Short-chain enoyl-CoA hydratase (ECHS1) is involved in amino acid and fatty acid catabolism in mitochondria and its deficiency causes Leigh syndrome or exercise-induced dystonia. More than 60 patients with this condition have been reported till date. The accumulation of intermediate metabolites of valine is assumed to be responsible for the cytotoxicity. Since protein restriction, including valine reportedly improves neurological symptoms, it is essential to consider the possible incidence of and diagnose ECHS1 syndrome in the earlier stages. This study reported the liquid chromatography with tandem mass spectrometry (LC-MS/MS) urine and plasma metabolite analysis in six cases, including four new cases with ECHS1 deficiency. The values of urine cysteine/cysteamine conjugates from valine metabolites, S-(2-carboxypropyl) cysteine/cysteamine from methacrylyl-CoA, and S-(2-carboxyethyl) cysteine/cysteamine from acryloyl-CoA were separated between six patients and six normal controls. The LC-MS/MS analysis revealed that these metabolites can be used for the early diagnosis and evaluation of diet therapy.

Among the branched-chain amino acids, leucine and isoleucine have been well studied for their roles in improving mitochondrial function and reducing oxidative stress. However, role of valine in mitochondrial function regulation and oxidative stress management remains elusive. This study investigated valine effect on mitochondrial function and oxidative stress in vitro. Valine increased expression of genes involved in mitochondrial biogenesis and dynamics. It upregulates mitochondrial function at complexes I, II, and IV levels of electron transport chain. Flow cytometry studies revealed, valine reduced oxidative stress by significantly lowering mitochondrial reactive oxygen species and protein expression of 4-hydroxynonenal. Functional role of valine against oxidative stress was analyzed by XFe96 Analyzer. Valine sustained oxidative phosphorylation and improved ATP generation rates during oxidative stress. In conclusion, our findings shed more light on the critical function of valine in protecting mitochondrial function thereby preventing mitochondrial/cellular damage induced by oxidative stress.

The emergence of multidrug-resistant bacteria presents a severe threat to public health and causes extensive losses in livestock husbandry and aquaculture. Effective strategies to control such infections are in high demand. Enhancing host immunity is an ideal strategy with fewer side effects than antibiotics. To explore metabolite candidates, we applied a metabolomics approach to investigate the metabolic profiles of mice after Klebsiella pneumoniae infection. Compared with the mice that died from K. pneumoniae infection, mice that survived the infection displayed elevated levels of l-valine. Our analysis showed that l-valine increased macrophage phagocytosis, thereby reducing the load of pathogens; this effect was not only limited to K. pneumoniae but also included Escherichia coli clinical isolates in infected tissues. Two mechanisms are involved in this process: l-valine activating the PI3K/Akt1 pathway and promoting NO production through the inhibition of arginase activity. The NO precursor l-arginine is necessary for l-valine-stimulated macrophage phagocytosis. The valine-arginine combination therapy effectively killed K. pneumoniae and exerted similar effects in other Gram-negative (E. coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria. Our study extends the role of metabolism in innate immunity and develops the possibility of employing the metabolic modulator-mediated innate immunity as a therapy for bacterial infections.

In the present study, a series of L-valine derived boroxazolidones, previously synthesized and reported to have residual activity in a human epithelial cell line, have been evaluated in vitro for their anti-glioblastoma activity. A boroxazolidone derivative containing 2,4-difluorophenyl moieties (6) was found to have higher cytotoxicity than the standard drug, Temozolomide (TMZ). Compound 6 was found to exhibit dose-dependent growth inhibitory effects with an IC50 of 49 μM and 53 μM for LN229 and SNB19 cells, respectively. Additionally, 6 was assessed for its role in apoptosis, caspase 3/7 activation and oxidative stress in SNB19 and LN229 cells. SNB19 cells treated with 6 showed 45.3% apoptosis in the population, while TMZ had 24.7%. In LN229 cells, the percentage of apoptotic cells treated with compound 6 and TMZ were the same. Both 6 and TMZ induced apoptosis through the activation of caspase 3/7 in SNB19 and LN229 cells. Interestingly, 6 exhibited a higher effectivity in promoting reactive oxygen species production in LN229, while it was 6-fold less in SNB19. Boroxazolidone-treated GBM cell lines increased reactive oxygen species production, suggesting that such species may be interlinked with the observed programmed cell death. Additionally, the treatment of both GBM cell lines with 6 led to G2/M phase arrest. The magnitude of anti-GBM effect of 6 is significantly higher than the known chemotherapeutic agent TMZ. This work further demonstrates the anticancer properties of L-valine derived boroxazolidones, adding another potential derivative to the collection of promising chemotherapeutic agents for GBM treatment.

Many lepidopteran larvae are serious agricultural pests due to their feeding activity. Digestion of the plant diet occurs mainly in the midgut and is facilitated by the peritrophic matrix (PM), an extracellular sac-like structure, which lines the midgut epithelium and creates different digestive compartments. The PM is attracting increasing attention to control lepidopteran pests by interfering with this vital function. To identify novel PM components and thus potential targets for insecticides, we performed an immunoscreening with anti-PM antibodies using an expression library representing the larval midgut transcriptome of the tobacco hornworm, Manduca sexta. We identified three cDNAs encoding valine-rich midgut proteins of M. sexta (MsVmps), which appear to be loosely associated with the PM. They are members of a lepidopteran-specific family of nine VMP genes, which are exclusively expressed in larval stages in M. sexta. Most of the MsVMP transcripts are detected in the posterior midgut, with the highest levels observed for MsVMP1. To obtain further insight into Vmp function, we expressed MsVMP1 in insect cells and purified the recombinant protein. Lectin staining and glycosidase treatment indicated that MsVmp1 is highly O-glycosylated. In line with results from qPCR, immunoblots revealed that MsVmp1 amounts are highest in feeding larvae, while MsVmp1 is undetectable in starving and molting larvae. Finally using immunocytochemistry, we demonstrated that MsVmp1 localizes to the cytosol of columnar cells, which secrete MsVmp1 into the ectoperitrophic space in feeding larvae. In starving and molting larvae, MsVmp1 is found in the gut lumen, suggesting that the PM has increased its permeability. The present study demonstrates that lepidopteran species including many agricultural pests have evolved a set of unique proteins that are not found in any other taxon and thus may reflect an important adaptation in the highly specialized lepidopteran digestive tract facing particular immune challenges.

Hip fractures constitute a major health concern. An adequate supply of amino acids is crucial to ensure optimal acquisition and remodeling of bone. Circulating amino acid levels have been proposed as markers of bone mineral density, but data on their ability to predict incident fractures are scarce.

In this study, we synthesized the valine (Val)-conjugated amide prodrug of doxorubicin (DOX) by the formation of amide bonds between DOX and Val. The synthesis of the DOX-Val prodrug was identified by a proton nuclear magnetic resonance (¹H-NMR) assay. In the MCF-7 cells (human breast adenocarcinoma cell; amino acid transporter-positive cell), the cellular accumulation efficiency of DOX-Val was higher than that of DOX according to the flow cytometry analysis data. Using confocal laser scanning microscopy (CLSM) imaging, it was confirmed that DOX-Val as well as DOX was mainly distributed in the nucleus of cancer cells. DOX-Val was intravenously administered to rats at a dose of 4 mg/kg, and the plasma concentrations of DOX-Val (prodrug) and DOX (formed metabolite) were quantitatively determined. Based on the systemic exposure (represented as area under the curve (AUC) values) of DOX-Val (prodrug) and DOX (formed metabolite), approximately half of DOX-Val seemed to be metabolized into DOX. However, it is expected that the remaining DOX-Val may exert improved cellular uptake efficiency in cancer cells after its delivery to the cancer region.

Although deregulation of transfer RNA (tRNA) biogenesis promotes the translation of pro-tumorigenic mRNAs in cancers1,2, the mechanisms and consequences of tRNA deregulation in tumorigenesis are poorly understood. Here we use a CRISPR-Cas9 screen to focus on genes that have been implicated in tRNA biogenesis, and identify a mechanism by which altered valine tRNA biogenesis enhances mitochondrial bioenergetics in T cell acute lymphoblastic leukaemia (T-ALL). Expression of valine aminoacyl tRNA synthetase is transcriptionally upregulated by NOTCH1, a key oncogene in T-ALL, underlining a role for oncogenic transcriptional programs in coordinating tRNA supply and demand. Limiting valine bioavailability through restriction of dietary valine intake disrupted this balance in mice, resulting in decreased leukaemic burden and increased survival in vivo. Mechanistically, valine restriction reduced translation rates of mRNAs that encode subunits of mitochondrial complex I, leading to defective assembly of complex I and impaired oxidative phosphorylation. Finally, a genome-wide CRISPR-Cas9 loss-of-function screen in differential valine conditions identified several genes, including SLC7A5 and BCL2, whose genetic ablation or pharmacological inhibition synergized with valine restriction to reduce T-ALL growth. Our findings identify tRNA deregulation as a critical adaptation in the pathogenesis of T-ALL and provide a molecular basis for the use of dietary approaches to target tRNA biogenesis in blood malignancies.

Pseudomonas species strain SBV1 can rapidly grow on medium containing β-valine as a sole nitrogen source. The tertiary amine feature of β-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of β-valine, a PsSBV1 gene library was prepared and used to complement the β-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding β-valinyl-coenzyme A ligase (BvaA) and two genes encoding β-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate β-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only β-valine and to a lesser extent, 3-aminobutyrate and β-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate β-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.

Obesity is widespread and linked to various co-morbidities. Bariatric surgery has been identified as the only effective treatment, promoting sustained weight loss and the remission of co-morbidities.

Valine catabolism is known to be essential for cancer cells but the detailed mechanism remains unclear. This study is to explore the critical roles of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) in colorectal cancers (CRC) and to develop a new therapy returning valine metabolism homeostasis. High HIBCH expression was first confirmed to correlate with poor survival in patients with CRC, which was then linked to the increased cell growth, resistant apoptosis, and decreased autophagy in CRC cells. The functions of HIBCH in CRC were dependent on its mitochondrial localization. High HIBCH level was further demonstrated to promote the metabolism of tricarboxylic acid cycle as well as oxidative phosphorylation in CRC cells. Based on above findings, we further discovered a novel valine catabolism inhibitor SBF-1. The pharmacological blockade of HIBCH mitochondrial localization with SBF-1 resulted in decreased cancer cell growth and increased autophagy, collectively contributing to the antitumor effect both in vitro and in vivo. Moreover, anti-VEGF therapy with bevacizumab increased HIBCH level in CRC cells, which in turn caused the resistance to the therapy. The interference with HIBCH function by SBF-1 significantly increased the antitumor efficacy of bevacizumab and led to a robust survival benefit. The present study identified HIBCH as a critical enzyme of valine catabolism in CRC progression and resistance to anti-VEGF therapy. We also provided a novel HIBCH inhibitor SBF-1, which highlighted the combined therapy using valine catabolic inhibitor along with anti-VEGF drugs, to control progression of CRC.

Avian species including chickens are known to be hyperglycemic animals. Hyperglycemia promotes the glycation which at first forms Amadori products undergoing further complex reaction to form advanced glycation end products (AGEs). Our previous study revealed that AGEs derived from glucose and amino acids were predominantly incorporated into spleen, kidney and liver. However, it has not been elucidated whether Amadori products (glycated amino acids) can also be incorporated into cells or not. Therefore, in the present study, radioactive glycated-tryptophan and -valine were prepared and the incorporation of these glycated amino acids into various chicken embryonic cells was studied. Various embryonic cells prepared from muscle, liver, spleen and kidney of chicken embryos were incubated in Medium 199 supplemented with 14C-labeled glycated-tryptophan or -valine. After incubation, embryonic cells were well-rinsed and then the radioactivity incorporated into cells was measured. It was revealed that both glycated amino acids were incorporated into embryonic cells derived from muscle, liver, spleen and kidney. In muscular cells, the incorporation of glycated-tryptophan was higher than that of glycated-valine. On the other hand, in embryonic cells derived from liver and kidney, the amount of glycated-tryptophan incorporated into cells was almost the same to that of glycated-valine. In conclusion, it was supposed that both glycated-tryptophan and -valine could be incorporated into various cells derived from muscle, liver, spleen and kidney of chicken embryos and that the incorporation might have the organ specificity.

Toward the elucidation of the advanced mechanism of L-valine production by Corynebacterium glutamicum, a highly developed industrial strain VWB-1 was analyzed, employing the combination of transcriptomics and proteomics methods. The transcriptional level of 1155 genes and expression abundance of 96 proteins were changed significantly by the transcriptome and proteome comparison of VWB-1 and ATCC 13869. It was indicated that the key genes involved in the biosynthesis of L-valine, ilvBN, ilvC, ilvD, ilvE were up-regulated in VWB-1, which together made prominent contributions in improving the carbon flow towards L-valine. The L-leucine and L-isoleucine synthesis ability were weakened according to the down-regulation of leuB and ilvA. The up-regulation of the branched chain amino acid transporter genes brnFE promoted the L-valine secretion capability of VWB-1. The NADPH and ATP generation ability of VWB-1 were strengthened through the up-regulation of the genes involved in phosphate pentose pathway and TCA pathway. Pyruvate accumulation was achieved through the weakening of the L-lactate, acetate and L-alanine pathways. The up-regulation of the genes coding for elongation factors and ribosomal proteins were beneficial for L-valine synthesis in C. glutamicum. All information acquired were useful for the genome breeding of better industrial L-valine producing strains.

Background and Objectives: Considering atherosclerosis as one of the more challenging threats to healthcare worldwide, any novel therapy that counteracts the risks for developing it, provides new opportunities for the management of this process. Material and methods: We performed an experimental research in which we induced a hypercholesterolemia via a cholesterol-rich diet. Our aim was to demonstrate the antiatherogenic potential of two essential amino acids (valine and leucine). The experimental study was carried out over a period of 60 days. Male Wistar rats weighing between 250-280 g were used and divided into 4 groups, each group including 8 animals. Group I-control was fed with a standard diet. Group II received cholesterol, group III cholesterol and valine and group IV cholesterol and leucine. Blood samples were collected from the retro-orbital plexus, under anesthesia with 75 mg/kg of intraperitoneal ketamine, in three different moments (R0-1st day, R1-the 30th day, R2-the 60th day) in order to measure the levels of triglycerides. Results: In R0, there were no significant differences between the average levels of triglycerides across all the groups (p < 0.05). Compared to the group I, in R1 and R2, the average levels of triglycerides were significantly higher in all groups (p < 0.001). Also, in R1 and R2, the average triglycerides in group II receiving cholesterol (C) were significantly higher than those in group III receiving valine (C + V) as well as in group IV receiving leucine (C + L) (p < 0.001; p < 0.05). In R2, the average triglycerides in group III were significantly lower than in group IV (p < 0.001). Conclusions: Our data provides evidence that valine and leucine have a direct impact on the lipid metabolism parameters by lowering the level of triglycerides. The comparison of the two essential amino acids indicates that valine acts more promptly and rapidly than leucine.

Microorganisms can restructure their transcriptional output to adapt to environmental conditions by sensing endogenous metabolite pools. In this paper, an Agilent customized microarray representing 4,106 genes was used to study temporal transcript profiles of Bacillus subtilis in response to valine, glutamate and glutamine pulses over 24 h. A total of 673, 835, and 1135 amino-acid-regulated genes were identified having significantly changed expression at one or more time points in response to valine, glutamate, and glutamine, respectively, including genes involved in cell wall, cellular import, metabolism of amino-acids and nucleotides, transcriptional regulation, flagellar motility, chemotaxis, phage proteins, sporulation, and many genes of unknown function. Different amino acid treatments were compared in terms of both the global temporal profiles and the 5-minute quick regulations, and between-experiment differential genes were identified. The highlighted genes were analyzed based on diverse sources of gene functions using a variety of computational tools, including T-profiler analysis, and hierarchical clustering. The results revealed the common and distinct modes of action of these three amino acids, and should help to elucidate the specific signaling mechanism of each amino acid as an effector.

HT-29 cells, originating from a human colon carcinoma, can proliferate in standard culture conditions with an absolute requirement for polyamines. The major precursor provided in the culture medium for polyamine biosynthesis is L-arginine. L-Arginine conversion to L-ornithine by arginase is followed by stepwise conversion of this latter amino acid to putrescine, spermidine and spermine. The aim of the present work was to document the consequences of a total inhibition of L-arginine flux through arginase, resulting in a decreased L-ornithine availability, on HT-29 cell proliferation and polyamine metabolism. L-Valine, a known arginase inhibitor, when used at a high concentration, i.e., 100 mM, inhibits L-arginine flux through arginase almost totally. The addition in the culture medium of 100 mM L-valine or 50 mM NaCl used to mimic the L-valine induced increase in medium osmolality both reduced equally cellular growth. Cell viability, protein synthesis or oxidative metabolism measured in isolated cells were unaffected by the L-valine treatment, suggesting that decreased proliferation was not associated with an acute toxic effect of this aminoacid, but was rather due to the increase in the medium osmolality. L-Valine treated cells displayed an altered polyamine metabolism when compared with control cells grown in the absence of the amino acid. After 4 days of treatment with 100 mM L-valine, L-ornithine flux through ornithine decarboxylase was significantly higher as well as putrescine and spermidine cellular uptakes in treated cells. However, the changes in polyamine metabolism led to similar polyamine cell contents in untreated and L-valine treated cells. In conclusion, we propose that the observed alterations of polyamine metabolism may reflect an adaptative response of HT-29 cells to the presence of L-valine which contribute together with the low amount of L-ornithine present in the culture medium to polyamine homeostasis.

Bio-based isobutantol is a sustainable 'drop in' substitute for petroleum-based fuels. However, well-studied production routes, such as the Ehrlich pathway, have yet to be commercialized despite more than a century of research. The more versatile bacterial valine catabolism may be a competitive alternate route producing not only an isobutanol precursor but several carboxylic acids with applications as biomonomers, and building blocks for other advanced biofuels. Here, we transfer the first two committed steps of the pathway from pathogenic Pseudomonas aeruginosa PAO1 to yeast to evaluate their activity in a safer model organism. Genes encoding the heteroligomeric branched chain keto-acid dehydrogenase (BCKAD; bkdA1, bkdA2, bkdB, lpdV), and the homooligomeric acyl-CoA dehydrogenase (ACD; acd1) were tagged with fluorescence epitopes and targeted for expression in either the mitochondria or cytoplasm of S. cerevisiae. We verified the localization of our constructs with confocal fluorescence microscopy before measuring the activity of tag-free constructs. Despite reduced heterologous expression of mitochondria-targeted enzymes, their specific activities were significantly improved with total enzyme activities up to 138% greater than those of enzymes expressed in the cytoplasm. In total, our results demonstrate that the choice of protein localization in yeast has significant impact on heterologous activity, and suggests a new path forward for isobutanol production.

Leucine has been shown to acutely inhibit hepatic glucose production in rodents by a mechanism requiring its metabolism to acetyl-CoA in the mediobasal hypothalamus (MBH). In the early stages, all branched-chain amino acids (BCAA) are metabolized by a shared set of enzymes to produce a ketoacid, which is later metabolized to acetyl-CoA. Consequently, isoleucine and valine may also modulate glucose metabolism. To examine this possibility we performed intrahypothalamic infusions of isoleucine or valine in rats and assessed whole body glucose kinetics under basal conditions and during euglycemic pancreatic clamps. Furthermore, because high fat diet (HFD) consumption is known to interfere with central glucoregulation, we also asked whether the action of BCAAs was affected by HFD. We fed rats a lard-rich diet for a short interval and examined their response to central leucine. The results showed that both isoleucine and valine individually lowered blood glucose by decreasing liver glucose production. Furthermore, the action of the BCAA leucine was markedly attenuated by HFD feeding. We conclude that all three BCAAs centrally modulate glucose metabolism in the liver and that their action is disrupted by HFD-induced insulin resistance.

In 2011-2012, Northern Vietnam experienced its first large scale hand foot and mouth disease (HFMD) epidemic. In 2011, a major HFMD epidemic was also reported in South Vietnam with fatal cases. This 2011-2012 outbreak was the first one to occur in North Vietnam providing grounds to study the etiology, origin and dynamic of the disease. We report here the analysis of the VP1 gene of strains isolated throughout North Vietnam during the 2011-2012 outbreak and before.

Bacterial endospores can survive harsh environmental conditions and long-term dormancy in the absence of nutrients, but can rapidly germinate under favorable conditions. In the present study, we employed transposon sequencing (Tn-seq) to identify genes with previously uncharacterized roles in spore germination. Identified genes that encoded spore inner membrane proteins were chosen for study of defined mutants, which exhibited delayed germination in several assays in response to varying germinants. Significantly slowed release of DPA indicated that mutants were affected in Stage I of germination. Several mutants exhibited phenotypic traits consistent with failure of a GerA germinant receptor-mediated response, while others appeared to have a more general loss of response to varied germinants. Use of a gerA-lacZ transcriptional fusion and quantitative western blotting of GerAC allowed mutants to be classified based upon normal or decreased gerA transcription and normal or reduced GerA accumulation. Fourteen genes were identified to have newly described roles within Bacillus spore germination. A more complete understanding of this process can contribute to the development of better spore decontamination procedures.