RESUMEN
Glutathione (GSH) is an important antioxidant that is generated and degraded via the GSH cycle. Quantification of the main components in the GSH cycle is necessary to evaluate the process of GSH. In this study, a robust ultra-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of 10 components (GSH; γ-glutamylcysteine; cysteinyl-glycine; n-acetylcysteine; homocysteine; cysteine; cystine; methionine; glutamate; pyroglutamic acid) in GSH cycle was developed. The approach was optimized in terms of derivative, chromatographic, and spectrometric conditions as well as sample preparation. The unstable thiol groups of GSH, γ-glutamylcysteine, cysteinyl-glycine, n-acetylcysteine, cysteine, and homocysteine were derivatized by n-ethylmaleimide. The derivatized and underivatized analytes were separated on an amino column with gradient elution. The method was further validated in terms of selectivity (no interference), linearity (R2 > 0.99), precision (% relative standard deviation [RSD%] range from 0.57 to 10.33), accuracy (% relative error [RE%] range from -3.42 to 10.92), stability (RSD% < 5.68, RE% range from -2.54 to 4.40), recovery (RSD% range from 1.87 to 7.87) and matrix effect (RSD% < 5.42). The validated method was applied to compare the components in the GSH cycle between normal and oxidative stress cells, which would be helpful in clarifying the effect of oxidative stress on the GSH cycle.
Asunto(s)
Glutatión , Espectrometría de Masas en Tándem , Espectrometría de Masas en Tándem/métodos , Glutatión/análisis , Cromatografía Líquida de Alta Presión/métodos , Humanos , Homocisteína/análisis , Cisteína/análisis , Ácido Pirrolidona Carboxílico/análisis , Ácido Pirrolidona Carboxílico/química , Ácido Pirrolidona Carboxílico/metabolismo , Dipéptidos/análisis , Acetilcisteína/análisis , Acetilcisteína/química , Cistina/análisisRESUMEN
Large-scale cell death is commonly observed during organismal development and in human pathologies1-5. These cell death events extend over great distances to eliminate large populations of cells, raising the question of how cell death can be coordinated in space and time. One mechanism that enables long-range signal transmission is trigger waves6, but how this mechanism might be used for death events in cell populations remains unclear. Here we demonstrate that ferroptosis, an iron- and lipid-peroxidation-dependent form of cell death, can propagate across human cells over long distances (≥5 mm) at constant speeds (around 5.5 µm min-1) through trigger waves of reactive oxygen species (ROS). Chemical and genetic perturbations indicate a primary role of ROS feedback loops (Fenton reaction, NADPH oxidase signalling and glutathione synthesis) in controlling the progression of ferroptotic trigger waves. We show that introducing ferroptotic stress through suppression of cystine uptake activates these ROS feedback loops, converting cellular redox systems from being monostable to being bistable and thereby priming cell populations to become bistable media over which ROS propagate. Furthermore, we demonstrate that ferroptosis and its propagation accompany the massive, yet spatially restricted, cell death events during muscle remodelling of the embryonic avian limb, substantiating its use as a tissue-sculpting strategy during embryogenesis. Our findings highlight the role of ferroptosis in coordinating global cell death events, providing a paradigm for investigating large-scale cell death in embryonic development and human pathologies.
Asunto(s)
Retroalimentación Fisiológica , Ferroptosis , Especies Reactivas de Oxígeno , Animales , Embrión de Pollo , Humanos , Cistina/metabolismo , Retroalimentación Fisiológica/fisiología , Ferroptosis/fisiología , Glutatión/metabolismo , Hierro/metabolismo , Peroxidación de Lípido , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Desarrollo Embrionario , Extremidades/embriologíaRESUMEN
Disrupted gut microbiota homeostasis is an important cause of inflammatory colitis. Studies have shown that effective supplementation with probiotics can maintain microbial homeostasis and alleviate colitis. Here, to increase the viability of probiotics in the harsh gastrointestinal environments and enable targeted delivery, a redox-sensitive selenium hyaluronic acid (HA-Se) hydrogel encapsulating probiotics was developed. HA was modified with selenocystamine dihydrochloride and crosslinked by an amide reaction to generate a redox-sensitive hydrogel with stable mechanical properties, a low hemolysis rate and satisfactory biocompatibility. The HA-Se hydrogel exhibited suitable sensitivity to 10 mM GSH or 100 µM H2O2. The encapsulation of Limosilactobacillus reuteri (LR) in the HA-Se hydrogel (HA-Se-LR) significantly increased the survival rate of the probiotics in simulated gastric and intestinal fluid. HA-Se-LR administration increased the survival rate of mice with dextran sulfate sodium (DSS)-induced colitis, significantly alleviated oxidative stress and inflammation, and increased the effect of LR on microbiota α diversity. These results indicate that the HA-Se hydrogel constructed in this study can be used as a delivery platform to treat colitis, expanding the targeted applications of the natural polymer HA in disease treatment and the administration of probiotics as drugs to alleviate disease symptoms.
Asunto(s)
Colitis , Cistamina , Sulfato de Dextran , Modelos Animales de Enfermedad , Ácido Hialurónico , Hidrogeles , Limosilactobacillus reuteri , Oxidación-Reducción , Probióticos , Animales , Ácido Hialurónico/química , Hidrogeles/química , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Ratones , Cistamina/química , Compuestos de Organoselenio/farmacología , Compuestos de Organoselenio/química , Estrés Oxidativo/efectos de los fármacos , Microbioma Gastrointestinal/efectos de los fármacos , Cistina/análogos & derivadosRESUMEN
SLC7A11 is a cystine transporter and ferroptosis inhibitor. How the stability of SLC7A11 is coordinately regulated in response to environmental cystine by which E3 ligase and deubiquitylase (DUB) remains elusive. Here, we report that neddylation inhibitor MLN4924 increases cystine uptake by causing SLC7A11 accumulation, via inactivating Cullin-RING ligase-3 (CRL-3). We identified KCTD10 as the substrate-recognizing subunit of CRL-3 for SLC7A11 ubiquitylation, and USP18 as SLC7A11 deubiquitylase. Upon cystine deprivation, the protein levels of KCTD10 or USP18 are decreased or increased, respectively, contributing to SLC7A11 accumulation. By destabilizing or stabilizing SLC7A11, KCTD10, or USP18 inversely regulates the cystine uptake and ferroptosis. Biologically, MLN4924 combination with SLC7A11 inhibitor Imidazole Ketone Erastin (IKE) enhanced suppression of tumor growth. In human breast tumor tissues, SLC7A11 levels were negatively or positively correlated with KCTD10 or USP18, respectively. Collectively, our study defines how SLC7A11 and ferroptosis is coordinately regulated by the CRL3KCTD10/E3-USP18/DUB axis, and provides a sound rationale of drug combination to enhance anticancer efficacy.
Asunto(s)
Cistina , Ferroptosis , Pirimidinas , Ubiquitina Tiolesterasa , Animales , Femenino , Humanos , Ratones , Sistema de Transporte de Aminoácidos y+/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Ciclopentanos/metabolismo , Ciclopentanos/farmacología , Cistina/metabolismo , Células HEK293 , Piperazinas/farmacología , Pirimidinas/farmacología , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Gestational diabetes mellitus (GDM) disrupts glucolipid metabolism, endangering maternal and fetal health. Despite limited research on its pathogenesis and treatments, we conducted a study using serum samples from GDM-diagnosed pregnant women. We performed metabolic sequencing to identify key small molecule metabolites and explored their molecular interactions with FGF21. We also investigated FGF21's impact on GDM using blood samples from affected women. Our analysis revealed a novel finding: elevated levels of L-Cystine in GDM patients. Furthermore, we observed a positive correlation between L-Cystine and FGF21 levels, and found that L-Cystine induces NRF2 expression via FGF21 for a period of 96 h. Under high glucose (HG) conditions, FGF21 upregulates NRF2 and downstream genes NQO1 and EPHX1 via AKT phosphorylation induced by activation of IRS1, enhancing endothelial function. Additionally, we confirmed that levels of FGF21, L-Cystine, and endothelial function at the third trimester were effectively enhanced through appropriate exercise and diet during pregnancy in GDM patients (GDM + ED). These findings suggest FGF21 as a potential therapeutic agent for GDM, particularly in protecting endothelial cells. Moreover, elevated L-Cystine via appropriate exercise and diet might be a potential strategy to enhance FGF21's efficacy.
Asunto(s)
Cistina , Diabetes Gestacional , Factores de Crecimiento de Fibroblastos , Factor 2 Relacionado con NF-E2 , Transducción de Señal , Humanos , Diabetes Gestacional/metabolismo , Femenino , Embarazo , Factores de Crecimiento de Fibroblastos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Adulto , Transducción de Señal/efectos de los fármacos , Cistina/metabolismoRESUMEN
PURPOSE: Although several potential radioprotectants have been explored, radiation esophagitis is still difficult to control. Further development of supportive therapies is required. Our purpose was to investigate the efficacy and safety of cystine and theanine for esophagitis in non-small cell lung cancer (NSCLC) patients undergoing chemoradiotherapy (CRT). METHODS: This study is a prospective observational study. The participants were recruited from unresectable locally advanced NSCLC who had scheduled to receive weekly paclitaxel or nab-paclitaxel/carboplatin plus radiation therapy (60 Gy in 30 fractions) for 6 weeks. They took an oral amino acid supplement containing 700 mg cystine and 280 mg theanine once daily regardless of CRT timing from the start of CRT until completion. The primary endpoint was the incidence of any grade esophagitis. The secondary endpoints were quality of life (QoL) and adverse events (AEs). RESULTS: A total of 26 patients were evaluated. All participants completed 60 Gy of RT in 30 fractions. The overall incidence of esophagitis was 73%; however, no ≥ grade 3 was reported. There were no AEs likely to be related to cystine and theanine. The mean EuroQoL 5-Dimension 5-Level health index score before and after chemoradiotherapy was 0.952 ± 0.0591 and 0.952 ± 0.0515 (P = 0.89), and the mean Visual Analogue Scale scores before and after treatment were 67.9 ± 15.4 and 79.4 ± 13.2 (P = 0.0047), respectively. CONCLUSION: Our study showed no severe esophagitis, any AEs, nor QoL decrease in NSCLC patients receiving CRT. Cystine and theanine are potentially effective to reduce severe CRT-induced esophagitis. TRIAL REGISTRATION: UMIN000052622, 26 October 2023, retrospectively registered.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Quimioradioterapia , Cistina , Esofagitis , Glutamatos , Neoplasias Pulmonares , Calidad de Vida , Humanos , Carcinoma de Pulmón de Células no Pequeñas/terapia , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Estudios Prospectivos , Masculino , Femenino , Esofagitis/etiología , Quimioradioterapia/efectos adversos , Quimioradioterapia/métodos , Persona de Mediana Edad , Neoplasias Pulmonares/radioterapia , Neoplasias Pulmonares/terapia , Anciano , Cistina/administración & dosificación , Cistina/análogos & derivados , Glutamatos/administración & dosificación , Glutamatos/efectos adversos , Glutamatos/uso terapéuticoRESUMEN
Cell models of mitochondrial complex â (Câ ) deficiency display significant elevations in reactive oxygen species (ROS) levels and an increase in cellular apoptosis. However, the underlying mechanisms governing anti-apoptotic processes in Câ -deficient cells remain elusive. Here, we introduced a mutation in NDUFS7, a crucial subunit of CI, in HEK293T cells and found that the absence of NDUFS7 resulted in reduced cell proliferation, elevated cell death, and increased susceptibility to oxidative stress. Mechanismly, we revealed that the upregulation of SLC7A11 played a crucial role in mitigating cell death resulting from NDUFS7 deficiency. Specifically, the increased expression of SLC7A11 enhanced cystine import, which subsequently reduced cell death by promoting the biosynthesis of reduced glutathione (GSH). Collectively, our findings suggest that SLC7A11-mediated cystine import, representing a novel pathway independent of NADPH production, plays a vital role in protection against NDUFS7 deficiency-induced cell death. This novel pathway provides potential insights into the understanding of pathogenic mechanisms and the therapeutic management of mitochondrial disorders associated with Câ deficiency.
Asunto(s)
Sistema de Transporte de Aminoácidos y+ , Cistina , Complejo I de Transporte de Electrón , Humanos , Sistema de Transporte de Aminoácidos y+/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Apoptosis , Muerte Celular , Cistina/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/deficiencia , Glutatión/metabolismo , Células HEK293 , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
While cysteine (CysSH) is known to be exported into the extracellular space, its biological significance is not well understood. The present study examined the movement of extracellular CysSH using stable isotope-labeled cystine (CysSSCys), which is transported into cells and reduced to CysSH. Exposure of HepG2 cells to 100 µM stable isotope-labeled CysSSCys resulted in 70 µM labeled CysSH in cell medium 1 h after CysSSCys exposure. When the cell medium was collected and incubated with either hydrogen peroxide (H2O2) or atmospheric electrophiles, such as 1,2-naphthoquinone, 1,4-naphthoquinone and 1,4-benzoquinone, CysSH in the cell medium was almost completely consumed. In contrast, extracellular levels of CysSH were unaltered during exposure of HepG2 cells to H2O2 for up to 2 h, suggesting redox cycling of CysSSCys/CysSH in the cell system. Experiments with and without changing cell medium containing CysSH from HepG2 cells revealed that oxidative and electrophilic modifications of cellular proteins, caused by exposure to H2O2 and 1,2-naphthoquinone, were significantly repressed by CysSH in the medium. We also examined participation of enzymes and/or antioxidants in intracellular reduction of CysSSCys to CysSH. These results provide new findings that extracellular CysSH derived from CysSSCys plays a role in the regulation of oxidative and electrophilic stress.
Asunto(s)
Cisteína , Cistina , Peróxido de Hidrógeno , Estrés Oxidativo , Humanos , Cisteína/metabolismo , Cisteína/farmacología , Células Hep G2 , Estrés Oxidativo/efectos de los fármacos , Cistina/metabolismo , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/metabolismo , Naftoquinonas/farmacología , Oxidación-Reducción , Benzoquinonas/farmacologíaRESUMEN
It has remained unknown how cells reduce cystine taken up from the extracellular space, which is a required step for further utilization of cysteine in key processes such as protein or glutathione synthesis. Here, we show that the thioredoxin-related protein of 14 kDa (TRP14, encoded by TXNDC17) is the rate-limiting enzyme for intracellular cystine reduction. When TRP14 is genetically knocked out, cysteine synthesis through the transsulfuration pathway becomes the major source of cysteine in human cells, and knockout of both pathways becomes lethal in C. elegans subjected to proteotoxic stress. TRP14 can also reduce cysteinyl moieties on proteins, rescuing their activities as here shown with cysteinylated peroxiredoxin 2. Txndc17 knockout mice were, surprisingly, protected in an acute pancreatitis model, concomitant with activation of Nrf2-driven antioxidant pathways and upregulation of transsulfuration. We conclude that TRP14 is the evolutionarily conserved enzyme principally responsible for intracellular cystine reduction in C. elegans, mice, and humans.
Asunto(s)
Caenorhabditis elegans , Cisteína , Cistina , Ratones Noqueados , Oxidación-Reducción , Proteoma , Tiorredoxinas , Animales , Humanos , Ratones , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Cisteína/metabolismo , Cistina/metabolismo , Peroxirredoxinas/metabolismo , Peroxirredoxinas/genética , Proteoma/metabolismo , Tiorredoxinas/metabolismo , Tiorredoxinas/genéticaRESUMEN
Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1' site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets.
Asunto(s)
Dominio Catalítico , Serina Peptidasa A1 que Requiere Temperaturas Altas , Péptidos , Serina Peptidasa A1 que Requiere Temperaturas Altas/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas/genética , Humanos , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Biblioteca de Péptidos , Cristalografía por Rayos X , Unión Proteica , Cistina/química , Cistina/metabolismo , Modelos MolecularesRESUMEN
Cysteine, the rate-controlling amino acid in cellular glutathione synthesis is imported as cystine, by the cystine/glutamate antiporter, xCT, and subsequently reduced to cysteine. As glutathione redox is important in muscle regeneration in aging, we hypothesized that xCT exerts upstream control over skeletal muscle glutathione redox, metabolism and regeneration. Bioinformatic analyses of publicly available datasets revealed that expression levels of xCT and GSH-related genes are inversely correlated with myogenic differentiation genes. Muscle satellite cells (MuSCs) isolated from Slc7a11sut/sut mice, which harbour a mutation in the Slc7a11 gene encoding xCT, required media supplementation with 2-mercaptoethanol to support cell proliferation but not myotube differentiation, despite persistently lower GSH. Slc7a11sut/sut primary myotubes were larger compared to WT myotubes, and also exhibited higher glucose uptake and cellular oxidative capacities. Immunostaining of myogenic markers (Pax7, MyoD, and myogenin) in cardiotoxin-damaged tibialis anterior muscle fibres revealed greater MuSC activation and commitment to differentiation in Slc7a11sut/sut muscle compared to WT mice, culminating in larger myofiber cross-sectional areas at 21 days post-injury. Slc7a11sut/sut mice subjected to a 5-week exercise training protocol demonstrated enhanced insulin tolerance compared to WT mice, but blunted muscle mitochondrial biogenesis and respiration in response to exercise training. Our results demonstrate that the absence of xCT inhibits cell proliferation but promotes myotube differentiation by regulating cellular metabolism and glutathione redox. Altogether, these results support the notion that myogenesis is a redox-regulated process and may help inform novel therapeutic approaches for muscle wasting and dysfunction in aging and disease.
Asunto(s)
Sistema de Transporte de Aminoácidos y+ , Diferenciación Celular , Metabolismo Energético , Glutatión , Músculo Esquelético , Oxidación-Reducción , Animales , Ratones , Glutatión/metabolismo , Músculo Esquelético/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Sistema de Transporte de Aminoácidos y+/genética , Desarrollo de Músculos , Células Satélite del Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Cistina/metabolismoRESUMEN
Cystinosis is a rare, autosomal recessive, lysosomal storage disease caused by mutations in the gene CTNS, leading to cystine accumulation in the lysosomes. While cysteamine lowers the cystine levels, it does not cure the disease, suggesting that CTNS exerts additional functions besides cystine transport. This study investigated the impact of infantile and juvenile CTNS mutations with discrepant genotype/phenotype correlations on CTNS expression, and subcellular localisation and function in clinically relevant cystinosis cell models to better understand the link between genotype and CTNS function. Using CTNS-depleted proximal tubule epithelial cells and patient-derived fibroblasts, we expressed a selection of CTNSmutants under various promoters. EF1a-driven expression led to substantial overexpression, resulting in CTNS protein levels that localised to the lysosomal compartment. All CTNSmutants tested also reversed cystine accumulation, indicating that CTNSmutants still exert transport activity, possibly due to the overexpression conditions. Surprisingly, even CTNSmutants expression driven by the less potent CTNS and EFS promoters reversed the cystine accumulation, contrary to the CTNSG339R missense mutant. Taken together, our findings shed new light on CTNS mutations, highlighting the need for robust assessment methodologies in clinically relevant cellular models and thus paving the way for better stratification of cystinosis patients, and advocating for the development of more personalized therapy.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Cistinosis , Humanos , Cistina/metabolismo , Cistinosis/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Cisteamina , Mutación/genéticaRESUMEN
Cysteine (Cys) and its oxidized form, cystine (Cys2), play crucial roles in biological systems and have considerable applications in cell culture. However, Cys in cell culture media is easily oxidized to Cys2, leading to solubility issues. Traditional analytical methods struggle to maintain the oxidation states of Cys and Cys2 during analysis, posing a significant challenge to accurately measuring and controlling these compounds. To effectively control the Cys and Cys2 levels, a rapid and accurate analytical method is required. Here, we screened derivatizing reagents that can react with Cys even under acidic conditions to realize a novel analytical method for simultaneously determining Cys and Cys2 levels. Diethyl 2-methylenemalonate (EMM) was found to possess the desired traits. EMM, characterized by its dual electron-withdrawing attributes, allowed for a rapid reaction with Cys under acidic conditions, preserving intact information for understanding the functions of target compounds. Combined with LC-MS/MS and an internal standard, this method provided high analytical accuracy in a short analytical time of 9 min. Using the developed method, the rapid oxidation of Cys in cell culture media was observed with the headspace of the storage container considerably influencing Cys oxidation and Cys2 precipitation rates. The developed method enabled the direct and simplified analysis of Cys behavior in practical media samples and could be used in formulating new media compositions, ensuring quality assurance, and real-time analysis of Cys and Cys2 in cell culture supernatants. This novel approach holds the potential to further enhance the media performance by enabling the timely optimal addition of Cys.
Asunto(s)
Medios de Cultivo , Cisteína , Cistina , Compuestos de Sulfhidrilo , Espectrometría de Masas en Tándem , Cromatografía Liquida/métodos , Química Clic , Medios de Cultivo/química , Cisteína/química , Cisteína/análisis , Cistina/química , Cistina/análogos & derivados , Cistina/análisis , Cromatografía Líquida con Espectrometría de Masas , Malonatos/química , Oxidación-Reducción , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/análisis , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Cystine-depleting therapy in nephropathic cystinosis is currently monitored via the white blood cell cystine assay, although its application and usefulness are limited by practical and technical issues. Therefore, alternative biomarkers that are widely available, more economical and less technically demanding, while reliably reflecting long-term adherence to cysteamine treatment, are desirable. Recently, we proposed chitotriosidase enzyme activity as a potential novel biomarker for the therapeutic monitoring of cysteamine treatment in cystinosis. In this study, we aimed to validate our previous findings and to confirm the value of chitotriosidase in the management of cystinosis therapy. MATERIALS & METHODS: A retrospective study was conducted on 12 patients treated at the National Institutes of Health Clinical Center and followed up for at least 2 years. Plasma chitotriosidase enzyme activity was correlated with corresponding clinical and biochemical data. RESULTS: Plasma chitotriosidase enzyme activity significantly correlated with WBC cystine levels, cysteamine total daily dosage and a Composite compliance score. Moreover, plasma chitotriosidase was a significant independent predictor for WBC cystine levels, and cut-off values were established in both non-kidney transplanted and kidney transplanted cystinosis patients to distinguish patients with a good versus poor compliance with cysteamine treatment. Our observations are consistent with those of our previous study and validate our findings. CONCLUSIONS: Chitotriosidase enzyme activity is a valid potential alternative biomarker for monitoring cysteamine treatment in nephropathic cystinosis patients. SYNOPSIS: Chitotriosidase enzyme activity is a valid potential alternative biomarker for monitoring cysteamine treatment in nephropathic cystinosis patients.
Asunto(s)
Cisteamina , Cistina , Cistinosis , Hexosaminidasas , Humanos , Cisteamina/uso terapéutico , Masculino , Femenino , Cistinosis/tratamiento farmacológico , Cistinosis/sangre , Estudios Retrospectivos , Hexosaminidasas/sangre , Adolescente , Cistina/sangre , Niño , Adulto , Biomarcadores/sangre , Adulto Joven , Monitoreo de Drogas/métodos , Depletores de Cistina/uso terapéutico , Preescolar , Trasplante de RiñónRESUMEN
Neurotoxicity is a common side effect of certain types of therapeutic drugs, posing a major hurdle for their clinical application. Accumulating evidence suggests that ferroptosis is involved in the neurotoxicity induced by these drugs. Therefore, targeting ferroptosis is considered to be a reasonable approach to prevent such side effect. Arctigenin (ATG) is a major bioactive ingredient of Arctium lappa L., a popular medicinal plant in Asia, and has been reported to have multiple bioactivities including neuroprotection. However, the mechanisms underlying the neuroprotection of ATG has not been well elucidated. The purpose of this study was to investigate whether the neuroprotection of ATG was associated with its ability to protect neuronal cells from ferroptosis. Using neuronal cell ferroptosis model induced by either classic ferroptosis induces or therapeutic drugs, we demonstrated for the first time that ATG in the nanomolar concentration range effectively prevented neuronal cell ferroptosis induced by classic ferroptosis inducer sulfasalazine (SAS) and erastin (Era), or therapeutic drug oxaliplatin (OXA) and 5-fluorouracil (5-FU). Mechanistically, we uncovered that the anti-ferroptotic effect of ATG was attributed to its ability to activate SLC7A11-cystine-cysteine axis. The findings of the present study implicate that ATG holds great potential to be developed as a novel agent for preventing SLC7A11 inhibition-mediated neurotoxicity.
Asunto(s)
Antineoplásicos , Ferroptosis , Furanos , Lignanos , Síndromes de Neurotoxicidad , Humanos , Cisteína , Cistina , Fluorouracilo , Antineoplásicos/farmacología , Sistema de Transporte de Aminoácidos y+RESUMEN
Cystinosis is a rare autosomal recessive disorder caused by mutations in the CTNS gene that encodes cystinosin, a ubiquitous lysosomal cystine/H+ antiporter. The hallmark of the disease is progressive accumulation of cystine and cystine crystals in virtually all tissues. At the kidney level, human cystinosis is characterized by the development of renal Fanconi syndrome and progressive glomerular and interstitial damage leading to end-stage kidney disease in the second or third decade of life. The exact molecular mechanisms involved in the pathogenesis of renal disease in cystinosis are incompletely elucidated. We have previously shown upregulation of NLRP2 in human cystinotic proximal tubular epithelial cells and its role in promoting inflammatory and profibrotic responses. Herein, we have investigated the role of NLRP2 in vivo using a mouse model of cystinosis in which we have confirmed upregulation of Nlrp2 in the renal parenchyma. Our studies show that double knock out Ctns-/- Nlrp2-/- animals exhibit delayed development of Fanconi syndrome and kidney tissue damage. Specifically, we observed at 4-6 months of age that animals had less glucosuria and calciuria and markedly preserved renal tissue, as assessed by significantly lower levels of inflammatory cell infiltration, tubular atrophy, and interstitial fibrosis. Also, the mRNA expression of some inflammatory mediators (Cxcl1 and Saa1) and the rate of apoptosis were significantly decreased in 4-6-month old kidneys harvested from Ctns-/- Nlrp2-/- mice compared to those obtained from Ctns-/-mice. At 12-14 months of age, renal histological was markedly altered in both genetic models, although double KO animals had lower degree of polyuria and low molecular weight proteinuria and decreased mRNA expression levels of Il6 and Mcp1. Altogether, these data indicate that Nlrp2 is a potential pharmacological target for delaying progression of kidney disease in cystinosis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Cistinosis , Enfermedades Renales , Animales , Cistina/metabolismo , Cistinosis/genética , Cistinosis/metabolismo , Cistinosis/patología , Riñón/patología , Enfermedades Renales/patología , ARN Mensajero , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Modelos Animales de Enfermedad , RatonesRESUMEN
The engineered human cystathionine-γ-lyase (hCGL) resulting in enhanced activity toward both cysteine and cystine unveils a potential robust antitumor activity. However, the presence of cysteine residues has the potential to induce oligomerization or incorrect disulfide bonding, which may decrease the bioavailability of biopharmaceuticals. Through a meticulous design process targeting the cysteine residues within engineered hCGL, a set of potential beneficial mutants were obtained by virtual screening employing Rosetta and ABACUS. Experimental measurements have revealed that most of the mutants showed increased activity toward both substrates l-Cys and CSSC. Furthermore, mutants C109V and C229D demonstrated Tm value increases of 8.2 and 1.8 °C, respectively. After an 80 min incubation at 60 °C, mutant C229D still maintained high residual activity. Unexpectedly, mutant C109V, displaying activity approximately 2-fold higher than the activity of wild type (WT) for both substrates, showed disappointing instability in plasma, which suggests that computational design still requires further consideration. Analysis of their structure and molecular dynamics (MD) simulation revealed the impact of hydrophobic interaction, hydrogen bonds, and near-attack conformation (NAC) stability on activity and stability. This study acquired information about mutants that exhibit enhanced activity or thermal resistance and serve as valuable guidance for subsequent specific cysteine modifications.
Asunto(s)
Cistationina gamma-Liasa , Cisteína , Simulación de Dinámica Molecular , Ingeniería de Proteínas , Cisteína/química , Cisteína/metabolismo , Humanos , Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/química , Cistationina gamma-Liasa/metabolismo , Estabilidad de Enzimas , Cistina/química , Enlace de Hidrógeno , Mutación , CinéticaRESUMEN
Here, we report a proof-of-concept resistive pulse method for analyzing chiral amino acids utilizing metal-amino acid crystallization differences. This method involves introducing an amino acid sample solution into a micropipette through a pressure-driven flow. The sample then mixes with a metal ion solution inside the pipette, forming metal-amino acid crystals. The crystal size depends on the enantiomeric excess (x) of chiral amino acid samples. Large x values lead to large crystals. The crystal size difference is then reflected in the resistive pulse size as they block the ionic transport in a micropipette to different extents. We used Cd-cystine crystallization as a model system and found approximately five times the mean current pulse size difference for racemic (x = 0) and L-only (x = +1) cystine samples. A similar result was observed for aspartate. Our discovery opens up new opportunities for micro/nanoscopic chiral amino acid analysis, which can potentially be used in single-cell analysis.
Asunto(s)
Aminoácidos , Cristalización , Estereoisomerismo , Aminoácidos/química , Cistina/química , Cadmio/química , Ácido Aspártico/química , Metales/químicaRESUMEN
The induction of ferroptosis is promising for cancer therapy. However, the mechanisms enabling cancer cells to evade ferroptosis, particularly in low-cystine environments, remain elusive. Our study delves into the intricate regulatory mechanisms of Activating transcription factor 3 (ATF3) on Cystathionine ß-synthase (CBS) under cystine deprivation stress, conferring resistance to ferroptosis in colorectal cancer (CRC) cells. Additionally, our findings establish a positively correlation between this signaling axis and CRC progression, suggesting its potential as a therapeutic target. Mechanistically, ATF3 positively regulates CBS to resist ferroptosis under cystine deprivation stress. In contrast, the suppression of CBS sensitizes CRC cells to ferroptosis through targeting the mitochondrial tricarboxylic acid (TCA) cycle. Notably, our study highlights that the ATF3-CBS signaling axis enhances ferroptosis-based CRC cancer therapy. Collectively, the findings reveal that the ATF3-CBS signaling axis is the primary feedback pathway in ferroptosis, and blocking this axis could be a potential therapeutic approach for colorectal cancer.
Asunto(s)
Neoplasias Colorrectales , Ferroptosis , Humanos , Cistationina betasintasa/metabolismo , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Ferroptosis/genética , Cistina , Carcinogénesis/genética , Transformación Celular Neoplásica , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismoRESUMEN
The effects of cysteine (Cys), glutathione (GSH) and cystine (GCys) on sulfides and meaty aroma were studied based on concentration monitoring and metabolomics. In multi-component models, Cys and GSH demonstrated a greater capacity to decrease dimethyl trisulfide (DMTS) levels and increase the proportion of 2-methyl-3-furanthiol (MFT), compared with GCys. Moreover, no discernible difference between Cys and GSH in dynamic profiles of volatiles to further analyze the synergistic effect of both. Results of single factor experiment and optimization revealed that the optimal thermal processing was a second-order thermal procedure. Aroma profiles revealed that the addition of Cys and GSH mixture increased the meaty intensity during the optimal thermal processing. Metabolomics based on Encyclopedia of Genes and Genomes pathway annotation confirmed that Cys and GSH significantly affected the degradation of methionine and thiamine in amino acid and protein metabolic pathways, resulting in various amounts of DMTS and MFT. Research on effect and potentially metabolic mechanisms revealed that the combination of Cys and GSH at ratio of 3:7 had higher and more effective control capacity for free radical reaction of sulfides than either one alone during second-order thermal processing, which would lay theoretical foundation for the development of high-quality thermal process products.