Here we show that glioblastoma express high levels of branched-chain amino acid transaminase 1 (BCAT1), the enzyme that initiates the catabolism of branched-chain amino acids (BCAAs). Expression of BCAT1 was exclusive to tumors carrying wild-type isocitrate dehydrogenase 1 (IDH1) and IDH2 genes and was highly correlated with methylation patterns in the BCAT1 promoter region. BCAT1 expression was dependent on the concentration of α-ketoglutarate substrate in glioma cell lines and could be suppressed by ectopic overexpression of mutant IDH1 in immortalized human astrocytes, providing a link between IDH1 function and BCAT1 expression. Suppression of BCAT1 in glioma cell lines blocked the excretion of glutamate and led to reduced proliferation and invasiveness in vitro, as well as significant decreases in tumor growth in a glioblastoma xenograft model. These findings suggest a central role for BCAT1 in glioma pathogenesis, making BCAT1 and BCAA metabolism attractive targets for the development of targeted therapeutic approaches to treat patients with glioblastoma.

This review aims to provide a historical reference of branched-chain amino acid (BCAA) metabolism and provide a link between peripheral and central nervous system (CNS) metabolism of BCAAs. Leucine, isoleucine, and valine (Leu, Ile, and Val) are unlike most other essential amino acids (AA), being transaminated initially in extrahepatic tissues, and requiring interorgan or intertissue shuttling for complete catabolism. Within the periphery, BCAAs are essential AAs and are required for protein synthesis, and are key nitrogen donors in the form of Glu, Gln, and Ala. Leucine is an activator of the mammalian (or mechanistic) target of rapamycin, the master regulator of cell growth and proliferation. The tissue distribution and activity of the catabolic enzymes in the peripheral tissues as well as neurological effects in Maple Syrup Urine Disease (MSUD) show the BCAAs have a role in the CNS. Interestingly, there are significant differences between murine and human CNS enzyme distribution and activities. In the CNS, BCAAs have roles in neurotransmitter synthesis, protein synthesis, food intake regulation, and are implicated in diseases. MSUD is the most prolific disease associated with BCAA metabolism, affecting the branched-chain α-keto acid dehydrogenase complex (BCKDC). Mutations in the branched-chain aminotransferases (BCATs) and the kinase for BCKDC also result in neurological dysfunction. However, there are many questions of BCAA metabolism in the CNS (as well as the periphery) that remain elusive. We discuss areas of BCAA and BCKA metabolism that have yet to be researched adequately.

Human lung tryptase (HLT), a trypsin-like serine proteinase stored as an active enzyme in association with heparin in mast cell granules, is released into the extracellular environment when mast cells are activated. Tryptases are unusual in that they form tetramers and bind heparin. As there are no known endogenous tryptase inhibitors, loss of heparin and dissociation of the active tetrameric enzyme to inactive monomers has been proposed as the mechanism of control. Activity and intrinsic fluorescence were used to measure the stabilization of HLT by NaCl, glycerol, and heparin. At physiological salt concentrations in the absence of heparin, activity decayed rapidly (t1/2 = 1-4 min at 37 degrees C) to an intermediate that could be immediately reactivated by heparin. But protein structural changes, as measured by intrinsic fluorescence, were much slower (t1/2 = 16 min), indicating that the intermediate continued to exist as a tetramer that slowly changed to a monomer. HLT tetramers, either active or inactive, were stabilized by 2 M NaCl, 20% glycerol, and heparin. Maximum stabilization was obtained with approximately 1 mol of heparin per HLT subunit. Heparan sulfate also stabilized HLT activity and active HLT was bound to and recovered from cartilage. Subunits of the inactive intermediate appeared to be loosely associated as demonstrated by the rapid disappearance of the tetramer in gel filtration studies in 1 M NaCl (t1/2 = 1.8 min), but the tetramer was stable in lower ionic strength buffers containing heparin. Fluorescence anisotropy measurements in the absence of heparin were also consistent with a slow (t1/2 = 22 min) transition from tetramer to monomer, and native polyacrylamide gel electrophoresis provided additional evidence for a tetrameric intermediate. HLT monomers isolated by gel filtration were minimally active in the presence of heparin. These data show that heparin-free HLT rapidly converts to an "inactive", loose tetrameric intermediate that can be reactivated with heparin or slowly dissociate to less active monomers and that tryptase released from mast cells is likely to remain active in association with heparin or other extracellular components. Thus, tryptase affinity for glycosaminoglycans and substrate specificity limitations are the primary factors controlling the proteolytic functions of these enzymes.

ular injections of leucine are sufficient to suppress food intake, but it remains unclear whether brain leucine signaling represents a physiological signal of protein balance. We tested whether variations in dietary and circulating levels of leucine, or all three branched-chain amino acids (BCAAs), contribute to the detection of reduced dietary protein. Of the essential amino acids (EAAs) tested, only intracerebroventricular injection of leucine (10 g) was sufficient to suppress food intake. Isocaloric low-(9% protein energy; LP) or normal-(18% protein energy) protein diets induced a divergence in food intake, with an increased consumption of LP beginning on day 2 and persisting throughout the study (P Ͻ 0.05). Circulating BCAA levels were reduced the day after LP diet exposure, but levels subsequently increased and normalized by day 4, despite persistent hyperphagia. Brain BCAA levels as measured by microdialysis on day 2 of diet exposure were reduced in LP rats, but this effect was most prominent postprandially. Despite these diet-induced changes in BCAA levels, reducing dietary leucine or total BCAAs independently from total protein was neither necessary nor sufficient to induce hyperphagia, while chronic infusion of EAAs into the brain of LP rats failed to consistently block LP-induced hyperphagia. Collectively, these data suggest that circulating BCAAs are transiently reduced by dietary protein restriction, but variations in dietary or brain BCAAs alone do not explain the hyperphagia induced by a low-protein diet.branched-chain amino acids; protein restriction; hypothalamus; macronutrient; food intake ALTHOUGH THE STUDY OF ingestive behavior historically has focused largely on the regulation of energy homeostasis, the consumption of adequate amounts of protein, specifically essential amino acids (EAAs), is also central to health and survival. Therefore, it seems likely that physiological systems exist to ensure sufficient protein intake. Alterations in dietary protein content can have profound effects on food intake, with diets high in protein suppressing food intake and diets moderately low in protein increasing food intake (2,11,18,23,38,39). Furthermore, when given the choice between diets that differ in protein content, many species will self-select between diets to ensure adequate consumption of protein (16,24,27), often at the expense of carbohydrate and fat (9,19,31).Despite these behavioral observations, the mechanism regulating protein intake is largely unknown (21). Recent work has focused on the branched-chain amino acid (BCAA) leucine as a potential protein signal. Intracerebroventricular injections of leucine suppress food intake and regulate key signaling systems (mTOR/AMPK) within hypothalamic neurons, while increased dietary leucine content reproduces the anorectic effects of a high-protein diet (3,5,23,29). While these data demonstrate that administration of excess leucine either to the diet or the brain is sufficient to suppress food intake, it remains unclear whether physiological fluctua...

A method for quantifying active cysteine proteinases in mammalian cells has been developed using an active-site-directed inhibitor. Fluoren-9-ylmethoxycarbonyl(di-iodotyrosylalanyl)-diaz omethane (Fmoc-[I2]Tyr-Ala-CHN2) was prepared and shown to react irreversibly with cathepsins B and L, but not with cathepsin S. The non- and mono-iodo forms of the inhibitor reacted with all three enzymes. These results demonstrate that, unlike cathepsins B and L, cathepsin S has a restricted S2-binding site that cannot accommodate the bulky di-iodotyrosine. Fmoc-[I2]Tyr-Ala-CHN2 was able to penetrate cells and react with active enzymes within the cells. A radiolabelled form of the inhibitor was synthesized and the concentration of functional inhibitor was established by titration with papain. This inhibitor was used to quantify active cysteine proteinases in cultured cells. Active cathepsin B was found to be expressed by all of the cells studied, consistently with a housekeeping role for this enzyme. Active forms of cathepsin L were also expressed by all of the cells, but in different quantities. Two additional proteins were labelled in some of the cells, and these may represent other non-characterized proteinases. Higher levels of active cathepsins B and L, and an unidentified protein of Mr 39000, were found in breast tumour cells that are invasive, compared with those that are not invasive. From the data obtained, it can be calculated that the concentrations of both active cathepsins B and L in lysosomes can be as high as 1 mM, each constituting up to 20% of total protein in the organelle. This new technique provides a more direct procedure for determining the proteolytic potential of cellular lysosomes.