Cytosolic Branched-chain Aminotransferase Research Articles

Abstract 5154: The cytosolic branched chain aminotransferase is an important metabolic checkpoint of T cell function in the lymphoma microenvironment

Abstract Rapidly growing lymphoma cells deplete the tumor microenvironment (TME) of nutrients needed to maintain anti-tumor T cell immunity. This metabolic disadvantage leads to diminished T cell function and reduced clearance of tumor cells. The branched chain amino acids (BCAAs) are required for growth of tumor and immune cells. In the TME, BCAAs are preferentially taken up by tumor cells for protein synthesis or energy production. The first step in BCAA degradation is reversible transamination catalyzed by the cytosolic branched chain aminotransferase, BCATc. Recent investigations suggest that BCATc is immunosuppressive in the TME. We found BCATc is induced upon T cell activation, during which time, BCATc negatively regulates T cell metabolism. We hypothesized that a loss of BCATc from T cells restores effector T cell function and limits the immunosuppressive Treg cells in the TME. Tumor studies were conducted using mice with BCATc deleted from CD4+ and CD8+ T cells (T-BCATcKO mice) and corresponding controls expressing the floxed transgene of BCATc in T cells (T-BCATcfl/fl mice). Mice were subcutaneously injected with 2.5 × 105 murine EL-4 lymphoma cells [tumor-injected mice, n=8-9/group] or phosphate-buffered saline [vehicle-injected mice, n=4-6/group]. Tumor growth, body weight, and food intake were monitored for 15 days followed by collection of tumors and organs. Splenocytes were purified and stained for expression of Foxp3 or used to isolate CD4+ and CD8+ T cells that were activated with anti-CD3 and anti-CD28, and, after initial expansion with cytokine IL-2, reactivated to hypotolerant or fully activated CD4+ Th1 cells, or expanded to effector CD8+T cells for 96h, in the presence or the absence of the leucine antagonist, N-acetyl-leucine amide (NALA). Supernates were tested for secretion of IFNγ (CD4+T cells) or perforin and granzyme B (CD8+T cells). T-BCATcKO mice demonstrated significant delay in lymphoma appearance and a 50% reduction in tumor volumes when compared to control mice. The removal of BCATc from T cells did not cause systemic overactivation of the immune system, with no changes in size or appearance of the spleen, liver, or intestines. Splenic Foxp3 expression was reduced by 80% in lymphoma challenged T-BCATcKO mice when compared to T-BCATcfl/fl controls. Hypotolerant and fully activated T-BCATcKO CD4+ T cells released significantly more IFNγ when compared to T-BCATcfl/fl T cells. The addition of NALA reduced the IFNγ levels to those found in control cells. CD8+ T cells from T-BCATcKO mice released significantly more granzyme B and perforin compared to T-BCATcfl/fl T cells. These findings demonstrate that BCATc-deficient T cells provide better systemic response to lymphoma likely due to improved Th1 and effector CD8+ T cell with diminished Treg function. Thus, BCATc may act as an important metabolic checkpoint of the lymphoma TME Citation Format: Tanner J. Wetzel, Lucas Figueroa, Leighton Wheeler, Alexander Martin, Christie Adam, Michael Boyer, Elitsa Ananieva. The cytosolic branched chain aminotransferase is an important metabolic checkpoint of T cell function in the lymphoma microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5154.

Abstract 5147: Mice with T cells deficient in the Cytosolic Branched Chain Aminotransferase reduce the tumor burden of low and high immunogenic strains of Lymphoma

Abstract Current immunotherapies rely on modulating host immunity to induce an anti-tumor immune response. The efficacy of such a response depends on the immunogenicity of the tumor, which varies greatly between different cancers or the same cancer in different individuals. While most lymphomas are immunogenic, non-malignant T cells that infiltrate the lymphoma microenvironment are subjected to immunosuppressive signals and areas of nutrient depletion, which leads to T cell exhaustion and inhibition of T cell function. Metabolic enzymes, such as the cytosolic branched chain aminotransferase, BCATc, exert immunosuppressive effects limiting T cells ability to function. Previous research described BCATc as a negative regulator of T cell activation, thus identifying BCATc as a potential target for immunotherapeutic modulation in the tumor microenvironment (TME). In the current study we exploit a low (EL4) and high (EL4-OVA) immunogenic murine lymphoma strains to explore how a loss of BCATc in T cells impacts their ability to produce anti-lymphoma immunity in mice. We used 12 week old male mice with BCATc deleted from T cells (T-BCATcKO) and control T-BCATcfl/fl mice that express the transgenic floxed version of BCATc. Mice were injected with 2.5x105 cells belonging to the low (EL4) or high (EL4-OVA) immunogenic strains and tumor growth, body weight, and food intake were monitored for up to 15 days followed by collection of tumor tissues and organs. Tumors were then lysed, and the protein profile of each tumor was determined by monitoring changes in expression of protein markers of T cell exhaustion (TOX, CD39), apoptosis (BAX, PUMA), or signaling proteins, such as the ribosomal S6 protein, the protein kinase B (AKT), and the energy sensor the AMP-activated protein kinase (AMPK). T-BCATcKO mice showed significant reduction in tumor growth compared to control mice regardless of the lymphoma strain being tested. The mouse cohort with the biggest number of tumor free mice was the T-BCATcKO cohort injected with EL4 cells (13% of the mice were tumor free on day 9). However, EL4-OVA cells grew into smaller tumors across all cohorts. Mice had comparable food intake but T-BCATcKO mice, injected with EL4 cells, had a significantly lower body weight. In T-BCATcKO mice, the reduced EL4 tumor mass correlated with upregulated AMPK and AKT along with higher expression of PUMA. The reduced EL4-OVA tumor masses correlated with downregulated S6 and decreased TOX expression, but high expression of BAX compared to the corresponding tumors isolated from T-BCATcfl/fl mice. These findings suggest that T cells deficient in BCATc can reduce lymphoma growth regardless of the immunogenicity of the lymphoma stain likely by establishing catabolic state and inducing apoptosis. Thus, BCATc may play an immunosuppressive role in the lymphoma TME and the absence of BCATc from T cells may help T cells combat lymphoma. Citation Format: Lucas D. Figueroa, Tanner Wetzel, Leighton Wheeler, Christie Adam, Michael Boyer, Elitsa Ananieva. Mice with T cells deficient in the Cytosolic Branched Chain Aminotransferase reduce the tumor burden of low and high immunogenic strains of Lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5147.

Targeting the Cytosolic Branched Chain Aminotransferase (BCATc) for T Cell Driven Anti‐Lymphoma Immunotherapy

The lymphoma microenvironment is a dynamic network between lymphoma cells and non‐malignant components that may promote tumor growth and consequently drug resistance. Progress in T cell metabolism has demonstrated that T cells experience metabolic disadvantage in the tumor microenvironment, which commonly manifests in T cell exhaustion and jeopardizes their potential to destroy cancer cells. We identified the cytosolic branched chain aminotransferase (BCATc) as a novel metabolic checkpoint of T cell activation. BCATc catalyzes the first step in the degradation of the essential amino acid leucine, which is a well‐known activator of complex 1 of the mammalian target of rapamycin (mTORC1). In T cells, BCATc is induced upon T cell activation and is a part of a negative feedback loop regulation of mTORC1. Based on extensive earlier research, we proposed that BCATc exerts immunosuppressive properties in the tumor microenvironment and we hypothesized that deleting BCATc from T cells renders them effective in eradicating lymphoma tumors.By using our newly designed mouse model with BCATc deleted from CD4+ and CD8+ T cells (T‐BCATcKO mice), we performed tumor studies where male and female mice were subcutaneously injected with 2.5x105 murine EL‐4 lymphoma cells [n=8] or phosphate‐buffered saline [n=4‐6]. As controls, mice expressing the floxed BCATc gene (T‐BCATcfl/flmice, n=9) were used. Tumor growth, body weight, and food intake were monitored for up to 15 days followed by collection of tumor tissues and organs. In addition, CD4+ T cells were isolated from spleens of T‐BCATcfl/fl and T‐BCATcKO mice, activated with a plate‐bound anti‐CD3 and soluble anti‐CD28 and left untreated or treated with 5 mM gabapentin (a competitive inhibitor of BCATc) or 10 mM N‐acetyl‐leucine amide (NALA, a structural analog of leucine) for 24‐72 hours to measure changes in the secretion of interferon gamma (IFNγ) from T helper 1 (Th1) cells or the expression of Foxp3, a master activator of regulatory T cells (CD4+CD25+ T reg cells).Deletion of BCATc from T cells or inhibition of the BCATc activity by gabapentin caused a significant increase in the secretion of IFNγ from Th1 cells after 24‐72 hours of activation, while CD4+CD25+ T reg cells expressed reduced levels of Foxp3 in response to gabapentin. In contrast, NALA blocked the IFNγ secretion and caused an increase in the expression of Foxp3. Following tumor inoculation, the T‐BCATcKO mice demonstrated a significant delay in tumor appearance and growth (~50 % reduction in tumor volumes) and the final tumor masses were reduced by 25% compared to control mice. None of the animals differed in body, organ weights or food intake and deletion of BCATc from T cells did not lead to systemic overactivation of the immune system. This study revealed that deleting BCATc from T cells may offer an advantage to better fight lymphoma and identified BCATc as a new therapeutic target to improve the efficiency of checkpoint inhibitors and help increase the low rates of remission seen in lymphoma patients.

The BCAT1 CXXC Motif Provides Protection against ROS in Acute Myeloid Leukaemia Cells.

The cytosolic branched-chain aminotransferase (BCAT1) has received attention for its role in myeloid leukaemia development, where studies indicate metabolic adaptations due to BCAT1 up-regulation. BCAT1, like the mitochondria isoform (BCAT2), shares a conserved CXXC motif ~10 Å from the active site. This CXXC motif has been shown to act as a ‘redox-switch’ in the enzymatic regulation of the BCAT proteins, however the response to reactive oxygen species (ROS) differs between BCAT isoforms. Studies indicate that the BCAT1 CXXC motif is several orders of magnitude less sensitive to the effects of ROS compared with BCAT2. Moreover, estimation of the reduction mid-point potential of BCAT1, indicates that BCAT1 is more reductive in nature and may possess antioxidant properties. Therefore, the aim of this study was to further characterise the BCAT1 CXXC motif and evaluate its role in acute myeloid leukaemia. Our biochemical analyses show that purified wild-type (WT) BCAT1 protein could metabolise H2O2 in vitro, whereas CXXC motif mutant or WT BCAT2 could not, demonstrating for the first time a novel antioxidant role for the BCAT1 CXXC motif. Transformed U937 AML cells over-expressing WT BCAT1, showed lower levels of intracellular ROS compared with cells over-expressing the CXXC motif mutant (CXXS) or Vector Controls, indicating that the BCAT1 CXXC motif may buffer intracellular ROS, impacting on cell proliferation. U937 AML cells over-expressing WT BCAT1 displayed less cellular differentiation, as observed by a reduction of the myeloid markers; CD11b, CD14, CD68, and CD36. This finding suggests a role for the BCAT1 CXXC motif in cell development, which is an important pathological feature of myeloid leukaemia, a disease characterised by a block in myeloid differentiation. Furthermore, WT BCAT1 cells were more resistant to apoptosis compared with CXXS BCAT1 cells, an important observation given the role of ROS in apoptotic signalling and myeloid leukaemia development. Since CD36 has been shown to be Nrf2 regulated, we investigated the expression of the Nrf2 regulated gene, TrxRD1. Our data show that the expression of TrxRD1 was downregulated in transformed U937 AML cells overexpressing WT BCAT1, which taken with the reduction in CD36 implicates less Nrf2 activation. Therefore, this finding may implicate the BCAT1 CXXC motif in wider cellular redox-mediated processes. Altogether, this study provides the first evidence to suggest that the BCAT1 CXXC motif may contribute to the buffering of ROS levels inside AML cells, which may impact ROS-mediated processes in the development of myeloid leukaemia.

Molecular Docking Studies of Dihydropyridazin-3(2H)-one Derivatives as Anticonvulsant Agents

Molecular docking is the identification of ligand’s correct binding geometry i.e. pose in the binding site and estimation of its binding affinity for rational design of drug molecule. The current study endeavored the high throughput in silico screening of 56 derivatives of dihydropyridazin-3(2H)-one docked with human cytosolic branched chain amino transferase using PyRx-virtual screening tool. Out of 56 compounds, almost all the test compounds showed very good binding affinity score. Gabapentin was used as standard drug which shows binding affinity of -6.2. On the basis of H-bond interactions, compounds 3, 9, 11, 25, 26, 31, 34, 39, 47, 48, 51, 54, 56 were found to be potent outcome for anticonvulsant activity. Compounds 11, 25, 39, 56 showed excellent H-bond interactions with protein active site, Among which compound 11 showed the outstanding interactions with acceptable bond length 2.34, 2.57, 2.62, 3.03 Å.

BCATc modulates crosstalk between the PI3K/Akt and the Ras/ERK pathway regulating proliferation in triple negative breast cancer.

The cytosolic branched chain aminotransferase (BCATc) protein has been found to be highly expressed in breast cancer subtypes, including triple negative breast cancer (TNBC), compared with normal breast tissue. The catabolism of branched-chain amino acids (BCAAs) by BCATc leads to the production of glutamate and key metabolites which further drive the TCA cycle, important for cellular metabolism and growth. Upregulation of BCATc has been associated with increased cell proliferation, cell cycle progression and metastasis in several malignancies including breast, gliomas, ovarian and colorectal cancer but the underlying mechanisms are unclear. As nutrient levels of BCAAs, substrates of BCATc, regulate the PI3K/Akt pathway we hypothesized that increased expression of BCATc would contribute to tumour cell growth through upregulation of the insulin/IGF-1 signalling pathway. This pathway is known to potentiate proliferation and metastasis of malignant cells through the activation of PI3K/Akt and the RAS/ERK signalling cascades. Here we show that knockdown of BCATc significantly reduced insulin and IGF-1-mediated proliferation, migration and invasion of TNBC cells. An analysis of this pathway showed that when overexpressed BCATc regulates proliferation through the PI3K/Akt axis, whilst simultaneously attenuating the Ras/Erk pathway indicating that BCATc acts as a conduit between these two pathways. This ultimately led to an increase in FOXO3a, a key regulator of cell proliferation and Nrf2, which mediates redox homeostasis. Together this data indicates that BCATc regulates TNBC cell proliferation, migration and invasion through the IGF-1/insulin PI3K/Akt pathway, culminating in the upregulation of FOXO3a and Nrf2, pointing to a novel therapeutic target for breast cancer treatment.

Leucine and branched-chain amino acid metabolism contribute to the growth of bone sarcomas by regulating AMPK and mTORC1 signaling.

Osteosarcoma and chondrosarcoma are sarcomas of the bone and the cartilage that are primarily treated by surgical intervention combined with high toxicity chemotherapy. In search of alternative metabolic approaches to address the challenges in treating bone sarcomas, we assessed the growth dependence of these cancers on leucine, one of the branched-chain amino acids (BCAAs), and BCAA metabolism. Tumor biopsies from bone sarcoma patients revealed differential expression of BCAA metabolic enzymes. The cytosolic branched-chain aminotransferase (BCATc) that is commonly overexpressed in cancer cells, was down-regulated in chondrosarcoma (SW1353) in contrast with osteosarcoma (143B) cells that expressed both BCATc and its mitochondrial isoform BCATm. Treating SW1353 cells with gabapentin, a selective inhibitor of BCATc, further revealed that these cells failed to respond to gabapentin. Application of the structural analog of leucine, N-acetyl-leucine amide (NALA) to disrupt leucine uptake, indicated that all bone sarcoma cells used leucine to support their energy metabolism and biosynthetic demands. This was evident from the increased activity of the energy sensor AMP-activated protein kinase (AMPK), down-regulation of complex 1 of the mammalian target of rapamycin (mTORC1), and reduced cell viability in response to NALA. The observed changes were most profound in the 143B cells, which appeared highly dependent on cytosolic and mitochondrial BCAA metabolism. This study thus demonstrates that bone sarcomas rely on leucine and BCAA metabolism for energy and growth; however, the differential expression of BCAA enzymes and the presence of other carbon sources may dictate how efficiently these cancer cells take advantage of BCAA metabolism.

Anticonvulsant evaluation of 2-pyrazolines carrying naphthyl moiety: An insight into synthesis and molecular docking study

A series of N-substituted-3-(napthalen-2-yl)-5-substituted phenyl-4,5-dihydropyrazole-1-carbothioamide derivatives (4a-n) were synthesized with the view of structural requirements of pharmacophore for potential anticonvulsant agents. The synthesized compounds were assayed intraperitoneally (i.p.) and subcutaneously (s.c.) in mice against seizures induced by MES and scPTZ methods, respectively. Neurologic deficit was evaluated by rotarod method. Among the tested compounds, 4g, 4i, 4j and 4n emerged as the most active molecule in the MES model at a dose of 30 mg/kg at 0.5h comparable to standardscarbamazepine and phenytoin. In the scPTZ test,4e and 4l were found to be most active compounds at the lowest dose of 30 mg/kg at 0.5h, in the management of the convulsive disorder. Molecular docking studies of the titled compounds were also donewith 3D crystal structure of human cytosolic branched chain amino transferase (hBCATc) enzyme and compound 4e was found to have five hydrogen bond interactions with the most important active site residues.In neurotoxicity studies, except compounds 4b, 4c, 4h and 4k, rest of the compounds showed no sign of toxicity.

Identification and Characterisation of a Novel Antioxidant Activity for the BCAT1 Cxxc Motif: Implications for Myeloid Leukaemia Development

Recently the human cytosolic branched-chain amino transferase (BCAT1) has been implicated in the development of myeloid leukaemia. Previously studies identified a redox active CXXC motif for BCAT1 analogous to the mitochondrial isoform (BCAT2). Oxidation of this CXXC motif can regulate aminotransferase activity in both isoforms. Interestingly the reduction mid-point potential (Em) of the BCAT1 CXXC motif is around 80mV lower compared with BCAT2. This suggests that the BCAT1 CXXC motif is more reducing in nature i.e. has antioxidant capacity. Further evidence to support this notion comes from the observation that BCAT1 is significantly less sensitive to inactivation mediated by hydrogen peroxide (H2O2). This is an important observation, since H2O2 is a reactive oxygen species (ROS) implicated in myeloid leukaemia development. Here we provide the first evidence to show that the BCAT1 CXXC can metabolise H2O2. By using CXXC motif mutant constructs, we also evaluate the role of the BCAT1 CXXC motif in myeloid leukaemia cells.To investigate the antioxidant capacity of the BCAT1 CXXC motif, cDNA (accession NM_001178094.1) was cloned into a pET28a vector for overexpression and purification in E.coli BL21(DE3) cells. Site directed mutagenesis of the CXXC motif subsequently followed creating three constructs: 1) BCAT1-WT (unmutated), 2) BCAT1-CXXS (C-terminal Cys → Ser mutant) and 3) BCAT1-SXXS (N/C-terminal Cys → Ser double mutant). Mutation of the BCAT1 CXXC motif was confirmed at the protein level by 5,5'-ditho-bis-(2-nitrobenzoic acid) titration. WT and CXXC motif mutant BCAT1 protein was added to 5mM H2O2 & monitored at λ240nm for the disappearance of H2O2. This was compared with 1U of catalase as a positive control. Our data show that 15μg of purified BCAT1-WT could metabolise H2O2 at a rate of 0.14±0.02 μmol/min. The BCAT1 CXXC motif mutants lacked capacity to do this. To verify our findings, BCAT1-WT treated with N-ethylmaleimide also lost the capacity to metabolise H2O2. This confirms that the antioxidant activity observed is Cys mediated. This novel finding for the BCAT1 CXXC motif may therefore be important in myeloid leukaemia development.To evaluate the BCAT1 CXXC motif in myeloid leukaemia cells, WT and CXXS mutant BCAT1 was subcloned into a pLENTI-C-Myc-DDK lentiviral vector prior to transduction of U937 cells. Empty vector (EV) control transgenic U937 cells were also generated. Stable expression of BCAT1 was achieved and confirmed by western-blot and qPCR; BCAT1-WT (6.5±1.7 fold increase) & BCAT1-CXXS (5.1±2.6 fold increase). The BCAT1 and EV transgenic U937 cells were subjected to H2O2 mediated oxidative stress and monitored for cell viability after 24h. A significant difference in the LD50 between BCAT1-WT, BCAT1-CXXS and EV control cells was observed; 620±28mM, 306±37mM, 251±26mM respectively (p<0.05). This finding suggests that the BCAT1 CXXC provides protection against H2O2 mediated cell death. To corroborate these findings, the same cells treated with rotenone observed a similar pattern in LD50; 1.05mM, 0.06mM, 0.12mM respectively.ROS are implicated in many cellular processes, including differentiation. Thus, we next asked whether the BCAT1 CXXC motif could suppress U937 cell differentiation mediated by phorbol 12-myristate 13-acetate (PMA). This compound was selected since ROS feature in PMA mediated differentiation. U937 cells were monitored for the expression of CD11b and CD36 following 48h PMA incubation. Our data show a significant reduction in the frequency of CD11b+/CD36+ U937 cells for BCAT1-WT (17.5±6.9%) compared with EV controls (54.0±10.5%, p<0.05). BCAT1-CXXS also showed a reduction (30.6±4.9%), but not to the same extent as BCAT1-WT. This finding was mirrored by the expression level of CD11b, where the MFI was significantly less for BCAT1-WT (88.0±16.3) compared with EV control (251.5±54.7, p<0.001) and BCAT1-CXXS (152.5±6.3, p<0.05). Analysis of intracellular ROS using 2′,7′-Dichlorodihydrofluorescein diacetate revealed a reduction in cellular ROS for BCAT1-WT cells (5929±13.8 MFI), compared with EV controls (8527±196 MFI) and BCAT1-CXXS (7879±18.4 MFI). This shows that the BCAT1 CXXC motif may control cellular ROS levels in myeloid leukaemia cells.In summary, this study identifies a novel antioxidant role for the BCAT1 CXXC which confers protection against ROS mediated cell death and differentiation of myeloid leukaemia cells. DisclosuresNo relevant conflicts of interest to declare.

Branched-chain amino acid metabolism in cancer.

Purpose of reviewThe current review aims to provide an update on the recent biomedical interest in oncogenic branched-chain amino acid (BCAA) metabolism, and discusses the advantages of using BCAAs and expression of BCAA-related enzymes in the treatment and diagnosis of cancers.Recent findingsAn accumulating body of evidence demonstrates that BCAAs are essential nutrients for cancer growth and are used by tumors in various biosynthetic pathways and as a source of energy. In addition, BCAA metabolic enzymes, such as the cytosolic branched-chain aminotransferase 1 (BCAT1) and mitochondrial branched-chain aminotransferase 2, have emerged as useful prognostic cancer markers. BCAT1 expression commonly correlates with more aggressive cancer growth and progression, and has attracted substantial scientific attention in the past few years. These studies have found the consequences of BCAT1 disruption to be heterogeneous; not all cancers share the same requirements for BCAA metabolites and the function of BCAT1 appears to vary between cancer types.SummaryBoth oncogenic mutations and cancer tissue-of-origin influence BCAA metabolism and expression of BCAA-associated metabolic enzymes. These new discoveries need to be taken into consideration during the development of new cancer therapies that target BCAA metabolism.

PO15DECREASED EXPRESSION OF THE MITOCHONDRIAL BCAT PROTEIN CORRELATES WITH IMPROVED PATIENT SURVIVAL IN IDH WILD-TYPE GLIOMAS

INTRODUCTION: Recent studies indicated that the human cytosolic branched chain aminotransferase protein (hBCATc), which metabolises the branched chain amino acids (BCAA), was significantly upregulated in IDH1/2 wild type (WT) glioblastomas and is associated with a worse prognosis compared with IDH mutant gliomas. The diagnostic and prognostic significance of markers of BCAA metabolism is currently under investigation. METHOD: 64 glioma tumour samples were compared for hBCATc, hBCATm and BCKDC expression using western blotting and IHC. DNA was extracted from fresh frozen tissue. Sanger sequencing of the p.Arg132 region of IDH1 and p.Arg172 region of IDH2 was undertaken. RESULTS: In IDH WT tumours, like hBCATc (p = 0.007), the mitochondrial isoform (hBCATm) is significantly (p = 0.036) expressed relative to IDH mutant gliomas. hBCATm additionally shows a more significant correlation with patient survival than hBCATc. In IDH WT tumours, low hBCATm expression is a positive prognostic factor (p = 0.003). hBCATm expression also correlated with WHO grade. Although previous reports indicate that increased hBCATc occurs exclusively in IDH-WT tumours, our studies demonstrate that 30% of IDH mutant tumours express hBCATc. Although hBCATc alone has been suggested as a putative therapeutic target, it is important to evaluate the expression of hBCATm as its expression may impact the efficacy of new treatments targeting hBCATc. CONCLUSION: IDH WT high grade gliomas traditionally have a poor prognosis. However we demonstrate for the first time that relatively low hBCATm may select for a better performing clinical cohort and may be a possible candidate target for drug therapy.

The cytosolic branched-chain aminotransferases of Arabidopsis thaliana influence methionine supply, salvage and glucosinolate metabolism.

Arabidopsis thaliana possesses six branched-chain aminotransferases (BCAT1-6). Previous studies revealed that some members of this protein family are involved in the biosynthesis of branched-chain amino acids and/or in the Met chain elongation pathway, the initial steps towards the biosynthesis of Met-derived glucosinolates. We now analyzed branched-chain aminotransferase 6 (BCAT6). In vivo GFP-tagging experiments strongly suggest this enzyme to be localized to the cytosol. Substrate specificity assays performed with recombinant enzyme revealed that BCAT6 transaminates Val, Leu and Ile as well as the corresponding 2-oxo acids but also transaminates Met and its cognate ketoacid 4-methyl-2-oxobutanoate. We established single (bcat6-1), double (bcat4-2/bcat6-1) and triple (bcat3-1/bcat4-2/bcat6-1) mutants involving BCAT6 with the latter exhibiting a clear macroscopic phenotype with smaller plants and abnormal leaf morphology. Metabolite profiling of these mutants demonstrated that BCAT6 can contribute to Met chain elongation with the triple mutant line lacking BCAT3, 4 and 6 showing a dramatic reduction of Met-derived glucosinolate species down to 32 and 14% of wild-type levels in plant foliage and seeds, respectively. This drop in glucosinolate levels is accompanied by a 46-fold increase of free Met, demonstrating the important role of the three branched-chain aminotransferases in converting Met to its 2-oxo acid for glucosinolate chain elongation. In addition, we determined the relative amounts of 5'-deoxy-5'-methylthioadenosine, an intermediate of the Met recycling pathway. This metabolite accumulated to relative high amounts in the absence of the cytosolic BCAT4 and BCAT6, suggesting that cytosolic Met salvage also contributes to the biosynthesis of glucosinolates.

New insights into the role of the branched-chain aminotransferase proteins in the human brain.

The human cytosolic branched-chain aminotransferase (hBCATc) enzyme is strategically located in glutamatergic neurons, where it is thought to provide approximately 30% of de novo nitrogen for brain glutamate synthesis. In health, glutamate plays a dominant role in facilitating learning and memory. However, in patients with Alzheimer's disease (AD), synaptic levels of glutamate become toxic, resulting in a direct increase in postsynaptic neuronal calcium, causing a cascade of events that contributes to the destruction of neuronal integrity and cell death, pathological features of AD. Our group is the first to map the hBCAT proteins to the human brain, where cell-specific compartmentation indicates key roles for these proteins in regulating glutamate homeostasis. Moreover, increased expression of hBCAT was observed in the brains of patients with AD relative to matched controls. We reflect on the importance of the redox-active CXXC motif, which confers novel roles for the hBCAT proteins, particularly with respect to substrate channeling and protein folding. This implies that, in addition to their role in glutamate metabolism, these proteins have additional functional roles that might impact redox cell signaling. This review discusses how these proteins behave as potential neuroprotectors during periods of oxidative stress. These findings are particularly important because an increase in misfolded proteins, linked to increased oxidative stress, occurs in several neurodegenerative conditions. Together, these studies give an overview of the diverse role that these proteins play in brain metabolism, in which a dysregulation of their expression may contribute to neurodegenerative conditions such as AD.

Cytosolic Branched Chain Aminotransferase (BCATc) Regulates mTORC1 Signaling and Glycolytic Metabolism in CD4+ T Cells

Here we show that expression of the cytosolic branched chain aminotransferase (BCATc) is triggered by the T cell receptor (TCR) of CD4(+) T cells. Induction of BCATc correlates with increased Leu transamination, whereas T cells from the BCATc(-/-) mouse exhibit lower Leu transamination and higher intracellular Leu concentrations than the cells from wild type (WT) mice. Induction of BCATc by TCR in WT cells is prevented by the calcineurin-nuclear factor of activated T cells (NFAT) inhibitor, cyclosporin A (CsA), suggesting that NFAT controls BCATc expression. Leu is a known activator of the mammalian target of rapamycin complex 1 (mTORC1). mTOR is emerging as a critical regulator of T cell activation, differentiation, and metabolism. Activated T cells from BCATc(-/-) mice show increased phosphorylation of mTORC1 downstream targets, S6 and 4EBP-1, indicating higher mTORC1 activation than in T cells from WT mice. Furthermore, T cells from BCATc(-/-) mice display higher rates of glycolysis, glycolytic capacity, and glycolytic reserve when compared with activated WT cells. These findings reveal BCATc as a novel regulator of T cell activation and metabolism and highlight the important role of Leu metabolism in T cells.

Prospects of Using Platelets as Peripheral Marker to Study the Role of GABA in Autism

Literature indicated that platelets could be used as a model for neuronal receptors such as γ-amino butyric acid (GABA) and serotonin. Research work exhibited the presence of low levels of GABA and high levels of serotonin concentration in the platelets of autistic children as compare to their healthy counter parts. There are also other evidences pointing out to the significant role of GABA in autism such as association of g-band frequency with the cortical concentration of GABA and gabapentin (GABA analogue) specifically inhibits the cytosolic branched chain amino transferase (BCATc); an enzyme responsible to modulate glutamate availability for the synthesis of GABA.

Dietary branched chain amino acids ameliorate injury-induced cognitive impairment

Neurological dysfunction caused by traumatic brain injury results in profound changes in net synaptic efficacy, leading to impaired cognition. Because excitability is directly controlled by the balance of excitatory and inhibitory activity, underlying mechanisms causing these changes were investigated using lateral fluid percussion brain injury in mice. Although injury-induced shifts in net synaptic efficacy were not accompanied by changes in hippocampal glutamate and GABA levels, significant reductions were seen in the concentration of branched chain amino acids (BCAAs), which are key precursors to de novo glutamate synthesis. Dietary consumption of BCAAs restored hippocampal BCAA concentrations to normal, reversed injury-induced shifts in net synaptic efficacy, and led to reinstatement of cognitive performance after concussive brain injury. All brain-injured mice that consumed BCAAs demonstrated cognitive improvement with a simultaneous restoration in net synaptic efficacy. Posttraumatic changes in the expression of cytosolic branched chain aminotransferase, branched chain ketoacid dehydrogenase, glutamate dehydrogenase, and glutamic acid decarboxylase support a perturbation of BCAA and neurotransmitter metabolism. Ex vivo application of BCAAs to hippocampal slices from injured animals restored posttraumatic regional shifts in net synaptic efficacy as measured by field excitatory postsynaptic potentials. These results suggest that dietary BCAA intervention could promote cognitive improvement by restoring hippocampal function after a traumatic brain injury.

S-Nitrosoglutathione Inactivation of the Mitochondrial and Cytosolic BCAT Proteins: S-Nitrosation and S-Thiolation

Specific proteins with reactive thiol(ate) groups are susceptible to nitric oxide (NO) modification, which can result in S-nitrosation, S-thiolation, or disulfide bond formation. In the present study the effect of NO modification on the functionality of human mitochondrial and cytosolic branched-chain aminotransferases (hBCATm and hBCATc, respectively) was investigated. Here, the NO reactive agents, S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-dl-penacillamine, and sodium nitroprusside, inactivated both isoforms in a dose-dependent manner. Furthermore, low concentrations of GSNO caused a time-dependent loss in BCAT activity (50 +/- 3% and 77 +/- 2% for hBCATc and hBCATm, respectively) correlating with the loss of four and one to two thiol groups, respectively, confirming the thiols as targets for NO modification. Analysis of GSNO-modified hBCATc by quadrupole time-of-flight mass spectrometry identified a major peak containing three NO adducts and a minor peak equivalent to two NO adducts and one glutathione (GSH) molecule, the latter confirmed by Western blot analysis. Moreover, prolonged exposure or increased levels of GSNO caused increased S-glutathionylation and partial dimerization of hBCATc, suggesting a possible shift from regulation by NO to one of adaptation during nitrosated stress. Although GSNO inactivated hBCATm, neither S-nitrosation, S-glutathionylation, nor dimerization could be detected, suggesting differential mechanisms of regulation through NO between isoforms in the mitochondria and cytosol. Reversal of GSNO-modified hBCAT using GSH alone was only partial, and complete reactivation was only possible using the glutaredoxin/GSH system (97 +/- 4% and 91 +/- 3% for hBCATc and hBCATm, respectively), implicating the importance of a full physiological redox system for activation/inactivation. To conclude, these results clearly demonstrate distinct functional/mechanistic responses to GSNO modification between BCAT isoforms and offer intriguing comparisons between the BCAT proteins and the respective cytosolic and mitochondrial hTrx and hGrx proteins.

Regulatory Control of Human Cytosolic Branched-Chain Aminotransferase by Oxidation and S-Glutathionylation and Its Interactions with Redox Sensitive Neuronal Proteins

Redox regulation of proteins through oxidation and S-thiolation are important regulatory processes, acting in both a protective and adaptive role in the cell. In the current study, we investigated the sensitivity of the neuronal human cytosolic branched-chain aminotransferase (hBCATc) protein to oxidation and S-thiolation, with particular attention focused on functionality and modulation of its CXXC motif. Thiol specific reagents showed significant redox cycling between the reactive thiols and the TNB anion, and using NEM, four of the six reactive thiols are critical to the functionality of hBCATc. Site-directed mutagenesis studies supported these findings where a reduced kcat (ranging from 50-70% of hBCATc) for C335S, C338S, C335/8S, and C221S, respectively, followed by a modest effect on C242S was observed. However, only the thiols of the CXXC motif (C335 and C338) were directly involved in the reversible redox regulation of hBCATc through oxidation (with a loss of 40-45% BCAT activity on air oxidation alone). Concurrent with these findings, under air oxidation, the X-ray crystallography structure of hBCATc showed a disulphide bond between C335 and C338. Further oxidation of the other four thiols was not evident until levels of hydrogen peroxide were elevated. S-thiolation experiments of hBCATc exposed to GSH provided evidence for significant recycling between GSH and the thiols of hBCATc, which implied that under reducing conditions GSH was operating as a thiol donor with minimal S-glutathionylation. Western blot analysis of WT hBCATc and mutant proteins showed that as the ratio of GSH:GSSG decreased significant S-glutathionylation occurred (with a further loss of 20% BCAT activity), preferentially at the thiols of the CXXC motif, suggesting a shift in function toward a more protective role for GSH. Furthermore, the extent of S-glutathionylation increased in response to oxidative stress induced by hydrogen peroxide potentially through a C335 sulfenic acid intermediate. Deglutathionylation of hBCATc-SSG using the GSH/glutaredoxin system provides evidence that this protein may play an important role in cellular redox regulation. Moreover, redox associations between hBCATc and several neuronal proteins were identified using targeted proteomics. Thus, our data provides strong evidence that the reactive thiol groups, in particular the thiols of the CXXC motif, play an integral role in redox regulation and that hBCATc has redox mediated associations with several neuronal proteins involved in G-protein cell signaling, indicating a novel role for hBCATc in cellular redox control.

Pyridoxal 5-Phosphate Enzymes as Targets for Therapeutic Agents

The vitamin B(6)-derived pyridoxal 5'-phosphate (PLP) is the cofactor of enzymes catalyzing a large variety of chemical reactions mainly involved in amino acid metabolism. These enzymes have been divided in five families and fold types on the basis of evolutionary relationships and protein structural organization. Almost 1.5% of all genes in prokaryotes code for PLP-dependent enzymes, whereas the percentage is substantially lower in eukaryotes. Although about 4% of enzyme-catalyzed reactions catalogued by the Enzyme Commission are PLP-dependent, only a few enzymes are targets of approved drugs and about twenty are recognised as potential targets for drugs or herbicides. PLP-dependent enzymes for which there are already commercially available drugs are DOPA decarboxylase (involved in the Parkinson disease), GABA aminotransferase (epilepsy), serine hydroxymethyltransferase (tumors and malaria), ornithine decarboxylase (African sleeping sickness and, potentially, tumors), alanine racemase (antibacterial agents), and human cytosolic branched-chain aminotransferase (pathological states associated to the GABA/glutamate equilibrium concentrations). Within each family or metabolic pathway, the enzymes for which drugs have been already approved for clinical use are discussed first, reporting the enzyme structure, the catalytic mechanism, the mechanism of enzyme inactivation or modulation by substrate-like or transition state-like drugs, and on-going research for increasing specificity and decreasing side-effects. Then, PLP-dependent enzymes that have been recently characterized and proposed as drug targets are reported. Finally, the relevance of recent genomic analysis of PLP-dependent enzymes for the selection of drug targets is discussed.

Expression of cytosolic branched chain aminotransferase (BCATc) mRNA in the developing mouse brain

Branched-chain aminotransferase (BCAT) catalyzes the transamination of essential branched-chain amino acids (BCAAs: leucine, isoleucine and valine) with α-ketoglutarate. Through this reaction, BCAAs provide nitrogen for the synthesis of glutamate, the predominant excitatory neurotransmitter. Two BCAT isoforms have been identified: one cytosolic (BCATc) and one mitochondrial (BCATm). In adult rodents, BCATc is expressed in a wide variety of structures of the central nervous system (CNS), in neurons. So far, no data were available about the expression of BCATc in the developing CNS. Here, we analyse the expression profile of BCATc mRNA in the mouse brain from embryonic day 12.5 to adult age. BCATc mRNA gradually appears in different brain regions starting from early stages of neural development, and is maintained until adulthood. BCATc mRNA is predominantly present in the cerebral cortex, hippocampus, thalamus, ventral midbrain, raphe, cerebellum and precerebellar system. This study represents the first detailed analysis of BCATc mRNA expression in the developing mouse brain.