6), an enigmatic and understudied caspase isoform. Starting from disulfide ligands identified in a cysteine trapping screen, we used a structure-informed covalent ligand design to produce potent, irreversible inhibitors (3a) and chemoproteomic probes (13-t) of C6 that exhibit unprecedented selectivity over other caspase family members and high proteome selectivity. This approach and the new tools described will enable rigorous interrogation of the role of caspase-6 in developmental biology and in inflammatory and neurodegenerative diseases.

Macrocyclisation provides a means of stabilising the conformation of peptides, often resulting in improved stability, selectivity, affinity, and cell permeability. In this work we report a new approach to peptide macrocyclisation using a cyanobenzothiazole-containing amino acid that we show can be incorporated into peptides by both in vitro translation and solid phase peptide synthesis, meaning it should be applicable to peptide discovery by mRNA display. This cyclisation proceeds rapidly, with minimal by-products, is selective over other amino acids including non N-terminal cysteines, and is compatible with further peptide elaboration exploiting such an additional cysteine in bicyclisation and derivatisation reactions. Using molecular dynamics simulation we show that the new cyclisation group is likely to influence the peptide conformation as compared to previous thioether-based approaches, through rigidity and intramolecular aromatic interactions, illustrating their complementarity.

Oriented ligand immobilization is one of the most effective strategies used in the design and construction of a high-capacity protein A chromatography. In this work, cysteine was introduced as anchoring sites by substituting a specific residue on Helix I, II, and at C-terminus of antibody binding domain Z from protein A, respectively, to investigate structural evolution and binding behavior of protein A ligands at liquid-solid interfaces. Among the three affinity dextran-coated Fe3O4 magnetic nanoparticles (Fe3O4@Dx MNPs), affinity MNPs with the immobilized ligand via N11C on Helix I (Fe3O4@Dx-Z1 MNPs) had the highest helical content, and MNPs with the immobilized ligand via G29C on Helix II (Fe3O4@Dx-Z2 MNPs) had the lowest helical content at the same pHs. It was attributed to less electrostatic attraction of ligand to negatively charged surface on Fe3O4@Dx-Z1 MNPs because of less positive charged residues on Helix I (K6) than Helix II (R27/K35). Among the three affinity MNPs, moreover, the highest affinity to immunoglobulin G (IgG) binding was observed on Fe3O4@Dx-Z1 MNPs in isothermal titration calorimetry measurement, further validating greater structural integrity of the ligand on Fe3O4@Dx-Z1 MNPs. Finally, the study of IgG binding on MNPs and 96-well plates showed that anchoring sites for ligand immobilization had distinct influences on IgG binding and IgG-mediated antigen binding. This work illustrated that anchoring sites of the ligands had a striking significance for the molecular structure of the ligand at liquid-solid interfaces and raised an important implication for the design and optimization of protein A chromatography and protein A-based immunoassay analysis.

The nucleocapsid (NC) domain of the retroviral Gag polyproteins mediates the incorporation of the viral genomic RNA into virions. Although SIV is widely used as a model for human immunodeficiency virus type 1 (HIV-1) infections, the SIV NC has been the subject of few studies which have provided discrepant data on the relative contribution of the two NC zinc finger motifs to genomic RNA encapsidation. Here, we demonstrate that mutations affecting the first cysteine in the distal zinc finger motif (C33S) or the N-terminal NC basic domain (R7A/K8A) drastically impair virion assembly and viral RNA binding. By contrast, amino acid substitutions targeting the first cysteine of the proximal zinc finger (C12S) or the basic region connecting both zinc fingers (R29A/R30A) allow substantial particle production and genomic RNA encapsidation. Our results help define the relative contribution of the SIV NC zinc finger motifs and basic regions to the NC biological properties.

Nanomedicine has emerged as an area of research that has earned a lot of attention in the past decade along with frequent and sustained advancements leading to its extensive plethora of biomedical applications. Following the same, the drug carrier systems like gold nanoparticles (AuNPs) have become the success stories with many marketed formulations now and has proved to be a most suitable answer to the drug delivery concers due to their exclusive electrical and optical properties. They serve as one of the best surfaces to either adsorb, attach, or conjugate and exhibit remarkable optoelectronic, physicochemical, morphological and functional properties. In this study, Gabapentin (GBP) a known FDA approved anti-epileptic drug is selected for improving its pharmaceutical limitations and we have synthesized monodispersed AuNPs for GBP conjugation using citrate reduction method. Here the research group has modified AuNP surfaces with three different thiol moieties namely, Mercaptopropionic acid (3-MPA), 11-Mercaptoundecanoic acid (11-MUA) and the amino acid L-Cysteine (L-Cys) and then attached the GBP with these linkers. The synthesized GBP–AuNP nano-conjugate moieties were further characterized and tested for their in-vitro cytotoxicity, release kinetics and stability properties. The in-vitro release kinetics and cytotoxicity (MTT-Assay) analysis of the formulated GBP–AuNP nano-conjugates along with all the 3 types of linkers was carried out and after the data analysis for the same, it was observed that the GBP–AuNP nano-conjugate with L-Cys linker exhibited the sustained release pattern and non- significant cytotoxicity followed by MUA and MPA, respectively. Moreover, the assessment of GBP-AuNPs evaluated through the comprehensive release kinetics analysis, reinforced the GBP-thiol linkers as potentially more effective and competent than GBP alone. Furthermore, all the experimental data was validated by statistical method, and it can be concluded from the present study that the symmetrical monodispersed AuNPs formulation coated with stable functionalized thiol layer were synthesized with minimal cytotoxicity and can be further explored as an efficient drug delivery system for therapeutic application.

Maintaining optimal one-carbon metabolism (OCM) is essential for health and pregnancy. In this cross-sectional study, folate status was assessed based on 5-methyltetrahydrofolate (5-MTHF) levels, and the association between 5-MTHF and OCM-related metabolites was investigated in 227 female Japanese university students aged 18–25 years. The participants were divided into high and low 5-MTHF groups based on their folate status. Serum samples of the participants were collected while they were fasting, and 18 OCM-related metabolites were measured using stable-isotope dilution liquid chromatography–electrospray tandem mass spectrometry. The association between serum 5-MTHF and OCM-related metabolite concentrations was assessed using Spearman’s rank correlation coefficient. Serum 5-MTHF concentrations were negatively correlated with total homocysteine (tHcy) concentrations and positively correlated with S-adenosylmethionine (SAM) and total cysteine (tCys) concentrations. Serum 5-MTHF concentrations demonstrated a stronger negative correlation with tHcy/tCys than with tHcy alone. The negative correlation between betaine and tHcy concentrations was stronger in the low 5-MTHF group than in the high 5-MTHF group. The 5-MTHF status could be linked to Hcy flux into the transsulfuration pathway via SAM. Therefore, the tHcy/tCys ratio may be a more sensitive indicator of the 5-MTHF status than tHcy alone. Furthermore, a low 5-MTHF status can enhance Hcy metabolism via betaine.

Transmission blocking interventions can stop malaria parasite transmission from mosquito to human by inhibiting parasite infection in mosquitos. One of the most advanced candidates for a malaria transmission blocking vaccine is Pfs230. Pfs230 is the largest member of the 6-cysteine protein family with 14 consecutive 6-cysteine domains and is expressed on the surface of gametocytes and gametes. Here, we present the crystal structure of the first two 6-cysteine domains of Pfs230. We identified high affinity Pfs230-specific nanobodies that recognized gametocytes and bind to distinct sites on Pfs230, which were isolated from immunized alpacas. Using two non-overlapping Pfs230 nanobodies, we show that these nanobodies significantly blocked P. falciparum transmission and reduced the formation of exflagellation centers. Crystal structures of the transmission blocking nanobodies with the first 6-cysteine domain of Pfs230 confirm that they bind to different epitopes. In addition, these nanobodies bind to Pfs230 in the absence of the prodomain, in contrast with the binding of known Pfs230 transmission blocking antibodies. These results provide additional structural insight into Pfs230 domains and elucidate a mechanism of action of transmission blocking Pfs230 nanobodies.

The aim of this study was the synthesis and evaluation of entirely S-protected thiolated hydroxyethylcellulose (HEC) with low and high viscosity, as well as thiolated poly-L-lysine (poly-L-Lys) used as dual-acting ionic as well as thiol-disulfide exchange mediated cross-linking hydrogel.Bis(mercaptosuccinic acid) was covalently attached to low and high viscous HECs via Fisher esterification, obtaining S-protected polymers. Poly-L-Lys-cysteine was synthesized via amidation of poly-L-Lys-HBr with cysteine (Cys). Thiolated polymers were examined in terms of cytotoxicity and rheological behavior of hydrogels containing these thiomers was evaluated with a cone-plate rheometer.Thiomers showed less cytotoxicity compared to the corresponding unmodified polymers. Rheological studies showed that cross-linking occurred between the two polymers via thiol-disulfide exchange reactions facilitated by the complementary charges. Employing poly-L-Lys-Cys in a concentration of either 0.5 or 5% (m/v) resulted in a 34.5-fold or 17.3-fold as well as a 53.6-fold or 29.6-fold improvement in dynamic viscosity within 5 min at 37 °C on S-protected thiolated low and high viscous HEC, compared to the corresponding unmodified HECs, respectively.By the combination of anionic S-protected thiolated polymers with a cationic thiolated polymer, dual-acting hydrogels exhibiting a time dependent increase in viscosity can be designed.

Onions are a significant source of organosulfur compounds with promising biological effects. However, the effects of different cooking methods on organosulfur composition and biological properties in onions remain unclear. Therefore, the objective of this study was to determine the composition of organosulfur compounds and the antiplatelet effect of six different onion varieties processed using different cooking methods (boiling, stir-frying, and steaming).The organosulfur compounds were quantified by measuring the S-Alk(en)ey-L-cysteine sulfoxides (ACSOs) contents utilizing and high-pressure liquid chromatography (HPLC). Results showed no significant differences in ACSOs contents among onion varieties. However, ‘Guigum' (GG) and ‘Turbo’ (TB) varieties showed the highest amount of ACSOs at 100.6 and 98.0 mg/100 g fresh weight (FW), respectively. Total ACSOs contents in the other four varieties were 62.7∼72.4 mg/100g FW, showing similar contents. Stir-fried (without the addition of water) onions retained the highest content of total ACSO, followed by steamed treated onions. However, with the boiling method, sulfur compounds were eluted and lost in the cooking water, resulted in a 46.8% loss of ACSOs. Onion extracts showed significant inhibition of the collagen-induced aggregation of rat blood platelets. The highest effect of platelet aggregation was observed in stir-fried onion extracts, whereas the lowest was in boiled extracts. Results demonstrated that stir-frying is an effective cooking method to retain the highest amount of health-beneficial organosulfur compounds in onion.

In mammals, six interleukin-17 (IL-17) genes, as potent pro-inflammatory cytokines, all accelerate the inflammatory responses. In teleosts, seven IL-17 genes have been found in various species, but little is known about the function of teleost-specific IL-17N. In this study, teleost IL-17N and IL-17A/F2 genes all had six conserved cysteine residues forming three intrachain disulfide bridges, the length of three exons of teleost IL-17N gene was similar to that of teleost IL-17A/F2 gene, and the neighbor-joining (NJ) phylogenetic tree showed that teleost IL-17N was clustered with vertebrate IL-17A/F, implying that teleost IL-17N gene may be a paralog of teleost IL-17A/F gene. Pelteobagrus fulvidraco (Pf) IL-17N gene was highly expressed in the blood, brain and kidney of healthy yellow catfish. Pf_IL-17N transcript and protein were notably up-regulated in the spleen, head kidney, gill and kidney detected after Edwardsiella ictaluri infection. Lipopolysaccharides (LPS), polyinosinic-polycytidylic acid (Poly I:C) and peptidoglycan (PGN) also remarkably induced the expression of Pf_IL-17N in the isolated peripheral blood leucocytes (PBLs) of yellow catfish. These results reveal that Pf_IL-17N may play important roles in preventing the invasion of pathogens. Furthermore, the recombinant (r) Pf_IL-17N protein could significantly induce the mRNA expressions of inflammatory cytokines, chemokines and antimicrobial peptide genes in yellow catfish in vivo and in vitro, and it also notably promoted the phagocytosis of myeloid cells in the PBLs and the chemotaxis of the PBLs and gill leucocytes (GLs) in yellow catfish. Besides, though the rPf_IL-17N protein could induce and aggravate inflammation infiltration in the kidney of yellow catfish, it did not effectively and notably increase the survival rate of yellow catfish after E. ictaluri infection. Furthermore, the rPf_IL-17N protein could induce the mRNA expressions of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signal pathways related genes, and the inhibitor of NF-κB and MAPK signal pathways could restrain the rPf_IL-17N protein-induced inflammatory response. This study provides crucial evidence that the Pf_IL-17N may mediate inflammatory response to eliminate invasive pathogens.

Selenophosphate synthetases use selenium and ATP to synthesize selenophosphate. This is required for biological utilization of selenium, most notably for the synthesis of the non-canonical amino acid selenocysteine (Sec). Therefore, selenophosphate synthetases underlie all functions of selenoproteins, which include redox homeostasis, protein quality control, hormone regulation, metabolism, and many others. This protein family comprises two groups, SelD/SPS2 and SPS1. The SelD/SPS2 group represent true selenophosphate synthetases, enzymes central to selenium metabolism which are present in all Sec-utilizing organisms across the tree of life. Notably, many SelD/SPS2 proteins contain Sec as catalytic residue in their N-terminal flexible selenium-binding loop, while others replace it with cysteine (Cys). The SPS1 group comprises proteins originated through gene duplications of SelD/SPS2 in metazoa in which the Sec/Cys-dependent catalysis was disrupted. SPS1 proteins do not synthesize selenophosphate and are not required for Sec synthesis. They have essential regulatory functions related to redox homeostasis and pyridoxal phosphate, which affect signaling pathways for growth and differentiation. In this review, we summarize the knowledge about the selenophosphate synthetase family acquired through decades of research, encompassing their structure, mechanism, function, and evolution.

Excessive dietary carbohydrate commonly impairs the functions of liver and intestine in carnivorous fish. In the present study, a 10-week feeding trial was carried out to explore the regulation of biotin on the hepatic and intestinal inflammation and apoptosis in turbot (Scophthalmus maximus L.) fed with high carbohydrate diets. Three isonitrogenous and isolipidic experimental diets were designed as follows: the CC diet with 18.6% of carbohydrate and 0.04 mg/kg of biotin, the HC diet with 26.9% of carbohydrate and 0.05 mg/kg of biotin, and the HCB diet with 26.9% of carbohydrate and 1.62 mg/kg of biotin. Results showed that high dietary carbohydrate (HC diet) impaired the morphology of liver and intestine, however, inclusion of dietary biotin (HCB diet) normalized their morphology. Inflammation-related gene expression of nuclear factor κB p65 (nf-κb p65), tumor necrosis factor α (tnf-α), interleukin-1β (il-1β), il-6 and il-8, and the protein expression of NF-κB p65 in the liver and intestine were significantly up-regulated in the HC group compared to those in the CC group (P 0.05), the HCB diet decreased their expression compared to the HC group (P 0.05). The gene expression of il-10 and transforming growth factor-β (tgf-β) in the liver and intestine were significantly decreased in the HC group compared to the CC group (P 0.05), and inclusion of dietary biotin increased the il-10 and tgf-β expression in the liver and intestine (P 0.05). Moreover, compared to the CC group, the HC group had a stronger degree of DNA fragmentation and more TUNEL-positive cells in the liver and intestine, and the HCB group had a slighter degree of DNA fragmentation and fewer TUNEL-positive cells compared to the HC group. Meanwhile, the gene expression of B-cell lymphoma protein-2-associated X protein (bax) and executor apoptosis-related cysteine peptidase 3 (caspase-3) were significantly up-regulated and the gene expression of B-cell lymphoma-2 (bcl-2) was significantly down-regulated both in the liver and intestine in the HC group compared with those in the CC group (P 0.05). Inclusion of dietary biotin significantly decreased the bax and caspase-3 mRNA levels and increased bcl-2 mRNA level in the liver and intestine (P 0.05). In conclusion, high dietary carbohydrate (26.9% vs 18.6%) induced inflammation and apoptosis in liver and intestine. Supplementation of biotin (1.62 mg/kg vs 0.05 mg/kg) in diet can alleviate the high-dietary-carbohydrate-induced hepatic and intestinal inflammation as well as inhibit apoptosis in turbot. The present study provides basic data for the application of biotin into feed, especially the high-carbohydrate feed for turbot.

Diverse naturally occurring events relevant to proteins, including processing and post-translational modification, provide significant clues enabling advances in peptide/protein chemistry. Our motivation to synthesize large proteins prompted us to seek scientific bases utilized in synthetic experiments on the intein-mediated protein processing system. This account describes peptide/protein thioester-producing protocols whose design is based on acyl transfer reactions observed in the intein system, and the development of the stimulus-responsive amide cleavage and its application to the modulation of peptide function. Finally, several findings derived from nature-inspired research efforts, including peptide mimetic synthesis and S-protected cysteine chemistry, are described.

Expression of mucin MUC2, a component of the colonic mucus layer, plays a crucial role in intestinal homeostasis. Here, we describe a new regulator of MUC2 expression, the deubiquitinase ZRANB1 (Trabid). A ZRANB1 mutation changing cysteine to serine in amino acid position 443, affects ubiquitination. To analyze ZRANB1 function in the intestine, we generated Zranb1 C443S mutant knock-in (Zranb1

This study aimed to identify the sources of volatile sulphur compounds (VSCs) and evaluate their mitigation by ferric oxide (Fe

In the present study, the zinc finger aspartate-histidine-histidine-cysteine (DHHC)-type containing 1 (ZDHHC1) gene was identified in a commercial fish, the Chinese perch Siniperca chuatsi. The ZDHHC1 has five putative transmembrane motifs and conserved DHHC domain, showing high amino-acid identity with other teleost fish, and vertebrate ZDHHC1 loci are conserved from fish to human. In vivo expression analysis indicated that ZDHHC1 gene was constitutively transcribed in all the examined organs/tissues, and was induced following infectious spleen and kidney necrosis virus (ISKNV) infection. It is further observed that ZDHHC1 interacts with MITA and the overexpression of ZDHHC1 in cells resulted in the upregulated expression of ISGs, such as Mx, RSAD2, IRF3 and type I IFNs such as IFNh and IFNc, exhibiting its antiviral function in fish as reported in mammals.

Inflammation is generally thought to be involved in the occurrence and development of preeclampsia (PE), but its specific effect on PE remains unclear. In the present study, the expression levels of 92 inflammation-related proteins were measured in the late pregnancy maternal plasma from patients with PE (

Nanocolloids that are cumulatively referred to as nanocarbons, attracted significant attention during the last decade because of facile synthesis methods, water solubility, tunable photoluminescence, easy surface modification, and high biocompatibility. Among the latest development in this reserach area are chiral nanocarbons exemplified by chiral carbon dots (CDots). They are expected to have applications in sensing, catalysis, imaging, and nanomedicine. However, the current methods of CDots synthesis show often contradictory chemical/optical properties and structural information that required a systematic study with careful structural evaluation. Here, we investigate and optimize chiroptical activity and photoluminescence of L- and D-CDots obtained by hydrothermal carbonization of L- and D-cysteine, respectively. Nuclear magnetic resonance spectroscopy demonstrates that they are formed via gradual dehydrogenation and condensation reactions of the starting amino acid leading to particles with a wide spectrum of functional groups including aromatic cycles. We found that the chiroptical activity of CDots has an inverse correlation with the synthesis duration and temperature, whereas the photoluminescence intensity has a direct one, which is associated with degree of carbonization. Also, our studies show that the hydrothermal synthesis of cysteine in the presence of boric acid leads to the formation of CDots rather than boron nitride nanoparticles as was previously proposed in several reports. These results can be used to design chiral carbon-based nanoparticles with optimal chemical, chiroptical, and photoluminescent properties.

Linaridins and lanthipeptides are two classes of natural products belonging to the ribosomally synthesized and posttranslationally modified peptide (RiPP) superfamily. Although these two RiPP classes share similar structural motifs such as dehydroamino acids and thioether-based cross-links, the biosynthesis of linaridins and lanthipeptides involved distinct sets of enzymes. Here, we report the identification of a novel lanthipeptide cypepeptin from a recombinant strain of

Diabetic nephropathy (DN) is one microvascular complication of diabetes. About 30% of diabetic patients can develop DN, which is closely related to the high incidence and mortality of heart diseases, and then develop end-stage renal diseases. Therefore, early detection and screening of high-risk patients with DN is important. Herein, we explored the differences of serum transcriptomics between DN and non-DN in type II diabetes mellitus (T2DM) patients. We obtained 110 target genes using weighted correlation network analysis. Gene Ontology enrichment analysis indicates these target genes are mainly related to membrane adhesion, alpha-amino acid biosynthesis, metabolism, and binding, terminus, inhibitory synapse, clathrinid-sculpted vesicle, kinase activity, hormone binding, receptor activity, and transporter activity. Kyoto Encyclopedia of Genes and Genomes analysis indicates the process of DN in diabetic patients can involve synaptic vesicle cycle, cysteine and methionine metabolism, N-Glycan biosynthesis, osteoclast differentiation, and cAMP signaling pathway. Next, we detected the expression levels of hub genes in a retrospective cohort. Then, we developed a risk score tool included in the prediction model for early DN in T2DM patients. The prediction model was well applied into clinical practice, as confirmed by internal validation and several other methods. A novel DN risk model with relatively high prediction accuracy was established based on clinical characteristics and hub genes of serum detection. The estimated risk score can help clinicians develop individualized intervention programs for DN in T2DM. External validation data are required before individualized intervention measures.

The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 3C-like protease (3CL

Nanoparticles can promote the accumulation of drugs in tumors. However, they find limited clinical applications because they cannot easily penetrate the stroma of cancer tissues, and it is difficult to control drug release. We developed a multiresponse multistage drug-delivery nanogel with improved tumor permeability and responsiveness to the tumor microenvironment for the controlled delivery of anticancer agents. For this purpose, ∼100 nm multistage drug delivery nanogels with pH, redox, near-infrared stimulation, and enzyme responsiveness were grown in situ using 20 nm gold nanoparticles (AuNPs) via an emulsion-aiding crosslinking technique with cysteine crosslinker. An alginate cysteine AuNP (ACA) nanocarrier can efficiently load the cationic drug doxorubicin (DOX) to produce a multistage drug delivery nanocarrier (DOX@ACA). DOX@ACA can maintain the slow release of DOX and reduce its toxicity. In cancer tissues, the high pH and reductase microenvironment combined with the in vitro delivery of alginate and near-infrared light drove drug release. The developed nanoparticles effectively inhibited cancer cells, and in vivo evaluations showed that they effectively enhanced antitumor activity while having negligible in vivo toxicity to major organs.

Herein, we have synthesized a series of three-photon fluorescent Pt(II) complexes targeting a tumor-associated biothiol, cysteine (Cys), which allows it to be detected without any interference from other intracellular proteins. We focused on how to significantly improve the fluorescence response of Cys

ConspectusThe structural boundaries of living cells are composed of numerous membrane-forming lipids. Lipids not only are crucial for the cellular compartmentalization but also are involved in cell signaling as well as energy storage. Abnormal lipid levels have been linked to severe human diseases such as cancer, multiple sclerosis, neurodegenerative diseases, as well as lysosomal storage disorders. Given their biological significance, there is immense interest in studying lipids and their effect on cells. However, limiting factors include the low solubility of lipids, their structural complexity, and the challenge of using genetic techniques to directly manipulate lipid structure. Current methods to study lipids rely mostly on lipidomics, which analyzes the composition of lipid extracts using mass spectrometry. Although, these efforts have successfully catalogued and profiled a great number of lipids in cells, many aspects about their exact functional role and subcellular distribution remain enigmatic.In this Account, we outline how our laboratory developed and applied different bioconjugation strategies to study the role of lipids and lipid modifications in cells. Inspired by our ongoing work on developing lipid bioconjugation strategies to generate artificial cell membranes, we developed a ceramide synthesis method in live cells using a salicylaldehyde ester that readily reacts with sphingosine in form of a traceless ceramide ligation. Our study not only confirmed existing knowledge about the association of ceramides with cell death, but also gave interesting new findings about the structure-function relationship of ceramides in apoptosis. Our initial efforts led us to investigate probes that detect endogenous sphingolipids using live cell imaging. We describe the development of a fluorogenic probe that reacts chemoselectively with sphingosine in living cells, enabling the detection of elevated endogenous levels of this biomarker in human disease. Building on our interest in the fluorescence labeling of lipids, we have also explored the use of bioorthogonal reactions to label chemically synthesized lipid probes. We discuss the development of photocaged dihydrotetrazine lipids, where the initiation of the bioorthogonal reaction can be triggered by visible light, allowing for live cell modification of membranes with spatiotemporal control.Finally, proteins are often post-translationally modified by lipids, which have important effects on protein subcellular localization and function. Controlling lipid modifications with small molecule probes could help reveal the function of lipid post-translational modifications and could potentially inspire novel therapeutic strategies. We describe how our previous studies on synthetic membrane formation inspired us to develop an amphiphilic cysteine derivative that depalmitoylates membrane-bound S-acylated proteins in live cells. Ultimately, we applied this amphiphile mediated depalmitoylation (AMD) in studies investigating the palmitoylation of cancer relevant palmitoylated proteins in healthy and diseased cells.

Biocontainment is a key methodology to reduce environmental risk through the deliberate release of genetically modified microorganisms. Previously, we developed a phosphite (HPO

Melanoma is a kind of aggressive skin neoplasms with high mortality. The purpose of this present research was to investigate the effects and potential mechanisms of long-noncoding RNA (lncRNA) MSC antisense RNA 1 (MSC-AS1) in melanoma. MSC-AS1, miR-330-3p and YAP1 expression levels in melanoma tissues and cells were assessed by quantitative real-time polymerase chain reaction. Melanoma cells were evaluated using cell count kit-8, clone formation and ELISA in vitro . The relationship among MSC-AS1, YAP1 and miR-330-3p was validated by pull-down and luciferase reporter assays. Finally, the role of MSC-AS1 in vivo was determined by the xenograft model. Results showed that lncRNA MSC-AS1 was upregulated in melanoma tissues and cells. High expression of MAS-AS1 was positively correlated with a poor prognosis. Pull-down and luciferase reporter demonstrated that miR-330-3p specifically binds directly to YAP1 and MSC-AS1, respectively. MSC-AS1 promoted the expression of YAP1 by downregulating miR-330-3p. Functional experiments suggested that knockdown of MSC-AS1 suppressed the proliferation of melanoma cells and decreased the levels of glutamine, glutamate and α-ketoglutarate, glutaminase and glutamine transporter alanine-serine-cysteine transporter 2. Upregulation of miR-330-3p alleviated the promotion effect of MSC-AS1 overexpression on the proliferation and glutaminolysis of melanoma cells. The above changes could be reversed by YAP1 overexpression. In addition, knockdown of MSC-AS1 dramatically restrained the growth of melanoma cells in xenograft model. In conclusion, our results revealed that MSC-AS1 facilitated the proliferation and glutaminolysis of melanoma cells by regulating miR-330-3p/ YAP1 pathway, suggesting that MSC-AS1 could provide a new idea for the treatment of melanoma.

The ligand-directed (LD) chemistry provides powerful tools for site-specific modification of proteins. We utilized a peptide with an appended methionine (Met) as a ligand; then, the Met thioether was modified into sulfonium which enabled a proximity induced group transfer onto protein cysteine in the vicinity upon peptide-target binding. The sulfonium warhead could be easily constructed with unprotected peptides, and the transferable group scope was conducted on model protein PDZ and its ligand peptides. In addition, a living cell labeling was successfully achieved.

For proper intracellular vesicle transport, it is essential for transport vesicle membranes to fuse with the appropriate target membranes. Ykt6 is a SNARE protein with functions in diverse vesicle transport pathways, including secretory, endocytotic and autophagic pathways. To exert these functions, the association of Ykt6 with vesicle membranes and the change of its conformation from closed to open play key roles. Recent studies have revealed regulatory mechanisms involved in Ykt6 membrane association and conformation change. When in the cytosol, the vicinal cysteine residues within the C-terminal CCAIM sequence of Ykt6 undergo diprenylation (farnesylation of the distal cysteine residues by farnesyltransferase; this is followed by geranylgeranylation of the proximal cysteine residue by geranylgeranyltransferase-III). Phosphorylation of a serine residue within the SNARE domain triggers the conversion of the Ykt6 conformation from closed to open, allowing Ykt6 membrane association. In this commentary, I briefly summarize and discuss the recently revealed regulatory mechanisms of Ykt6 function by diprenylation and phosphorylation.

Cysteine side chains can exist in distinct oxidation states depending on the pH and redox potential of the environment, and cysteine oxidation plays important yet complex regulatory roles. Compared with the effects of post-translational modifications such as phosphorylation, the effects of oxidation of cysteine to sulfenic, sulfinic, and sulfonic acid on protein structure and function remain relatively poorly characterized. We present an analysis of the role of cysteine reactivity as a regulatory factor in proteins, emphasizing the interplay between electrostatics and redox potential as key determinants of the resulting oxidation state. A review of current computational approaches suggests underdeveloped areas of research for studying cysteine reactivity through molecular simulations.

The lipid peroxidation end product, 4-hydroxy-2-nonenal (HNE), is a secondary mediator of oxidative stress due to its strong ability to form adducts to the side chains of lysine, histidine, and cysteine residues (Cys) at increasing reactivities. This reaction can take place in various cellular environments and may be dependent on solvent. Moreover, approximately 10% of cysteine residues within the cells exist as the negatively charged cysteinate, which may also have a distinct reactivity toward HNE. In this study, quantum chemical calculations are used to investigate the reactivity of HNE toward Cys and cysteinate in three distinct solvent environments to mimic the aqueous, polar, and hydrophobic regions within the cell. Water enhances the reactivity of HNE to cysteine compared to that of the polar and hydrophobic solvents, and the reactivity of HNE is further augmented when Cys is first ionized to cysteinate. This is also confirmed by the transition state rate constant calculations. This study reveals the role of solvent polarity in these reactions and how cysteinate can account for the seemingly high reactivity of HNE toward Cys compared to other amino acid residues and demonstrates how a strong nucleophile can enhance the reactivity of an antioxidant analogue of the Cys residue.

Mercury (Hg) is a heavy metal widespread in all environmental compartments as one of the most hazardous pollutants. Human exposure to this natural element is detrimental for several cellular types including erythrocytes (RBC) that accumulate Hg mainly bound to the SH groups of different cellular components, including protein cysteine residues. The cellular membrane represents a major target of Hg-induced damage in RBC with loss of physiological phospholipid asymmetry, due to phosphatidylserine (PS) exposure to the external membrane leaflet. To investigate Hg-induced cytotoxicity at the molecular level, the possible interaction of this heavy metal with RBC membrane proteins was investigated. Furthermore, Hg-induced alterations in band 3 protein (B3p) transport function, PS-exposing macrovesicle (MVs) formation and morphological changes were assessed. For this aim, human RBC were treated in vitro with different HgCl 2 concentrations (range 10-40 µM) and the electrophoretic profile of membrane proteins as well as the expression levels of Ankyrin and Flottilin-2 evaluated by SDS-PAGE and Western blot, respectively. The effect of alterations in these proteins on RBC morphology was evaluated by digital holographic microscopy and anionic transport efficiency of B3p was evaluated as sulphate uptake. Finally, PS- bearing MVs were quantified by annexin-V binding using FACS analysis. Findings presented in this paper indicate that RBC exposure to HgCl 2 induces modifications in the electrophoretic profile of membrane protein fraction. Furthermore, our study reveals the Hg induced alterations of specific membrane proteins, such as Ankyrin, a protein essential for membrane-cytoskeleton linkage and Flotillin-2, a major integral protein of RBC lipid rafts, likely responsible for decreased membrane stability and increased fragmentations. Accordingly, under the same experimental conditions, RBC morphological changes and PS-bearing MVs release are observed. Finally, RBC treatment significantly affects the B3p-mediated anionic transport, that we report reduced upon HgCl 2 treatment in a dose dependent manner. Altogether, the findings reported in this paper confirm that RBC are particularly vulnerable to Hg toxic effect and provide new insight in the Hg-induced protein modification in human RBC affecting the complex biological system of cellular membrane. In particular, Hg could induce dismantle of vertical cohesion between the plasma membrane and cytoskeleton as well as destabilization of lateral linkages of functional domains. Consequently, decreased membrane deformability could impair RBC capacity to deal with the shear forces in the circulation increasing membrane fragmentations. Furthermore, findings described in this paper have also significant implication in RBC physiology, particularly related to gas exchanges.

International audience; Iron−sulfur (Fe−S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Biosynthesis of Fe−S clusters into the ISCU scaffold protein is initiated by ferrous iron insertion, followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear. We show here by SEC, circular dichroism (CD), and Mossbauer spectroscopies that iron binds to the assembly site of the monomeric form of prokaryotic and eukaryotic ISCU proteins via either one or two cysteines, referred to the 1-Cys and 2-Cys forms, respectively. The latter predominated at pH 8.0 and correlated with the Fe−S cluster assembly activity, whereas the former increased at a more acidic pH, together with free iron, suggesting that it constitutes an intermediate of the iron insertion process. Iron not binding to the assembly site was non-specifically bound to the aggregated ISCU, ruling out the existence of a structurally defined auxiliary site in ISCU. Characterization of the 2-Cys form by site-directed mutagenesis, CD, NMR, X-ray absorption, Mossbauer, and electron paramagnetic resonance spectroscopies showed that the iron center is coordinated by four strictly conserved amino acids of the assembly site, Cys35, Asp37, Cys61, and His103, in a tetrahedral geometry. The sulfur receptor Cys104 was at a very close distance and apparently bound to the iron center when His103 was missing, which may enable iron-dependent sulfur acquisition. Altogether, these data provide the structural basis to elucidate the Fe−S cluster assembly process and establish that the initiation of Fe−S cluster biosynthesis by insertion of a ferrous iron in the assembly site of ISCU is a conserved mechanism.

Chemical reactivity in droplets is often assumed to mimic reactivity in bulk, continuous water. Here, we study the catalytic oxidation of cysteine by electrogenerated hexacyanoferrate(III) in microliter droplets. These droplets are adsorbed onto glassy carbon macroelectrodes and placed into an immiscible 1,2-dichloroethane phase. We combined cyclic voltammetry, optical microscopy, and finite element simulations to quantify the apparent bimolecular rate constant, k(c,app), in microdroplets and bulk water. Statistical analyses reveal that the apparent bimolecular rate constant (k(c,app)) values formicrodroplets are larger than those in the continuous phase. Reactant adsorption to the droplet boundary has previously been implicated as the cause of such rate accelerations. Finite element modeling of this system suggests that molecular adsorption to the liquid|liquid interface cannot alone account for our observations, implicating kinetics of the bimolecular reaction either at the boundary or throughout the microliter volume. Our results indicate that cysteine oxidation by electrogenerated hexacyanoferrate(III) can be accelerated within a microenvironment, which may have profound implications on understanding biological processes within a cell.

During bacterial cell division, filaments of tubulin-like FtsZ form the Z-ring, which is the cytoplasmic scaffold for divisome assembly. In Escherichia coli, the actin homologue FtsA anchors the Z-ring to the membrane and recruits divisome components, including bitopic FtsN. FtsN regulates the periplasmic peptidoglycan synthase FtsWI. To characterize how FtsA regulates FtsN, we applied electron microscopy to show that E. coli FtsA forms antiparallel double filaments on lipid monolayers when bound to the cytoplasmic tail of FtsN. Using X-ray crystallography, we demonstrate that Vibrio maritimus FtsA crystallizes as an equivalent double filament. We identified an FtsA-FtsN interaction site in the IA-IC interdomain cleft of FtsA using X-ray crystallography and confirmed that FtsA forms double filaments in vivo by site-specific cysteine cross-linking. FtsA-FtsN double filaments reconstituted in or on liposomes prefer negative Gaussian curvature, like those of MreB, the actin-like protein of the elongasome. We propose that curved antiparallel FtsA double filaments together with treadmilling FtsZ filaments organize septal peptidoglycan synthesis in the division plane.

Protein structures are decisive for their activities and interactions with other molecules. Global analysis of protein structures and conformational changes cannot be achieved by commonly used abundance-based proteomics. Here, we integrated cysteine covalent labeling, selective enrichment, and quantitative proteomics to study protein structures and structural changes on a large scale. This method was applied to globally investigate protein structures in HEK293T cells and protein structural changes in the cells with the tunicamycin (Tm)-induced endoplasmic reticulum (ER) stress. We quantified several thousand cysteine residues, which contain unprecedented and valuable information of protein structures. Combining this method with pulsed stable isotope labeling by amino acids in cell culture, we further analyzed the folding state differences between pre-existing and newly synthesized proteins in cells under the Tm treatment. Besides newly synthesized proteins, unexpectedly, many pre-existing proteins were found to become unfolded upon ER stress, especially those related to gene transcription and protein translation. Furthermore, the current results reveal that N-glycosylation plays a more important role in the folding process of the tertiary and quaternary structures than the secondary structures for newly synthesized proteins. Considering the importance of cysteine in protein structures, this method can be extensively applied in the biological and biomedical research fields.

Efficient methodologies for synthesizing enantiopure α-deuterated derivatives of serine, cysteine, selenocysteine, and 2,3-diaminopropanoic acid have been developed. H/D exchange was achieved by deprotonation of a chiral bicyclic serine equivalent followed by selective deuteration. Additionally, diastereoselective additions of thiols, selenols, and amines to a chiral bicyclic dehydroalanine in deuterated alcohols allowed site-selective deuteration at the Cα atom of cysteine, selenocysteine, and 2,3-diaminopropanoic acid derivatives. A deuterated analogue of carbocysteine, a drug for the treatment of bronchiectasis, was synthesized.

Peptide toxins secreted by venomous animals bind to mammalian ion channel proteins and modulate their function. The high specificity of these toxins for their target ion channels enables them to serve as powerful tools for ion channel biology. Toxins labeled with fluorescent dyes are employed for the cellular imaging of channels and also for studying toxin-channel and toxin-membrane interactions. Several of these toxins are cysteine-rich, rendering the production of properly folded fluorescently labeled toxins technically challenging. Herein, we evaluate a variety of site-specific protein bioconjugation approaches for producing fluorescently labeled double-knot toxin (DkTx), a potent TRPV1 ion channel agonist that contains an uncommonly large number of cysteines (12 out of a total of 75 amino acids present in the protein). We find that popular cysteine-mediated bioconjugation approaches are unsuccessful as the introduction of a non-native cysteine residue for thiol modification leads to the formation of misfolded toxin species. Moreover,iN/i-terminal aldehyde-mediated bioconjugation approaches are also not suitable as the resultant labeled toxin lacks activity. In contrast to these approaches,iC/i-terminal bioconjugation of DkTx via the sortase bioconjugation technology yields functionally active fluorescently labeled DkTx. We employ this labeled toxin for imaging rat TRPV1 heterologously expressed iniXenopus laevis/ioocytes, as well as for performing membrane binding studies on giant unilamellar vesicles composed of different lipid compositions. Our studies set the stage for using fluorescent DkTx as a tool for TRPV1 biology and provide an informative blueprint for labeling cysteine-rich proteins.

Celastrol (CEL), a pentacyclic triterpene compound, has been proven to have a definite antipulmonary fibrosis effect. However, its direct targets for antipulmonary fibrosis remain unknown. In this study, we designed and synthesized a series of celastrol-based probes to identify the direct targets in human pulmonary fibroblasts using an activity-based protein profiling strategy. Among many fished targets, we identified a key protein, cullin-associated and neddylation-dissociated 1 (CAND1), which was involved in fibroblast-myofibroblast transformation (FMT). More importantly, we found that the inhibitory effect of celastrol on FMT is dependent on CAND1, through improving the interactions between CAND1 and Cullin1 to promote the activity of Skp1/Cullin1/F-box ubiquitin ligases. In silico studies and cysteine mutation experiments further demonstrated that Cys264 of CAND1 is the site for conjugation of celastrol. This reveals a new mechanism of celastrol against pulmonary fibrosis and may provide a novel therapeutic option for antipulmonary fibrosis.

Tauopathies are characterised by intracellular deposits of fibrillar tau tangles. However, the interneuronal spread of pathological tau species precedes the development of major tau burdens. Two amyloid motifs, VQIINK in repeat 2 and VQIVYK in repeat 3, of tau repeat domain, assemble into β-sheet-rich fibrils on their own but alone do not form seed-competent fibrils. In contrast, the entire R3 region self-aggregates and forms seed-competent fibrils. Our study aimed to identify the minimal regions in the tau repeat domain that define seeding and its impact on intracellular tau phosphorylation and aggregation. Using peptides of individual repeats, we show that R2, like R3, forms seed-competent fibrils when assembled in the presence of heparin. However, R3, but not R2, forms seed-competent fibrils when assembled without heparin, even though both R2 and R3 have identical N-terminal hexapeptide and cysteine residue sequences. Moreover, cysteine to alanine substitution in R3 abrogates its self-aggregation and seeding potency. Tau RD P301S biosensor cells and Tau P301L (0N4R)-expressing HEK293 cells seeded with R2 and R3 fibrils show the induction of pathological phosphorylation of tau at Ser262/Ser396/Ser404 positions and oligomerisation of native tau. Protein fractions of biosensor cells seeded with R2 and R3 fibrils reseed endogenous tau aggregation when introduced into a fresh set of biosensor cells. Our findings suggest that R3 may be the minimal region for pathological seed generation under physiological conditions, whereas R2 might need polyanionic cofactors to generate pathogenic seeds. Lastly, R2 and R3 fibrils induce template-induced misfolding and pathological hyperphosphorylation of intracellular tau, making intracellular tau seed-competent.

Current methods for the screening of viral infections in clinical settings, such as reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), are expensive, time-consuming, require trained personnel and sophisticated instruments. Therefore, novel sensors that can save time and cost are required specially in remote areas and developing countries that may lack the advanced scientific infrastructure for this task. In this work, we present a sensitive, and highly specific biosensing approach for the detection of harmful viruses that have cysteine residues within the structure of their cell surface proteins. We utilized new method for the rapid screening of SARS-CoV-2 virus in biological fluids through its S1 protein by surface enhanced Raman spectroscopy (SERS). The protein is captured from aqueous solutions and biological specimens using a target-specific extractor substrate. The structure of the purified protein is then modified to convert it into a bio-thiol by breaking the disulfide bonds and freeing up the sulfhydryl (SH) groups of the cysteine residues. The formed biothiol chemisorbs favourably onto a highly sensitive plasmonic sensor and probed by a handheld Raman device in few seconds. The new method was used to screen the S1 protein in aqueous medium, spiked human blood plasma, mucus, and saliva samples down to 150 fg/L. The label-free SERS biosensing method has strong potential for the fingerprint identification many viruses (e.g. the human immunodeficiency virus, the human polyomavirus, the human papilloma virus, the adeno associated viruses, the enteroviruses) through the cysteine residues of their capsid proteins. The new method can be applied at points of care (POC) in remote areas and developing countries lacking sophisticated scientific infrastructure.

The mechanisms underlying the disruption of excitation-contraction (EC) coupling in dystrophin-deficient muscles are not well understood. Here, using animal models for Duchenne muscular dystrophies (DMD), we show a Ca2+-dependent protease (calpain-1)-mediated proteolysis of SH3 and cysteine-rich domain 3 (STAC3) and junctophilin 1 (JP1), essential EC coupling proteins, in dystrophin-deficient muscle, and highlighting the dissociation of STAC3 and JP1 from dihydropyridine receptor as a causative factor in EC uncoupling of dystrophic muscles.

Serine acetyltransferase (SAT) catalyzes the acetylation of l-serine in the first step of the two-step pathway to synthesize L-cysteine in bacteria, protozoans and plants. L-cysteine is known to be involved in feedback regulation of SAT. However, in E. histolytica, SAT exists in three isoforms where third isoform SAT3 is nearly insensitive to feedback inhibition. Here, we explored the previously unknown precise mechanism of the insensitivity of EhSAT3 to L-cysteine. The C-terminal deletion mutants of EhSAT3 were inhibited completely by L-cysteine in contrast to the wildtype EhSAT3. The crystal structure of EhSAT3ΔC22 in complex with cysteine revealed that C-terminal region swaps over the neighboring monomer in the trimer. This structure combined with the modeled C-terminal residues suggests that EhSAT3 C-terminal end interacts with the active site and play crucial role in feedback inhibition. The interacting distances between sulfur of cysteine and protein indicate cysteine is in deprotonated (S

Sulfenic acids are important intermediates in the oxidation of cysteine thiol groups in proteins by reactive oxygen species. The mechanism is influenced heavily by the presence of polar groups, other thiol groups, and solvent, all of which determines the need to compute precisely the energies involved in the process. Surprisingly, very scarce experimental information exists about a very basic property of sulfenic acids, the enthalpies of formation. In this Article, we use high level quantum chemical methods to derive the enthalpy of formation at 298.15 K of methane-, ethene-, ethyne-, and benzenesulfenic acids, the only ones for which some experimental information exists. The methods employed were tested against well-known experimental data of related species and extensive CCSD(T) calculations. Our best results consistently point out to a much lower enthalpy of formation of methanesulfenic acid, CH3SOH (ΔfH0(298.15K) = -35.1 ± 0.4 kcal mol-1), than the one reported in the NIST thermochemical data tables. The enthalpies of formation derived for ethynesulfenic acid, HC≡CSOH, +32.9 ± 1.0 kcal/mol, and benzenesulfenic acid, C6H5SOH, -2.6 ± 0.6 kcal mol-1, also differ markedly from the experimental values, while the enthalpy of formation of ethenesulfenic acid CH2CHSOH, not available experimentally, was calculated as -11.2 ± 0.7 kcal mol-1.

ConspectusIn recent years, considerable progress has been made toward elucidating the geometric and electronic structures of thiol dioxygenases (TDOs). TDOs catalyze the conversion of substrates with a sulfhydryl group to their sulfinic acid derivatives via the addition of both oxygen atoms from molecular oxygen. All TDOs discovered to date belong to the family of cupin-type mononuclear nonheme Fe(II)-dependent metalloenzymes. While most members of this enzyme family bind the Fe cofactor by two histidines and one carboxylate side chain (2-His-1-carboxylate) to provide a monoanionic binding motif, TDOs feature a neutral three histidine (3-His) facial triad. In this Account, we present a bioinformatics analysis and multiple sequence alignment that highlight the significance of the secondary coordination sphere in tailoring the substrate specificity and reactivity among the different TDOs. These insights provide the framework within which important structural and functional features of the distinct TDOs are discussed.The best studied TDO is cysteine dioxygenase (CDO), which catalyzes the conversion of cysteine to cysteine sulfinic acid in both eukaryotes and prokaryotes. Crystal structures of resting and substrate-bound mammalian CDOs revealed two surprising structural motifs in the first- and second coordination spheres of the Fe center. The first is the presence of the abovementioned neutral 3-His facial triad that coordinates the Fe ion. The second is the existence of a covalent cross-link between the sulfur of Cys93 and an ortho carbon of Tyr157 (mouse CDO numbering scheme). While the exact role of this cross-link remains incompletely understood, various studies established that it is needed for proper substrate Cys positioning and gating solvent access to the active site. Intriguingly, bacterial CDOs lack the Cys-Tyr cross-link; yet, they are as active as cross-linked eukaryotic CDOs.The other known mammalian TDO is cysteamine dioxygenase (ADO). Initially, it was believed that ADO solely catalyzes the oxidation of cysteamine to hypotaurine. However, it has recently been shown that ADO additionally oxidizes N-terminal cysteine (Nt-Cys) peptides, which indicates that ADO may play a much more significant role in mammalian physiology than was originally anticipated. Though predicted on the basis of sequence alignment, site-directed mutagenesis, and spectroscopic studies, it was not until last year that two crystal structures, one of wild-type mouse ADO (solved by us) and the other of a variant of nickel-substituted human ADO, finally provided direct evidence that this enzyme also features a 3-His facial triad. These structures additionally revealed several features that are unique to ADO, including a putative cosubstrate O

Up to 40% of pheochromocytomas (PCCs) and paragangliomas (PGLs) are hereditary. Germline mutations/deletions in fumarate hydratase ( FH ) cause hereditary leiomyomatosis and renal cell carcinoma syndrome which manifests predominantly with FH-deficient uterine/cutaneous leiomyomas and renal cell carcinomas (RCCs)-tumors characterized by loss of immunohistochemical (IHC) expression of FH and/or positive staining for S-(2-succino)-cysteine. Occasional patients develop PCC/PGL. We investigated the incidence, morphologic, and clinical features of FH-deficient PCC/PGL. We identified 589 patients with PCC/PGLs that underwent IHC screening for FH and/or S-(2-succino)-cysteine. Eight (1.4%) PCC/PGLs were FH deficient (1.1% in an unselected population). The median age for FH-deficient cases was 55 (range: 30 to 77 y) with 50% arising in the adrenal. All 4 with biochemical data were noradrenergic. Two (25%) metastasized, 1 dying of disease after 174 months. Germline testing was performed on 7 patients, 6 of whom had FH missense mutations. None were known to have a significant family history before presentation or developed cutaneous leiomyomas, or FH-deficient RCC at extended follow-up. The patient wild-type for FH on germline testing was demonstrated to have somatic FH mutation and loss of heterozygosity corresponding to areas of subclonal FH deficiency in her tumor. One patient did not undergo germline testing, but FH mutation was demonstrated in his tumor. We conclude that FH-deficient PCC/PGL are underrecognized but can be identified by IHC. FH-deficient PCC/PGL are strongly associated with germline missense mutations but are infrequently associated with leiomyoma or RCC, suggesting there may be a genotype-phenotype correlation. FH-deficient PCC/PGL may have a higher metastatic risk.

Hypertrophic cardiomyopathy (HCM) is an underdiagnosed genetic heart disease worldwide. The management and prognosis of obstructive HCM (HOCM) and non-obstructive HCM (HNCM) are quite different, but it also remains challenging to discriminate these two subtypes. HCM is characterized by dysmetabolism, and myocardial amino acid (AA) metabolism is robustly changed. The present study aimed to delineate plasma AA and derivatives profiles, and identify potential biomarkers for HCM.Plasma samples from 166 participants, including 57 cases of HOCM, 52 cases of HNCM, and 57 normal controls (NCs), who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020, were collected and analyzed by high-performance liquid chromatography-mass spectrometry based on targeted AA metabolomics. Three separate classification algorithms, including random forest, support vector machine, and logistic regression, were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.The univariate analysis showed that the serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels in the HCM group were significantly different from those in the NC group. Four AAs and derivatives (Panel A; proline, glycine, cysteine, and choline) were screened out by multiple feature selection algorithms for discriminating HCM patients from NCs. The receiver operating characteristic (ROC) analysis in Panel A yielded an area under the ROC curve (AUC) of 0.83 (0.75-0.91) in the training set and 0.79 (0.65-0.94) in the validation set. Moreover, among 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between HOCM and HNCM, 3 AAs (Panel B; arginine, proline, and ornithine) were selected to differentiate the two subgroups. The AUC values in the training and validation sets for Panel B were 0.83 (0.74-0.93) and 0.82 (0.66-0.98), respectively.The plasma AA and derivatives profiles were distinct between the HCM and NC groups. Based on the differential profiles, the two established screening models have potential value in assisting HCM screening and identifying whether it is obstructive.

Once considered potential liabilities, the modern era witnesses a renaissance of interest in covalent inhibitors in drug discovery. The available toolbox of electrophilic warheads is limited by constraints on tuning reactivity and selectivity. Following our work on a class of ferroptotic agents termed CETZOLEs, we discovered new tunable heterocyclic electrophiles which are capable of inducing ferroptosis. The biological evaluation demonstrated that thiazoles with an alkyne electrophile at the 2-position selectively induce ferroptosis with high potency. Density functional theory calculations and NMR kinetic studies demonstrated the ability of our heterocycles to undergo thiol addition, an apparent prerequisite for cytotoxicity. Chemoproteomic analysis indicated several potential targets, the most prominent among them being GPX4 protein. These results were further validated by western blot analysis and the cellular thermal shift assay. Incorporation of these heterocycles into appropriate pharmacophores generated highly cytotoxic agents such as the analogue

A practically useful protocol for the asymmetric synthesis of artificial β-aryl-substituted cysteine derivatives was developed through sequential Pd(II)-catalyzed Heck cross-coupling with aryl iodides and hydrothiolation reaction with various alkyl thiols in the presence of triethylamine taking place in the ligand sphere of a robust and bench-stable chiral dehydroalanine Ni(II) complex. The subsequent acidic decomposition of the single diastereomeric Ni(II) complexes led to the target enantiopure cysteine derivatives.

Symbiotic partnerships with rhizobial bacteria enable legumes to grow without nitrogen fertilizer because rhizobia convert atmospheric nitrogen gas into ammonia via nitrogenase. After Sinorhizobium meliloti penetrate the root nodules that they have elicited in Medicago truncatula, the plant produces a family of about 700 nodule cysteine-rich (NCR) peptides that guide the differentiation of endocytosed bacteria into nitrogen-fixing bacteroids. The sequences of the NCR peptides are related to the defensin class of antimicrobial peptides, but have been adapted to play symbiotic roles. Using a variety of spectroscopic, biophysical and biochemical techniques, we show here that the most extensively characterized NCR peptide, 24 amino acid NCR247, binds haem with nanomolar affinity. Bound haem molecules and their iron are initially made biologically inaccessible through the formation of hexamers (6 haem/6 NCR247) and then higher-order complexes. We present evidence that NCR247 is crucial for effective nitrogen-fixing symbiosis. We propose that by sequestering haem and its bound iron, NCR247 creates a physiological state of haem deprivation. This in turn induces an iron-starvation response in rhizobia that results in iron import, which itself is required for nitrogenase activity. Using the same methods as for L-NCR247, we show that the D-enantiomer of NCR247 can bind and sequester haem in an equivalent manner. The special abilities of NCR247 and its D-enantiomer to sequester haem suggest a broad range of potential applications related to human health.