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1.
Eur Rev Med Pharmacol Sci ; 28(4): 1471-1479, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38436181

RESUMO

OBJECTIVE: Thiols are organic compounds containing sulfhydryl groups that exert antioxidant effects via dynamic thiol-disulfide homeostasis. The shift towards disulfide indicates the presence of an oxidative environment. Different modes of delivery can affect thiol-disulfide homeostasis. Accordingly, we planned this research to evaluate the effects of the mode of delivery on thiol-disulfide homeostasis in both maternal serum and fetal cord blood samples. PATIENTS AND METHODS: We conducted a prospective case-control study involving two groups: vaginal delivery (n=50) and elective cesarean section (CS) (n=45). The vaginal delivery group exclusively comprised uncomplicated term deliveries, while the CS group included pregnant individuals with scheduled cesarean deliveries due to the absence of spontaneous labor onset. Maternal serum and fetal cord blood samples were collected, and thiol-disulfide exchanges were analyzed using an automated method capable of measuring both aspects of the thiol-disulfide balance. RESULTS: The levels of native thiol (-SH) and total thiol in both maternal serum and fetal cord blood samples were significantly higher in the vaginal delivery group than those in the CS group. An important discovery of our study was that fetal cord disulfide (-SS) level, which may reflect oxidative stress, was higher in newborns born via vaginal delivery when examined alone. However, in both maternal and fetal cord blood, the combined ratios, SS/SH ratio (%), SS/Total thiol ratio (%), and SH/Total thiol ratio (%) were observed to be similar between the groups in both maternal and fetal cord blood. It was observed that as the mother's weight gained during pregnancy increased, SS/SH and SS/total thiol increased (positive correlation), while SH/total thiol decreased (negative correlation). CONCLUSIONS: Our results showed that the dynamic thiol-disulfide homeostasis was greatly influenced by the way of delivery and supported the idea that vaginally-delivered infants may have more oxidative stress.


Assuntos
Cesárea , Parto Obstétrico , Recém-Nascido , Gravidez , Lactente , Humanos , Feminino , Estudos de Casos e Controles , Dissulfetos , Homeostase , Estresse Oxidativo , Compostos de Sulfidrila
2.
Methods Mol Biol ; 2778: 101-115, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478274

RESUMO

Membrane-embedded ß-barrels are the major building blocks of the Gram-negative outer membrane and are involved in antibiotic resistance, virulence, and the maintenance of bacterial cell physiology. The increased frequency of multidrug resistant Gram-negative infections warrants the sharing of accessible methods for the study of ß-barrels. One such method is "in vivo disulfide-bond crosslinking" which is a highly informative and cost-effective approach to study the structure, topology, dynamicity, and function of ß-barrels in situ. The approach can also be used to identify and finely map both stable or transient interactions between ß-barrels and other interacting proteins. In this chapter, I describe the conceptual basis of in vivo disulfide-bond crosslinking and the potential pitfalls in experimental design. I also provide a general protocol for high-efficiency in vivo disulfide-bond crosslinking and modified protocols as examples for how the method can be adapted to different scenarios.


Assuntos
Proteínas da Membrana Bacteriana Externa , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/metabolismo , Modelos Moleculares , Bactérias/metabolismo , Dissulfetos
3.
Metallomics ; 16(3)2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38425033

RESUMO

The tuberculosis (TB) emergency has been a pressing health threat for decades. With the emergence of drug-resistant TB and complications from the COVID-19 pandemic, the TB health crisis is more serious than ever. Mycobacterium tuberculosis (Mtb), the causative agent of TB, requires iron for its survival. Thus, Mtb has evolved several mechanisms to acquire iron from the host. Mtb produces two siderophores, mycobactin and carboxymycobactin, which scavenge for host iron. Mtb siderophore-dependent iron acquisition requires the export of apo-siderophores from the cytosol to the host environment and import of iron-bound siderophores. The export of Mtb apo-siderophores across the inner membrane is facilitated by two mycobacterial inner membrane proteins with their cognate periplasmic accessory proteins, designated MmpL4/MmpS4 and MmpL5/MmpS5. Notably, the Mtb MmpL4/MmpS4 and MmpL5/MmpS5 complexes have also been implicated in the efflux of anti-TB drugs. Herein, we solved the crystal structure of M. thermoresistibile MmpS5. The MmpS5 structure reveals a previously uncharacterized, biologically relevant disulfide bond that appears to be conserved across the Mycobacterium MmpS4/S5 homologs, and comparison with structural homologs suggests that MmpS5 may be dimeric.


Assuntos
Mycobacteriaceae , Mycobacterium tuberculosis , Tuberculose , Humanos , Pandemias , Mycobacterium tuberculosis/metabolismo , Tuberculose/microbiologia , Sideróforos/metabolismo , Ferro/metabolismo , Dissulfetos/metabolismo , Proteínas de Bactérias/metabolismo
4.
Helicobacter ; 29(2): e13064, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38459689

RESUMO

BACKGROUND: Helicobacter pylori (H. pylori) infection is the most extensively studied risk factor for gastric cancer. As with any bacteria, H. pylori will release distinctive odors that result from an emission of volatile metabolic byproducts in unique combinations and proportions. Effectively capturing and identifying these volatiles can pave the way for the development of innovative and non-invasive diagnostic methods for determining infection. Here we characterize the H. pylori volatilomic signature, pinpoint potential biomarkers of its presence, and evaluate the variability of volatilomic signatures between different H. pylori isolates. MATERIALS AND METHODS: Using needle trap extraction, volatiles in the headspace above H. pylori cultures were collected and, following thermal desorption at 290°C in a splitless mode, were analyzed using gas chromatography-mass spectrometry. The resulting volatilomic signatures of H. pylori cultures were compared to those obtained from an analysis of the volatiles in the headspace above the cultivating medium only. RESULTS: Amongst the volatiles detected, 21 showed consistent differences between the bacteria cultures and the cultivation medium, with 11 compounds being elevated and 10 showing decreased levels in the culture's headspace. The 11 elevated volatiles are four ketones (2-pentanone, 5-methyl-3-heptanone, 2-heptanone, and 2-nonanone), three alcohols (2-methyl-1-propanol, 3-methyl-1-butanol, and 1 butanol), one aromatic (styrene), one aldehyde (2-ethyl-hexanal), one hydrocarbon (n-octane), and one sulfur compound (dimethyl disulfide). The 10 volatiles with lower levels in the headspace of the cultures are four aldehydes (2-methylpropanal, benzaldehyde, 3-methylbutanal, and butanal), two heterocyclic compounds (2-ethylfuran and 2-pentylfuran), one ketone (2-butanone), one aromatic (benzene), one alcohol (2-butanol) and bromodichloromethane. Of the volatile species showing increased levels, the highest emissions are found to be for 3-methyl-1-butanol, 1-butanol and dimethyl disulfide. Qualitative variations in their emissions from the different isolates was observed. CONCLUSIONS: The volatiles emitted by H. pylori provide a characteristic volatilome signature that has the potential of being developed as a tool for monitoring infections caused by this pathogen. Furthermore, using the volatilome signature, we are able to differentiate different isolates of H. pylori. However, the volatiles also represent potential confounders for the recognition of gastric cancer volatile markers.


Assuntos
Dissulfetos , Infecções por Helicobacter , Helicobacter pylori , Pentanóis , Neoplasias Gástricas , Humanos , Álcoois
5.
Mikrochim Acta ; 191(4): 190, 2024 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-38460000

RESUMO

Golgi protein 73 (GP73) is a new serum marker associated with early diagnosis and postoperative assessment of hepatocellular carcinoma (HCC). Herein, an electrochemical/fluorescence dual-signal biosensor was designed for determination of GP73 based on molybdenum disulfide/ferrocene/palladium nanoparticles (MoS2-Fc-PdNPs) and nitrogen-doped graphene quantum dots (NGQDs). GP73 aptamer (Apt) was labeled with NGQDs to form the NGQDs-Apt fluorescence probe. MoS2-Fc-PdNPs served not only as the fluorescence quencher but also as electrochemical enhancer. The sensing platform (NGQDs-Apt/MoS2-Fc-PdNPs) was formed based on the fluorescence resonance energy transfer (FRET) mechanism. In the presence of GP73, the specific binding of NGQDs-Apt to GP73 interrupted FRET, restoring the fluorescence of NGQDs-Apt at λex/em = 348/438 nm and enhancing the oxidation current of Fc in MoS2-Fc-PdNPs at 0.04 V through differential pulse voltammetry (DPV). Under the optimal conditions, the DPV current change and fluorescence recovery have a good linear relationship with GP73 concentration from 1.00 to 10.0 ng/mL. The calibration equation for the fluorescence mode was Y1 = (0.0213 ± 0.00127)X + (0.0641 ± 0.00448) and LOD was 0.812 ng/mL (S/N = 3). The calibration equation of the electrochemical mode was Y2 = (3.41 ± 0.111)X + (1.62 ± 0.731), and LOD of 0.0425 ng/mL (S/N = 3). The RSDs of fluorescence mode and electrochemical mode after serum detection were 1.62 to 5.21% and 0.180 to 6.62%, respectively. By combining the electrochemical and fluorescence assay, more comprehensive and valuable information for GP73 was provided. Such dual-mode detection platform shows excellent reproducibility, stability, and selectivity and has great application potential.


Assuntos
Carcinoma Hepatocelular , Dissulfetos , Grafite , Neoplasias Hepáticas , Nanopartículas Metálicas , Pontos Quânticos , Humanos , Molibdênio , Paládio , Nitrogênio , Reprodutibilidade dos Testes , Metalocenos
6.
ACS Nano ; 18(11): 7945-7958, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38452275

RESUMO

Ferroptosis is a form of regulated cell death accompanied by lipid reactive oxygen species (ROS) accumulation in an iron-dependent manner. However, the efficiency of tumorous ferroptosis was seriously restricted by intracellular ferroptosis defense systems, the glutathione peroxidase 4 (GPX4) system, and the ubiquinol (CoQH2) system. Inspired by the crucial role of mitochondria in the ferroptosis process, we reported a prodrug nanoassembly capable of unleashing potent mitochondrial lipid peroxidation and ferroptotic cell death. Dihydroorotate dehydrogenase (DHODH) inhibitor (QA) was combined with triphenylphosphonium moiety through a disulfide-containing linker to engineer well-defined nanoassemblies (QSSP) within a single-molecular framework. After being trapped in cancer cells, the acidic condition provoked the structural disassembly of QSSP to liberate free prodrug molecules. The mitochondrial membrane-potential-driven accumulation of the lipophilic cation prodrug was delivered explicitly into the mitochondria. Afterward, the thiol-disulfide exchange would occur accompanied by downregulation of reduced glutathione levels, thus resulting in mitochondria-localized GPX4 inactivation for ferroptosis. Simultaneously, the released QA from the hydrolysis reaction of the adjacent ester bond could further devastate mitochondrial defense and evoke robust ferroptosis via the DHODH-CoQH2 system. This subcellular targeted nanoassembly provides a reference for designing ferroptosis-based strategy for efficient cancer therapy through interfering antiferroptosis systems.


Assuntos
Ferroptose , Compostos Organofosforados , Pró-Fármacos , Pró-Fármacos/farmacologia , Pró-Fármacos/metabolismo , Di-Hidro-Orotato Desidrogenase , Peroxidação de Lipídeos , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Dissulfetos/metabolismo
7.
NPJ Biofilms Microbiomes ; 10(1): 30, 2024 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-38521769

RESUMO

Biofilms are surface-associated communities of bacteria that grow in a self-produced matrix of polysaccharides, proteins, and extracellular DNA (eDNA). Sub-minimal inhibitory concentrations (sub-MIC) of antibiotics induce biofilm formation, potentially as a defensive response to antibiotic stress. However, the mechanisms behind sub-MIC antibiotic-induced biofilm formation are unclear. We show that treatment of Pseudomonas aeruginosa with multiple classes of sub-MIC antibiotics with distinct targets induces biofilm formation. Further, addition of exogenous eDNA or cell lysate failed to increase biofilm formation to the same extent as antibiotics, suggesting that the release of cellular contents by antibiotic-driven bacteriolysis is insufficient. Using a genetic screen for stimulation-deficient mutants, we identified the outer membrane porin OprF and the ECF sigma factor SigX as important. Similarly, loss of OmpA - the Escherichia coli OprF homolog - prevented sub-MIC antibiotic stimulation of E. coli biofilms. Our screen also identified the periplasmic disulfide bond-forming enzyme DsbA and a predicted cyclic-di-GMP phosphodiesterase encoded by PA2200 as essential for biofilm stimulation. The phosphodiesterase activity of PA2200 is likely controlled by a disulfide bond in its regulatory domain, and folding of OprF is influenced by disulfide bond formation, connecting the mutant phenotypes. Addition of reducing agent dithiothreitol prevented sub-MIC antibiotic biofilm stimulation. Finally, activation of a c-di-GMP-responsive promoter follows treatment with sub-MIC antibiotics in the wild-type but not an oprF mutant. Together, these results show that antibiotic-induced biofilm formation is likely driven by a signaling pathway that translates changes in periplasmic redox state into elevated biofilm formation through increases in c-di-GMP.


Assuntos
Antibacterianos , Infecções por Pseudomonas , Humanos , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Pseudomonas aeruginosa/fisiologia , Escherichia coli/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Diester Fosfórico Hidrolases , Dissulfetos/metabolismo
8.
Protein Sci ; 33(4): e4949, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38511500

RESUMO

Primary defects in folding of mutant proinsulin can cause dominant-negative proinsulin accumulation in the endoplasmic reticulum (ER), impaired anterograde proinsulin trafficking, perturbed ER homeostasis, diminished insulin production, and ß-cell dysfunction. Conversely, if primary impairment of ER-to-Golgi trafficking (which also perturbs ER homeostasis) drives misfolding of nonmutant proinsulin-this might suggest bi-directional entry into a common pathological phenotype (proinsulin misfolding, perturbed ER homeostasis, and deficient ER export of proinsulin) that can culminate in diminished insulin storage and diabetes. Here, we've challenged ß-cells with conditions that impair ER-to-Golgi trafficking, and devised an accurate means to assess the relative abundance of distinct folded/misfolded forms of proinsulin using a novel nonreducing SDS-PAGE/immunoblotting protocol. We confirm abundant proinsulin misfolding upon introduction of a diabetogenic INS mutation, or in the islets of db/db mice. Whereas blockade of proinsulin trafficking in Golgi/post-Golgi compartments results in intracellular accumulation of properly-folded proinsulin (bearing native disulfide bonds), impairment of ER-to-Golgi trafficking (regardless whether such impairment is achieved by genetic or pharmacologic means) results in decreased native proinsulin with more misfolded proinsulin. Remarkably, reversible ER-to-Golgi transport defects (such as treatment with brefeldin A or cellular energy depletion) upon reversal quickly restore the ER folding environment, resulting in the disappearance of pre-existing misfolded proinsulin while preserving proinsulin bearing native disulfide bonds. Thus, proper homeostatic balance of ER-to-Golgi trafficking is linked to a more favorable proinsulin folding (as well as trafficking) outcome.


Assuntos
Diabetes Mellitus , Células Secretoras de Insulina , Camundongos , Animais , Proinsulina/genética , Proinsulina/química , Dobramento de Proteína , Insulina/química , Retículo Endoplasmático , Homeostase , Dissulfetos/química
9.
ACS Sens ; 9(3): 1410-1418, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38456391

RESUMO

Dimethyl disulfide (DMDS) is a common odor pollutant with an extremely low olfactory threshold. Highly sensitive and selective detection of DMDS in ambient humid air background, by metal oxide semiconductor (MOS) sensors, is highly desirable to address the increased public concern for health risk. However, it has still been a critical challenge up to now. Herein, p-type delafossite CuGaO2 has been proposed as a promising DMDS sensing material owing to its striking hydrophobicity (revealed by water contact angle measurement) and excellent partial catalytic oxidation properties (indicated by mass spectroscopy). The present CuGaO2 sensor shows a selective DMDS response, with satisfied humidity resistance performance and long-term stability at a relatively low operation temperature of 140 °C. An ultrahigh response of 100 to 10 ppm DMDS and a low limit of detection of 3.3 ppb could be achieved via a pulsed temperature modulation strategy. A smart sensing system based on a CuGaO2 sensor has been developed, which could precisely monitor DMDS vapor in ambient humid air, even with the presence of multiple interfering gases, demonstrating the practical application capability of MOS sensors for environmental odor monitoring.


Assuntos
Dissulfetos , Gases , Óxidos/química , Temperatura
10.
J Neuroinflammation ; 21(1): 70, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38515139

RESUMO

Myeloperoxidase (MPO) plays critical role in the pathology of cerebral ischemia-reperfusion (I/R) injury via producing hypochlorous acid (HOCl) and inducing oxidative modification of proteins. High-mobility group box 1 (HMGB1) oxidation, particularly disulfide HMGB1 formation, facilitates the secretion and release of HMGB1 and activates neuroinflammation, aggravating cerebral I/R injury. However, the cellular sources of MPO/HOCl in ischemic brain injury are unclear yet. Whether HOCl could promote HMGB1 secretion and release remains unknown. In the present study, we investigated the roles of microglia-derived MPO/HOCl in mediating HMGB1 translocation and secretion, and aggravating the brain damage and blood-brain barrier (BBB) disruption in cerebral I/R injury. In vitro, under the co-culture conditions with microglia BV cells but not the single culture conditions, oxygen-glucose deprivation/reoxygenation (OGD/R) significantly increased MPO/HOCl expression in PC12 cells. After the cells were exposed to OGD/R, MPO-containing exosomes derived from BV2 cells were released and transferred to PC12 cells, increasing MPO/HOCl in the PC12 cells. The HOCl promoted disulfide HMGB1 translocation and secretion and aggravated OGD/R-induced apoptosis. In vivo, SD rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) plus different periods of reperfusion. Increased MPO/HOCl production was observed at the reperfusion stage, accomplished with enlarged infarct volume, aggravated BBB disruption and neurological dysfunctions. Treatment of MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH) and HOCl scavenger taurine reversed those changes. HOCl was colocalized with cytoplasm transferred HMGB1, which was blocked by taurine in rat I/R-injured brain. We finally performed a clinical investigation and found that plasma HOCl concentration was positively correlated with infarct volume and neurological deficit scores in ischemic stroke patients. Taken together, we conclude that ischemia/hypoxia could activate microglia to release MPO-containing exosomes that transfer MPO to adjacent cells for HOCl production; Subsequently, the production of HOCl could mediate the translocation and secretion of disulfide HMGB1 that aggravates cerebral I/R injury. Furthermore, plasma HOCl level could be a novel biomarker for indexing brain damage in ischemic stroke patients.


Assuntos
Lesões Encefálicas , Isquemia Encefálica , Proteína HMGB1 , AVC Isquêmico , Traumatismo por Reperfusão , Humanos , Ratos , Animais , Ácido Hipocloroso , Microglia/metabolismo , Proteína HMGB1/metabolismo , Ratos Sprague-Dawley , Lesões Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Barreira Hematoencefálica/metabolismo , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/patologia , Neurônios/metabolismo , Traumatismo por Reperfusão/metabolismo , Peroxidase/metabolismo , Taurina , Dissulfetos
11.
Int J Biol Sci ; 20(3): 1042-1044, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38322120

RESUMO

Disulfidptosis occurs as a result of the accumulation of intracellular cystine followed by disulfide stress in actin cytoskeleton proteins due to a reduction of NADPH produced through the pentose phosphate pathway in cells with high expression of SLC7A11. It is a cell death caused by the redox imbalance resulting from the disruption of amino acid metabolism and glucose metabolism. The discovery of disulfidptosis has sparked immense enthusiasm, but there are numerous unresolved issues that need to be addressed. Solutions to these riddles will provide insights into the detailed mechanisms and the pathophysiological relevance of disulfidptosis and utilizing disulfidptosis as an actionable therapeutic target.


Assuntos
Dissulfetos , Proteínas dos Microfilamentos , Morte Celular , NADP
12.
J Mater Chem B ; 12(9): 2334-2345, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38327236

RESUMO

Intracellular bacteria often lead to chronic and recurrent infections; however, most of the known antibiotics have poor efficacy against intracellular bacteria due to their poor cell membrane penetration efficiency into the cytosol. Here, a thiol-mediated nanodrug delivery system, named Van-DM NPs, was developed to improve vancomycin's penetration efficiency and intracellular antibacterial activities. Van-DM NPs were prepared through self-assembly of vancomycin (Van) and the disulfide molecule (DM) in NaOH buffer solution. On the one hand, the disulfide exchange reaction between Van-DM NPs and the bacterial surface enhances vancomycin accumulation in bacteria, increasing the local concentration of vancomycin. On the other hand, the disulfide exchange reaction between Van-DM NPs and the mammalian cell membrane triggered the translocation of Van-DM NPs across the mammalian cell membrane into the cell cytosol. These dual mechanisms promote antibacterial activities of vancomycin against both extracellular and intracellular bacteria S. aureus. Furthermore, in an intravenous S. aureus infection mouse model, Van-DM NPs exhibited high antibacterial capability and efficiently reduced the bacterial load in liver and spleen, where intracellular bacteria tend to reside. Altogether, the reported Van-DM NPs would be highly promising against intracellular pathogenic infections.


Assuntos
Nanopartículas , Vancomicina , Animais , Camundongos , Vancomicina/farmacologia , Staphylococcus aureus , Dissulfetos , Antibacterianos/farmacologia , Bactérias , Mamíferos
13.
J Am Chem Soc ; 146(8): 5204-5214, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38358897

RESUMO

We report piperazine-fused six-membered-cyclic disulfides as redox substrates that unlock best-in-class bioreduction probes for live cell biology, since their self-immolation after reduction is unprecedentedly rapid. We develop scalable, diastereomerically pure, six-step syntheses that access four key cis- and trans-piperazine-fused cyclic dichalcogenides without chromatography. Fluorogenic redox probes using the disulfide piperazines are activated >100-fold faster than the prior art monoamines, allowing us to deconvolute reduction and cyclization rates during activation. The cis- and trans-fused diastereomers have remarkably different reductant specificities, which we trace back to piperazine boat/chair conformation effects: the cis-fused disulfide C-DiThia is activated only by strong vicinal dithiol reductants, but the trans-disulfide T-DiThia is activated even by moderate concentrations of monothiols such as GSH. Thus, in cellular applications, cis-disulfide probes selectively report on the reductive activity of the powerful thioredoxin proteins, while trans-disulfides are rapidly but promiscuously reactive. Finally, we showcase late-stage diversifications of the piperazine-disulfides, promising their broad applicability as redox-cleavable cores for probes and prodrugs that interface powerfully with cellular thiol/disulfide redox biology, for solid phase synthesis and purification, and for stimulus-responsive linkers in bifunctional reagents and antibody-drug conjugates - in addition to their dithiols' potential as high-performance reducing agents.


Assuntos
Dissulfetos , Compostos de Sulfidrila , Dissulfetos/química , Compostos de Sulfidrila/química , Reagentes de Ligações Cruzadas , Piperazina , Tiorredoxinas/metabolismo , Oxirredução , Biologia
14.
Nat Commun ; 15(1): 1733, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38409212

RESUMO

Glutaredoxins catalyze the reduction of disulfides and are key players in redox metabolism and regulation. While important insights were gained regarding the reduction of glutathione disulfide substrates, the mechanism of non-glutathione disulfide reduction remains highly debated. Here we determined the rate constants for the individual redox reactions between PfGrx, a model glutaredoxin from Plasmodium falciparum, and redox-sensitive green fluorescent protein 2 (roGFP2), a model substrate and versatile tool for intracellular redox measurements. We show that the PfGrx-catalyzed oxidation of roGFP2 occurs via a monothiol mechanism and is up to three orders of magnitude faster when roGFP2 and PfGrx are fused. The oxidation kinetics of roGFP2-PfGrx fusion constructs reflect at physiological GSSG concentrations the glutathionylation kinetics of the glutaredoxin moiety, thus allowing intracellular structure-function analysis. Reduction of the roGFP2 disulfide occurs via a monothiol mechanism and involves a ternary complex with GSH and PfGrx. Our study provides the mechanistic basis for understanding roGFP2 redox sensing and challenges previous mechanisms for protein disulfide reduction.


Assuntos
Glutarredoxinas , Glutationa , Proteínas de Fluorescência Verde/metabolismo , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Oxirredução , Dissulfetos/metabolismo , Catálise , Dissulfeto de Glutationa/metabolismo
15.
Anim Sci J ; 95(1): e13917, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38323750

RESUMO

Allicin is a sulfur-containing compound extracted from raw garlic (Allium sativum L.). We compared the effect of allicin addition on growth performance, serum biochemical parameters, and rumen microbiota of goats compared to monensin. Twenty-four Anhui white goats were assigned randomly to one of three dietary treatments: 1) a basal diet (CON); 2) the basal diet with allicin addition at 750 mg per head per day (AC); 3) the basal diet with monensin addition at 30 mg per kg of diet (MS). Animals were fed for 8 weeks. Results showed the average daily gain, and feed efficiency was increased with allicin and monensin addition. Serum levels of IgG, total superoxide dismutase, and glutathione peroxidase were higher in the AC group than those in the CON and MS groups. The microbiota analysis revealed that monensin addition mainly affected genera related to carbohydrate and protein metabolism, and allicin mainly affected genera related to energy metabolism and intestinal health. In conclusion, allicin could improve growth performance and have advantages over monensin in improving the antioxidant capacity and immune function of goats. Allicin may be a potential alternative to monensin.


Assuntos
Dissulfetos , Alho , Microbiota , Ácidos Sulfínicos , Animais , Ração Animal/análise , Antioxidantes/metabolismo , Dieta/veterinária , Suplementos Nutricionais/análise , Cabras/metabolismo , Monensin/farmacologia , Rúmen/metabolismo
16.
Talanta ; 272: 125727, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38364570

RESUMO

Disulfide rebridging methods are emerging recently as new ways to specifically modify antibody-based entities and produce future conjugates. Briefly, the solvent-accessible disulfide bonds of antibodies or antigen-binding fragments (Fab) thereof are reduced under controlled conditions and further covalently attached with a rebridging agent allowing the incorporation of one payload per disulfide bond. There are many examples of successful rebridging cases providing homogeneous conjugates due to the use of symmetrical reagents, such as dibromomaleimides. However, partial rebridging due to the use of unsymmetrical ones, containing functional groups with different reactivity, usually leads to the development of heterogeneous species that cannot be identified by a simple sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) due to its lack of sensitivity, resolution and low mass accuracy. Mass spectrometry coupled to liquid chromatography (LC-MS) approaches have already been demonstrated as highly promising alternatives for the characterization of newly developed antibody-drug-conjugate (ADC) and monoclonal antibody (mAb)-based formats. We report here the in-depth characterization of covalently rebridged antibodies and Fab fragments in-development, using size-exclusion chromatography hyphenated to mass spectrometry in denaturing conditions (denaturing SEC-MS, dSEC-MS). DSEC-MS was used to monitor closely the rebridging reaction of a conjugated trastuzumab, in addition to conjugated Fab fragments, which allowed an unambiguous identification of the covalently rebridged products along with the unbound species. This all-in-one approach allowed a straightforward analysis of the studied samples with precise mass measurement; critical quality attributes (CQAs) assessment along with rebridging efficiency determination.


Assuntos
Anticorpos Monoclonais , Imunoconjugados , Anticorpos Monoclonais/química , Trastuzumab , Cromatografia Líquida/métodos , Fragmentos Fab das Imunoglobulinas , Imunoconjugados/química , Dissulfetos/química
17.
Int J Biol Sci ; 20(4): 1125-1141, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38385081

RESUMO

Previous studies have demonstrated that diallyl disulfide (DADS) exhibits potent anti-tumor activity. However, the pharmacological actions of DADS in inhibiting the growth of colorectal cancer (CRC) cells have not been clarified. Herein, we show that DADS treatment impairs the activation of the pentose phosphate pathway (PPP) to decrease PRPP (5-phosphate ribose-1-pyrophosphate) production, enhancing DNA damage and cell apoptosis, and inhibiting the growth of CRC cells. Mechanistically, DADS treatment promoted POU2F1 K48-linked ubiquitination and degradation by attenuating the PI3K/AKT signaling to up-regulate TRIM21 expression in CRC cells. Evidently, TRIM21 interacted with POU2F1, and induced the K272 ubiquitination of POU2F1. The effects of DADS on the enhanced K272 ubiquitination of POU2F1, the PPP flux, PRPP production, DNA damage and cell apoptosis as well as the growth of CRC tumors in vivo were significantly mitigated by TRIM21 silencing or activating the PI3K signaling in CRC cells. Conversely, the effects of DADS were enhanced by TRIM21 over-expression or inhibiting the PI3K/AKT signaling in CRC cells. Collectively, our findings reveal a novel mechanism by which DADS suppresses the growth of CRC by promoting POU2F1 ubiquitination, and may aid in design of novel therapeutic intervention of CRC.


Assuntos
Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/análogos & derivados , Compostos Alílicos , Neoplasias Colorretais , Dissulfetos , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Apoptose/genética , Compostos Alílicos/farmacologia , Compostos Alílicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Dano ao DNA , Fator 1 de Transcrição de Octâmero/genética
18.
Int J Mol Sci ; 25(4)2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38396632

RESUMO

Chromatin status is critical for sperm fertility and reflects spermatogenic success. We tested a multivariate approach for studying pig sperm chromatin structure to capture its complexity with a set of quick and simple techniques, going beyond the usual assessment of DNA damage. Sperm doses from 36 boars (3 ejaculates/boar) were stored at 17 °C and analyzed on days 0 and 11. Analyses were: CASA (motility) and flow cytometry to assess sperm functionality and chromatin structure by SCSA (%DFI, DNA fragmentation; %HDS, chromatin maturity), monobromobimane (mBBr, tiol status/disulfide bridges between protamines), chromomycin A3 (CMA3, protamination), and 8-hydroxy-2'-deoxyguanosine (8-oxo-dG, DNA oxidative damage). Data were analyzed using linear models for the effects of boar and storage, correlations, and multivariate analysis as hierarchical clustering and principal component analysis (PCA). Storage reduced sperm quality parameters, mainly motility, with no critical oxidative stress increases, while chromatin status worsened slightly (%DFI and 8-oxo-dG increased while mBBr MFI-median fluorescence intensity-and disulfide bridge levels decreased). Boar significantly affected most chromatin variables except CMA3; storage also affected most variables except %HDS. At day 0, sperm chromatin variables clustered closely, except for CMA3, and %HDS and 8-oxo-dG correlated with many variables (notably, mBBr). After storage, the relation between %HDS and 8-oxo-dG remained, but correlations among other variables disappeared, and mBBr variables clustered separately. The PCA suggested a considerable influence of mBBr on sample variance, especially regarding storage, with SCSA and 8-oxo-dG affecting between-sample variability. Overall, CMA3 was the least informative, in contrast with results in other species. The combination of DNA fragmentation, DNA oxidation, chromatin compaction, and tiol status seems a good candidate for obtaining a complete picture of pig sperm nucleus status. It raises many questions for future molecular studies and deserves further research to establish its usefulness as a fertility predictor in multivariate models. The usefulness of CMA3 should be clarified.


Assuntos
Biofilmes , Compostos Bicíclicos com Pontes , Cromatina , Suínos , Masculino , Animais , Citometria de Fluxo , 8-Hidroxi-2'-Desoxiguanosina , Sêmen , Reatores Biológicos , Espermatozoides , DNA/genética , Fragmentação do DNA , Dissulfetos
19.
Eur Rev Med Pharmacol Sci ; 28(2): 757-762, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38305617

RESUMO

OBJECTIVE: Obesity, a prevalent chronic disease, results from an imbalance between energy intake and expenditure. The oxidative stress associated with obesity stems from an imbalance between reactive oxygen species and the cell's antioxidant defense system. Oxidative stress can cause many diseases. The assessment of thiol/disulfide balance, a biochemical test, can be used to detect oxidative stress. The aim of this study is to determine the changes in oxidative stress associated with obesity after obesity surgery by assessing the thiol/disulfide levels. PATIENTS AND METHODS: The study was conducted with 40 volunteer patients with a body mass index (BMI) above 40 who underwent obesity surgery at Harran University Hospital General Surgery. Thiol and disulfide levels and other blood parameters were measured from the preoperative and postoperative 2nd and 6th-month blood samples of the patients. BMI was calculated by recording the weights and heights of the cases. Patients with diseases that could affect oxidative stress measurements and those using medication were excluded from the study, and the analyses were performed accordingly. RESULTS: The results showed a statistically significant decrease in native thiol, disulfide, reduced thiol, oxidized thiol, glucose, ALT (alanine aminotransferase), ALP (alkaline phosphatase), total cholesterol, HDL (high-density lipoprotein), triglyceride, and BMI values between the preoperative, 2-month postoperative, and 6-month postoperative measurements (p<0.05). CONCLUSIONS: Restrictive methods such as sleeve gastrectomy in individuals with morbid obesity led to weight control and a decrease in adipose tissue, reducing oxidative stress and increasing antioxidant response.


Assuntos
Laparoscopia , Obesidade Mórbida , Humanos , Antioxidantes , Obesidade Mórbida/cirurgia , Obesidade Mórbida/complicações , Dissulfetos , Compostos de Sulfidrila , Gastrectomia/métodos , Laparoscopia/métodos
20.
Anal Chem ; 96(10): 4057-4066, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38407829

RESUMO

Disulfide bridges in peptides and proteins play an essential role in maintaining their conformation, structural integrity, and consequently function. Despite ongoing efforts, it is still not possible to detect disulfide bonds and the connectivity of multiply bridged peptides directly through a simple and sufficiently validated protein sequencing or peptide mapping method. Partial or complete reduction and chemical cysteine modification are required as initial steps, followed by the application of a proper detection method. Edman degradation (ED) has been used for primary sequence determination but is largely neglected since the establishment of mass spectrometry (MS)-based protein sequencing. Here, we evaluated and thoroughly characterized the phenyl thiohydantoin (PTH) cysteine derivatives PTH-S-methyl cysteine and PTH-S-carbamidomethyl cysteine as bioanalytical standards for cysteine detection and quantification as well as for the elucidation of the disulfide connectivity in peptides by ED. Validation of the established derivatives was performed according to the guidelines of the International Committee of Harmonization on bioanalytical method validation, and their analytical properties were confirmed as reference standards. A series of model peptides was sequenced to test the usability of the PTH-Cys-derivatives as standards, whereas the native disulfide-bonded peptides CCAP-vil, µ-conotoxin KIIIA, and human insulin were used as case studies to determine their disulfide bond connectivity completely independent of MS analysis.


Assuntos
Cisteína , Dissulfetos , Humanos , Cisteína/química , Dissulfetos/química , Peptídeos/química , Proteínas , Sequência de Aminoácidos
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