Mechanism and structural dynamics of sulfur transfer during de novo [2Fe-2S] cluster assembly on ISCU2.

Maturation of iron-sulfur proteins in eukaryotes is initiated in mitochondria by the core iron-sulfur cluster assembly (ISC) complex, consisting of the cysteine desulfurase sub-complex NFS1-ISD11-ACP1, the scaffold protein ISCU2, the electron donor ferredoxin FDX2, and frataxin, a protein dysfunctional in Friedreich's ataxia. The core ISC complex synthesizes [2Fe-2S] clusters de novo from Fe and a persulfide (SSH) bound at conserved cluster assembly site residues. Here, we elucidate the poorly understood Fe-dependent mechanism of persulfide transfer from cysteine desulfurase NFS1 to ISCU2. High-resolution cryo-EM structures obtained from anaerobically prepared samples provide snapshots that both visualize different stages of persulfide transfer from Cys381NFS1 to Cys138ISCU2 and clarify the molecular role of frataxin in optimally positioning assembly site residues for fast sulfur transfer. Biochemical analyses assign ISCU2 residues essential for sulfur transfer, and reveal that Cys138ISCU2 rapidly receives the persulfide without a detectable intermediate. Mössbauer spectroscopy assessing the Fe coordination of various sulfur transfer intermediates shows a dynamic equilibrium between pre- and post-sulfur-transfer states shifted by frataxin. Collectively, our study defines crucial mechanistic stages of physiological [2Fe-2S] cluster assembly and clarifies frataxin's molecular role in this fundamental process.

A review on L-methioninase in cancer therapy: Precision targeting, advancements and diverse applications for a promising future.

Cancer remains a global health challenge, demanding novel therapeutic options due to the debilitating side effects of conventional treatments on healthy tissues. The review highlights the potential of L-methioninase, a pyridoxal-5-phosphate (PLP)-dependent enzyme, as a promising avenue in alternative cancer therapy. L-methioninase offers a unique advantage, its ability to selectively target and inhibit the growth of cancer cells without harming healthy cells. This selectivity arises because tumor cells lack an essential enzyme called methionine synthase, which healthy cells use to make the vital amino acid L-methionine. Several sources harbor L-methioninase, including bacteria, fungi, plants, and protozoa. Future research efforts can explore and exploit this diverse range of sources to improve the therapeutic potential of L-methioninase in the fight against cancer. Despite challenges, research actively explores microbial L-methioninase for its anticancer potential. This review examines the enzyme's side effects, advancements in combination therapies, recombinant technologies, polymer conjugation and novel delivery methods like nanoparticles, while highlighting the success of oral administration in preclinical trials. Beyond its promising role in cancer therapy, L-methioninase holds potential applications in food science, antioxidants, and various health concerns like diabetes, cardiovascular issues, and neurodegenerative diseases. This review provides a piece of current knowledge and future prospects of L-methioninase, exploring its diverse therapeutic potential.

Reduction of Tumor Biomarkers from very High to Normal and Extensive Metastatic Lesions to Undetectability in a Patient With Stage IV HER2-positive Breast Cancer Treated With Low-dose Trastuzumab Deruxtecan in Combination With Oral Recombinant Methioninase and a Low-methionine Diet.

BACKGROUND/AIM: Breast cancer is the most common and the deadliest cancer among women in the world. Treatment options for HER2-positive metastatic breast cancer patients are limited. Trastuzumab deruxtecan (T-DXd), an antibody-drug conjugate (ADC), has recently been introduced as second-line chemotherapy for HER2-positive metastatic breast cancer. The aim of the present study was to evaluate the efficacy of methionine restriction with oral recombinant methioninase (o-rMETase) and a low-methionine diet combined with T-DXd, on a patient with HER2-positive recurrent stage IV breast cancer. CASE REPORT: A 66-year-old female was diagnosed with HER2-positive metastatic breast cancer. Computed tomography (CT) indicated peritoneal dissemination, thickening of the sigmoid colon and splenic flexure and widespread bone metastases. The patient was previously treated with fulvestrant, trastuzumab, pertuzumab, paclitaxel and capecitabine which were ineffective. T-DXd was administered as a second-line chemotherapy. Since the patient experienced strong side effects, the dose of T-Dxd was decreased. The patient began methionine restriction using o-rMETase and a low-methionine diet along with T-DXd. After the start of the combined treatment, CA15-3 and CA27.29, tumor markers for breast cancer, decreased rapidly from a very high level. The levels of both tumor markers are currently normal. Additionally, peritoneal-dissemination nodules, ascites and the thickness of the sigmoid colon and splenic flexure are no longer detected on CT. The patient maintains a high performance status, without severe side effects of the combination treatment. CONCLUSION: Methionine restriction consisting of o-rMETase and a low-methionine diet, in combination with T-DXd as second-line chemotherapy, was highly effective in a patient with HER2-positive stage IV breast cancer.

PLA inhibits TNF-α-induced PANoptosis of prostate cancer cells through metabolic reprogramming.

Previous studies have shown that phenyllactic acid (alpha-Hydroxyhydrocinnamic acid, 2-Hydroxy-3-phenylpropionic acid, PLA), a type of organic acid metabolite, has excellent diagnostic efficacy when used to differentiate between prostate cancer, benign prostatic hyperplasia, and prostatitis. This research aims to explore the molecular mechanism by which PLA influences the PANoptosis of prostate cancer (PCa) cell lines. First, we found that PLA was detected in all prostate cancer cell lines (PC-3, PC-3 M, DU145, LNCAP). Further experiments showed that the addition of PLA to prostate cancer cells could promote ATP generation, enhance cysteine desulfurase (NFS1) expression, and reduce tumor necrosis factor alpha (TNF-α) levels, thereby inhibiting apoptosis in prostate cancer cells. Notably, overexpression of NFS1 can inhibit the binding of TNF-α to serpin mRNA binding protein 1 (SERBP1), suggesting that NFS1 competes with TNF-α for binding to SERBP1. Knockdown of SERBP1 significantly reduced the level of small ubiquity-related modifier (SUMO) modification of TNF-α. This suggests that NFS1 reduces the SUMO modification of TNF-α by competing with SERBP1, thereby reducing the expression and stability of TNF-α and ultimately inhibiting apoptosis in prostate cancer cell lines. In conclusion, PLA inhibits TNF-α induced panapoptosis of prostate cancer cells through metabolic reprogramming, providing a new idea for targeted treatment of prostate cancer.

Impact of LuxS on virulence and pathogenicity in Klebsiella pneumoniae exhibiting varied mucoid phenotypes.

How the LuxS/AI-2 quorum sensing (QS) system influences the pathogenicity of K. pneumoniae is complicated by the heterogeneity of the bacterial mucoid phenotypes. This study aims to explore the LuxS-mediated regulation of the pathogenicity of K. pneumoniae with diverse mucoid phenotypes, including hypermucoid, regular-mucoid, and nonmucoid. The wild-type, luxS knockout, and complemented strains of three K. pneumoniae clinical isolates with distinct mucoid phenotypes were constructed. The results revealed the downregulation of virulence genes of regular-mucoid, and nonmucoid but not hypermucoid strains. The deletion of luxS reduced the pathogenicity of the regular-mucoid, and nonmucoid strains in mice; while in hypermucoid strain, luxS knockout reduced virulence in late growth but enhanced virulence in the early growth phase. Furthermore, the absence of luxS led the regular-mucoid and nonmucoid strains to be more sensitive to the host cell defense, and less biofilm-productive than the wild-type at both the low and high-density growth state. Nevertheless, luxS knockout enhanced the resistances to adhesion and phagocytosis by macrophage as well as serum-killing, of hypermucoid K. pneumoniae at its early low-density growth state, while it was opposite to those in its late high-density growth phase. Collectively, our results suggested that LuxS plays a crucial role in the pathogenicity of K. pneumoniae, and it is highly relevant to the mucoid phenotypes and growth phases of the strains. LuxS probably depresses the capsule in the early low-density phase and promotes the capsule, biofilm, and pathogenicity during the late high-density phase, but inhibits lipopolysaccharide throughout the growth phase, in K. pneumoniae.IMPORTANCECharacterizing the regulation of physiological functions by the LuxS/AI-2 quorum sensing (QS) system in Klebsiella pneumoniae strains will improve our understanding of this important pathogen. The genetic heterogeneity of K. pneumoniae isolates complicates our understanding of its pathogenicity, and the association of LuxS with bacterial pathogenicity has remained poorly addressed in K. pneumoniae. Our results demonstrated strain and growth phase-dependent variation in the contributions of LuxS to the virulence and pathogenicity of K. pneumoniae. Our findings provide new insights into the important contribution of the LuxS/AI-2 QS system to the networks that regulate the pathogenicity of K. pneumoniae. Our study will facilitate our understanding of the regulatory mechanisms of LuxS/AI-2 QS on the pathogenicity of K. pneumoniae under the background of their genetic heterogeneity and help develop new strategies for diminished bacterial virulence within the clinical K. pneumoniae population.

Characterization of AI-2/LuxS quorum sensing system in biofilm formation, pathogenesis of Streptococcus equi subsp. zooepidemicus.

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of both humans and animals. Quorum sensing (QS) plays an important role in the regulation of bacterial group behaviors. The aim of this study was to characterize the LuxS in SEZ and evaluate its impact on biofilm formation, pathogenesis and gene expression. The wild-type SEZ and its LuxS mutant (ΔluxS) were examined for growth, biofilm formation, virulence factors, and transcriptomic profiles. Our results showed that LuxS deficiency did not affect SEZ hemolytic activity, adhesion or capsule production. For biofilm assay demonstrated that mutation in the luxS gene significantly enhances biofilm formation, produced a denser biofilm and attached to a glass surface. RAW264.7 cell infection indicated that ΔluxS promoted macrophage apoptosis and pro-inflammatory responses. In mice infection, there was no significant difference in mortality between SEZ and ΔluxS. However, the bacterial load in the spleen of mice infected with ΔluxS was significantly higher than in those infected with SEZ. And the pathological analysis further indicated that spleen damage was more severe in the ΔluxS group. Moreover, transcriptomics analysis revealed significant alterations in carbon metabolism, RNA binding and stress response genes in ΔluxS. In summary, this study provides the first evidence of AI-2/LuxS QS system in SEZ and reveals its regulatory effects on biofilm formation, pathogenicity and gene expression.

Selective Synergy of Rapamycin Combined With Methioninase on Cancer Cells Compared to Normal Cells.

BACKGROUND/AIM: Rapamycin and recombinant methioninase (rMETase) have both shown efficacy to target cancer cells. Rapamycin prevents cancer-cell growth by inhibition of the mTOR protein kinase. rMETase, by degrading methionine, targets the methionine addiction of cancer and has been shown to improve the efficacy of chemotherapy drugs. In the present study, we aimed to determine if a synergy exists between rapamycin and rMETase when used in combination against a colorectal-carcinoma cell line, compared to normal fibroblasts, in vitro. MATERIALS AND METHODS: The half-maximal inhibitory concentrations (IC50) of rapamycin alone and rMETase alone against the HCT-116 human colorectal-cancer cell line and Hs-27 human fibroblasts were determined using the CCK-8 Cell Viability Assay. After calculating the IC50 of each drug, we determined the efficacy of rapamycin and rMETase combined on both HCT-116 and Hs-27. RESULTS: Hs-27 normal fibroblasts were more sensitive to rapamycin than HCT-116 colon-cancer cells (IC50=0.37 nM and IC50=1.38 nM, respectively). HCT-116 cells were more sensitive to rMETase than Hs-27 cells (IC50 0.39 U/ml and IC50 0.96 U/ml, respectively). The treatment of Hs-27 cells with the combination of rapamycin (IC50=0.37 nM) and rMETase (IC50=0.96 U/ml) showed no significant difference in their effect on Hs-27 cell viability compared to the two drugs being used separately. However, the treatment of HCT-116 cells with the combination of rapamycin (IC50=1.38 nM) and rMETase (IC50=0.39 U/ml) was able to decrease cancer-cell viability significantly more than either single-drug treatment. CONCLUSION: Rapamycin and rMETase, when used in combination against colorectal-cancer cells, but not normal fibroblasts, in vitro, have a cancer-specific synergistic effect, suggesting that the combination of these drugs can be used as an effective, targeted cancer therapy.

Extensive Synergy Between Recombinant Methioninase and Eribulin Against Fibrosarcoma Cells But Not Normal Fibroblasts.

BACKGROUND/AIM: The aim of the present study was to determine the synergy of recombinant methioninase (rMETase) and the anti-tubulin agent eribulin on fibrosarcoma cells, in comparison to normal fibroblasts, in vitro. MATERIALS AND METHODS: HT1080 human fibrosarcoma cells and HS27 human fibroblasts were used for in vitro experiments. Four groups were analyzed in vitro: No-treatment control; eribulin; rMETase; eribulin plus rMETase. Dual-color HT1080 cells which express red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nuclei were used to visualize cytoplasmic and nuclear dynamics during treatment. RESULTS: Eribulin combined with rMETase greatly decreased the viability of HT 1080 cells. In contrast, eribulin combined with rMETase did not show synergy on Hs27 normal fibroblasts. Eribulin combined with rMETase also caused more fragmentation of the nucleus than all other treatments. CONCLUSION: The combination treatment of eribulin plus rMETase demonstrated efficacy on fibrosarcoma cells in vitro. In contrast, normal fibroblasts were resistant to this combination, indicating the potential clinical applicability of the treatment.

Conjugation to gold nanoparticles of methionine gamma-lyase, a cancer-starving enzyme. Physicochemical characterization of the nanocomplex for prospective nanomedicine applications.

The pyridoxal 5'-dependent enzyme methionine γ-lyase (MGL) catalyzes the degradation of methionine. This activity has been profitable to develop an antitumor agent exploiting the strict dependence of most malignant cells on the availability of methionine. Indeed, methionine depletion blocks tumor proliferation and leads to an increased susceptibility to anticancer drugs. Here, we explore the conjugation of MGL to gold nanoparticles capped with citrate (AuNPs) as a novel strategy to deliver MGL to cancer cells. Measurements of Transmission Electron Microscopy, Dynamic Light Scattering, Asymmetrical Flow Field-Flow Fractionation, X-ray Photoelectron Spectroscopy, and Circular Dichroism allowed to achieve an extensive biophysical and biochemical characterization of the MGL-AuNP complex including particle size, size distribution, MGL loading yield, enzymatic activity, and impact of gold surface on protein structure. Noticeably, we found that activity retention was improved over time for the enzyme adsorbed to AuNPs with respect to the enzyme free in solution. The acquired body of knowledge on the nanocomplex properties and this encouraging stabilizing effect upon conjugation are the necessary basis for further studies aimed at the evaluation of the therapeutic potential of MGL-AuNP complex in a biological milieu.

Methionine gamma lyase fused with S3 domain VGF forms octamers and adheres to tumor cells via binding to EGFR.

Methods for targeting enzymes exhibiting anticancer properties, such as methionine γ-lyase (MGL), have not yet been sufficiently developed. Here, we present the data describing the physico-chemical properties and cytotoxic effect of fusion protein MGL-S3 - MGL from Clostridium sporogenes translationally fused to S3 domain of the viral growth factor of smallpox. MGL-S3 has methioninase activity comparable to native MGL. In solution, MGL-S3 protein primarily forms octamers, whereas native MGL, on the contrary, usually forms tetramers. MGL-S3 binds to the surface of the neuroblastoma SH-SY5Y and epidermoid carcinoma A431 cells and, unlike native MGL, remains there and retains its cytotoxic effect after media removal. In HEK293T cells lacking EGFRs, no adhesion was recorded. Confocal fluorescence microscopy confirms the preferential adhesion of MGL-S3 to tumor cells, while it avoids getting into lysosomes. Both MGL and MGL-S3 arrest cell cycle of SH-SY5Y cells mainly in the G1 phase, while only MGL-S3 retains this ability after washing the cells.

Exogenous C-S Lyase Enzyme, a Potential Tool To Release Aromas in Wine or Beer?

Varietal thiols have an impact on the overall aroma of many white, rosé, and red wines and beers. They originate from the metabolism of non-odorant aroma precursors by yeast during the fermentation step, via an intrinsic enzyme, the carbon-sulfur ß-lyase (CSL, EC 4.4.1.13). However, this metabolism is directly dependent upon efficient internalization of aroma precursors and intracellular CSL activity. Consequently, the overall CSL activity converts on average only 1% of the total precursors available. To improve the conversion of thiol precursors during winemaking or brewing, we investigated the possibility of using an exogenous CSL enzyme from Lactobacillus delbrueckii subsp. bulgaricus produced in Escherichia coli. We first implemented a reliable spectrophotometric method to monitor its activity on different related aroma precursors and studied its activity in the presence of various competing analogues and at different pH values. This study allowed us to highlight the parameters to define CSL activity and structural insights for the recognition of the substrate, which pave the way for the use of exogenous CSL for the release of aromas in beer and wine.

Recombinant Methioninase Decreased the Effective Dose of Irinotecan by 15-fold Against Colon Cancer Cells: A Strategy for Effective Low-toxicity Treatment of Colon Cancer.

BACKGROUND/AIM: Irinotecan (IRN), a topoisomerase I inhibitor and pro-drug of SN-38, is first-line treatment of colon cancer as part of FOLFIRI and FOLFOXIRI combination chemotherapy. However, IRN causes dose-limiting adverse events such as neutropenia and diarrhea. Dose reductions are sometimes required, which reduce efficacy. Recombinant methioninase (rMETase) targets the fundamental basis of cancer, methionine addiction, known as the Hoffman effect, and enhances the efficacy of numerous chemotherapy drugs. The present study determined the efficacy of rMETase when administered in combination with IRN. MATERIALS AND METHODS: Cell viability was assessed by cultivating the HCT-116 human colorectal cancer cell line in 96-well plates at 1×103 cells per well in Dulbecco's modified Eagle's medium (DMEM). Subsequently, HCT-116 cells were treated with increasing concentrations of SN-38, the active form of IRN, ranging from 0.5 nM to 32 nM, and/or rMETase ranging from 0.125 to 8 U/ml. After treatment for 72 h, the half-maximal inhibitory concentration (IC50) of SN-38 alone and rMETase alone for HCT-116 cells were determined. Using the IC50 concentration of rMETase, we determined the IC50 of SN-38 in combination with rMETase. Cell viability was determined with the cell-counting Kit-8 with the WST-8 reagent.. RESULTS: The IC50 of rMETase alone for the HCT-116 cells was 0.55 U/ml, and the IC50 of IRN (SN-38) alone was 3.50 nM. rMETase at 0.55 U/ml lowered the IC50 of SN-38 to 0.232 nM (p<0.0001), a 15-fold reduction. CONCLUSION: rMETase and IRN are strongly synergistic, giving rise to the possibility of lowering the effective dose of IRN for the treatment of patients with colon cancer, thereby reducing its severe toxicity. This new strategy will allow more patients with cancer to be effectively treated with IRN.

Roles of luxS in regulation of probiotic characteristics and inhibition of pathogens in Lacticaseibacillus paracasei S-NB.

Lactic acid bacteria (LAB) have excellent tolerance to the gastrointestinal environment and high adhesion ability to intestinal epithelial cells, which could be closely related to the LuxS/AI-2 Quorum sensing (QS) system. Here, the crucial enzymes involved in the synthesis of AI-2 was analyzed in Lacticaseibacillus paracasei S-NB, and the luxS deletion mutant was constructed by homologous recombination based on the Cre-lox system. Afterwards, the effect of luxS gene on the probiotic activities in L. paracasei S-NB was investigated. Notably, the tolerance of simulated gastrointestinal digestion, AI-2 production, ability of auto-aggregation and biofilm formation significantly decreased (p < 0.05 for all) in the S-NB△luxS mutant. Compared to the wild-type S-NB, the degree of reduction in the relative transcriptional level of the biofilm -related genes in Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 was diminished when co-cultured with S-NB△luxS. Furthermore, the inhibitory effect of S-NB△luxS on the adhesion (competition, exclusion and displacement) of E. coli ATCC 25922 and S. aureus ATCC 25923 to Caco-2 cells markedly decreased. Therefore, comprehensive analysis of the role by luxS provides an insight into the LuxS/AI-2 QS system of L. paracasei S-NB in the regulation of strain characteristics and inhibition of pathogens.

Resveratrol drives ferroptosis of acute myeloid leukemia cells through Hsa-miR-335-5p/NFS1/ GPX4 pathway in a ROS-dependent manner.

To explore the potential target to induce ferroptosis for treating acute myeloid leukemia (AML) as well as its mechanism and latent drugs. Using the keyword "acute myelogenous leukemia", the related dataset in TCGA and GEO were used for searching differentially expressed genes. After the filtrate by ROC curve, AUC values, and survival analysis, RT-qPCR as well as Western-blot analysis were performed to verify the high expression level of NFS1 in AML-193 and OCI-AML-3 cells. After CCK-8 detection with and without various cell death inhibitors, ferroptosis were further detected by the expression level of GPX4. After taking the intersection in Starbase and TargetScan, the upstream regulatory miRNA of NFS1 was found. Then the relation of hsa-miR-335-5p, NFS1, as well as GPX4, was ascertained by knockdown and overexpression study in AML-193 and OCI-AML-3 cells. In addition, cellular ROS was detected by DCFH-DA. Finally, resveratrol was used to intensify ferroptosis of AML-193 and OCI-AML-3 cells. NFS1 was highly expressed in AML cells, positively associated with AML-related mortality, and can be used to diagnose AML. Knockout of NFS1 facilitated ROS accumulation and ferroptosis-associated labile iron pool increase. si-NFS1 can inhibit the expression level of GPX4, facilitate ROS accumulation and induce ferroptosis-associated labile iron pool increase. Besides, overexpressed GPX4 can lead to down-regulated cell death after si-NFS1 treatment. Hsa-miR-335-5p was found as the upstream regulator of NFS1. The expression of NFS1 can be up-regulated by sh-hsa-miR-335-5p transfection and can be inhibited by hsa-miR-335-5p transfection. Resveratrol was found can increase the expression level of hsa-miR-335-5p and decrease the expression of NFS1 and GPX4. Resveratrol can intensify ferroptosis of AML cells via Hsa-miR-335-5p/NFS1/ GPX4 pathway through a ROS-dependent manner.

Effects of luxS gene on growth characteristics, biofilm formation, and antimicrobial resistance of multi-antimicrobial-resistant Vibrio parahaemolyticus Vp2015094 isolated from shellfish.

AIMS: Vibrio parahaemolyticus is an important foodborne pathogen worldwide, which can cause gastroenteritis. This study aimed to investigate the effect of quorum sensing system LuxS/AI-2-related gene luxS on the biological characteristics and antimicrobial resistance of V. parahaemolyticus Vp2015094 from shellfish, which carried a multi-antimicrobial-resistant plasmid. METHODS AND RESULTS: The critical gene luxS related to the synthesis of AI-2 in V. parahaemolyticus Vp2015094 was knocked out by homologous recombination with suicide plasmid. The effect of luxS on the biological characteristics of V. parahaemolyticus was determined by comparing the growth, AI-2 activity, motility, biofilm formation ability, and antibiotic resistance between the wildtype strain and the luxS deletion mutant. Compared with wildtype strain, the production of AI-2, the motility and biofilm formation ability, antimicrobial resistance, and conjugation frequency of luxS deletion mutant strain were decreased. The transcriptome sequencing showed that the transcriptional levels of many genes related to motility, biofilm formation, antimicrobial resistance, and conjugation were significantly downregulated after luxS deletion. CONCLUSIONS: Quorum sensing system LuxS/AI-2-related gene luxS in V. parahaemolyticus Vp2015094 played an important role in growth characteristics, biofilm formation, antimicrobial resistance, and resistance genes' transfer.

Direct Cysteine Desulfurase Activity Determination by NMR and the Study of the Functional Role of Key Structural Elements of Human NFS1.

The mitochondrial cysteine desulfurase NFS1 is an essential PLP-dependent enzyme involved in iron-sulfur cluster assembly. The enzyme catalyzes the desulfurization of the l-Cys substrate, producing a persulfide and l-Ala as products. In this study, we set the measurement of the product l-Ala by NMR in vitro by means of 1H NMR spectra acquisition. This methodology provided us with the possibility of monitoring the reaction in both fixed-time and real-time experiments, with high sensitivity and accuracy. By studying I452A, W454A, Q456A, and H457A NFS1 variants, we found that the C-terminal stretch (CTS) of the enzyme is critical for function. Specifically, mutation of the extremely conserved position W454 resulted in highly decreased activity. Additionally, we worked on two singular variants: "GGG" and C158A. In the former, the catalytic Cys-loop was altered by including two Gly residues to increase the flexibility of this loop. This variant had significantly impaired activity, indicating that the Cys-loop motions are fine-tuned in the wild-type enzyme. In turn, for C158A, we found an unanticipated increase in l-Cys desulfurase activity. Furthermore, we carried out molecular dynamics simulations of the supercomplex dedicated to iron-sulfur cluster biosynthesis, which includes NFS1, ACP, ISD11, ISCU2, and FXN subunits. We identified CTS as a key element that established interactions with ISCU2 and FXN concurrently; we found specific interactions that are established when FXN is present, reinforcing the idea that FXN not only forms part of the iron-sulfur cluster assembly site but also modulates the internal motions of ISCU2.

Effects of metformin on Streptococcus suis LuxS/AI-2 quorum sensing system and biofilm formation.

Streptococcus suis (S. suis) regulates biofilm formation through LuxS/AI-2 quorum sensing system, increasing drug resistance and exacerbating infection. The anti-hyperglycaemic agent metformin has anti-bacterial and anti-biofilm activities. This study aimed to investigate the anti-biofilm and anti-quorum sensing activity of metformin in S. suis. We first determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of metformin on S. suis. The results indicated that metformin showed no obvious inhibitory or bactericidal effect. Crystal violet staining showed that metformin significantly inhibited the formation of S. suis biofilm at sub-MIC concentration, which was also confirmed by scanning electron microscopy. Then, we quantified the AI-2 signal molecules in S. suis, and the results showed that metformin had a significant inhibitory effect on the production of AI-2 signal in S. suis. Inhibition of enzyme activity and molecular docking experiments showed that metformin has a significant binding activity to LuxS protein. In addition, qRT-PCR results showed that metformin significantly down-regulated the expression of AI-2 synthesis-related genes luxS and pfs, and adhesion-related genes luxS, pfs, gapdh, sly, fbps, and ef. Western blotting also showed that metformin significantly reduced the expression of LuxS protein. Our study suggests that metformin seems to be a suitable candidate for the inhibition of S. suis LuxS/AI-2 QS system and prevention of biofilm formation, which provided a new idea for the prevention and control of S. suis.

Structural and Biochemical Characterization of Mycobacterium tuberculosis Zinc SufU-SufS Complex.

Iron-sulfur (Fe-S) clusters are inorganic prosthetic groups in proteins composed exclusively of iron and inorganic sulfide. These cofactors are required in a wide range of critical cellular pathways. Iron-sulfur clusters do not form spontaneously in vivo; several proteins are required to mobilize sulfur and iron, assemble and traffic-nascent clusters. Bacteria have developed several Fe-S assembly systems, such as the ISC, NIF, and SUF systems. Interestingly, in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), the SUF machinery is the primary Fe-S biogenesis system. This operon is essential for the viability of Mtb under normal growth conditions, and the genes it contains are known to be vulnerable, revealing the Mtb SUF system as an interesting target in the fight against tuberculosis. In the present study, two proteins of the Mtb SUF system were characterized for the first time: Rv1464(sufS) and Rv1465(sufU). The results presented reveal how these two proteins work together and thus provide insights into Fe-S biogenesis/metabolism by this pathogen. Combining biochemistry and structural approaches, we showed that Rv1464 is a type II cysteine-desulfurase enzyme and that Rv1465 is a zinc-dependent protein interacting with Rv1464. Endowed with a sulfurtransferase activity, Rv1465 significantly enhances the cysteine-desulfurase activity of Rv1464 by transferring the sulfur atom from persulfide on Rv1464 to its conserved Cys40 residue. The zinc ion is important for the sulfur transfer reaction between SufS and SufU, and His354 in SufS plays an essential role in this reaction. Finally, we showed that Mtb SufS-SufU is more resistant to oxidative stress than E. coli SufS-SufE and that the presence of zinc in SufU is likely responsible for this improved resistance. This study on Rv1464 and Rv1465 will help guide the design of future anti-tuberculosis agents.

Uncovering the link between the SpnIII restriction modification system and LuxS in Streptococcus pneumoniae meningitis isolates.

Streptococcus pneumoniae is capable of randomly switching their genomic DNA methylation pattern between six distinct bacterial subpopulations (A-F) via recombination of a type 1 restriction-modification locus, spnIII. These pneumococcal subpopulations exhibit phenotypic changes which favor carriage or invasive disease. In particular, the spnIIIB allele has been associated with increased nasopharyngeal carriage and the downregulation of the luxS gene. The LuxS/AI-2 QS system represent a universal language for bacteria and has been linked to virulence and biofilm formation in S. pneumoniae. In this work, we have explored the link between spnIII alleles, the luxS gene and virulence in two clinical pneumococcal isolates from the blood and cerebrospinal fluid (CSF) of one pediatric meningitis patient. The blood and CSF strains showed different virulence profiles in mice. Analysis of the spnIII system of these strains recovered from the murine nasopharynx showed that the system switched to different alleles commensurate with the initial source of the isolate. Of note, the blood strain showed high expression of spnIIIB allele, previously linked with less LuxS protein production. Importantly, strains with deleted luxS displayed different phenotypic profiles compared to the wildtype, but similar to the strains recovered from the nasopharynx of infected mice. This study used clinically relevant S. pneumoniae strains to demonstrate that the regulatory network between luxS and the type 1 restriction-modification system play a key role in infections and may support different adaptation to specific host niches.

Hydrogen sulfide alleviates mitochondrial damage and ferroptosis by regulating OPA3-NFS1 axis in doxorubicin-induced cardiotoxicity.

Ferroptosis is a major cause of cardiotoxicity induced by doxorubicin (DOX). Previous studies have shown that hydrogen sulfide (H2S) inhibits ferroptosis in cardiomyocytes and myoblasts, but the underlying mechanism has not been fully elucidated. In this study, we investigated the role of H2S in protecting against DOX-induced cardiotoxicity both in vivo and in vitro, and elucidated the potential mechanisms involved. We found that DOX downregulated the expression of glutathione peroxidase 4 (GPX4) and NFS1, and upregulated the expression of acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) expression level, resulting in increased lipid peroxidation and ferroptosis. Additionally, DOX inhibited MFN2 expression and increased DRP1 and FIS1 expression, leading to abnormal mitochondrial structure and function. In contrast, exogenous H2S inhibited DOX-induced ferroptosis by restoring GPX4 and NFS1 expression, and reducing lipid peroxidation in H9C2 cells. This effect was similar to that of the ferroptosis antagonist ferrostatin-1 (Fer-1) in protecting against DOX-induced cardiotoxicity. We further demonstrated that the protective effect of H2S was mediated by the key mitochondrial membrane protein optic atrophy 3 (OPA3), which was downregulated by DOX and restored by exogenous H2S. Overexpression of OPA3 alleviated DOX-induced mitochondrial dysfunction and ferroptosis both in vivo and in vitro. Mechanistically, NFS1 has an inhibitory effect on ferroptosis, and NFS1 deficiency increases the susceptibility of cardiomyocytes to ferroptosis. OPA3 is involved in the regulation of ferroptosis by interacting with NFS1. Post-translationally, DOX promoted OPA3 ubiquitination, while exogenous H2S antagonized OPA3 ubiquitination by promoting OPA3 s-sulfhydration. In summary, our findings suggested that H2S protects against DOX-induced cardiotoxicity by inhibiting ferroptosis via targeting the OPA3-NFS1 axis. This provides a potential therapeutic strategy for the treatment of DOX-induced cardiotoxicity.