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1.
Protein Sci ; 33(3): e4912, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38358254

RESUMO

Outer membrane proteins perform essential functions in uptake and secretion processes in bacteria. MspA is an octameric channel protein in the outer membrane of Mycobacterium smegmatis and is structurally distinct from any other known outer membrane protein. MspA is the founding member of a family with more than 3000 homologs and is one of the most widely used proteins in nanotechnological applications due to its advantageous pore structure and extraordinary stability. While a conserved C-terminal signal sequence is essential for folding and protein assembly in the outer membrane of Gram-negative bacteria, the molecular determinants of these processes are unknown for MspA. In this study, we show that mutation and deletion of methionine 183 in the highly conserved C-terminus of MspA and mutation of the conserved tryptophan 40 lead to a complete loss of protein in heat extracts of M. smegmatis. Swapping these residues partially restores the heat stability of MspA indicating that methionine 183 and tryptophan 40 form a conserved sulfur-π electron interaction, which stabilizes the MspA monomer. Flow cytometry showed that all MspA mutants are surface-accessible demonstrating that oligomerization and membrane integration in M. smegmatis are not affected. Thus, the conserved C-terminus of MspA is essential for its thermal stability, but it is not required for protein assembly in its native membrane, indicating that this process is mediated by a mechanism distinct from that in Gram-negative bacteria. These findings will benefit the rational design of MspA-like pores to tailor their properties in current and future applications.


Assuntos
Mycobacterium , Triptofano , Triptofano/metabolismo , Porinas/química , Porinas/genética , Porinas/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Metionina/metabolismo
2.
Artigo em Russo | MEDLINE | ID: mdl-38334732

RESUMO

OBJECTIVE: To study 11C-methionine (MET) metabolism in gliomas using CNS tumor biobank imaging data. MATERIAL AND METHODS: MRI and 11C-MET PET/CT were performed in 225 patients (49±14 years, M/F=84/101) according to standard protocols with analysis of 11C-MET accumulation index and volumetric parameters (V_FLAIR, V_PET and V_PET/FLAIR). These results were compared with molecular genetic testing and 2-year overall survival. RESULTS: We examined 225 patients with gliomas (97 glioblastomas, 70 astrocytomas, 58 oligodendrogliomas). Accumulation index and volume of 11C-MET in glioblastomas were significantly higher in the general group (AI=2.90, Se 69%, Sp 76%, AUC 0.76; V_PET=24.3 cm3, Se 67%, Sp 60%, AUC 0.65; V_PET/FLAIR 0.46, Se 60%, Sp 69%, AUC 0.67) and within the group of astrocytomas (AI=2.93, Se 68%, Sp 89%, AUC 0.84; V_PET=8.06 cm3, Se 91%, Sp 35%, AUC 0.66; V_PET/FLAIR 0.27, Se 77%, Sp 60%, AUC 0.71). The median 2-year overall survival in patients with glioblastomas was 13 months that was significantly lower compared to IDH «+¼ gliomas (p<0.0001). There was a relationship between high accumulation index of 11C-MET and shorter overall survival in patients with glioblastomas. Significantly higher AI >3.59 (Se 89%, Sp 67%, AUC 0.79) was additionally obtained in subgroup of patients with glioblastomas >50 years (n=34) for EGFR «+¼ tumors. CONCLUSION: We found variable 11C-MET metabolism in WHO 2021 gliomas and confirmed significant difference in metabolic activity and volume of 11C-MET accumulation in glioblastomas compared to IDH «+¼ gliomas. Moreover, we revealed the relationship between high accumulation index and shorter survival. Analysis of 11C-MET metabolism in patients over 50 years old revealed higher accumulation index in the EGFR «+¼ group. Further comparison of these imaging methods and assessment of other significant mutations are necessary to identify the anatomical and metabolic patterns of IDH «+¼ gliomas.


Assuntos
Astrocitoma , Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/genética , Tomografia por Emissão de Pósitrons/métodos , Radioisótopos de Carbono , Glioma/diagnóstico por imagem , Glioma/genética , Encéfalo/patologia , Metionina , Receptores ErbB
3.
BMC Endocr Disord ; 24(1): 19, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38311763

RESUMO

BACKGROUND: We assessed the value of positron emission tomography/computed tomography (PET/CT) with [13N]N-ammonia ([13N]N-NH3) and [11C]C-methionine ([11C]C-MET) for the evaluation and management of recurrent secreting pituitary adenoma, which could not be detected by magnetic resonance imaging (MRI) or fluorine-18 fluorodeoxyglucose ([18F]F-FDG) PET. METHODS: Nine consecutive patients with biochemical and clinical evidence of active recurrent tumor not detected by MRI and [18F]F-FDG PET were enrolled in this study. All of the patients underwent [13N]N-NH3 and [11C]C-MET PET/CT, after which the pattern of tracer uptake was studied, the tumor position was located, and a clinical decision was made. RESULTS: In general, [11C]C-MET had a higher uptake in pituitary adenomas (PAs) than that in pituitary tissues, while [13N]N-NH3 had a higher uptake in pituitary tissue than in pituitary adenomas. Increased [11C]C-MET uptake was observed in all nine PAs and three pituitary tissues, while all pituitary tissues and only one pituitary adenoma showed increased [13N]N-NH3 uptake. Four patients had concordant imaging and surgical findings indicative of biochemical remission without hypopituitarism after treatment. Radiotherapy was adopted in two patients, medication in another two, and follow-up observation in one case. CONCLUSION: Combined [11C]C-MET and [13N]N-NH3 PET/CT is effective in the differentiation of PAs from pituitary tissue in recurrent functional PAs with negative MRI or [18F]F-FDG PET. These results provide a valuable reference for further disease management.


Assuntos
Adenoma , Neoplasias Hipofisárias , Humanos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Fluordesoxiglucose F18 , Neoplasias Hipofisárias/diagnóstico por imagem , Neoplasias Hipofisárias/patologia , Metionina , Amônia , Recidiva Local de Neoplasia/diagnóstico por imagem , Adenoma/diagnóstico por imagem , Adenoma/patologia , Tomografia por Emissão de Pósitrons/métodos , Racemetionina , Imageamento por Ressonância Magnética/métodos , Compostos Radiofarmacêuticos
4.
Neurosurg Focus ; 56(2): E6, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38301247

RESUMO

OBJECTIVE: Surgery is the mainstay of treatment for low-grade glioma (LGG)-related epilepsy. However, the goal of achieving both oncological radical resection and seizure freedom can be challenging. PET with [11C]methionine (MET) has been recently introduced in clinical practice for the management of patients with LGGs, not only to monitor the response to treatments, but also as a preoperative tool to define the metabolic tumor extent and to predict tumor grading, type, and prognosis. Still, its role in defining tumor-related epilepsy and postoperative seizure outcomes is limited. The aim of this preliminary study was to investigate the role of MET PET in defining preoperative seizure characteristics and short-term postoperative seizure control in a cohort of patients with newly diagnosed temporal lobe low-grade gliomas (tLGGs). METHODS: Patients with newly diagnosed and histologically proven temporal lobe grade 2/3 gliomas (2021 WHO CNS tumor classification) who underwent resection at the authors' institution between July 2011 and March 2021 were included in this retrospective study. MET PET images were acquired, fused with MRI scans, and qualitatively and semiquantitatively analyzed. Any eventual PET/MRI involvement of the temporomesial area, seizure characteristics, and 1-year seizure outcomes were reported. RESULTS: A total of 52 patients with tLGGs met the inclusion criteria. MET PET was positive in 41 (79%) patients, with a median metabolic tumor volume of 14.56 cm3 (interquartile range [IQR] 6.5-28.2 cm3). The median maximum and mean tumor-to-background ratio (TBRmax, TBRmean) were 2.24 (IQR 1.58-2.86) and 1.53 (IQR 1.37-1.70), respectively. The metabolic tumor volume was found to be related to the presence of seizures at disease onset, but only in noncodeleted tumors (p = 0.014). Regarding patients with uncontrolled seizures at surgery, only the temporomesial area PET involvement showed a statistical correlation both in the univariate (p = 0.058) and in the multivariate analysis (p = 0.030). At 1-year follow-up, seizure control was correlated with MET PET-derived semiquantitative data. Particularly, higher TBRmax (p = 0.0192) and TBRmean (p = 0.0128) values were statistically related to uncontrolled seizures 1 year after surgery. CONCLUSIONS: This preliminary study suggests that MET PET may be used as a preoperative tool to define seizure characteristics and outcomes in patients with tLGGs. These findings need to be further validated in larger series with longer epileptological follow-ups.


Assuntos
Neoplasias Encefálicas , Epilepsia do Lobo Temporal , Epilepsia , Glioma , Humanos , Metionina , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/cirurgia , Estudos Retrospectivos , Radioisótopos de Carbono , Glioma/complicações , Glioma/diagnóstico por imagem , Glioma/cirurgia , Convulsões/diagnóstico por imagem , Convulsões/etiologia , Convulsões/cirurgia , Racemetionina , Lobo Temporal/diagnóstico por imagem , Lobo Temporal/cirurgia , Tomografia por Emissão de Pósitrons , Resultado do Tratamento , Epilepsia do Lobo Temporal/diagnóstico por imagem , Epilepsia do Lobo Temporal/cirurgia
5.
J Immunother Cancer ; 12(2)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38302417

RESUMO

BACKGROUND: Although immune checkpoint inhibitor (ICI)-based therapy is advantageous for patients with advanced melanoma, resistance and relapse are frequent. Thus, it is crucial to identify effective drug combinations and develop new therapies for the treatment of melanoma. SGN1, a genetically modified Salmonella typhimurium species that causes the targeted deprivation of methionine in tumor tissues, is currently under investigation in clinical trials. However, the inhibitory effect of SGN1 on melanoma and the benefits of SGN1 in combination with ICIs remain largely unexplored. Therefore, this study aims to investigate the antitumor potential of SGN1, and its ability to enhance the efficacy of antibody-based programmed cell death-ligand 1 (PD-L1) inhibitors in the treatment of murine melanoma. METHODS: The antitumor activity of SGN1 and the effect of SGN1 on the efficacy of PD-L1 inhibitors was studied through murine melanoma models. Further, The Cancer Genome Atlas-melanoma cohort was clustered using ConsensusClusterPlus based on the methionine deprivation-related genes, and immune characterization was performed using xCell, Microenvironment Cell Populations-counter, Estimation of Stromal and Immune cells in MAlignant Tumor tissues using Expression data, and immunophenoscore (IPS) analyses. The messenger RNA data on programmed death-1 (PD-1) immunotherapy response were obtained from the Gene Expression Omnibus database. Gene Set Enrichment Analysis of methionine deprivation-up gene set was performed to determine the differences between pretreatment responders and non-responders. RESULTS: This study showed that both, the intratumoral and the intravenous administration of SGN1 in subcutaneous B16-F10 melanomas, suppress tumor growth, which was associated with an activated CD8+T-cell response in the tumor microenvironment. Combination therapy of SGN1 with systemic anti-PD-L1 therapy resulted in better antitumor activity than the individual monotherapies, respectively, and the high therapeutic efficacy of the combination was associated with an increase in the systemic level of tumor-specific CD8+ T cells. Two clusters consisting of methionine deprivation-related genes were identified. Patients in cluster 2 had higher expression of methionine_deprivation_up genes, better clinical outcomes, and higher immune infiltration levels compared with patients in cluster 1. Western blot, IPS analysis, and immunotherapy cohort study revealed that methionine deficiency may show a better response to ICI therapy CONCLUSIONS:: This study reports Salmonella-based SGN1 as a potent anticancer agent against melanoma, and lays the groundwork for the potential synergistic effect of ICIs and SGN1 brought about by improving the immune microenvironment in melanomas.


Assuntos
Inibidores de Checkpoint Imunológico , Melanoma Experimental , Humanos , Camundongos , Animais , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Linfócitos T CD8-Positivos , Metionina , Estudos de Coortes , Recidiva Local de Neoplasia , Melanoma Experimental/tratamento farmacológico , Salmonella , Microambiente Tumoral
6.
Biochem Biophys Res Commun ; 701: 149589, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38309152

RESUMO

OBJECTIVE: To evaluate the role of PRDX2 in nonalcoholic steatohepatitis (NASH). METHODS: NASH was induced in wild-type (WT) mice and liver-specific PRDX2 knockout (PRDX2 LKO) mice that were fed a methionine-choline deficient diet (MCD) for 5 weeks. Assessments of PRDX2 LKO's impact on the pathogenesis of NASH include histological analyses, quantitative PCR (q-PCR), western blotting (WB), and RNA sequencing (RNA-Seq). RESULTS: PRDX2 LKO mice exhibited a significant increase in hepatic lipid accumulation and inflammation compared to WT mice after MCD feeding. PRDX2 KO markedly elevated circulating levels of aspartate aminotransferase (AST) and the pro-inflammatory signaling pathways within the liver. There was a notable increase in the activities of signal transducer and activator of transcription 1 (STAT1) and nuclear factor kappa B (NF-кB). We also found that PRDX2 KO significantly increased the extent of lipid peroxidation in the liver, most likely owing to the impaired peroxidase activity of PRDX2. Of interest, these findings were observed only in MCD-fed female mice, suggesting the sexual dimorphism of PRDX2 KO in MCD-induced NASH. CONCLUSION: PRDX2 deficiency increases MCD-induced NASH in female mice, suggesting a protective role for PRDX2.


Assuntos
Deficiência de Colina , Hepatopatia Gordurosa não Alcoólica , Camundongos , Feminino , Animais , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Colina/metabolismo , Metionina/metabolismo , Deficiência de Colina/metabolismo , Fígado/metabolismo , Racemetionina/metabolismo , Dieta , Camundongos Knockout , Camundongos Endogâmicos C57BL
7.
Int J Mol Sci ; 25(3)2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38338668

RESUMO

Non-alcoholic steatohepatitis (NASH) is an inflammatory form of non-alcoholic fatty liver disease (NAFLD), closely associated with disease progression, cirrhosis, liver failure, and hepatocellular carcinoma. Time-restricted feeding (TRF) has been shown to decrease body weight and adiposity and improve metabolic outcomes; however, the effect of TRF on NASH has not yet been fully understood. We had previously reported that inositol polyphosphate multikinase (IPMK) mediates hepatic insulin signaling. Importantly, we have found that TRF increases hepatic IPMK levels. Therefore, we investigated whether there is a causal link between TRF and IPMK in a mouse model of NASH, i.e., methionine- and choline-deficient diet (MCDD)-induced steatohepatitis. Here, we show that TRF alleviated markers of NASH, i.e., reduced hepatic steatosis, liver triglycerides (TG), serum alanine transaminase (ALT) and aspartate aminotransferase (AST), inflammation, and fibrosis in MCDD mice. Interestingly, MCDD led to a significant reduction in IPMK levels, and the deletion of hepatic IPMK exacerbates the NASH phenotype induced by MCDD, accompanied by increased gene expression of pro-inflammatory chemokines. Conversely, TRF restored IPMK levels and significantly reduced gene expression of proinflammatory cytokines and chemokines. Our results demonstrate that TRF attenuates MCDD-induced NASH via IPMK-mediated changes in hepatic steatosis and inflammation.


Assuntos
Deficiência de Colina , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/metabolismo , Metionina/metabolismo , Colina/metabolismo , Deficiência de Colina/complicações , Deficiência de Colina/metabolismo , Fígado/metabolismo , Racemetionina/metabolismo , Dieta , Inflamação/metabolismo , Quimiocinas/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças
8.
J Am Chem Soc ; 146(6): 3710-3720, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38308759

RESUMO

1/2H and 13C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp2 C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57Fe, 13C, 15N, and 2H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]2+ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]2+ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.


Assuntos
Proteínas Ferro-Enxofre , S-Adenosilmetionina , S-Adenosilmetionina/metabolismo , Metionina , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Domínio Catalítico , Racemetionina , Radicais Livres/química , Proteínas Ferro-Enxofre/química
9.
10.
Mol Genet Genomics ; 299(1): 7, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38349549

RESUMO

Traditional maize grain is deficient in methionine, an essential amino acid required for proper growth and development in humans and poultry birds. Thus, development of high methionine maize (HMM) assumes great significance in alleviating malnutrition through sustainable and cost-effective approach. Of various genetic loci, aspartate kinase2 (ask2) gene plays a pivotal role in regulating methionine accumulation in maize. Here, we sequenced the entire ask2 gene of 5394 bp with 13 exons in five wild and five mutant maize inbreds to understand variation at nucleotide level. Sequence analysis revealed that an SNP in exon-13 caused thymine to adenine transversion giving rise to a favourable mutant allele associated with leucine to glutamine substitution in mutant ASK2 protein. Gene-based diversity analysis with 11 InDel markers grouped 48 diverse inbreds into three major clusters with an average genetic dissimilarity of 0.570 (range, 0.0-0.9). The average major allele frequency, gene diversity and PIC are 0.693, 0.408 and 0.341, respectively. A total of 45 haplotypes of the ask2 gene were identified among the maize inbreds. Evolutionary relationship analysis performed among 22 orthologues grouped them into five major clusters. The number of exons varied from 7 to 17, with length varying from 12 to 495 bp among orthologues. ASK2 protein with 565 amino acids was predicted to be in homo-dimeric state with lysine and tartaric acid as binding ligands. Amino acid kinase and ACT domains were found to be conserved in maize and orthologues. The study depicted the presence of enough genetic diversity in ask2 gene in maize, and development of HMM can be accelerated through introgression of favourable allele of ask2 into the parental lines of elite hybrids using molecular breeding.


Assuntos
Ácido Aspártico , Zea mays , Humanos , Zea mays/genética , Haplótipos , Aminoácidos , Metionina/genética , Racemetionina
11.
Sci Rep ; 14(1): 3093, 2024 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-38326523

RESUMO

In this study, we have examined the feasibility of using elemental sulfur content of soybean seeds as a proxy for the overall sulfur amino acid content of soybean seeds. Earlier, we have identified by high throughput ionomic phenotyping several high and low sulfur containing soybean lines from the USDA Soybean Germplasm Collection. Here, we measured the cysteine and methionine content of select soybean lines by high-performance liquid chromatography. Our results demonstrate that those soybean lines which had high elemental sulfur content also had a higher cysteine and methionine content when compared to soybean lines with low elemental sulfur. SDS-PAGE and immunoblot analysis revealed that the accumulation of Bowman Birk protease inhibitor and lunasin in soybean seeds may only be marginally correlated with the elemental sulfur levels. However, we found a positive correlation between the levels of trypsin and chymotrypsin inhibitor activities and elemental sulfur and sulfur amino acid content of the seeds. Thus, elemental sulfur content and/or protease inhibitor activity measurement can be utilized as a rapid and cost-effective method to predict the overall sulfur amino acid content of soybean seeds. Our findings will benefit breeders in their endeavors to develop soybean cultivars with enhanced sulfur amino acid content.


Assuntos
Aminoácidos Sulfúricos , Inibidor da Tripsina de Soja de Bowman-Birk , Cisteína/metabolismo , Inibidor da Tripsina de Soja de Bowman-Birk/química , Análise Custo-Benefício , Aminoácidos Sulfúricos/metabolismo , Metionina/metabolismo , Sementes/metabolismo , Inibidores de Proteases/metabolismo
12.
J Med Chem ; 67(1): 543-554, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38166392

RESUMO

Small molecules capable of modulating methionine adenosyltransferase 2A (MAT2A) are of significant interest in precise cancer therapeutics. Herein, we raised the hole-electron Coulombic attraction as a reliable molecular descriptor for predicting the reactive oxygen generation capacity of MAT2A inhibitors, based on which we discovered compound H3 as a sonically activated degrader of MAT2A. Upon sonication, H3 can generate reactive oxygen species to specifically degrade cellular MAT2A via rapid oxidative reactions. Combination of H3 and sonication induced 87% MAT2A depletion in human colon cancer cells, thus elevating its antiproliferation effects by 8-folds. In vivo, H3 had a favorable pharmacokinetic profile (bioavailability = 77%) and ADME properties. Owing to the MAT2A degradation merits, H3 at a dosage of 10 mg/kg induced 31% tumor regression in xenograft colon tumor models. The significantly boosted antitumor potency can potentially alleviate the toxicity of high-dose MAT2A inhibitors to normal cells and tissues, especially to the liver.


Assuntos
Neoplasias Hepáticas , Metionina Adenosiltransferase , Humanos , Metionina Adenosiltransferase/metabolismo , Elétrons , Neoplasias Hepáticas/metabolismo , S-Adenosilmetionina/metabolismo , Metionina
13.
Clin Epigenetics ; 16(1): 1, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38167534

RESUMO

BACKGROUND: The objective of this study was to examine and analyze differential methylation profiles in order to investigate the influence of hyper-methioninemia (HM) on the development of diabetic nephropathy (DN). Male Wistar rats, aged eight weeks and weighing 250-300 g, were randomly assigned into four groups: a control group (Healthy, n = 8), streptozocin-induced rats (STZ group, n = 8), HM + STZ group (n = 8), and the Tangshen Formula (TSF) treatment group (TSF group, n = 8). Blood glucose levels and other metabolic indicators were monitored before treatment and at four-week intervals until 12 weeks. Total DNA was extracted from the aforementioned groups, and DNA methylation landscapes were analyzed via reduced representative bisulfite sequencing. RESULTS: Both the STZ group and HM + STZ group exhibited increased blood glucose levels and urinary albumin/creatinine ratios in comparison with the control group. Notably, the HM + STZ group exhibited a markedly elevated urinary albumin/creatinine ratio (411.90 ± 88.86 mg/g) compared to the STZ group (238.41 ± 62.52 mg/g). TSF-treated rats demonstrated substantial reductions in both blood glucose levels and urinary albumin/creatinine ratios in comparison with the HM + STZ group. In-depth analysis of DNA methylation profiles revealed 797 genes with potential therapeutic effects related to TSF, among which approximately 2.3% had been previously reported as homologous genes. CONCLUSION: While HM exacerbates DN through altered methylation patterns at specific CpG sites, TSF holds promise as a viable treatment for DN by restoring abnormal methylation levels. The identification of specific genes provides valuable insights into the underlying mechanisms of DN pathogenesis and offers potential therapeutic targets for further investigation.


Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Ratos , Masculino , Animais , Nefropatias Diabéticas/induzido quimicamente , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/genética , Glicemia , Metionina/metabolismo , Estreptozocina/metabolismo , Estreptozocina/farmacologia , Estreptozocina/uso terapêutico , Creatinina/metabolismo , Creatinina/farmacologia , Creatinina/uso terapêutico , Ratos Wistar , Metilação de DNA , Rim/metabolismo , Racemetionina/metabolismo , Racemetionina/farmacologia , Albuminas/metabolismo
14.
Nat Cell Biol ; 26(1): 72-85, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38168768

RESUMO

A growing body of evidence indicates that gut microbiota influence brain function and behaviour. However, the molecular basis of how gut bacteria modulate host nervous system function is largely unknown. Here we show that vitamin B12-producing bacteria that colonize the intestine can modulate excitatory cholinergic signalling and behaviour in the host Caenorhabditis elegans. Here we demonstrate that vitamin B12 reduces cholinergic signalling in the nervous system through rewiring of the methionine (Met)/S-adenosylmethionine cycle in the intestine. We identify a conserved metabolic crosstalk between the methionine/S-adenosylmethionine cycle and the choline-oxidation pathway. In addition, we show that metabolic rewiring of these pathways by vitamin B12 reduces cholinergic signalling by limiting the availability of free choline required by neurons to synthesize acetylcholine. Our study reveals a gut-brain communication pathway by which enteric bacteria modulate host behaviour and may affect neurological health.


Assuntos
S-Adenosilmetionina , Vitamina B 12 , Animais , Vitamina B 12/metabolismo , S-Adenosilmetionina/metabolismo , Caenorhabditis elegans/metabolismo , Colina/metabolismo , Bactérias/metabolismo , Metionina/metabolismo , Vitaminas/metabolismo , Colinérgicos/metabolismo
15.
Drug Des Devel Ther ; 18: 29-41, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38225973

RESUMO

Background: The preclinical diagnosis of tumors is of great significance to cancer treatment. Near-infrared fluorescence imaging technology is promising for the in-situ detection of tumors with high sensitivity. Methods: Here, a fluorescent probe was synthesized on the basis of Au nanoclusters with near-infrared light emission and applied to fluorescent cancer cell labeling. Near-infrared methionine-N-Hydroxy succinimide Au nanoclusters (Met-NHs-AuNCs) were prepared successfully by one-pot synthesis using Au nanoclusters, methionine, and N-Hydroxy succinimide as frameworks, reductants, and stabilizers, respectively. The specific fluorescence imaging of tumor cells or tissues by fluorescent probe was studied on the basis of SYBYL Surflex-DOCK simulation model of LAT1 active site of overexpressed receptor on cancer cell surface. The results showed that Met-NHs-AuNCs interacted with the surface of LAT1, and C_Score scored the conformation of the probe and LAT1 as five. Results: Characterization and in vitro experiments were conducted to explore the Met-NHs-AuNCs targeted uptake of cancer cells. The prepared near-infrared fluorescent probe (Met-NHs-AuNCs) can specifically recognize the overexpression of L-type amino acid transporter 1 (LAT1) in cancer cells so that it can show red fluorescence in cancer cells. Meanwhile, normal cells (H9c2) have no fluorescence. Conclusion: The fluorescent probe demonstrates the power of targeting and imaging cancer cells.


Assuntos
Nanopartículas Metálicas , Neoplasias , Humanos , Corantes Fluorescentes , Neoplasias/metabolismo , Imagem Óptica/métodos , Metionina/química , Racemetionina , Succinimidas , Ouro/química
16.
Chemphyschem ; 25(4): e202300565, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38175858

RESUMO

Methionine side chains are flexible entities which play important roles in defining hydrophobic interfaces. We utilize deuterium static solid-state NMR to assess rotameric inter-conversions and other dynamic modes of the methionine in the context of a nine-residue random-coil peptide (RC9) with the low-complexity sequence GGKGMGFGL. The measurements in the temperature range of 313 to 161 K demonstrate that the rotameric interconversions in the hydrated solid powder state persist to temperatures below 200 K. Removal of solvation significantly reduces the rate of the rotameric motions. We employed 2 H NMR line shape analysis, longitudinal and rotation frame relaxation, and chemical exchange saturation transfer methods and found that the combination of multiple techniques creates a significantly more refined model in comparison with a single technique. Further, we compare the most essential features of the dynamics in RC9 to two different methionine-containing systems, characterized previously. Namely, the M35 of hydrated amyloid-ß1-40 in the three-fold symmetric polymorph as well as Fluorenylmethyloxycarbonyl (FMOC)-methionine amino acid with the bulky hydrophobic group. The comparison suggests that the driving force for the enhanced methionine side chain mobility in RC9 is the thermodynamic factor stemming from distributions of rotameric populations, rather than the increase in the rate constant.


Assuntos
Peptídeos beta-Amiloides , Metionina , Temperatura , Espectroscopia de Ressonância Magnética , Peptídeos beta-Amiloides/química , Racemetionina , Ressonância Magnética Nuclear Biomolecular
17.
mBio ; 15(2): e0207323, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38179948

RESUMO

Tuberculosis (TB) is a significant global public health threat. Despite the long-standing use of para-aminosalicylic acid (PAS) as a second-line anti-TB drug, its resistance mechanism remains unclear. In this study, we isolated 90 mutants of PAS-resistant Mycobacterium tuberculosis (MTB) H37Ra in 7H11 solid medium and performed whole-genome sequencing, gene overexpression, transcription level comparison and amino acid level determination in MTB, and promoter activity by ß-galactosidase assays in Mycobacterium smegmatis to elucidate the mechanism of PAS resistance. Herein, we found that 47 of 90 (52.2%) PAS-resistant mutants had nine different mutations in the intergenic region of metM (Rv3253c) and Rv3254. Beta-galactosidase assays confirmed that mutations increased promoter activity only for metM but not Rv3254. Interestingly, overexpression of MetM or its M. smegmatis homolog (MSMEI_1796) either by its promoter in metM's direction or by exogenous expression in MTB induced PAS resistance in a methionine-dependent manner. Therefore, drug susceptibility results for the metM promoter mutants can be misleading when using standard 7H10 or 7H9 medium, which lacks methionine. At the metabolism level, PAS treatment led to higher intracellular methionine levels in the mutants than the wild type, antagonizing PAS and conferring resistance. Furthermore, 12 different mutations in the metM promoter were identified in clinical MTB strains. In summary, we found a novel mechanism of PAS resistance in MTB. Mutations in the metM (Rv3253c) promoter upregulate metM transcription and elevate intracellular methionine, which antagonize PAS. Our findings shed new light on the mechanism of PAS resistance in MTB and highlight issues with the current PAS susceptibility culture medium.IMPORTANCEAlthough para-aminosalicylic acid (PAS) has been used to treat TB for more than 70 years, the understanding of PAS resistance mechanisms is still vague, living gaps in our ability to predict resistance and apply PAS effectively in clinical practice. This study aimed to address this knowledge gap by inducing in vitro PAS resistance in Mycobacterium tuberculosis (MTB) using 7H11 medium and discovering a new PAS resistance mechanism. Our research revealed that spontaneous mutations occurring in the promoter region of the methionine transporting gene, metM, can upregulate the expression of metM, resulting in increased intracellular transport of methionine and consequently high-level resistance of Mycobacterium tuberculosis to PAS. Notably, this resistance phenotype cannot be observed when using the commonly recommended 7H10 medium, possibly due to the lack of additional methionine supply compared with that when using the 7H11 medium. Mutations on the regulatory region of metM were also found in some clinical MTB strains. These findings may have important implications for the unexplained PAS resistance observed in clinical settings and provide insight into the failures of PAS treatment. Additionally, they underscore the importance of considering the choice of culture media when conducting drug susceptibility testing for MTB.


Assuntos
Ácido Aminossalicílico , Mycobacterium tuberculosis , Ácido Aminossalicílico/farmacologia , Ácido Aminossalicílico/metabolismo , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana/genética , Antituberculosos/farmacologia , Mutação , Metionina/metabolismo , beta-Galactosidase/genética
18.
Nat Cancer ; 5(1): 131-146, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38168934

RESUMO

Availability of the essential amino acid methionine affects cellular metabolism and growth, and dietary methionine restriction has been implicated as a cancer therapeutic strategy. Nevertheless, how liver cancer cells respond to methionine deprivation and underlying mechanisms remain unclear. Here we find that human liver cancer cells undergo irreversible cell cycle arrest upon methionine deprivation in vitro. Blocking methionine adenosyl transferase 2A (MAT2A)-dependent methionine catabolism induces cell cycle arrest and DNA damage in liver cancer cells, resulting in cellular senescence. A pharmacological screen further identified GSK3 inhibitors as senolytics that selectively kill MAT2A-inhibited senescent liver cancer cells. Importantly, combined treatment with MAT2A and GSK3 inhibitors therapeutically blunts liver tumor growth in vitro and in vivo across multiple models. Together, methionine catabolism is essential for liver tumor growth, and its inhibition can be exploited as an improved pro-senescence strategy for combination with senolytic agents to treat liver cancer.


Assuntos
Quinase 3 da Glicogênio Sintase , Neoplasias Hepáticas , Humanos , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Metionina/farmacologia , Metionina Adenosiltransferase/metabolismo
19.
Biochem Biophys Res Commun ; 695: 149418, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38176171

RESUMO

Glioma is a highly recalcitrant disease with a 5-year survival of 6.8 %. Temozolomide (TMZ), first-line therapy for glioma, is more effective in O6-methylguanine-DNA methyltransferase (MGMT)-negative gliomas than in MGMT-positive gliomas as MGMT confers resistance to TMZ. Methionine restriction is effective for many cancers in mouse models including glioma. The concern is that methionine restriction could induce MGMT by decreasing DNA methylation and confer resistance to TMZ. In the present study, we investigated the efficacy of combining methionine restriction with TMZ for the treatment of MGMT-negative glioma, and whether methionine restriction induced MGMT. Human MGMT-negative U87 glioma cells were used to determine the efficacy of TMZ combined with methionine restriction. Recombinant methioninase (rMETase) inhibited U87 glioma growth without induction of MGMT in vitro. The combination of rMETase and TMZ inhibited U87 cell proliferation more than either agent alone in vitro. In the orthotopic nude-mouse model, the combination of TMZ and a methionine-deficient diet was much more effective than TMZ alone: two mice out of five were cured of glioma by the combination. No mice died during the treatment period. Methionine restriction enhanced the efficacy of TMZ in MGMT-negative glioma without inducing MGMT, demonstrating potential clinical promise for improved outcome of a currently incurable disease.


Assuntos
Neoplasias Encefálicas , Glioma , Temozolomida , Animais , Humanos , Camundongos , Antineoplásicos Alquilantes/uso terapêutico , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Metilases de Modificação do DNA/farmacologia , Metilases de Modificação do DNA/uso terapêutico , Enzimas Reparadoras do DNA/genética , Resistencia a Medicamentos Antineoplásicos , Glioma/tratamento farmacológico , Glioma/genética , Metionina/farmacologia , Camundongos Nus , O(6)-Metilguanina-DNA Metiltransferase , Racemetionina/farmacologia , Temozolomida/uso terapêutico , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/genética
20.
Microbiol Spectr ; 12(2): e0280323, 2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38230928

RESUMO

Streptococcus suis (S. suis) has been increasingly recognized as a porcine zoonotic pathogen that threatens the health of both pigs and humans. Multidrug-resistant Streptococcus suis is becoming increasingly prevalent, and novel strategies to treat bacterial infections caused by these organisms are desperately needed. In the present study, an untargeted metabolomics analysis showed that the significant decrease in methionine content and the methionine biosynthetic pathway were significantly affected by the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis in drug-resistant S. suis. The addition of L-methionine restored the bactericidal activity of macrolides, doxycycline, and ciprofloxacin on S. suis in vivo and in vitro. Further studies showed that the exogenous addition of methionine affects methionine metabolism by reducing S-adenosylmethionine synthetase activity and the contents of S-adenosylmethionine, S-adenosyl homocysteine, and S-ribose homocysteine. Methionine can decrease the total methylation level and methylesterase activity in multidrug resistant S. suis. The drug transport proteins and efflux pump genes were significantly downregulated in S. suis by exogenous L-methionine. Moreover, the exogenous addition of methionine can reduce the survival of S. suis by affecting oxidative stress and metal starvation in bacteria. Thus, L-methionine may influence the development of resistance in S. suis through methyl metabolism and metal starvation. This study provides a new perspective on the mitigation of drug resistance in S. suis.IMPORTANCEBacterial antibiotic resistance has become a severe threat to human and animal health. Increasing the efficacy of existing antibiotics is a promising strategy against antibiotic resistance. Here, we report that L-methionine enhances the efficacy of macrolides, doxycycline, and ciprofloxacin antibiotics in killing Streptococcus suis, including multidrug-resistant pathogens. We investigated the mechanism of action of exogenous methionine supplementation in restoring macrolides in Streptococcus suis and the role of the methionine cycle pathway on methylation levels, efflux pump genes, oxidative stress, and metal starvation in Streptococcus suis. It provides a theoretical basis for the rational use of macrolides in clinical practice and also identifies a possible target for restoring drug resistance in Streptococcus suis.


Assuntos
Infecções Estreptocócicas , Streptococcus suis , Humanos , Animais , Suínos , Streptococcus suis/genética , Macrolídeos/uso terapêutico , Metionina/metabolismo , Metionina/uso terapêutico , Doxiciclina/uso terapêutico , Infecções Estreptocócicas/microbiologia , Antibacterianos/uso terapêutico , Ciprofloxacina , Homocisteína/metabolismo , Homocisteína/uso terapêutico
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