Branched-chain Aminotransferase Research Articles

IMMU-58. BCAT1 TARGETED METABOLIC REPROGRAMMING PLAYS CRITICAL ROLES IN MODULATING MYELOID DERIVED SUPPRESSOR CELL FUNCTION IN GLIOBLASTOMA

Abstract A key component that has limited the efficacy of immunotherapy in glioblastoma (GBM) is the significant infiltration of immunosuppressive myeloid cells. Our previous work characterized myeloid derived suppressor cells (MDSCs) unique to GBM that drive tumor growth and immune suppression. Transcriptomic and flow cytometric analyses demonstrated that the most induced programs in these MDSCs are dominated by metabolic and mTOR pathways. Therefore, we aimed to understand the role of metabolic reprogramming in MDSC differentiation and immunosuppressive function. Using single cell RNA sequencing, we identified overexpression of enzymes and transporters involved in branched-chain amino acid (BCAA) metabolism, notably branched chain aminotransferase 1 (BCAT1), the first enzyme in the metabolic pathway. Intriguingly, this was only found in specific populations of early and monocytic MDSCs (E-MDSCs, M-MDSCs), and not polymorphonuclear MDSCs (PMN-MDSCs). Using established MDSC modeling systems, we found that M-MDSC differentiation and T cell suppressive function are dependent on BCAAs and can be hindered by pharmacological inhibition of BCAT1 activity or genetic knockout of BCAT1. We also establish that the influence of BCAA on M-MDSC function is specifically reliant on the BCAT1-mediated step of BCAA metabolism. To determine the underlying mechanism driving BCAT1’s impact on M-MDSC function, we evaluated the activity of signaling pathways known to be critical to M-MDSCs including the mTORC1 pathway. We found that the impact of BCAA and BCAT1 activity on M-MDSC function is abrogated by inhibition of mTORC1 pathway with reduction in M-MDSC differentiation and activity. Consistent with our single cell RNA sequencing analysis, modulation of BCAA levels and BCAT1 activity had no impact on PMN-MDSCs. Our results underscore the critical role of BCAT1 in preferentially modulating M-MDSC activity through mTORC1 signaling. Our finding paves the way for discovery of a targetable therapeutic approach to harness immunometabolism in myeloid cells to develop novel immune therapies.

CTNI-37. INTERIM SAFETY ANALYSIS OF THE PHASE IB/II RANDOMIZED ANTI-GLUTAMATERGIC DRUG REPURPOSING TRIAL GLUGLIO IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA (NCT05664464)

Abstract The advent of cancer neuroscience has significantly advanced our understanding of brain tumor biology, yet the clinical implications remain largely unexplored. Glutamate, the most abundant excitatory neurotransmitter, presents a potential therapeutic target in brain tumors. Glioblastoma cells synthesize and secrete large amounts of glutamate, driving epilepsy, neuronal death, tumor growth, and invasion. Several brain-penetrating drugs, approved for other conditions, can inhibit glutamate synthesis, secretion, and signaling. These drugs include: (i) gabapentin, an anti-seizure medication that inhibits the glutamate synthesis enzyme branched-chain amino acid transaminase 1; (ii) sulfasalazine, an anti-inflammatory drug that blocks the cystine-glutamate exchanger system Xc, reducing glutamate secretion; and (iii) memantine, a cognitive enhancer that prevents glutamate-driven, calcium-induced neuronal death and tumor cell invasion by blocking N-methyl-D-aspartate type glutamate receptors. The multi-center, open-label, parallel-group, two-arm, 1:1 randomized phase Ib/II trial GLUGLIO is investigating the efficacy of a daily oral anti-glutamatergic triple combination of gabapentin, sulfasalazine, and memantine until tumor progression, in conjunction with standard chemoradiotherapy, compared to chemoradiotherapy alone. The primary endpoint is progression-free survival at 6 months (PFS-6). A sample size of 120 patients was determined to detect an increase in PFS-6 from 50% to 70%, with 80% power, using exploratory hypothesis testing at a one-sided significance level of 10%. Secondary endpoints include overall survival, seizure-free survival, response rate, quality of life for patients and caregivers, symptom burden, cognitive functioning, tumor glutamate levels (measured by magnetic resonance spectroscopy), and the use of anti-convulsants and steroids. Each drug is dosed up to the maximum approved amount. Results from the phase Ib part of the trial, including an interim safety analysis, will be presented at the conference.

Characterization of an aminotransferase TlBCAT from Trichoderma longibrachiatum UN32 involved in dendrobine-type total alkaloids biosynthesis.

Trichoderma longibrachiatum UN32 is an industrially important fungus capable of producing Dendrobine-Type Total alkaloids (DTTAs). Several reports have pointed out that branched-chain amino acid aminotransferases (BCATs) participate in backbone modification or promote the production of secondary metabolites. We previously proposed that cobalt chloride increased DTTAs production in T. longibrachiatum UN32, which was associated with enhanced expression of the gene BCAT, TlBCAT (Genbank accession No. PP465542). Following cloning and characterization, the cDNA of TlBCAT was found to consists of 1191bp, encoding a protein of 397 amino acid residues. The molecular mass of TlBCAT was about 42kDa through SDS-PAGE analysis. The predicted pI value was 5.54. Recombinant TlBCAT can catalyze L-leu with a catalytic efficiency of 15.91 mM- 1S- 1. In the binding pocket, residues interacting with PLP, including Tyr68, Arg93, Tyr136, and Lys194, are highly conserved. TlBCAT exhibits a broad spectrum of substrate specificity, typical for catalyzing the transamination reaction of various branched-chain and hydrophobic amino acids, with α-ketoglutarate as the amino acceptor. Exogenous branched-chain amino acids (BCAAs) positively affect Trichoderma growth and DTTAs production. These findings offer insights into the physiological significance of BCAAs and present a novel approach for enhancing DTTAs production in Trichoderma mycelium cultures.

Oligonol®, an Oligomerized Polyphenol from Litchi chinensis, Enhances Branched-Chain Amino Acid Transportation and Catabolism to Alleviate Sarcopenia.

Branched-chain amino acids (BCAAs) are essential for muscle protein synthesis and are widely acknowledged for mitigating sarcopenia. Oligonol® (Olg), a low-molecular-weight polyphenol from Litchi chinensis, has also been found to attenuate sarcopenia by improving mitochondrial quality and positive protein turnover. This study aims to investigate the effect of Olg on BCAA-stimulated protein synthesis in sarcopenia. In sarcopenic C57BL/6 mice and senescence-accelerated mouse-prone 8 (SAMP8) mice, BCAAs were significantly decreased in skeletal muscle but increased in blood serum. Furthermore, the expressions of membrane L-type amino acid transporter 1 (LAT1) and branched-chain amino acid transaminase 2 (BCAT2) in skeletal muscle were lower in aged mice than in young mice. The administration of Olg for 8 weeks significantly increased the expressions of membrane LAT1 and BCAT2 in the skeletal muscle when compared with non-treated SAMP8 mice. We further found that BCAA deprivation via LAT1-siRNA in C2C12 myotubes inhibited the signaling of protein synthesis and facilitated ubiquitination degradation of BCAT2. In C2C12 cells mimicking sarcopenia, Olg combined with BCAA supplementation enhanced mTOR/p70S6K activity more than BCAA alone. However, blocked LAT1 by JPH203 reversed the synergistic effect of the combination of Olg and BCAAs. Taken together, changes in LAT1 and BCAT2 during aging profoundly alter BCAA availability and nutrient signaling in aged mice. Olg increases BCAA-stimulated protein synthesis via modulating BCAA transportation and BCAA catabolism. Combining Olg and BCAAs may be a useful nutritional strategy for alleviating sarcopenia.

Integrated Analysis of Metabolome and Transcriptome Reveals the Effect of Burdock Fructooligosaccharide on the Quality of Chinese Cabbage (Brassica rapa L. ssp. Pekinensis).

Burdock fructooligosaccharide (BFO) is fructose with a low polymerization degree, which could improve the immunity to pathogens, quality, and stress resistance of vegetables. Still, there are no studies on applying BFO in Chinese cabbage. In this study, the effects of exogenous BFO sprayed with different concentrations (0, 5, 10, 20, 30 g·L-1) on the growth and soluble sugar content of Chinese cabbage seedlings were determined. The result showed that 10 g·L-1 was the appropriate spraying concentration. Based on metabolome analysis, a total of 220 differentially accumulated metabolites (DAMs) were found, among which flavonoid metabolites, glucosinolate metabolites, and soluble sugar-related metabolites were the key metabolites involved in improving the quality of Chinese cabbage caused by BFO. Further combination analysis with transcriptome, trans-cinnamate 4-monooxygenase (CYP73A5), and chalcone synthase 1 (CHS1) were more closely associated with the DAMs of flavonoid biosynthesis. Sulfotransferases 18 (SOT18), Branched-chain amino acid amino transferases 6 (BCAT6), and cytochrome P450 monooxygenase (CYP83A1) were the key genes in glucosinolate biosynthesis. Hexokinase (HxK1), beta-glucosidase 8 (BGL08), invertase 3 (INV3), beta-glucosidase 3B (BGL3B), and sucrose phosphate synthase 1 (SPS1) were significantly upregulated, potentially playing crucial roles in the soluble sugar metabolism. In conclusion, these results provided an understanding of the effects of BFO on the expression of genes and the accumulation of metabolites related to quality formation in Chinese cabbage.

Ultrasensitive Amplification-Free Quantification of a Methyl CpG-Rich Cancer Biomarker by Single-Molecule Kinetic Fingerprinting.

The most well-studied epigenetic marker in humans is the 5-methyl modification of cytosine in DNA, which has great potential as a disease biomarker. Currently, quantification of DNA methylation relies heavily on bisulfite conversion followed by PCR amplification and NGS or microarray analysis. PCR is subject to potential bias in differential amplification of bisulfite-converted methylated versus unmethylated sequences. Here, we combine bisulfite conversion with single-molecule kinetic fingerprinting to develop an amplification-free assay for DNA methylation at the branched-chain amino acid transaminase 1 (BCAT1) promoter. Our assay selectively responds to methylated sequences with a limit of detection below 1 fM and a specificity of 99.9999%. Evaluating complex genomic DNA matrices, we reliably distinguish <5% DNA methylation at the BCAT1 promoter in whole blood DNA from completely unmethylated whole-genome amplified DNA. Taken together, these results demonstrate the feasibility and sensitivity of our amplification-free, single-molecule quantification approach to improve the early detection of methylated cancer DNA biomarkers.

BCAT1 contributes to the development of TKI-resistant CML.

Although most of chronic myeloid leukemia (CML) patients can be effectively treated by the tyrosine kinase inhibitors (TKIs), such as Imatinib, TKI-resistance still occurs in approximately 15-17% of cases. Although many studies indicate that branched chain amino acid (BCAA) metabolism may contribute to the TKI resistance in CML, the detailed mechanisms remains largely unknown. The cell proliferation, colony formation and in vivo transplantation were used to determined the functions of BCAT1 in leukemogenesis. Quantitative real-time PCR (RT-PCR), western blotting, RNA sequencing, BCAA stimulation in vitro were applied to characterize the underlying molecular mechanism that control the leukemogenic activity of BCAT1-knockdown cells. In this report, we revealed that branched chain amino acid transaminase 1 (BCAT1) is highly enriched in both mouse and human TKI-resistant CML cells. Leukemia was almost completely abrogated upon BCAT1 knockdown during transplantation in a BCR-ABLT315I-induced murine TKI-resistant CML model. Moreover, knockdown of BCAT1 led to a dramatic decrease in the proliferation of TKI-resistant human leukemia cell lines. BCAA/BCAT1 signaling enhanced the phosphorylation of CREB, which is required for maintenance of TKI-resistant CML cells. Importantly, blockade of BCAA/BCAT1 signaling efficiently inhibited leukemogenesis both in vivo and in vitro. These findings demonstrate the role of BCAA/BCAT1 signaling in cancer development and suggest that targeting BCAA/BCAT1 signaling is a potential strategy for interfering with TKI-resistant CML.

Multi-omics analysis identifies BCAT2 as a potential pan-cancer biomarker for tumor progression and immune microenvironment modulation.

Branched-chain amino acid transaminase 2 (BCAT2) encodes a crucial protein involved in the initial catalysis of branched-chain amino acid (BCAA) catabolism, with emerging evidence suggesting its association with tumor progression. This study explores BCAT2 in a pan-cancer multi-omics context and evaluates its prognostic significance. We utilized a multi-database approach, analyzing cBioPortal for genetic alterations, RNA-Seq data from TCGA and GTEx for expression patterns, and RSEM for transcript analysis. Protein expression and interaction networks were assessed using the Human Protein Atlas, UniProt, and STRING. Prognostic value was determined through Cox regression analysis of TCGA clinical survival data, while immune cell infiltration across various cancers was examined using TCGA data and the TIMER2 platform. Our results revealed that BCAT2 alterations are primarily amplifications and is upregulated in various tumors, correlating with poor survival rates in several tumor types, including GBMLGG, LGG, and UVM. Elevated BCAT2 protein levels were common in pan-cancer, interacting with a range of metabolic enzymes. Additionally, BCAT2 expression significantly influenced CD4+ T cells, CD8+ T cells, and Treg cells infiltration, with varied correlations across cancer types. These findings indicate BCAT2 as a potential biomarker for cancer diagnosis and therapy, potentially regulating key metabolic and immune factors to mediate tumor progression and the microenvironment.

Inhibition of BCAT1 expression improves recurrent miscarriage by regulating cellular dysfunction and inflammation of trophoblasts.

Sustained or chronic inflammation in the placenta can result in placental insufficiency, leading to adverse reproductive outcomes such as pregnancy loss. Branched-chain amino acid transaminase 1 (BCAT1) expresses in the placenta and is involved in the pathological inflammatory response, but its role in recurrent miscarriage (RM) has not been fully investigated. In the present study, we delved into the effects of BCAT1 on trophoblast inflammation induced by lipopolysaccharide (LPS) and a mouse model of pregnancy loss induced by LPS. In vitro, after the HTR-8/SVneo cells were treated with LPS and BCATc inhibitor 2 (a selective BCAT inhibitor), the cell apoptosis was verified by TUNEL assay, and the activity of caspase-3 and caspase-9 was detected. Real-time PCR, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) were used to determine the expression of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and inflammasomes (NLRP3 and ASC) in LPS-treated trophoblast cells. Western blot analysis was performed to verify the expression of phospho-IκBα (p-IκBα) in cells and NF-κB p65 in the nuclei. IF staining was used to detect the nuclear translocation of NF-κB p65. The DNA binding activity of NF-κB was detected by an electrophoretic mobility shift assay (EMSA). The results demonstrated that inhibition of BCAT1 reduced trophoblast apoptosis, suppressed the release of proinflammatory cytokines, and prevented NLRP3 inflammasome activation in response to LPS. Additionally, BCAT1 inhibition blocked the activation of the NF-κB pathway in trophoblasts. This study highlights the potential therapeutic role of targeting BCAT1 in preventing adverse reproductive outcomes associated with chronic placental inflammation.

Small-molecule targeting BCAT1-mediated BCAA metabolism inhibits the activation of SHOC2-RAS-ERK to induce apoptosis of Triple-negative breast cancer cells

IntroductionTriple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer with the worst prognosis. Exploring novel carcinogenic factors and therapeutic drugs for TNBC remains a focus to improve prognosis. Branched-chain amino acid transaminase 1 (BCAT1), a crucial enzyme in branched-chain amino acid (BCAA) metabolism, has been linked to various tumor developments, but its carcinogenic function and mechanism in TNBC remain unclear. Eupalinolide B (EB) is a naturally-derived small-molecule with anti-tumor activity, but its role in TNBC remains unknown. ObjectivesBy exploring the targets and pharmacological mechanisms of EB in inhibiting TNBC, this study aimed to discover novel therapeutic targets and potential inhibitors for TNBC, and elucidate novel pathogenic mechanisms of TNBC. MethodsThe inhibitory effect of EB on TNBC was investigated using mouse models and cellular phenotypic experiments. Activity-based protein profiling (ABPP) technology, pull down-WB, CETSA-WB and MST were utilized to discover and validate the targets of EB. The oncogenic role of BCAT1 was determined through clinical data analysis and biochemical experiments. To elucidate the mechanism by which EB inhibited TNBC, many methods, including but not limited to HPLC and proteomic sequencing were used. ResultsWe found that EB significantly inhibited TNBC progression. We identified BCAT1 as the direct target of EB and confirmed that BCAT1 was critical for TNBC development. EB inhibited BCAT1-involved BCAA metabolism to reduce the synthesis of BCAAs (including Leu, Ile, and Val), thereby inhibiting SHOC2 (a Leu-rich repeat protein) expression and the downstream SHOC2-participating RAS-ERK signaling pathway, ultimately leading to apoptosis of TNBC cells. ConclusionCollectively, this study not only elucidates the oncogenic role of BCAT1 and its downstream SHOC2-RAS-ERK signaling axis in TNBC progression but also opens up avenues for potential therapies targeting BCAT1 or BCAA metabolism (using EB alone or in combination with its inhibitor candesartan) for TNBC treatment.

Integrated Metabolomics and Proteomics Analysis Provides Insights into the Formation of Volatile Compounds in Three Different Polyembryonic Mango Cultivars.

Understanding volatile compound formation is critical for enhancing the flavor quality of mangoes. Integrated untargeted metabolomics and proteomics were employed to explore volatile compound formation in three different polyembryonic mango cultivars ("Ah Ping," "Rosa," and "Rosigold"). A total of 87 volatile compounds were identified using SPME-GC-MS. Untargeted metabolomics and proteomics resulted in identification of 508 metabolites and 4481 proteins, respectively. Integrative analysis revealed that the volatile compound formation was influenced by fatty acids, amino acids, pentose, and hexose, as well as terpenoid metabolisms. Specifically, upward expression of core enzymes in lipoxygenanse pathway was responsible for the higher levels of some C6 and C9 volatile compounds in "Ah Ping." The differential expression of key enzymes in fatty acid degradation facilitates the varied contents of straight-chain volatile compounds. The upregulation of glutamate decarboxylase and branched-chain amino acid aminotransferase upstream of butanoate metabolism led to the highest levels of butyl esters in "Ah Ping." Furthermore, the different levels of volatile furan and pyran compounds might be attributed to differential expression of critical enzymes in pentose and hexose metabolism. These findings established a metabolic and proteomic map unraveling the biosynthesis of specific volatile compounds and provided insights into understanding the characteristic flavor of mango.

Metabolic rewiring during bone development underlies tRNA m7G-associated primordial dwarfism.

Translation of mRNA to protein is tightly regulated by transfer RNAs (tRNAs), which are subject to various chemical modifications that maintain structure, stability, and function. Deficiency of tRNA N7-methylguanosine (m7G) modification in patients causes a type of primordial dwarfism, but the underlying mechanism remains unknown. Here we report that the loss of m7G rewires cellular metabolism, leading to the pathogenesis of primordial dwarfism. Conditional deletion of the catalytic enzyme Mettl1 or missense mutation of the scaffold protein Wdr4 severely impaired endochondral bone formation and bone mass accrual. Mechanistically, Mettl1 knockout decreased abundance of m7G-modified tRNAs and inhibited translation of mRNAs relating to cytoskeleton and Rho GTPase signaling. Meanwhile, Mettl1 knockout enhanced cellular energy metabolism despite incompetent proliferation and osteogenic commitment. Further exploration revealed that impairment of Rho GTPase signaling upregulated the level of branched-chain amino acid transaminase 1 (BCAT1) that rewired cell metabolism and restricted intracellular α-ketoglutarate (αKG). Supplementation of αKG ameliorated the skeletal defect of Mettl1-deficient mice. In addition to the selective translation of metabolism-related mRNAs, we further revealed that Mettl1 knockout globally regulated translation via integrated stress response (ISR) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Restoring translation by targeting either ISR or mTORC1 aggravated bone defects of Mettl1-deficient mice. Overall, our study unveils a critical role of m7G tRNA modification in bone development by regulation of cellular metabolism and indicates suspension of translation initiation as a quality control mechanism in response to tRNA dysregulation.

A 2-hydroxybutyrate-mediated feedback loop regulates muscular fatigue

Several metabolites have been shown to have independent and at times unexpected biological effects outside of their metabolic pathways. These include succinate, lactate, fumarate, and 2-hydroxyglutarate. 2-Hydroxybutyrate (2HB) is a byproduct of endogenous cysteine synthesis, produced during periods of cellular stress. 2HB rises acutely after exercise; it also rises during infection and is also chronically increased in a number of metabolic disorders. We show here that 2HB inhibits branched-chain aminotransferase enzymes, which in turn triggers a SIRT4-dependent shift in the compartmental abundance of protein ADP-ribosylation. The 2HB-induced decrease in nuclear protein ADP-ribosylation leads to a C/EBPβ-mediated transcriptional response in the branched-chain amino acid degradation pathway. This response to 2HB exposure leads to an improved oxidative capacity in vitro. We found that repeated injection with 2HB can replicate the improvement to oxidative capacity that occurs following exercise training. Together, we show that 2-HB regulates fundamental aspects of skeletal muscle metabolism.

A 2-hydroxybutyrate-mediated feedback loop regulates muscular fatigue.

Several metabolites have been shown to have independent and at times unexpected biological effects outside of their metabolic pathways. These include succinate, lactate, fumarate, and 2-hydroxyglutarate. 2-Hydroxybutyrate (2HB) is a byproduct of endogenous cysteine synthesis, produced during periods of cellular stress. 2HB rises acutely after exercise; it also rises during infection and is also chronically increased in a number of metabolic disorders. We show here that 2HB inhibits branched-chain aminotransferase enzymes, which in turn triggers a SIRT4-dependent shift in the compartmental abundance of protein ADP-ribosylation. The 2HB-induced decrease in nuclear protein ADP-ribosylation leads to a C/EBPβ-mediated transcriptional response in the branched-chain amino acid degradation pathway. This response to 2HB exposure leads to an improved oxidative capacity in vitro. We found that repeated injection with 2HB can replicate the improvement to oxidative capacity that occurs following exercise training. Together, we show that 2-HB regulates fundamental aspects of skeletal muscle metabolism.

Differential Expression of Branched-Chain Aminotransferase in the Rat Ocular Tissues.

Branched-chain amino acids (BCAAs) play vital roles in metabolic and physiological processes, with their catabolism initiated by two branched-chain aminotransferase isozymes: cytosolic (BCATc) and mitochondrial (BCATm). These enzymes have tissue and cell-specific compartmentalization and are believed to shuttle metabolites between cells and tissues. Although their expression and localization have been established in most tissues, ocular tissues remain unknown. In this study, we used immunohistochemical analyses to investigate the expression and localization of BCAT enzymes in the normal eye tissues. As expected, BCATc was highly expressed in the neuronal cells of the retina, particularly in the ganglion cell layers, inner nuclear layer, and plexiform layer, with little to no expression in Müller cells. BCATc was also present in the cornea, retinal pigment epithelium (RPE), choroid, ciliary body, and iris but not in the lens. In contrast, BCATm was expressed across all ocular tissues, with strong expression in the Muller cells of the retina, the endothelial and epithelial layers of the cornea, the choroid and iris, and the epithelial cells at the lens's front. The extensive expression and distribution of BCAT isozymes in the ocular tissue, suggests that BCAA transamination is widespread in the eye, potentially aiding in metabolite transport between ocular tissues. The findings provide new insights into the physiological role of BCATs in the eye, particularly within the neuronal retina.

The Soybean Cyst Nematode Effector Cysteine Protease 1 (CPR1) Targets a Mitochondrial Soybean Branched-Chain Amino Acid Aminotransferase (GmBCAT1).

The soybean cyst nematode (SCN; Heterodera glycines) facilitates infection by secreting a repertoire of effector proteins into host cells to establish a permanent feeding site composed of a syncytium of root cells. Among the diverse proteins secreted by the nematode, we were specifically interested in identifying proteases to pursue our goal of engineering decoy substrates that elicit an immune response when cleaved by an SCN protease. We identified a cysteine protease that we named Cysteine Protease 1 (CPR1), which was predicted to be a secreted effector based on transcriptomic data obtained from SCN esophageal gland cells, presence of a signal peptide, and lack of transmembrane domains. CPR1 is conserved in all isolates of SCN sequenced to date, suggesting it is critical for virulence. Transient expression of CPR1 in Nicotiana benthamiana leaves suppressed cell death induced by a constitutively active nucleotide binding leucine-rich repeat protein, RPS5, indicating that CPR1 inhibits effector-triggered immunity. CPR1 localizes in part to the mitochondria when expressed in planta. Proximity-based labeling in transgenic soybean roots, co-immunoprecipitation, and cleavage assays identified a branched-chain amino acid aminotransferase from soybean (GmBCAT1) as a substrate of CPR1. Consistent with this, GmBCAT1 also localizes to mitochondria. Silencing of the CPR1 transcript in the nematode reduced penetration frequency in soybean roots while the expression of CPR1 in soybean roots enhanced susceptibility. Our data demonstrates that CPR1 is a conserved effector protease with a direct target in soybean roots, highlighting it as a promising candidate for decoy engineering.

Branched-chain amino acid transaminase 1 confers EGFR-TKI resistance through epigenetic glycolytic activation

Third-generation EGFR tyrosine kinase inhibitors (TKIs), exemplified by osimertinib, have demonstrated promising clinical efficacy in the treatment of non-small cell lung cancer (NSCLC). Our previous work has identified ASK120067 as a novel third-generation EGFR TKI with remarkable antitumor effects that has undergone New Drug Application (NDA) submission in China. Despite substantial progress, acquired resistance to EGFR-TKIs remains a significant challenge, impeding the long-term effectiveness of therapeutic approaches. In this study, we conducted a comprehensive investigation utilizing high-throughput proteomics analysis on established TKI-resistant tumor models, and found a notable upregulation of branched-chain amino acid transaminase 1 (BCAT1) expression in both osimertinib- and ASK120067-resistant tumors compared with the parental TKI-sensitive NSCLC tumors. Genetic depletion or pharmacological inhibition of BCAT1 impaired the growth of resistant cells and partially re-sensitized tumor cells to EGFR TKIs. Mechanistically, upregulated BCAT1 in resistant cells reprogrammed branched-chain amino acid (BCAA) metabolism and promoted alpha ketoglutarate (α-KG)-dependent demethylation of lysine 27 on histone H3 (H3K27) and subsequent transcriptional derepression of glycolysis-related genes, thereby enhancing glycolysis and promoting tumor progression. Moreover, we identified WQQ-345 as a novel BCAT1 inhibitor exhibiting antitumor activity both in vitro and in vivo against TKI-resistant lung cancer with high BCAT1 expression. In summary, our study highlighted the crucial role of BCAT1 in mediating resistance to third-generation EGFR-TKIs through epigenetic activation of glycolysis in NSCLC, thereby supporting BCAT1 as a promising therapeutic target for the treatment of TKI-resistant NSCLC.

CHIP-mediated ubiquitin degradation of BCAT1 regulates glioma cell proliferation and temozolomide sensitivity

Glioma, a malignant and infiltrative neoplasm of the central nervous system, poses a significant threat due to its high mortality rates. Branched-chain amino acid transaminase 1 (BCAT1), a key enzyme in branched-chain amino acid (BCAA) catabolism, exhibits elevated expression in gliomas and correlates strongly with poor prognosis. Nonetheless, the regulatory mechanisms underlying this increased BCAT1 expression remains incompletely understood. In this study, we reveal that ubiquitination at Lys360 facilitates BCAT1 degradation, with low ubiquitination levels contributing to high BCAT1 expression in glioma cells. The Carboxyl terminus of Hsc70-interacting protein (CHIP), an E3 ubiquitin ligase, interacts with BCAT1 via its coiled-coil (CC) domain, promoting its K48-linkage ubiquitin degradation through proteasomal pathway. Moreover, CHIP-mediated BCAT1 degradation induces metabolic reprogramming, and impedes glioma cell proliferation and tumor growth both in vitro and in vivo. Furthermore, a positive correlation is observed between low CHIP expression, elevated BCAT1 levels, and unfavorable prognosis among glioma patients. Additionally, we show that the CHIP/BCAT1 axis enhances glioma sensitivity to temozolomide by reducing glutathione (GSH) synthesis and increasing oxidative stress. These findings underscore the critical role of CHIP/BCAT1 axis in glioma cell proliferation and temozolomide sensitivity, highlighting its potential as a diagnostic marker and therapeutic target in glioma treatment.

BCAT1 promotes cell proliferation, migration, and invasion via the PI3K-Akt signaling pathway in oral squamous cell carcinoma.

To analyze the expression, biological function of branched chain amino-acid transaminase 1 (BCAT1) in oral squamous cell carcinoma (OSCC). Real-time PCR and immunohistochemistry were used to analyze the expression of BCAT1 protein in OSCC and normal oral tissues. Based on the clinicopathological information of patients, the relationship between the expression of BCAT1 protein and other clinicopathological factors was analyzed. Real-time PCR and western blot assays were used to analyze the expression of BCAT1 gene and protein in normal human oral keratinocytes (HOK) and human OSCC cells, respectively. After BCAT1 overexpression or knockdown, the proliferation, cell cycle, migration, and invasion of human OSCC cells were analyzed by CCK8, flow cytometry, wound healing, and transwell invasion assays, respectively. After adding the BCAT1 inhibitor EGR240 to OSCC cells, the changes in cell proliferation, migration, and invasion ability in OSCC cells were analyzed. Based on the TCGA database, the involved signal pathway in BCAT1-related and BCAT1-binding genes was obtained for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, verified by western blot assays. After inhibiting PI3K, the effect of BCAT1 on the expression of the downstream phosphorylated protein of the PI3K-Akt signaling pathway was analyzed by western blot assays. The relationship between the expression of BCAT1 and EMT-related protein of OSCC cells was also analyzed. The expression of BCAT1 gene and protein were upregulated in OSCC tissue, which positively correlated with the pathological grade of patients with OSCC. Compared with normal oral keratinocytes, BCAT1 gene and protein were upregulated in OSCC cells. BCAT1 overexpression promoted the proliferation, migration, and invasion of OSCC cells. BCAT1 knockdown or inhibition could reduce the proliferation, migration, and invasion abilities of OSCC cells. The results of bioinformatics analysis and Western bolt showed that BCAT1 could regulate the activation of PI3K-Akt signaling pathway, and promote epithelial-mesenchymal transition (EMT) of OSCC cells. BCAT1 could promote the proliferation, migration, and invasion of OSCC cells via PI3K-Akt signaling pathway, which is a potential therapeutic target for OSCC.

Transcriptional Regulation of the Genes Encoding Branched-Chain Aminotransferases in Kluyveromyces lactis and Lachancea kluyveri Is Independent of Chromatin Remodeling

Transcriptional Regulation of the Genes Encoding Branched-Chain Aminotransferases in Kluyveromyces lactis and Lachancea kluyveri Is Independent of Chromatin Remodeling