Branched-chain Aminotransferase Research Articles

Insights into the metabolic profiling of Polygonati Rhizoma fermented by Lactiplantibacillus plantarum under aerobic and anaerobic conditions using a UHPLC-QE-MS/MS system.

Polygonati Rhizoma is a multi-purpose food with medicinal uses. Fermentation of Polygonati Rhizoma by lactic acid bacteria could provide new insights into the development of Polygonati Rhizoma products. In this study, Lactiplantibacillus plantarum was fermented with Polygonati Rhizoma extracts in a bioreactor under aerobic and anaerobic conditions with pH and DO real-time detection. Metabolic profiling was determined by UHPLC-QE-MS/MS system. Principal component analysis and orthogonal partial least-squares discriminant analysis were used to perform multivariate analysis. A total of 98 differential metabolites were identified in broth after fermentation, and 36 were identified between fermentation under aerobic and anaerobic conditions. The main metabolic pathways in the fermentation process are ABC transport and amino acid biosynthesis. Most of the compounds such as L-arginine, L-aspartic acid, leucine, L-lysine, citrate, inosine, carnitine, betaine, and thiamine were significantly increased during fermentation, playing a role in enhancing food flavor. Compared with anaerobic fermentation, aerobic conditions led to a significant rise in the levels of some compounds such as valine, isoleucine, and glutamate; this increase was mainly related to branched-chain amino acid transaminase, isocitrate dehydrogenase, and glutamate dehydrogenase. Aerobic fermentation is more beneficial for the fermentation of Polygonati Rhizoma by L. plantarum to produce flavor and functional substances. This study is the first report on the fermentation of Polygonati Rhizoma by L. plantarum and provides insights that would be applicable in the development of Polygonati Rhizoma fermented products.

The role of branched-chain aminotransferase 1 in driving glioblastoma cell proliferation and invasion varies with tumor subtype.

Branched-chain aminotransferase 1 (BCAT1) has been proposed to drive proliferation and invasion of isocitrate dehydrogenase (IDH) wild-type glioblastoma cells. However, the Cancer Genome Atlas (TCGA) dataset shows considerable variation in the expression of this enzyme in glioblastoma. The aim of this study was to determine the role of BCAT1 in driving the proliferation and invasion of glioblastoma cells and xenografts that have widely differing levels of BCAT1 expression and the mechanism responsible. The activity of BCAT1 was modulated in IDH wild-type patient-derived glioblastoma cell lines, and in orthotopically implanted tumors derived from these cells, to examine the effects of BCAT1 expression on tumor phenotype. In cells with constitutively high BCAT1 expression and a glycolytic metabolic phenotype, inducible shRNA knockdown of the enzyme resulted in reduced proliferation and invasion by increasing the concentration of α-ketoglutarate, leading to reduced DNA methylation, HIF-1α destabilization, and reduced expression of the transcription factor Forkhead box protein M1 (FOXM1). Conversely, overexpression of the enzyme increased HIF-1α expression and promoted proliferation and invasion. However, in cells with an oxidative phenotype and very low constitutive expression of BCAT1 increased expression of the enzyme had no effect on invasion and reduced cell proliferation. This occurred despite an increase in HIF-1α levels and could be explained by decreased TCA cycle flux. There is a wide variation in BCAT1 expression in glioblastoma and its role in proliferation and invasion is dependent on tumor subtype.

The emerging role of the branched chain aminotransferases, BCATc and BCATm, for anti-tumor T-cell immunity.

Challenges regarding successful immunotherapy are associated with the heterogeneity of tumors and the complex interactions within the surrounding tumor microenvironment (TME), particularly those between immune and tumor cells. Of interest, T cells receive a myriad of environmental signals to elicit differentiation to effector subtypes, which is accompanied by metabolic reprogramming needed to satisfy the high energy and biosynthetic demands of their activated state. However, T cells are subjected to immunosuppressive signals and areas of oxygen and nutrient depletion in the TME, which causes T-cell exhaustion and helps tumor cells escape immune detection. The cytosolic and mitochondrial branched chain amino transferases, BCATc and BCATm, respectively, are responsible for the first step of the branched chain amino acid (BCAA) degradation, of which, metabolites are shunted into various metabolic processes. In recent years, BCAT isoenzymes have been investigated for their role in a variety of cancers found throughout the body; however, a gap of knowledge exists regarding the role BCAT isoenzymes play within immune cells of the TME. The aim of this review is to summarize recent findings about BCAAs and their catabolism at the BCAT step during T-cell metabolic reprogramming and to discuss the BCAT putative role in the anti-tumor immunity of T cells. Not only does this review acknowledges gaps pertaining to BCAA metabolism in the TME but it also identifies the practical application of BCAA metabolism in T cells in response to cancer and spotlights a potential target for pharmacological intervention.

METTL16 drives leukemogenesis and leukemia stem cell self-renewal by reprogramming BCAA metabolism.

N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, is involved in many pathological processes. METTL16 is a recently identified m6A methyltransferase. However, its role in leukemia has yet to be investigated. Here, we show that METTL16 is a highly essential gene for the survival of acute myeloid leukemia (AML) cells via CRISPR-Cas9 screening and experimental validation. METTL16 is aberrantly overexpressed in human AML cells, especially in leukemia stem cells (LSCs) and leukemia-initiating cells (LICs). Genetic depletion of METTL16 dramatically suppresses AML initiation/development and maintenance and significantly attenuates LSC/LIC self-renewal, while moderately influencing normal hematopoiesis in mice. Mechanistically, METTL16 exerts its oncogenic role by promoting expression of branched-chain amino acid (BCAA) transaminase 1 (BCAT1) and BCAT2 in an m6A-dependentmanner and reprogramming BCAA metabolism in AML. Collectively, our results characterize theMETTL16/m6A/BCAT1-2/BCAA axis in leukemogenesis and highlight the essential role of METTL16-mediatedm6A epitranscriptome and BCAA metabolism reprograming in leukemogenesis and LSC/LIC maintenance.

Chromosome-level assembly of triploid genome of Sichuan pepper (Zanthoxylum armatum)

As an important spice species in Rutaceae, the Sichuan pepper (Zanthoxylum armatum) can provide pungent and numbing taste, as well as aroma in its mature fruit. Here we assembled a chromosome-level genome of green prickly ash which was widely cultivated in a major production area including Chongqing and Sichuan province, China. We generated 712 Gb (∼112 × ) PacBio long reads and 511 Gb (∼82 × ) Hi-C data, and yielded an assembly of 99 pseudochromosomes with total size of 5.32 Gb and contig N50 of 796 kb. The genomic analyses and cytogenetic experiments both indicated that the cultivar ‘Zhuye Huajiao’ was a triploid. We identified a Zanthoxylum-specific whole genome duplication event emerging about 24.8 million years ago (Mya). We also detected a transposition burst event (0.3–0.4 Mya) responsible for the large genome size of Z. armatum. Metabolomic analysis of the Zanthoxylum fruits during development stages revealed profiles of 39 volatile aroma compounds and 528 secondary metabolites, from which six types of sanshools were identified. Based on metabolomic and transcriptomic network analysis, we screened candidate genes encoding long chain acyl-CoA synthetase, fatty acid desaturase, branched-chain amino acid aminotransferase involved in sanshool biosynthesis and three genes encoding terpene synthase during fruit development. The multi-omics data provide insights into the evolution of Zanthoxylum and molecular basis of numbing and aroma flavor of Sichuan pepper.

DROUGHT-INDUCED BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE enhances drought tolerance in rice.

Plants accumulate several metabolites in response to drought stress, including branched-chain amino acids (BCAAs). However, the roles of BCAAs in plant drought responses and the underlying molecular mechanisms for BCAA accumulation remain elusive. Here, we demonstrate that rice (Oryza sativa) DROUGHT-INDUCED BRANCHED-CHAIN AMINO ACID AMINOTRANSFERASE (OsDIAT) mediates the accumulation of BCAAs in rice in response to drought stress. An in vitro enzyme activity assay indicated that OsDIAT is a branched-chain amino acid aminotransferase, and subcellular localization analysis revealed that OsDIAT localizes to the cytoplasm. The expression of OsDIAT was induced in plants upon exposure to abiotic stress. OsDIAT-overexpressing (OsDIATOX) plants were more tolerant to drought stress, whereas osdiat plants were more susceptible to drought stress compared with nontransgenic (NT) plants. Amino acid analysis revealed that BCAA levels were higher in OsDIATOX but lower in osdiat compared with in NT plants. Finally, the exogenous application of BCAAs improved plant tolerance to osmotic stress compared with that in control plants. Collectively, these findings suggest that OsDIAT mediates drought tolerance by promoting the accumulation of BCAAs.

Reduced branched-chain aminotransferase activity alleviates metabolic vulnerability caused by dim light exposure at night in Drosophila

The rhythmic pattern of biological processes controlled by light over 24 h is termed the circadian rhythm. Disturbance of circadian rhythm due to exposure to light at night (LAN) disrupts the sleep-wake cycle and can promote cardiovascular disease, diabetes, cancer, and metabolic disorders in humans. We studied how dim LAN affects the circadian rhythm and metabolism using male Drosophila. Wild-type flies exposed to the dim light of 10 lux at night displayed altered 24 h sleep-wake behavior and expression patterns of circadian rhythm genes. In addition, the flies became more vulnerable to metabolic stress, such as starvation. Whole-body metabolite analysis revealed decreased amounts of branched-chain amino acids (BCAAs), such as isoleucine and valine. The dim light exposure also increased the expression of branched-chain amino acid aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase (BCKDC) enzyme complexes that regulate the metabolism of BCAAs. Flies with the Bcat heterozygous mutation were not vulnerable to starvation stress, even when exposed to dim LAN, and hemolymph BCAA levels did not decrease in these flies. Furthermore, the vulnerability to starvation stress was also suppressed when the Bcat expression level was reduced in the whole body, neurons, or fat body during adulthood using conditional GAL4 and RNA interference. Finally, the metabolic vulnerability was reversed when BCAAs were fed to wild-type flies exposed to LAN. Thus, short-term dim light exposure at night affects the expression of circadian genes and BCAA metabolism in Drosophila, implying a novel function of BCAAs in suppressing metabolic stress caused by disrupted circadian rhythm.

Evaluation of therapeutic strategies targeting BCAA catabolism using a systems pharmacology model.

Background: Abnormal branched-chained amino acids (BCAA) accumulation in cardiomyocytes is associated with cardiac remodeling in heart failure. Administration of branched-chain α-keto acid dehydrogenase (BCKD) kinase inhibitor BT2 has been shown to reduce cardiac BCAA levels and demonstrated positive effects on cardiac function in a preclinical setting. The current study is focused on evaluating the impact of BT2 on the systemic and cardiac levels of BCAA and their metabolites as well as activities of BCAA catabolic enzymes using a quantitative systems pharmacology model. Methods: The model is composed of an ordinary differential equation system characterizing BCAA consumption with food, disposal in the proteins, reversible branched-chain-amino-acid aminotransferase (BCAT)-mediated transamination to branched-chain keto-acids (BCKA), followed by BCKD-mediated oxidation. Activity of BCKD is regulated by the balance of BCKDK and protein phosphatase 2Cm (PP2Cm) activities, affected by BT2 treatment. Cardiac BCAA levels are assumed to directly affect left ventricular ejection fraction (LVEF). Biochemical characteristics of the enzymes are taken from the public domains, while plasma and cardiac BCAA and BCKA levels in BT2 treated mice are used to inform the model parameters. Results: The model provides adequate reproduction of the experimental data and predicts synchronous BCAA responses in the systemic and cardiac space, dictated by rapid BCAA equilibration between the tissues. The model-based simulations indicate maximum possible effect of BT2 treatment on BCAA reduction to be 40% corresponding to 12% increase in LVEF. Model sensitivity analysis demonstrates strong impact of BCKDK and PP2Cm activities as well as total BCKD and co-substrate levels (glutamate, ketoglutarate and ATP) on BCAA and BCKA levels. Conclusion: Model based simulations confirms using of plasma measurements as a marker of cardiac BCAA changes under BCKDK inhibition. The proposed model can be used for optimization of preclinical study design for novel compounds targeting BCAA catabolism.

Metabolon formation regulates branched-chain amino acid oxidation and homeostasis.

The branched-chain aminotransferase isozymes BCAT1 and BCAT2, segregated into distinct subcellular compartments and tissues, initiate the catabolism of branched-chain amino acids (BCAAs). However, whether and how BCAT isozymes cooperate with downstream enzymes to control BCAA homeostasis in an intact organism remains largely unknown. Here, we analyse system-wide metabolomic changes in BCAT1- and BCAT2-deficient mouse models. Loss of BCAT2 but not BCAT1 leads to accumulation of BCAAs and branched-chain α-keto acids (BCKAs), causing morbidity and mortality that can be ameliorated by dietary BCAA restriction. Through proximity labelling, isotope tracing and enzymatic assays, we provide evidence for the formation of a mitochondrial BCAA metabolon involving BCAT2 and branched-chain α-keto acid dehydrogenase. Disabling the metabolon contributes to BCAT2 deficiency-induced phenotypes, which can be reversed by BCAT1-mediated BCKA reamination. These findings establish a role for metabolon formation in BCAA metabolism in vivo and suggest a new strategy to modulate this pathway in diseases involving dysfunctional BCAA metabolism.

High Expression of BCAT1 Affects DNA Damage Response By Inhibiting α-KG-Dependent Histone Demethylase

Branched-chain amino acids (BCAA) are essential for tumor growth and proliferation. Branched-chain aminotransferase 1(BCAT1) correlates with AML aggression, not only that, it has been reported that overexpression of BCAT1 in leukemia cells decreases intracellular αKG and impairs the activities of αKG-dependent dioxygenase. In this study, we firstly analyzed the prognostic significance of BCAT1 expression in Cytogenetically normal AML(CN-AML) patients. We found that patients with high levels of BCAT1 had poor prognosis, the median overall survival time is shorter in BCAT1 high group (356 days versus 570 days,p=0.004)(Figure A).Next, we performed gene knockout in THP-1 and MV4-11 cells and gene overexpressing in OCI-AML3 cells. In summary, BCAT1 suppression of BCAT1 markedly limited cell proliferation and colony-forming ability by blocking cell circle at G0/G1 phase(Figure B,C),whereas overexpression contributed to colony-forming and cell growth(Figure D,E). In vivo experiments confirmed that BCAT1 knockout can reduce the tumor burden of xenograft mice, while BCAT1 overexpression remarkably increased tumor burden (Figure F).Survival analysis showed that the BCAT1 OE group significantly shortened survival time(22.5 days vs 26.0 days), and the median survival time of the knockout group was 2.5 days longer than control(26.0 days vs 28.5 day ).(Figure G). To further clarify the mechanism by which BCAT1 affects the proliferation of AML cells, we collected specimens from 87 AML patients and performed RNA-Seq analysis. Through Gene Set Enrichment Analysis, we found that the DNA Genes down-regulated in response to ultraviolet (UV) radiation pathway was enriched in the high expression group of BCAT1(Figure H). Therefore, we further examined the effect of BCAT1 expression level on DNA damage repair in vitro.DNA damage foci studies were performed in control and BCAT1 OE cells. More γ-H2AX focis in BCAT1 overexpressed cells were observed. About 48.20% of BCAT1 OE cell were obeseved ＞5 γ-H2AX foci, which was much higher than the control group(13.6%). And significant regression of γ-H2AX levels in BCAT1 overexpressing cells after the addition of DM-αKG(21%)(Figure I).We used western blot to detect DSB repair-related proteins and found that ATM was also down-regulated in BCAT1 overexpressing cells, along with an upregulation γ-H2AX and the changes of these proteins can also be rescued by exogenous supplementation of DM-aKG(Figure J). High expression of BCAT1 reduces the level of intracellular α-KG and inhibits the activity of α-KG-dependent dioxygenases. Histone demethylase KDM2-7 are αKG-dependent dioxygenase, so we speculated that the decrease in a-KG caused by high BCAT1 expression would limit the activity of histone demethylase, thereby affecting the cellular histone methylation levels. We detected several classic histone methylation markers after BCAT1 overexpression, results showed that the levels of H3K9me3 were significantly up-regulated after BCAT1 overexpression, which could be reversed by supplementation of DM-α-KG(Figure K). Therefore, we believe that the regulation of cellular histone methylation levels by BCAT1 is affected by α-KG levels. H3K9me3 is an epigenetic suppressive marker. IDH1mut cells inhibited ATM expression by upregulating histone H3K9 methylation levels and impede cellular DNA damage repair. The elevated DNA damage level in BCAT1 overexpression condition may be therapeutically exploited using therapies that induce or augment DNA damage, such as chemotherapy and PARP inhibitor, Next, we tried to compare the responses of BCAT1 overexpression and control AML cells to the PARP inhibitor BMN673(Talazoparib). After treatment with BMN673, the survival fraction of BCAT1 overexpressing AML cells was lower than that of negative control in MTS assay. Vice versa, The IC50 of BMN673 in BCAT1 knockout AML cells is much larger than that of the control(Figure L). In summary, this study verified BCAT1 high expression is an independent prognostic predictor of CN-AML.BCAT1 reduces intracellular αKG levels, inhibits αKG-dependent histone demethylation enzymes to inhibit H3K9me3 demethylation, thereby suppressing ATM expression and inhibiting DNA damage response in AML cells. Therefore, BCAT1 high expressing AML cells have higher sensitivity to PARP inhibitors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CTNI-23. PRELIMINARY SAFETY AND PHARMACOKINETICS DATA FOR A PHASE 1B TRIAL OF TELAGLENASTAT IN COMBINATION WITH RADIATION THERAPY AND TEMOZOLOMIDE IN PATIENTS WITH IDH-MUTANT GRADE 2/3 ASTROCYTOMA (NCI-10218)

Abstract IDH mutant gliomas depend on glutaminase for glutamate/glutathione generation from glutamine because R-2-hydroxyglutarate inhibits branched chain amino acid transaminase mediated glutamate biosynthesis. Telaglenastat (CB-839 HCl) is a potent glutaminase-1 specific inhibitor which depletes tumor glutamate in orthotopic IDH mutant glioma PDX models and extends survival in these orthotopic models when added to radiation/temozolomide. NCI-10218 (NCT03528642) is a phase I clinical trial investigating the safety and tolerability of telaglenastat administered orally concurrently with standard doses of radiation (50.4 Gy, grade 2; 59.4 Gy, grade 3) and temozolomide (75 mg/m2 orally daily) in patients (age 16+) with previously untreated IDH mutant grade 2/3 astrocytoma. Telaglenastat dose was escalated in cohorts (400-800 mg twice daily) based on a standard 3 + 3 design to determine the recommended phase 2 dose (RP2D). Toxicities were graded per CTCAE v5.0. An expansion cohort additionally incorporated a seven-day run-in of telaglenastat monotherapy at RP2D prior to radiation to evaluate the pharmacodynamic impact of telaglenastat on plasma and tumor metabolites. 23 patients with IDH mutant astrocytoma (WHO grade 2, n = 5; WHO grade 3, n = 18) were accrued between December 2018 and January 2022 (Dose Escalation: 16; Dose Expansion: 7). Median age was 32 years (range 23-69 years). 61% were male and 70% were ECOG 0. No dose-limiting toxicities were observed. Grade 3/4 adverse events (independent of attribution) included: lymphopenia (3), neutropenia (2), leukocytosis (2), alanine transaminase elevation (2), thrombocytopenia (1), leukopenia (1), maculopapular rash (1), hyperglycemia (1), hyponatremia (1). The RP2D of concurrent telaglenastat was defined as 800 mg twice daily. Following peak absorption on Day 15 at RP2D, the mean (%CV) terminal elimination half-life in the plasma was 4.2 (53.5%) hours (range 2.1-7.1 hours). The Cmax, Tmax, oral clearance, and AUC were 1496 ng/mL, 4.0 hr, 93.6 L/hr/m2, and 7430 ng/mL*hr, respectively.

TMET-23. GLIOBLASTOMA EXHIBIT SEX DIFFERENCES IN BRANCHED-CHAIN AMINO ACID METABOLISM

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. It is more prevalent in males and female patients exhibit a survival advantage. Understanding the molecular mechanisms that underlie those sex differences could support novel treatment strategies. Amino acids, such as glutamine and the branched-chain amino acids (leucine, isoleucine, valine; BCAAs), play important roles in GBM as they are involved in biosynthesis, energetics, redox homeostasis, and mTOR signaling. Using murine transformed astrocytes, we recently discovered that male cells are more dependent on glutamine and utilize more glutamine to replenish biosynthetic pathways, including BCAA regeneration. BCAA regeneration (i.e., re-amination of branched-chain ketoacids to BCAA) is catalyzed by branched-chain aminotransferases (BCATs) and requires glutamate as a nitrogen donor. Importantly, BCAA regeneration has been linked to cancer progression and poor outcome. Using stable isotope glutamine labeling we found that male transformed astrocytes exhibit greater nitrogen transfer from glutamate onto branched-chain ketoacids to regenerate BCAAs. In line with increased glutamate utilization for BCAA regeneration via BCAT, we found that male transformed astrocytes exhibit greater BCAT1 protein expression levels. Concordantly, male transformed astrocytes are more sensitive to BCAA deprivation compared to female cells, indicating that BCAA metabolism may play a sex-specific role in GBM. Using human GBM surgical specimens, we found greater BCAA metabolite abundance in male GBM. Importantly, our findings on sex differences in BCAA abundance and dependency in GBM are in line with previous reports that show that BCAA abundance and utilization differ between males and females under normal physiological conditions. Together, these data suggest that male GBM are more dependent on BCAAs and that BCAT1 may play unique roles in male and female GBM metabolism. Our data highlight that GBM metabolism differs between males and females and underline the importance of considering sex in metabolic targeting approaches.

BCAT1, as a prognostic factor for HCC, can promote the development of liver cancer through activation of the AKT signaling pathway and EMT.

More and more studies have shown that Branched chain amino acid transaminase 1 (BCAT1) is involved in the occurrence and development of a variety of tumors. However, the mechanism of its occurrence and development in hepatocellular carcinoma (HCC) remains unclear.Here, we demonstrated the relationship between BCAT1 and AKT signaling pathway, as well as EMT, and the clinical significance of BCAT1 by using BCAT1 expression in 5 cell lines and 113 liver cancer and non-liver cancer tissue samples.The results showed that the expression of AKT was positively correlated with BCAT1 in HCC tissues, and BCAT1 could promote the progression of HCC cells through the AKT signaling pathway. Clinical analysis and Bioinformatics technology analysis revealed that BCAT1 was correlated with poor prognosis, and BCAT1 expression in the HCC tissues was evidently correlated with tumor number, vascular invasion, Edmondson grade and TNM stage (P < 0.05).In vitro studies showed that BCAT1 increased the invasion and migration of in MHCC-97H cells a d Huh7 cells. By inhibiting the expression of the BCAT1 gene, we detected the corresponding changes in the expression levels of Twist, E-cadherin and Vimentin, confirming that BCAT1 may promote the invasion and migration of HCC cells through epithelial-mesenchymal transformation (EMT). Overall, BCAT1 can activate AKT signaling pathway and EMT to promote the development and metastasis of HCC cells. this study may provide new ideas and directions for cancer diagnosis and treatment.

BCATc inhibitor 2 ameliorated mitochondrial dysfunction and apoptosis in oleic acid-induced non-alcoholic fatty liver disease model.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent hepatic disease in the world. Disorders of branched chain amino acid (BCAA) metabolism is involved in various diseases. In this study, we aim to explore the role of BCAA metabolism in the development of NAFLD and the protective effect of BCATc Inhibitor 2, an inhibitor of cytosolic branched chain amino acid transaminase, against NAFLD as well as its underlying mechanism. It was found that oleic acid induced lipid accumulation and apoptosis in HepG2 and LO2 cells. Supplementation of BCAAs further aggravated oleic acid-induced lipid accumulation and apoptosis. In contrast, treatment of BCATc Inhibitor 2 ameliorated oleic acid-induced lipid accumulation and apoptosis. Molecularly, supplementation of BCAAs or treatment of BCATc Inhibitor 2 up-regulated or down-regulated the expression of SREBP1 and lipogenesis-related genes without affecting lipolysis-related genes. BCATc Inhibitor 2 maintained mitochondrial function by ameliorating oleic acid-induced mitochondrial ROS generation and mitochondrial membrane potential disruption. In addition, BCATc Inhibitor 2 treatment alleviated oleic acid-induced activation of JNK and AKT signaling pathway and Bcl2/Bax/Caspase axis. In conclusion, our results indicate BCAA metabolism is involved in NAFLD and BCATc Inhibitor 2 protects against oleic acid-induced lipid accumulation and apoptosis. These findings suggest that BCATc Inhibitor 2 is a promising candidate drug for the treatment of NAFLD.

BAY-069, a Novel (Trifluoromethyl)pyrimidinedione-Based BCAT1/2 Inhibitor and Chemical Probe.

The branched-chain amino acid transaminases (BCATs) are enzymes that catalyze the first reaction of catabolism of the essential branched-chain amino acids to branched-chain keto acids to form glutamate. They are known to play a key role in different cancer types. Here, we report a new structural class of BCAT1/2 inhibitors, (trifluoromethyl)pyrimidinediones, identified by a high-throughput screening campaign and subsequent optimization guided by a series of X-ray crystal structures. Our potent dual BCAT1/2 inhibitor BAY-069 displays high cellular activity and very good selectivity. Along with a negative control (BAY-771), BAY-069 was donated as a chemical probe to the Structural Genomics Consortium.

Microbial diversity and functional genes of red vinasse acid based on metagenome analysis.

Red vinasse acid has a distinct flavor and a vivid red color that are directly tied to the intricate metabolic activities of microorganisms that produce it. In this study, metagenomic technology was used to mine its functional genes and examine the microbial diversity of red vinasse acid. The findings revealed the identification of 2,609 species, 782 genera, and 63 phyla of microorganisms, and the dominant genus was Lactobacillus. Amino acid metabolism and carbohydrate metabolism were significant activities among the 16,093 and 49,652 genes that were annotated in the evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. In gluconeogenesis, red vinasse acid encodes 194 genes controlling the transporter protein systems of different sugars and has key enzyme genes that catalyze the conversion of intracellular sugars into glycolytic intermediates. In amino acid flavor formation, red vinasse acid contains 32 control genes for branched-chain aminotransferase (BCAT), 27 control genes for aromatic-amino-acid transaminase (ArAT), 60 control genes for keto acid invertase, 123 control genes for alcohol/aldehyde dehydrogenase, and 27 control genes for acetyl esterase, which have the basis for the formation of strong flavor substances from amino acids.

Discovery of inhibitors against mycobacterium branched-chain amino acid aminotransferases through in silico screening and experimental evaluation.

Tuberculosis (TB) is one of the most dangerous infectious diseases and is caused by Mycobacterium bovis (Mb) and Mycobacterium tuberculosis (Mt). Branched-chain amino acid aminotransferases (BCATs) were reported to be the key enzyme for methionine synthesis in Mycobacterium. Blocking the methionine synthesis in Mycobacterium can inhibit the growth of Mycobacterium. Therefore, in silico screening of inhibitors can be a good way to develop a potential drug for treating TB. A pyridoxal 5'-phosphate (PLP)-form of Mycobacterium bovis branched-chain amino acid aminotransferases (MbBCAT), an active form of MbBCAT, was constructed manually for docking approximately 150 000 compounds and the free energy was calculated in Autodock Vina. The 10 compounds which had the highest affinity to MbBCAT were further evaluated for their inhibitory effects against MbBCAT. Within the selected compounds, compound 4 (ZINC12359007) was found to be the best inhibitor against MbBCAT with the inhibitory constant Ki of 0·45 μmol l-1 and IC50 of 2·37 μmol l-1 . Our work provides potential candidates to develop effective drugs to prevent TB since the well-known structural information would be beneficial in the structure-based modification and design.

BCAT1 redox function maintains mitotic fidelity.

The metabolic enzyme branched-chain amino acid transaminase 1 (BCAT1) drives cell proliferation in aggressive cancers such as glioblastoma. Here, we show that BCAT1 localizes to mitotic structures and has a non-metabolic function as a mitotic regulator. Furthermore, BCAT1 is required for chromosome segregation in cancer and induced pluripotent stem cells and tumor growth in human cerebral organoid and mouse syngraft models. Applying gene knockout and rescue strategies, we show that the BCAT1 CXXC redox motif is crucial for controlling cysteine sulfenylation specifically in mitotic cells, promoting Aurora kinase B localization to centromeres, and securing accurate chromosome segregation. These findings offer an explanation for the well-established role of BCAT1 in promoting cancer cell proliferation. In summary, our data establish BCAT1 as a component of the mitotic apparatus that safeguards mitotic fidelity through a moonlighting redox functionality.

Reprograming of proteasomal degradation by branched chain amino acid metabolism.

Branched-chain amino acid (BCAA) metabolism is a central hub for energy production and regulation of numerous physiological processes. Controversially, both increased and decreased levels of BCAAs are associated with longevity. Using genetics and multi-omics analyses in Caenorhabditis elegans, we identified adaptive regulation of the ubiquitin-proteasome system (UPS) in response to defective BCAA catabolic reactions after the initial transamination step. Worms with impaired BCAA metabolism show a slower turnover of a GFP-based proteasome substrate, which is suppressed by loss-of-function of the first BCAA catabolic enzyme, the branched-chain aminotransferase BCAT-1. The exogenous supply of BCAA-derived carboxylic acids, which are known to accumulate in the body fluid of patients with BCAA metabolic disorders, is sufficient to regulate the UPS. The link between BCAA intermediates and UPS function presented here sheds light on the unexplained role of BCAAs in the aging process and opens future possibilities for therapeutic interventions.

Inhibitory Effects of Gabapentin on the Proliferation and Cell Motility of Hepatocellular Carcinoma Cells

Background: Gabapentin, originally an antiepileptic agent, was found to have anti-cancer activity on multiple cancer cells. However, its effects on hepatocellular carcinoma (HCC) and associated molecular mechanisms are unclear. Objectives: In this study, we investigated the anti-cancer effects of gabapentin against HCC cells in vitro and in vivo. Methods: Human HCC cells were inoculated with various levels of gabapentin for 24 and 48 h. We utilized the MTT assay to detect the proliferation of HCC cells after gabapentin treatment. The effect of gabapentin on the migration of HCC cells was detected by transwell migration assay. We established a model of subcutaneously transplanted HCC in nude mice and observed the impact of gabapentin on HCC cell tumorigenicity in vivo. The changes in RNA expression in gabapentin-treated HCC cells were evaluated by RNA sequencing analysis, and the results were analyzed and further validated by qRT-PCR. Results: Gabapentin significantly inhibited the proliferation of a variety of human HCC cells in a time- and dose-dependent approach. After treatment with 10 mM gabapentin for 12 h, the transendothelial migration of HCC cells via membrane remarkably reduced. Three weeks after the hypodermic transplanting of HCC in nude mice with Huh7 cell line, the gabapentin-treated group had a dramatic decrease in mean tumor volume and weight relative to the controls. Relative to the normal Huh7 cell line, the results of RNA sequencing of Huh7 cells treated with gabapentin for 24 h showed the differential enrichment of genes involved in "energy metabolism", "cancers", "signal transduction", and "folding, sorting, and degradation". The genes CDH11 and ARHGAP15 related to cell migration were further verified by qRT-PCR. Conclusions: Our results suggested that gabapentin has an inhibitory effect on the growth, migration, and tumor formation of hepatoma cells, and the mechanism of gabapentin’s inhibition on HCC cells may be related to some signaling pathways, which will lay a foundation for the future studies on branched-chain aminotransferase 1 (BCAT1) as a target for HCC treatment.