Search Results (9)

The enzyme γ-glutamyl transpeptidase (GGT), located on the surface of cellular membranes, hydrolyzes extracellular glutathione (GSH) to guarantee the recycling of cysteine and maintain intracellular redox homeostasis. High expression levels of GGT on tumor cells are associated with increased cell proliferation and resistance against chemotherapy. Therefore, GGT inhibitors have potential as adjuvants in treating GGT-positive tumors; however, most have been abandoned during clinical trials due to toxicity. Recent studies indicate marine-derived ovothiols as more potent non-competitive GGT inhibitors, inducing a mixed cell-death phenotype of apoptosis and autophagy in GGT-overexpressing cell lines, such as the chronic B leukemic cell HG-3, while displaying no toxicity towards non-proliferative cells. In this work, we characterize the activity of two synthetic ovothiol analogs, L-5-sulfanylhistidine and iso-ovothiol A, in GGT-positive cells, such as HG-3 and HL-60 cells derived from acute promyelocytic leukemia. The two compounds inhibit the activity of membrane-bound GGT, without altering cell vitality nor inducing cytotoxic autophagy in HG-3 cells. We provide evidence that a portion of L-5-sulfanylhistidine enters HG-3 cells and acts as a redox regulator, contributing to the increase in intracellular GSH. On the other hand, ovothiol A, which is mostly sequestered by external membrane-bound GGT, induces intracellular ROS increase and the consequent autophagic pathways. These findings provide the basis for developing ovothiol derivatives as adjuvants in treating GGT-positive tumors’ chemoresistance. Full article

Lentinula edodes (L. edodes) is a globally popular edible mushroom because of its characteristic sulfur-containing flavor compounds. However, the formation of the volatile sulfur-containing compounds in the mycelium of L. edodes has not been studied. We found that there were also sulfur-containing aroma compounds in the mycelium of L. edodes, and the content and composition varied at different stages of mycelial growth and development. The γ-glutamyl-transpeptidase (GGT) and cysteine sulfoxide lyase (C-S lyase) related to the generation of sulfur compounds showed the highest activities in the 15-day sample. Candidate genes for the metabolism of volatile sulfur compounds in mycelium were screened using transcriptome analysis, including encoding the GGT enzyme, C-S lyase, fatty acid oxidase, HSP20, and P450 genes. The expression patterns of Leggt3 and Leccsl3 genes were consistent with the measured activities of GGT and C-S lyase during the cultivation of mycelium and molecular dynamics simulations showed that they could stably bind to the substrate. Our findings provide insights into the formation of sulfur-containing flavor compounds in L. edodes. The mycelium of L. edodes is suggested for use as material for the production of sulfur-containing flavor compounds. Full article

Sortase A (SrtA) is a cysteine transpeptidase that binds to the periplasmic membrane and plays a crucial role in attaching surface proteins, including staphylococcal protein A (SpA), to the peptidoglycan cell wall. Six pentacyclic polyketides (1–6) were isolated from the marine sponge Xestospongia sp., and their structures were elucidated using spectroscopic techniques and by comparing them to previously reported data. Among them, halenaquinol (2) was found to be the most potent SrtA inhibitor, with an IC₅₀ of 13.94 μM (4.66 μg/mL). Semi-quantitative reverse transcription PCR data suggest that halenaquinol does not inhibit the transcription of srtA and spA, while Western blot analysis and immunofluorescence microscopy images suggest that it blocks the cell wall surface anchoring of SpA by inhibiting the activity of SrtA. The onset and magnitude of the inhibition of SpA anchoring on the cell wall surface in S. aureus that has been treated with halenaquinol at a value 8× that of the IC₅₀ of SrtA are comparable to those for an srtA-deletion mutant. These findings contribute to the understanding of the mechanism by which marine-derived pentacyclic polyketides inhibit SrtA, highlighting their potential as anti-infective agents targeting S. aureus virulence. Full article

Amyotrophic lateral sclerosis (ALS) is a progredient neurodegenerative disease characterized by a degeneration of the first and second motor neurons. Elevated levels of reactive oxygen species (ROS) and decreased levels of glutathione, which are important defense mechanisms against ROS, have been reported in the central nervous system (CNS) of ALS patients and animal models. The aim of this study was to determine the cause of decreased glutathione levels in the CNS of the ALS model wobbler mouse. We analyzed changes in glutathione metabolism in the spinal cord, hippocampus, cerebellum, liver, and blood samples of the ALS model, wobbler mouse, using qPCR, Western Blot, HPLC, and fluorometric assays. Here, we show for the first time a decreased expression of enzymes involved in glutathione synthesis in the cervical spinal cord of wobbler mice. We provide evidence for a deficient glutathione metabolism, which is not restricted to the nervous system, but can be seen in various tissues of the wobbler mouse. This deficient system is most likely the reason for an inefficient antioxidative system and, thus, for elevated ROS levels. Full article

Endogenous formaldehyde is generated as a normal metabolite via bio-catalysis of γ-glutamyl transpeptidase (GGT) and L-cysteine sulfoxide lyase (C-S lyase) during the growth and development of Lentinula edodes. In this study, we investigated the mRNA and protein expression levels, the activities of GGT and C-S lyase, and the endogenous formaldehyde content in L. edodes at different growth stages. With the growth of L. edodes, a decrease was found in the mRNA and protein expression levels of GGT, while an increase was observed in the mRNA and protein expression levels of C-S lyase as well as the activities of GGT and C-S lyase. Our results revealed for the first time a positive relationship of formaldehyde content with the expression levels of Csl (encoding Lecsl) and Lecsl (C-S lyase protein of Lentinula edodes) as well as the enzyme activities of C-S lyase and GGT during the growth of L. edodes. This research provided a molecular basis for understanding and controlling the endogenous formaldehyde formation in Lentinula edodes in the process of growth. Full article

Bacterial sortases are cysteine transpeptidases that regulate the covalent linkage of several surface protein virulence factors in Gram-positive bacteria. Virulence factors play significant roles in adhesion, invasion of host tissues, biofilm formation and immune evasion, mediating the bacterial pathogenesis and infectivity. Therefore, sortases are emerging as important targets for the design of new anti-infective agents. We employed a computational study, based on structure derived descriptors and molecular fingerprints, in order to develop simple classification methods which could allow predicting low active or high active SrtA inhibitors. Our results indicate that a highly active SrtA inhibitor has a molecular weight ranging between 180 and 600, contains one up to four nitrogen atoms, up to three oxygen atoms and under 18 hydrogen atoms. Also the hydrogen acceptor number and the molecular flexibility, as assessed by the number of rotatable bounds, have emerged as the most relevant descriptors for SrtA affinity. The Bemis-Murcko scaffolding revealed favoured scaffolds as containing at least two ring structures bonded directly or merged in a condensed cycle. This data represent a valuable tool for identifying new potent SrtA inhibitors, potential anti-virulence agents targeted against Gram-positive bacteria, including multiresistant strains. Full article

Staphylococcus aureus (S. aureus) is a major cause of infection in hospitals and communities. Widespread dissemination of multi-drug resistant S. aureus is a serious threat to the health of humans and animals. An anti-virulence strategy has been widely considered as an alternative therapeutic approach. Inhibitors of virulence factors are able to treat S. aureus infections without influencing the growth or viability of bacteria and rarely lead to bacterial resistance. Sortase A (SrtA) is a membrane-associated cysteine transpeptidase that catalyzes up to 25 surface proteins that covalently bind to cell wall peptidoglycans. In S. aureus, most of these surface proteins have been identified as important virulence factors that are vital in bacterial pathogenesis. In the present study, we show that acacetin, a natural flavonoid compound, inhibits the activity of SrtA in S. aureus (IC₅₀ = 36.46 ± 4.69 μg/mL, 128 μM) which affects the assembly of protein A (SpA) to cell walls and reduces the binding of S. aureus to fibrinogen (Fg). The mechanism of the interaction between acacetin and SrtA were preliminarily discussed using molecular dynamics simulations. The results suggested that acacetin adopted a compact conformation binding at the pocket of the SrtA via residues Arg-139 and Lys-140. By performing an animal infection model, we demonstrated that acacetin was able to protect mice from renal abscess formation induced by S. aureus and significantly increased survival rates. Taken together, these findings suggest that acacetin may be a promising candidate for the development of anti-S. aureus drugs. Full article

Sortase A (SrtA) is a cysteine transpeptidase of most Gram-positive bacteria that is responsible for the anchorage of many surface protein virulence factors to the cell wall layer. SrtA mutants are unable to display surface proteins and are defective in the establishment of infections without affecting microbial viability. In this study, we report that quercitrin (QEN), a natural compound that does not affect Staphylococcus aureus growth, can inhibit the catalytic activity of SrtA in fibrinogen (Fg) cell-clumping and immobilized fibronectin (Fn) adhesion assays. Molecular dynamics simulations and mutagenesis assays suggest that QEN binds to the binding sites of the SrtA G167A and V193A mutants. These findings indicate that QEN is a potential lead compound for the development of new anti-virulence agents against S. aureus infections. Full article

Gamma glutamyl transpeptidase (GGT) is a transferase, which is of great importance in sustaining intracellular cysteine and glutathione levels. The abnormal expression of GGT is significantly associated with features of many metabolic syndromes (e.g., hepatocellular carcinoma). Therefore, it is essential to develop methods to detect GGT so as to monitor the physiological or pathological phenomena related to this species. In this work, by making use of a complex formed by Cu²⁺ and glutathione, which may exhibit excellent voltammetric response, we have proposed a novel potential electrochemical method for the detection of the enzyme. Results show that in the presence of GGT, the formation of Cu²⁺-glutathione complex on a working electrode will be disrupted, resulting in greatly depressed electrochemical signals. The primary method exhibits some advantages, such as it being fast, cost-efficient, and conveniently operated. It also has the potential to be further developed as an effective method in the quantitative detection of GGT in real samples. Full article