Branched-chain Amino Acid Transferase Research Articles

Cross-talk between BCKDK-mediated phosphorylation and STUB1-dependent ubiquitination degradation of BCAT1 promotes GBM progression

Branched-chain amino acid transferase 1 (BCAT1) is highly expressed in multiple cancers and is associated with poor prognosis, particularly in glioblastoma (GBM). However, the post-translational modification (PTM) mechanism of BCAT1 is unknown. Here, we investigated the cross-talk mechanisms between phosphorylation and ubiquitination modifications in regulating BCAT1 activity and stability. We found that BCAT1 is phosphorylated by branched chain ketoacid dehydrogenase kinase (BCKDK) at S5, S9, and T312, which increases its catalytic and antioxidant activity and stability. STUB1 (STIP1 homology U-box-containing protein 1), the first we found and reported E3 ubiquitin ligase of BCAT1, can also be phosphorylated by BCKDK at the S19 site, which disrupts the interaction with BCAT1 and inhibits its degradation. In addition, we demonstrate through in vivo and in vitro experiments that BCAT1 phosphorylation inhibiting its ubiquitination at multiple sites is associated with GBM proliferation and that inhibition of the BCKDK-BCAT1 axis enhances the sensitivity to temozolomide (TMZ). Overall, we identified novel mechanisms for the regulation of BCAT1 modification and elucidated the importance of the BCKDK-STUB1-BCAT1 axis in GBM progression.

P338: BRANCHED CHAIN AMINO ACID TRANSAMINASE 1 ASSOCIATES WITH THE KU70/KU86 HETERODIMER AND MODULATES THE DNA DAMAGE RESPONSE IN T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA CELLS

Background: T-cell acute lymphoblastic leukemia (T-ALL) is a biologically and clinically heterogeneous disease mostly associated with NOTCH1 mutations that characterize over 60% of cases. Therapy-resistant or refractory T-ALL remains a major clinical challenge. Branched Chain Amino Acid Transferase 1 (BCAT1), a cytosolic aminotransferase converts BCAAs into their corresponding branched-chain α-keto acids and vice versa. Aberrant expression of BCAT1 has been demonstrated in different tumor models, including acute leukemia. However, its biological role in T-ALL remains to be elucidated. Aims: The purpose of this study was to dissect the functional role of BCAT1 in T-ALL, with particular emphasis on a putative link between BCAT1 and NOTCH1 in promoting T-ALL initiation and progression. Methods: We evaluated BCAT1 expression using RNAseq data from 264 T-ALL samples (ALL0434 protocol). BCAT1 transcript and protein levels were determined in T-ALL cell lines (n=13) and patient derived xenografts (PDX; n=12). Metabolomics analysis (using 13C6 labeled Leucine) was performed following NOTCH1 inhibition. Further, five PDX samples were treated with a γ secretase inhibitor (DBZ) to dampen NOTCH signaling. BCAT1 promoter occupancy by NOTCH1 was determined by Chromatin Immunoprecipitation (ChIP) coupled with qPCR. Tandem affinity purification (TAP) and mass spectrometry (MS) analysis were used to identify BCAT1 interacting proteins from CUTLL1 T-ALL cells. Kinetics of the DNA damage response (DDR) was evaluated in BCAT1 knock-down cells. Drug combination experiments using the DNA damaging agent (etoposide) and a specific BCAT inhibitor (ERG245) were performed in vitro and in vivo in PDX T-ALL models. Results: We found variable BCAT1 expression levels amongst the different T-ALL molecular subgroups, with higher expression levels in TLX1 (p<0.001), HOXA-TLX3 (p<0.05) and NKX2-1 (p<0.01) subtypes. Considering recurrent genetic alterations, we found BCAT1 expression to be significantly higher (p<0.01) in NOTCH1 mutated cases compared to un-mutated samples. Interestingly, NOTCH1 mutated PDX samples also showed higher BCAT1 expression compared to un-mutated cases. Metabolic studies disclosed that BCAAs oxidation is decreased following NOTCH1 inhibition (reduction in 3-methyl-2 oxovaleric acid). Further, inhibition of NOTCH1 signaling with DBZ decreased BCAT1 expression in numerous T-ALL models. ChIP analysis and luciferase reporter assays suggest that NOTCH1 may directly regulate BCAT1 expression. Functionally, TAP followed by MS analysis of BCAT1 interacting proteins disclosed that BCAT1 may be implicated in non-metabolic processes such as DNA replication and repair and rRNA processing. Indeed, we found Ku70/Ku86 proteins to interact with BCAT1. Evaluation of the DDR following DNA damage, disclosed that BCAT1 deficient cells present an accentuated but defective DDR which translates into marked DNA damage leading to pronounced cell death. Consistently, a marked synergistic therapeutic response was found between the DNA damaging drug etoposide and the BCAT specific inhibitor ERG245 in numerous T-ALL models. Summary/Conclusion: NOTCH1 may directly regulate BCAT1 expression in T-ALL cells. BCAT1 is involved in sustaining genomic integrity following DNA damage through its interaction with the Ku70/Ku86 heterodimer, known to mediate classical non-homologous end-joining. Our results identify BCAT1 as a novel therapeutic target and suggest that the combination between DNA double stand break inducing agents (such as etoposide) and a BCAT inhibitor could be particularly useful in NOTCH1-mutant T-ALL cases.

Chemotherapy Drugs Induce Cachexia and Alter Muscle Protein Metabolism

Chemotherapy is the main regimen to treat neoplastic tumours. Maintenance of muscle mass during treatment is associated with greater patient prognosis and reduces toxicity and severe side effects. However, chemotherapy induces cachexia, a wasting disorder characterized by muscle weakness and a loss of muscle mass. Although cancer, a disease treated with chemotherapy, is often diagnosed in older individuals, studies investigating chemotherapy‐induced cachexia are done exclusively in young animals at an age equivalent to a 20‐year‐old human. Therefore, studies investigating the effects of chemotherapy‐induced cachexia at an age range relevant to humans is needed. The objective of this study is to compare the severity and mechanisms of chemotherapy‐induced cachexia in 3‐month‐old adult (equivalent to a 20‐year‐old human) and 18‐month‐old (equivalent to a 56‐year‐old human) mice. Three‐month‐old adult CD2F1 male mice were treated with either folfiri (50mg/kg 5‐fluorouracil (5FU), 90mg/kg Leucovorin and 24mg/kg CPT11) (drug) or vehicle (10% DMSO in saline) for 6‐weeks. Male mice aged to 18‐months will undergo similar treatments. Adult mice treated with the drug cocktail exhibited significant body weight loss (~10%, n=4, p=0.0117) with no differences in food intake. Decreases in body weight were associated with a loss of muscle mass in the gastrocnemius (~25%, n=4, p=0.006), quadriceps (~30%, n=4, p=0.019) and tibialis anterior (~40%, n=4, p&lt;0.0001). Insulin tolerance tests at weeks 3 and 6 of treatment were also performed and glucose levels were found to be non‐significantly higher in drug‐treated mice. Signalling factors related to the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), protein synthesis, autophagy and the ubiquitin proteasome system will be measured. Key enzymes responsible for the breakdown of the branched chain amino acids (BCAAs), mainly branched‐chain alpha‐ketoacid dehydrogenase complex (BCKD) and branched‐chain amino acid transferase 2 (BCAT2) will also be measured in the skeletal muscle. Along this line, in cell culture, we have previously shown decreased (~20%) activity of the BCKD enzyme, corresponding with decreased amino acid concentrations of leucine (~40%), isoleucine (~60%), valine (~50%), alanine (~30%), arginine (~60%), phenylalanine (50%), serine (50%) and glutamate (50%) in chemotherapy‐treated myotubes. We also observed decreased (~65%) expression of amino acid transporters sodium‐coupled neutral amino acid transporter 1 (SNAT1) and large neutral amino acid transporter 1 (LAT1). Timepoint analysis showed SNAT1 expression to be decreased prior to decreases in BCKD activity, signifying that alterations in BCAA metabolism may be a result of decreased levels of BCAAs and amino acids in chemotherapy‐treated myotubes. Therefore, these findings stemmed our interest in how the BCAAs, and their metabolites are affected in‐vivofollowing chemotherapy treatment. Findings from this study will help to elucidate whether unique signatures of cachexia are experienced in older mice. Better understanding of the mechanisms underlying muscle mass regulation following chemotherapy treatment may help combat cachexia.

The key enzymes and flavor precursors involved in formation of characteristic flavor compounds of low-salt fermented common carp (Cyprinus carpio L.)

Unique flavor is the key quality of fermented fish. In this study, the precursors and key enzymes related to the flavor of Suanyu were analyzed. The results showed that 17 kinds of free amino acids and 22 kinds of free fatty acids were detected during the fermentation. According to variable importance for predictive components values (VIP), amino acids including aspartate, phenylalanine, methionine, glutamic acid, leucine, valine, isoleucine, cysteine and fatty acids including C16:0, C20:5, C20:4, C18:1, C22:6, C18:0 and C16:1 were screened as key precursors affecting the characteristic flavor of Suanyu. Moreover, the changes of enzyme activities in different metabolic processes of flavor were determined. Phosphofructokinase (PFK), pyruvate kinase (PK), lipoxygenase (LOX), hydroperoxidase lyase (HPL) and alcohol acyltransferase (AAT) activities reached the maximum in the first week of fermentation. However, hexokinase (HK), branched chain amino acid transferase (BCAT), aromatic amino acid transferase (ARAT) and esterase activities significantly decreased with the fermentation. Furthermore, ARAT, HPL, esterase, BCAT, AAT and HK were screened as the key enzymes that affected the characteristic flavor of Suanyu. All these results indicated that the control of key flavor precursors and enzymes during fermentation was a potential method to improve the flavor of fermented fish.

Dietary amylose:amylopectin ratio influences the expression of amino acid transporters and enzyme activities for amino acid metabolism in the gastrointestinal tract of goats.

This study was designed to investigate the effects of dietary starch structure on muscle protein synthesis and gastrointestinal amino acid (AA) transport and metabolism of goats. Twenty-seven Xiangdong black female goats (average body weight = 9·00 ± 1·12 kg) were randomly assigned to three treatments, i.e., fed a T1 (normal maize 100 %, high amylose maize 0 %), T2 (normal maize 50 %, high amylose maize 50 %) and T3 (normal maize 0 %, high amylose maize 100 %) diet for 35 d. All AA in the ileal mucosa were decreased linearly as amylose:amylopectin increased in diets (P < 0·05). The plasma valine (linear, P = 0·03), leucine (linear, P = 0·04) and total AA content (linear, P = 0·03) increased linearly with the increase in the ratio of amylose in the diet. The relative mRNA levels of solute carrier family 38 member 1 (linear, P = 0·01), solute carrier family 3 member 2 (linear, P = 0·02) and solute carrier family 38 member 9 (linear, P = 0·02) in the ileum increased linearly with the increase in the ratio of amylose in the diet. With the increase in the ratio of amylose:amylopectin in the diet, the mRNA levels of acetyl-CoA dehydrogenase B (linear, P = 0·04), branched-chain amino acid transferase 1 (linear, P = 0·02) and branched-chain α-keto acid dehydrogenase complex B (linear, P = 0·01) in the ileum decreased linearly. Our results revealed that the protein abundances of phosphorylated mammalian target of rapamycin (p-mTOR) (P < 0·001), phosphorylated 4E-binding protein 1 (P < 0·001) and phosphorylated ribosomal protein S6 kinases 1 (P < 0·001) of T2 and T3 were significantly higher than that of T1. In general, a diet with a high amylose ratio could reduce the consumption of AA in the intestine, allowing more AA to enter the blood to maintain higher muscle protein synthesis through the mTOR pathway.

High-throughput behavioral screen in C. elegans reveals Parkinson\u2019s disease drug candidates

We recently linked branched-chain amino acid transferase 1 (BCAT1) dysfunction with the movement disorder Parkinson’s disease (PD), and found that RNAi-mediated knockdown of neuronal bcat-1 in C. elegans causes abnormal spasm-like ‘curling’ behavior with age. Here we report the development of a machine learning-based workflow and its application to the discovery of potentially new therapeutics for PD. In addition to simplifying quantification and maintaining a low data overhead, our simple segment-train-quantify platform enables fully automated scoring of image stills upon training of a convolutional neural network. We have trained a highly reliable neural network for the detection and classification of worm postures in order to carry out high-throughput curling analysis without the need for user intervention or post-inspection. In a proof-of-concept screen of 50 FDA-approved drugs, enasidenib, ethosuximide, metformin, and nitisinone were identified as candidates for potential late-in-life intervention in PD. These findings point to the utility of our high-throughput platform for automated scoring of worm postures and in particular, the discovery of potential candidate treatments for PD.

Metformin rescues Parkinson’s disease phenotypes caused by hyperactive mitochondria

Metabolic dysfunction occurs in many age-related neurodegenerative diseases, yet its role in disease etiology remains poorly understood. We recently discovered a potential causal link between the branched-chain amino acid transferase BCAT-1 and the neurodegenerative movement disorder Parkinson's disease (PD). RNAi-mediated knockdown of Caenorhabditis elegans bcat-1 is known to recapitulate PD-like features, including progressive motor deficits and neurodegeneration with age, yet the underlying mechanisms have remained unknown. Using transcriptomic, metabolomic, and imaging approaches, we show here that bcat-1 knockdown increases mitochondrial respiration and induces oxidative damage in neurons through mammalian target of rapamycin-independent mechanisms. Increased mitochondrial respiration, or "mitochondrial hyperactivity," is required for bcat-1(RNAi) neurotoxicity. Moreover, we show that post-disease-onset administration of the type 2 diabetes medication metformin reduces mitochondrial respiration to control levels and significantly improves both motor function and neuronal viability. Taken together, our findings suggest that mitochondrial hyperactivity may be an early event in the pathogenesis of PD, and that strategies aimed at reducing mitochondrial respiration may constitute a surprising new avenue for PD treatment.

Transcriptome Sequencing Analysis of Birch (Betula platyphylla Sukaczev) under Low-Temperature Stress

Low temperature is one of the common abiotic stresses that adversely affect the growth and development of plants. In this study, we used RNA-Seq to identify low-temperature-responsive genes in birch and further analyzed the underlying molecular mechanism. Birch seedlings were treated by the low temperature (6 °C) for 0, 1, 1.5, 2, 2.5, and 3 h, respectively. A total of 3491 genes were differentially expressed after low-temperature stress. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) functional enrichment analysis were performed for the differentially expressed genes (DEGs). GO analysis indicated that 3491 DEGs were distributed into 1002 categories, and these DEGs were enriched in “cell process”, “metabolic process”, and “stimulus response”, under the “biological process” category; in “organelles” and “cell components”, under the “cell component” category; and in “catalytic activity” and “adhesion”, under the “molecular function” category. The KEGG enrichment indicated that 119 DEGs were involved in Ca2+ and plant hormone signal transduction; 205 DEGs were involved in secondary metabolic processes, such as lipid metabolism and phenylpropanoid biosynthesis pathway; and 20 DEGs were involved in photosynthesis. In addition, a total of 362 transcription factors (TFs) were differentially expressed under low-temperature stress, including AP2/ERF, C2H2, MYB-HB-like, WRKY, bHLH, WD40-like, and GRAS families. Gene Bpev01.c0480.g0081 (calmodulin-like CML38), Bpev01.c1074.g0005 (calmodulin-like CML25), Bpev01.c1074.g0001 (Calcium-binding EF-hand family protein), Bpev01.c2029.g0005 (calmodulin-like protein), Bpev01.c0154.g0008 (POD), Bpev01.c0015.g0143 (N-acetyl-1-glutamate synthase), and Bpev01.c0148.g0010 (branched chain amino acid transferase) were up-regulated at a high level, under low-temperature stress.

Prognostic significance of branched-chain amino acid transferase 1 and CD133 in triple-negative breast cancer

BackgroundPrevious studies have shown that branched-chain amino acid transferase 1 (BCAT1) is associated with tumour progression in triple-negative breast cancer (TNBC). Furthermore, CD133 has emerged as a novel cancer stem cell marker for indicating tumour progression. However, the prognostic significance of these two markers remains to be verified. This study was conducted to investigate the correlation between BCAT1 and CD133 expression and clinicopathological features, as well as the prognosis of patients with TNBC.MethodsThe study cohort included 291 patients with TNBC. Tissue microarrays were constructed for both cancer and normal tissues. The expression of BCAT1 and CD133 was detected by immunohistochemical staining, and the levels were evaluated using an H-scoring system. Cut-off points for BCAT1 and CD133 expression were determined using receiver operating characteristic curves.ResultsThe median follow-up time for the study participants was 68.73 months (range: 1.37–103.6 months). The 5-year disease-free survival (DFS) and overall survival (OS) rates of the 291 patients with TNBC were 72.51 and 82.47%, respectively. Higher levels of BCAT1 and CD133 expression independently indicated shorter DFS and OS. High levels of both BCAT1 and CD133 expression were detected in 36 (12.37%) patients, who had significantly shorter DFS and OS (both P < 0.001) compared to other patients.ConclusionBCAT1 and CD133 can be considered as biomarkers with prognostic significance for TNBC.

Metabolic balancing by miR-276 shapes the mosquito reproductive cycle and Plasmodium falciparum development

The blood-feeding behavior of Anopheles females delivers essential nutrients for egg development and drives parasite transmission between humans. Plasmodium growth is adapted to the vector reproductive cycle, but how changes in the reproductive cycle impact parasite development remains unclear. Here, we show that the bloodmeal-induced miR-276-5p fine-tunes the expression of branched-chain amino acid transferase to terminate the reproductive cycle. Silencing of miR-276 prolongs high rates of amino acid (AA) catabolism and increases female fertility, suggesting that timely termination of AA catabolism restricts mosquito investment into reproduction. Prolongation of AA catabolism in P. falciparum-infected females also compromises the development of the transmissible sporozoite forms. Our results suggest that Plasmodium sporogony exploits the surplus mosquito resources available after reproductive investment and demonstrate the crucial role of the mosquito AA metabolism in within-vector parasite proliferation and malaria transmission.

Expanding the genetic and phenotypic spectrum of branched-chain amino acid transferase 2 deficiency.

The first step in branched-chain amino acid (BCAA) catabolism is catalyzed by the two BCAA transferase isoenzymes, cytoplasmic branched-chain amino acid transferase (BCAT) 1, and mitochondrial BCAT2. Defects in the second step of BCAA catabolism cause maple syrup urine disease (MSUD), a condition which has been far more extensively investigated. Here, we studied the consequences of BCAT2 deficiency, an ultra-rare condition in humans. We present genetic, clinical, and functional data in five individuals from four different families with homozygous or compound heterozygous BCAT2 mutations which were all detected following abnormal biochemical profile results or familial mutation segregation studies. We demonstrate that BCAT2 deficiency has a recognizable biochemical profile with raised plasma BCAAs and, in contrast with MSUD, low-normal branched-chain keto acids (BCKAs) with undetectable l-allo-isoleucine. Interestingly, unlike in MSUD, none of the individuals with BCAT2 deficiency developed acute encephalopathy even with exceptionally high BCAA levels. We observed wide-ranging clinical phenotypes in individuals with BCAT2 deficiency. While one adult was apparently asymptomatic, three individuals had presented with developmental delay and autistic features. We show that the biochemical characteristics of BCAT2 deficiency may be amenable to protein-restricted diet and that early treatment may improve outcome in affected individuals. BCAT2 deficiency is an inborn error of BCAA catabolism. At present, it is unclear whether developmental delay and autism are parts of the variable phenotypic spectrum of this condition or coincidental. Further studies will be required to explore this.

Expression of genes related to energy metabolism and the unfolded protein response in dairy cow mammary cells is affected differently during dietary supplementation with energy from protein and fat

Secretory capacity of bovine mammary glands is enabled by a high number of secretory cells and their ability to use a range of metabolites to produce milk components. We isolated RNA from milk fat to measure expression of genes involved in energy-yielding pathways and the unfolded protein response in mammary glands of lactating cows given supplemental energy from protein (PT) and fat (FT) tested in a 2 × 2 factorial arrangement. We hypothesized that PT and FT would affect expression of genes in the branched-chain AA catabolic pathway and tricarboxylic acid (TCA) cycle based on the different energy types (aminogenic versus lipogenic) used to synthesize milk components. We also hypothesized that the response of genes related to endoplasmic reticulum (ER) homeostasis via the unfolded protein response would reflect the increase in milk production stimulated by PT and FT. Fifty-six multiparous Holstein-Friesian dairy cows were fed a basal total mixed ration (34% grass silage, 33% corn silage, 5% grass hay, and 28% concentrate on a dry matter basis) for a 28-d control period. Experimental rations were then fed for 28 d, consisting of (1) low protein, low fat (LP/LF); (2) high protein, low fat (HP/LF); (3) low protein, high fat (LP/HF); or (4) high protein and high fat (HP/HF). To obtain the high-protein (HP) and high-fat (HF) diets, intake of the basal ration was restricted and supplemented isoenergetically (net energy basis) with 2.0 kg/d rumen-protected protein (soybean + rapeseed, 50:50 mixture on dry matter basis) and 0.68 kg/d hydrogenated palm fatty acids on a dry matter basis. RNA from milk fat samples collected on d 27 of each period underwent real-time quantitative PCR. Energy from protein increased expression of BCAT1 (branched-chain amino acid transferase 1) mRNA, but only at the LF level, and tended to decrease expression of mRNA encoding the main subunit of the branched-chain keto-acid dehydrogenase complex. mRNA expression of malic enzyme, a proposed channeling route for AA though the TCA cycle, was decreased by PT, but only at the LF level. Expression of genes associated with de novo fatty acid synthesis was not affected by PT or FT. Energy from fat had no independent effect on genes related to ER homeostasis. At the LF level, PT activated XBP1 (X-box binding protein 1) mRNA. At the HF level, PT increased mRNA expression of the gene encoding GADD34 (growth arrest and DNA damage-inducible 34). These findings support our hypothesis that mammary cells use aminogenic and lipogenic precursors differently for milk component production when dietary intervention alters AA and fatty acid supply. They also suggest that mammary cells respond to increased AA supply through mechanisms of ER homeostasis, dependent on the presence of FT.

Construction of industrial brewing yeast for fermentation under high temperature and high gravity condition

As a new beer fermentation technology, high temperature and high gravity fermentation has brought many benefits to brewery industry, but there are also a series of problems such as the decrease of yeast flocculation ability at the end of fermentation and the high concentration of higher alcohols. To increase yeast flocculation ability and reduce the production of higher alcohols in high temperature and high gravity fermentation of beer, BAT2 was replaced by the FLO5 expression cassette to obtain the mutant strain S6-BF2. Real-time quantitative PCR showed that the relative transcriptional level of FLO5 in S6-BF2 improved 17.8 times compared with that in S6. The flocculation ability of mutant S6-BF2 heightened by 63% compared to that of the original strain S6, and the concentration of higher alcohols decreased from 175.58 mg/L to 159.58 mg/L in high temperature and high gravity fermentation of beer. Moreover, the activity of mitochondrial branched-chain amino acid transferase was repressed, resulting in the production of higher alcohols of 142.13 mg/L, reduced by 18.4% compared to that of the original strain S6, meanwhile, the flocculation ability of mutant S6-BF2B1 kept unchanged compared to the mutant S6-BF2. The determination result of flavor compounds showed that the higher alcohols/ester ratio in beer was reasonable. This research has suggested an effective strategy for enhancing yeast flocculation ability and decreasing production of higher alcohols in high-temperature and high-gravity brewing.

Branched chain amino acid transferase 1 and malignant tumors

As a key enzyme that catalyzes the metabolism of branched chain amino acids, branched chain amino acid transferase 1 (BCAT1) is often involved in a variety of biosynthetic pathways. Reaserches show that BCAT1 is highly expressed in many kinds of malignant tumors such as leukemia, glioma, nasopharyngeal carcinoma, gastric cancer and breast cancer, et al, suggesting a close relationship with the proliferation, invasion and metastasis of tumor cells. Thus, BCAT1 plays an important role in the genesis and progression of tumor, and may have the potential to be a new therapeutic target. Key words: Neoplasms; Branched chain amino acid transaminase 1; Branched chain amino acid

Gabapentin Can Suppress Cell Proliferation Independent of the Cytosolic Branched-Chain Amino Acid Transferase 1 (BCAT1).

The metabolism of branched-chain amino acids (BCAA) has recently been implicated in the growth of several cancer cell types. Gabapentin, a synthetic amino acid, is commonly used in high concentrations in this context to inhibit the cytosolic branched-chain amino acid transferase (BCAT1) enzyme. Here, we report that 10 mM gabapentin reduces the growth of HCT116 cells, which have an active branched-chain amino acid transferase but express very low levels of BCAT1, and presumably rely on the mitochondrial BCAT2 enzyme. Gabapentin did not affect transamination of BCAA to branched-chain keto acids (BCKA) in HCT116 cells, nor the reverse formation of BCAA from BCKA, indicating that the branched-chain amino acid transaminase is not inhibited. Moreover, the growth-inhibitory effect of gabapentin could not be rescued by supplementation with BCKA, and this was not due to the lack of uptake of BCKA, indicating that other effects of gabapentin are important. An untargeted LC-MS analysis of gabapentin-treated cells revealed a marked depletion of branched-chain carnitines. These results demonstrate that gabapentin at high concentrations can inhibit cell proliferation without affecting BCAT1 and may affect mitochondrial BCKA catabolism.

An integrative tissue-network approach to identify and test human disease genes.

Effective discovery of causal disease genes must overcome the statistical challenges of quantitative genetics studies and the practical limitations of human biology experiments. Here we developed diseaseQUEST, an integrative approach that combines data from human genome-wide disease studies with in silico network models of tissue- and cell-type-specific function in model organisms to prioritize candidates within functionally conserved processes and pathways. We used diseaseQUEST to predict candidate genes for 25 different diseases and traits, including cancer, longevity, and neurodegenerative diseases. Focusing on Parkinson's disease (PD), a diseaseQUEST-directed Caenhorhabditis elegans behavioral screen identified several candidate genes, which we experimentally verified and found to be associated with age-dependent motility defects mirroring PD clinical symptoms. Furthermore, knockdown of the top candidate gene, bcat-1, encoding a branched chain amino acid transferase, caused spasm-like 'curling' and neurodegeneration in C. elegans, paralleling decreased BCAT1 expression in PD patient brains. diseaseQUEST is modular and generalizable to other model organisms and human diseases of interest.

Multistage Ultraviolet Photodissociation Mass Spectrometry To Characterize Single Amino Acid Variants of Human Mitochondrial BCAT2.

Unraveling disease mechanisms requires a comprehensive understanding of how the interplay between higher-order structure and protein-ligand interactions impacts the function of a given protein. Recent advances in native mass spectrometry (MS) involving multimodal or higher-energy activation methods have allowed direct interrogation of intact protein complexes in the gas phase, allowing analysis of both composition and subunit connectivity. We report a multistage approach combining collisional activation and 193 nm ultraviolet photodissociation (UVPD) to characterize single amino acid variants of the human mitochondrial enzyme branched-chain amino acid transferase 2 (BCAT2), a protein implicated in chemotherapeutic resistance in glioblastoma tumors. Native electrospray ionization confirms that both proteins exist as homodimers. Front-end collisional activation disassembles the dimers into monomeric subunits that are further interrogated using UVPD to yield high sequence coverage of the mutated region. Additionally, holo (ligand-bound) fragment ions resulting from photodissociation reveal that the mutation causes destabilization of the interactions with a bound cofactor. This study demonstrates the unique advantages of implementing UVPD in a multistage MS approach for analyzing intact protein assemblies.

In search of druggable targets for GBM amino acid metabolism

BackgroundAmino acid (AA) pathways may contain druggable targets for glioblastoma (GBM). Literature reviews and GBM database (http://r2.amc.nl) analyses were carried out to screen for such targets among 95 AA related enzymes.MethodsFirst, we identified the genes that were differentially expressed in GBMs (3 datasets) compared to non-GBM brain tissues (5 datasets), or were associated with survival differences. Further, protein expression for these enzymes was also analyzed in high grade gliomas (HGGs) (proteinatlas.org). Finally, AA enzyme and gene expression were compared among the 4 TCGA (The Cancer Genome Atlas) subtypes of GBMs.ResultsWe detected differences in enzymes involved in glutamate and urea cycle metabolism in GBM. For example, expression levels of BCAT1 (branched chain amino acid transferase 1) and ASL (argininosuccinate lyase) were high, but ASS1 (argininosuccinate synthase 1) was low in GBM. Proneural and neural TCGA subtypes had low expression of all three. High expression of all three correlated with worse outcome. ASL and ASS1 protein levels were mostly undetected in high grade gliomas, whereas BCAT1 was high. GSS (glutathione synthetase) was not differentially expressed, but higher levels were linked to poor progression free survival. ASPA (aspartoacylase) and GOT1 (glutamic-oxaloacetic transaminase 1) had lower expression in GBM (associated with poor outcomes). All three GABA related genes -- glutamate decarboxylase 1 (GAD1) and 2 (GAD2) and 4-aminobutyrate aminotransferase (ABAT) -- were lower in mesenchymal tumors, which in contrast showed higher IDO1 (indoleamine 2, 3-dioxygenase 1) and TDO2 (tryptophan 2, 3-diaxygenase). Expression of PRODH (proline dehydrogenase), a putative tumor suppressor, was lower in GBM. Higher levels predicted poor survival.ConclusionsSeveral AA-metabolizing enzymes that are higher in GBM, are also linked to poor outcome (such as BCAT1), which makes them potential targets for therapeutic inhibition. Moreover, existing drugs that deplete asparagine and arginine may be effective against brain tumors, and should be studied in conjunction with chemotherapy. Last, AA metabolism is heterogeneous in TCGA subtypes of GBM (as well as medulloblastomas and other pediatric tumors), which may translate to variable responses to AA targeted therapies.

Improving Longevity with Metadichol® by Inhibiting the BCAT1 Gene

Metadichol® [1] is a Nanoemulsion of long-chain alcohols found in many foods. It is commonly called Policosanol and is present in foods such as rice, sugar cane, wheat, peanuts Metadichol acts as an inverse agonist on Nuclear Vitamin D receptors (VDR) that are present in cells throughout the body to stimulate the immune system and affects many biologIcal processes to modulate many diseases. Branched-chain amino acid transferase (BCAT1) catalyzes the reversible transamination of leucine, isoleucine, and valine branched-chain amino acids (BCAA) to their respective alpha-keto acids, liberating L-glutamate. When this gene is inhibited, the amino acid chains accumulated in the tissue triggering longevity in the nematodes. The health and longevity of the nematodes improved when BCAT1 was inhibited. Gabapentin has been shown to inhibit BCAT1, but IC50 is 10000 uM. Metadichol® inhibits BCAT1 with an IC50 of 3.3 um, 3000 times more potent than Gabapentin.

Abstract PR11: Tissue-of-origin dictates the metabolic fate of branched chain amino acids in mutant Kras-driven cancers

Abstract Introduction: Growth signaling is associated with changes in metabolism in cell culture, but the relatively homogenous nature of culture systems leaves open the question of how specific mutations might behave differently in different tissue contexts. Indeed, previous work has demonstrated that tumor metabolic gene expression most closely resembles the tissue of origin for that tumor1, and that the same oncogenic driver can cause different metabolic phenotypes in different tissues2. We recently found that pancreatic cancers (PDAC) are associated with increased plasma branched-chain amino acid (BCAA) levels resulting from increased whole body protein turnover, while a model of non-small cell lung cancer (NSCLC) driven by the same genetic lesions displays the opposite changes in plasma BCAA levels3. Based on these data, we hypothesized that changes in plasma BCAAs might reflect a difference in the utilization of BCAA by tumors arising in each tissue. Methods: C57B6/J male mice heterozygous for the lox-stop-lox (LSL)-KrasG12D allele and homozygous for the Trp53flox/flox allele were exposed to an inhaled viral Cre-recombinase or crossed to a pancreas-specific Pdx-1-cre to generate models of NSCLC and PDAC respectively. Cell lines previously derived from these mouse models were used for in vitro and allograft studies. To identify the metabolic fates of BCAAs in vitro, we performed tracing studies utilizing stable-isotope 13C-Leu or 15N-Leu in place of the abundant 12C or 14N isotopomer. Similarly, we used amino acid defined diets in which stable isotope BCAAs were included to trace amino acid fate in vivo. Data: We first confirmed that plasma BCAA levels change in opposite directions early in disease in the isogenic mouse models of PDAC and NSCLC. Consistent with different metabolic fates of BCAAs, 13C stable isotope tracing in vivo revealed increased incorporation of BCAAs into tissue protein in NSCLC tumors, with decreased incorporation observed in PDAC tumors. NSCLC tumors also displayed increased generation ofα-ketoisocaproate, the transamination product of leucine, suggesting an additional role for BCAAs a nitrogen source in NSCLC. Further supporting this possibility, we found increased expression of branched-chain amino acid transferase 2 (Bcat2), but not other BCAA catabolic genes, in NSCLC tumors. To directly examine the fate of BCAA-derived nitrogen, we used 15N-BCAA tracing studies and CRISPR/Cas9-mediated knockout of BCAA catabolic genes. Tracing in vitro revealed increased transamination of 15N-Leu in NSCLC cells compared with PDAC cells. We also generated NSCLC and PDAC cells deficient in Bcat isoforms to assess the effects of Bcat knockout in both allograft and autochthonous models. Conclusions: Despite sharing an identical combination of mutations, mouse models of NSCLC and PDAC exhibit distinct phenotypes with regards to BCAA metabolism in tumors. The different fates of BCAAs in each of these tumor types might explain previously observed alterations in plasma BCAA levels and provides further insight into how tumors can influence whole body metabolic phenotypes in early cancer. 1. Hu, J., et al. Heterogeneity of tumor-induced gene expression changes in the human metabolic network. Nature biotechnology 31, 522-529 (2013). 2. Yuneva, M.O., et al. The Metabolic Profile of Tumors Depends on Both the Responsible Genetic Lesion and Tissue Type. Cell Metabolism 15, 157-170 (2012). 3. Mayers, J.R., et al. Elevation of circulating branched-chain amino acids is an early event in human pancreatic adenocarcinoma development. Nature Medicine (2014). Citation Format: Jared R. Mayers, Margaret E. Torrence, Shawn M. Davidson, Thales Papagiannakopoulos, Allison N. Lau, Tyler Jacks, Matthew G. Vander Heiden. Tissue-of-origin dictates the metabolic fate of branched chain amino acids in mutant Kras-driven cancers. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstractnr PR11.