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Project description:Silencing branched-chain ketoacid dehydrogenase kinase exacerbates doxorubicin induced cytotoxicity in triple-negative breast cancer cells
| PRJNA685647 | ENA
Project description:The branched-chain ?-ketoacid dehydrogenase phosphatase (BDP) component of the human branched-chain ?-ketoacid dehydrogenase complex (BCKDC) has been expressed in Escherichia coli and purified in the soluble form. The monomeric BDP shows a strict dependence on Mn(2+) ions for phosphatase activity, whereas Mg(2+) and Ca(2+) ions do not support catalysis. Metal binding constants for BDP, determined by competition isothermal titration calorimetry, are 2.4 nm and 10 ?m for Mn(2+) and Mg(2+) ions, respectively. Using the phosphorylated decarboxylase component (p-E1b) of BCKDC as a substrate, BDP shows a specific activity of 68 nmol/min/mg. The Ca(2+)-independent binding of BDP to the 24-meric transacylase (dihydrolipoyl transacylase; E2b) core of BCKDC results in a 3-fold increase in the dephosphorylation rate of p-E1b. However, the lipoyl prosthetic group on E2b is not essential for BDP binding or E2b-stimulated phosphatase activity. Acidic residues in the C-terminal linker of the E2b lipoyl domain are essential for the interaction between BDP and E2b. The BDP structure was determined by x-ray crystallography to 2.4 Å resolution. The BDP structure is dominated by a central ?-sandwich. There are two protrusions forming a narrow cleft ?10 Å wide, which constitutes the active site. The carboxylate moieties of acidic residues Asp-109, Asp-207, Asp-298, and Asp-337 in the active-site cleft participate in binding two metal ions. Substitutions of these residues with alanine nullify BDP phosphatase activity. Alteration of the nearby Arg-104 increases the K(m) for p-E1b peptide by 60-fold, suggesting that this residue is critical for the recognition of the native p-E1b protein.
| S-EPMC3308798 | biostudies-literature
Project description:The mitochondrial branched-chain ?-ketoacid dehydrogenase complex (BCKDC) is negatively regulated by reversible phosphorylation.BCKDC kinase (BDK) inhibitors that augment BCKDC flux have been shown to reduce branched-chain amino acid (BCAA) concentrations in vivo. In the present study, we employed high-throughput screens to identify compound 3,6- dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) as a novel BDK inhibitor (IC(50) = 3.19 ?M). BT2 binds to the same site in BDK as other known allosteric BDK inhibitors, including (S)-?-cholorophenylproprionate ((S)-CPP). BT2 binding to BDK triggers helix movements in the N-terminal domain, resulting in the dissociation of BDK from the BCKDC accompanied by accelerated degradation of the released kinase in vivo. BT2 shows excellent pharmacokinetics (terminal T(1?2) = 730 min) and metabolic stability (no degradation in 240 min), which are significantly better than those of (S)-CPP. BT2, its analog 3-chloro-6-fluorobenzo[ b]thiophene-2-carboxylic acid (BT2F), and a prodrug of BT2 (i.e. N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[ b]thiophene-2-carboxamide (BT3)) significantly increase residual BCKDC activity in cultured cells and primary hepatocytes from patients and a mouse model of maple syrup urine disease. Administration of BT2 at 20 mg/kg/day to wild-type mice for 1 week leads to nearly complete dephosphorylation and maximal activation of BCKDC in heart, muscle, kidneys, and liver with reduction in plasma BCAA concentrations. The availability of benzothiophene carboxylate derivatives as stable BDK inhibitors may prove useful for the treatment of metabolic disease caused by elevated BCAA concentrations.
| S-EPMC4110271 | biostudies-literature
Project description:Maple syrup urine disease (MSUD) is an inherited error in the metabolism of branched-chain amino acids (BCAAs) caused by a severe deficiency of the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, which ultimately leads to neurological disorders. The limited therapies, including protein-restricted diets and liver transplants, are not as effective as they could be for the treatment of MSUD due to the current lack of molecular insights into the disease pathogenesis. To address this issue, we developed a Drosophila model of MSUD by knocking out the dDBT gene, an ortholog of the human gene encoding the dihydrolipoamide branched chain transacylase (DBT) subunit of BCKDH. The homozygous dDBT mutant larvae recapitulate an array of MSUD phenotypes, including aberrant BCAA accumulation, developmental defects, poor mobile behavior and disrupted L-glutamate homeostasis. Moreover, the dDBT mutation causes neuronal apoptosis during the developmental progression of larval brains. The genetic and functional evidence generated by in vivo depletion of dDBT expression in the eye indicates severe impairment of retinal rhabdomeres. Further, the dDBT mutant shows elevated oxidative stress and higher lipid peroxidation accumulation in the larval brain. Therefore, we conclude from in vivo evidence that the loss of dDBT results in oxidative brain damage that may lead to neuronal cell death and contribute to aspects of MSUD pathology. Importantly, when the dDBT mutants were administrated with Metformin, the aberrances in BCAA levels and motor behavior were ameliorated. This intriguing outcome strongly merits the use of the dDBT mutant as a platform for developing MSUD therapies.This article has an associated First Person interview with the joint first authors of the paper.
| S-EPMC7473638 | biostudies-literature
Project description:Branched-chain α-ketoacid dehydrogenase (BCKDH) catalyzes the critical step in the branched-chain amino acid (BCAA) catabolic pathway and has been the focus of extensive studies. Mutations in the complex disrupt many fundamental metabolic pathways and cause multiple human diseases including maple syrup urine disease (MSUD), autism, and other related neurological disorders. BCKDH may also be required for the synthesis of monomethyl branched-chain fatty acids (mmBCFAs) from BCAAs. The pathology of MSUD has been attributed mainly to BCAA accumulation, but the role of mmBCFA has not been evaluated. Here we show that disrupting BCKDH in Caenorhabditis elegans causes mmBCFA deficiency, in addition to BCAA accumulation. Worms with deficiency in BCKDH function manifest larval arrest and embryonic lethal phenotypes, and mmBCFA supplementation suppressed both without correcting BCAA levels. The majority of developmental defects caused by BCKDH deficiency may thus be attributed to lacking mmBCFAs in worms. Tissue-specific analysis shows that restoration of BCKDH function in multiple tissues can rescue the defects, but is especially effective in neurons. Taken together, we conclude that mmBCFA deficiency is largely responsible for the developmental defects in the worm and conceivably might also be a critical contributor to the pathology of human MSUD.
| S-EPMC4742758 | biostudies-literature
Project description:The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are elevated in maple syrup urine disease, heart failure, obesity, and type 2 diabetes. BCAA homeostasis is controlled by the mitochondrial branched-chain ?-ketoacid dehydrogenase complex (BCKDC), which is negatively regulated by the specific BCKD kinase (BDK). Here, we used structure-based design to develop a BDK inhibitor, (S)-?-chloro-phenylpropionic acid [(S)-CPP]. Crystal structures of the BDK-(S)-CPP complex show that (S)-CPP binds to a unique allosteric site in the N-terminal domain, triggering helix movements in BDK. These conformational changes are communicated to the lipoyl-binding pocket, which nullifies BDK activity by blocking its binding to the BCKDC core. Administration of (S)-CPP to mice leads to the full activation and dephosphorylation of BCKDC with significant reduction in plasma BCAA concentrations. The results buttress the concept of targeting mitochondrial BDK as a pharmacological approach to mitigate BCAA accumulation in metabolic diseases and heart failure.
| S-EPMC3683707 | biostudies-literature
Project description:Branched chain α-ketoacid dehydrogenase complex (BCKDC) is the rate-limiting enzyme in branched chain amino acid (BCAA) catabolism, a metabolic pathway with great importance for human health. BCKDC belongs to the mitochondrial α-ketoacid dehydrogenase complex family, which also includes pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex. Here, we revealed that BCKDC can be substantially inhibited by reactive nitrogen species (RNS) via a mechanism similar to what we recently discovered with pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex-RNS can cause inactivating covalent modifications of the lipoic arm on its E2 subunit. In addition, we showed that such reaction between RNS and the lipoic arm of the E2 subunit can further promote inhibition of the E3 subunits of α-ketoacid dehydrogenase complexes. We examined the impacts of this RNS-mediated BCKDC inhibition in muscle cells, an important site of BCAA metabolism, and demonstrated that the nitric oxide production induced by cytokine stimulation leads to a strong inhibition of BCKDC activity and BCAA oxidation in myotubes and myoblasts. More broadly, nitric oxide production reduced the level of functional lipoic arms across the multiple α-ketoacid dehydrogenases and led to intracellular accumulation of their substrates (α-ketoacids), decrease of their products (acyl-CoAs), and a lower cellular energy charge. In sum, this work revealed a new mechanism for BCKDC regulation, demonstrated that RNS can generally inhibit all α-ketoacid dehydrogenases, which has broad physiological implications across multiple cell types, and elucidated the mechanistic connection between RNS-driven inhibitory modifications on the E2 and E3 subunits of α-ketoacid dehydrogenases.
| S-EPMC10656228 | biostudies-literature
Project description:Branched chain amino acid (BCAA) catabolic impairments have been implicated in several diseases. Branched chain ketoacid dehydrogenase (BCKDH) controls the rate limiting step in BCAA degradation, the activity of which is inhibited by BCKDH kinase (BDK)-mediated phosphorylation. Screening efforts to discover BDK inhibitors led to identification of thiophene PF-07208254, which improved cardiometabolic endpoints in mice. Structure-activity relationship studies led to identification of a thiazole series of BDK inhibitors; however, these inhibitors did not improve metabolism in mice upon chronic administration. While the thiophenes demonstrated sustained branched chain ketoacid (BCKA) lowering and reduced BDK protein levels, the thiazoles increased BCKAs and BDK protein levels. Thiazoles increased BDK proximity to BCKDH-E2, whereas thiophenes reduced BDK proximity to BCKDH-E2, which may promote BDK degradation. Thus, we describe two BDK inhibitor series that possess differing attributes regarding BDK degradation or stabilization and provide a mechanistic understanding of the desirable features of an effective BDK inhibitor.
| S-EPMC10412597 | biostudies-literature
Project description:The mammalian mitochondrial branched-chain ketoacid dehydrogenase (BCKD) complex is a multienzyme complex involved in the catabolism of branched-chain amino acids. BCKD is regulated by the BCKD kinase, or BCKDK, which binds to the E2 subunit of BCKD, phosphorylates its E1 subunit, and inhibits enzymatic activity. Inhibition of the BCKD complex results in increased levels of branched-chain amino acids and branched-chain ketoacids, and this buildup has been associated with heart failure, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. To find BCKDK inhibitors for potential treatment of these diseases, we performed both NMR and virtual fragment screening and identified tetrazole-bearing fragments that bind BCKDK at multiple sites. Through structure-based virtual screening expanding from these fragments, the angiotensin receptor blocker class antihypertension drugs and angiotensin receptor blocker-like compounds were discovered to be potent BCKDK inhibitors, suggesting potential new avenues for heart failure treatment combining BCKDK inhibition and antihypertension.
| S-EPMC9976451 | biostudies-literature
Project description:The catabolism of branched chain amino acids (BCAAs) is mainly carried out in skeletal muscle myofibers. It is mediated by branched chain aminotransferase 2 and branched chain alpha ketoacid dehydrogenase (BCKDH) in mitochondria for energy supply, especially during exercise. BCKDH kinase (BCKDK) is a negative regulator of BCAAs catabolism by its inhibitory phosphorylation of the BCKDH E1a subunit. The data presented in this article are related to the research article that we previously have reported entitled "Energy metabolism profile of the effects of amino acid treatment on skeletal muscle cells: Leucine inhibits glycolysis of myotubes" (Suzuki et al., 2020)[1]. In this report, we have demonstrated that 1hour treatment of BT2, an inhibitor of BCKDK, decreased the glycolysis of C2C12 differentiated myotubes compared to the control. Although BCAAs metabolism is basically assumed to be carried out in differentiated myofibers, BCKDK is expressed in both undifferentiated myoblasts and differentiated myotubes, and the biological and physiological significance of BCAAs metabolism in myoblasts is still unclear. Present data demonstrate an in vitro assessment of BT2 on C2C12 myoblasts proliferation and differentiation. The data suggest that activation of BCAAs catabolism by the BCKDK inhibitor BT2 impairs C2C12 myoblasts proliferation and differentiation.
| S-EPMC7276516 | biostudies-literature