Gotowa bibliografia na temat „Valine Catabolism”

In Streptomyces ambofaciens, valine favored spiramycin biosynthesis by supplying aglycone precursors. The kinetics of valine consumption and isobutyrate production showed that isobutyrate accumulated in the cell during the growth phase, was excreted in the stationary phase, and then was reassimilated during spiramycin production. When valine was in excess, its deamination led to high ammonium excretion and to a significant drop in spiramycin production. We demonstrated that ammonium ions were the cause of the negative effect. Addition of a chelator agent, Ca3(PO4)2, improved spiramycin production by sixfold. In contrast, addition of ammonium, between 0 and 48 h, severely reduced spiramycin production. The negative effect of ammonium was reversed by addition of a catabolic intermediate of valine, isobutyrate. In addition to stimulating the specific growth rate, ammonium ions slowed down valine catabolism: the specific valine uptake rate, excretion, and reassimilation of isobutyrate were lowered by the pulse of ammonium. Our study showed that in addition to valine dehydrogenase, which provided the nitrogen necessary to the cell, ammonium ions repressed ketoisovalerate dehydrogenase, which introduced valine as carbon, energy, and aglycone precursor sources. However, valine dehydrogenase and ketoisovalerate dehydrogenase did not constitute the principal enzymatic targets of the negative effect of ammonium in spiramycin production.Key words: spiramycin, Streptomyces ambofaciens, valine catabolism, ammonium.

Background: Branched-chain amino acids (BCAAs), essential nutrients including leucine, isoleucine, and valine, serve as a resource for energy production and the regulator of important nutrient and metabolic signals. Recent studies have suggested that dysfunction of BCAA catabolism is associated with the risk of cardiovascular disease. Platelets play an important role in cardiovascular disease, but the functions of BCAA catabolism in platelets remain unknown. Methods: The activity of human platelets from healthy subjects before and after ingestion of BCAAs was measured. Protein phosphatase 2Cm specifically dephosphorylates branched-chain α-keto acid dehydrogenase and thereby activates BCAA catabolism. Protein phosphatase 2Cm–deficient mice were used to elucidate the impacts of BCAA catabolism on platelet activation and thrombus formation. Results: We found that ingestion of BCAAs significantly promoted human platelet activity (n=5; P <0.001) and arterial thrombosis formation in mice (n=9; P <0.05). We also found that the valine catabolite α-ketoisovaleric acid and the ultimate oxidation product propionyl-coenzyme A showed the strongest promotion effects on platelet activation, suggesting that the valine/α-ketoisovaleric acid catabolic pathway plays a major role in BCAA-facilitated platelet activation. Protein phosphatase 2Cm deficiency significantly suppresses the activity of platelets in response to agonists (n=5; P <0.05). Our results also suggested that BCAA metabolic pathways may be involved in the integrin αIIbβ3–mediated bidirectional signaling pathway that regulates platelet activation. Mass spectrometry identification and immunoblotting revealed that BCAAs enhanced propionylation of tropomodulin-3 at K255 in platelets or Chinese hamster ovary cells expressing integrin αIIbβ3. The tropomodulin-3 K255A mutation abolished propionylation and attenuated the promotion effects of BCAAs on integrin-mediated cell spreading, suggesting that K255 propionylation of tropomodulin-3 is an important mechanism underlying integrin αIIbβ3–mediated BCAA-facilitated platelet activation and thrombosis formation. In addition, the increased levels of BCAAs and the expression of positive regulators of BCAA catabolism in platelets from patients with type 2 diabetes mellitus are significantly correlated with platelet hyperreactivity. Lowering dietary BCAA intake significantly reduced platelet activity in ob/ob mice (n=4; P <0.05). Conclusions: BCAA catabolism is an important regulator of platelet activation and is associated with arterial thrombosis risk. Targeting the BCAA catabolism pathway or lowering dietary BCAA intake may serve as a novel therapeutic strategy for metabolic syndrome–associated thrombophilia.

SUMMARYAll of the enzymes of proline catabolism were present in Heligmosomoides polygyrus and Panagrellus redivivus and the activities were, in general, similar to those found in rat liver. Both nematodes were also shown to be able to catabolize the branched-chain amino acids leucine, isoleucine and valine, by pathways similar to those found in mammalian liver. There were no significant differences in amino acid catabolism between the animal-parasitic and free-living species of nematode.

During valine catabolism in muscle both 2-oxoisovalerate and 3-hydroxyisobutyrate can be released into the circulation. 3-Hydroxyisobutyrate is a good gluconeogenic substrate in isolated cortical tubules and hepatocytes. The maximal rate of gluconeogenesis from 3-hydroxyisobutyrate was greater than from 2-oxoisovalerate. We propose that 3-hydroxyisobutyrate is an inter-organ metabolite by which the gluconeogenic potential of valine, whose catabolism has been initiated in muscle, may be conserved.

DPD (dihydropyrimidine dehydrogenase) constitutes the first step of the pyrimidine degradation pathway, in which the pyrimidine bases uracil and thymine are catabolized to β-alanine and the R-enantiomer of β-AIB (β-aminoisobutyric acid) respectively. The S-enantiomer of β-AIB is predominantly derived from the catabolism of valine. It has been suggested that an altered homoeostasis of β-alanine underlies some of the clinical abnormalities encountered in patients with a DPD deficiency. In the present study, we demonstrated that only a slightly decreased concentration of β-alanine was present in the urine and plasma, whereas normal levels of β-alanine were present in the cerebrospinal fluid of patients with a DPD deficiency. Therefore the metabolism of β-alanine-containing peptides, such as carnosine, may be an important factor involved in the homoeostasis of β-alanine in patients with DPD deficiency. The mean concentration of β-AIB was approx. 2–3-fold lower in cerebrospinal fluid and urine of patients with a DPD deficiency, when compared with controls. In contrast, strongly decreased levels (10-fold) of β-AIB were present in the plasma of DPD patients. Our results demonstrate that, under pathological conditions, the catabolism of valine can result in the production of significant amounts of β-AIB. Furthermore, the observation that the R-enantiomer of β-AIB is abundantly present in the urine of DPD patients suggests that significant cross-over exists between the thymine and valine catabolic pathways.

Branched chain amino acids (BCAAs), leucine, isoleucine and valine, are essential amino acids widely studied for their crucial role in the regulation of protein synthesis mainly through the activation of the mTOR signaling pathway and their emerging recognition as players in the regulation of various physiological and metabolic processes, such as glucose homeostasis. BCAA supplementation is primarily used as a beneficial nutritional intervention in chronic liver and kidney disease as well as in muscle wasting disorders. However, downregulated/upregulated plasma BCAAs and their defective catabolism in various tissues, mainly due to altered enzymatic activity of the first two enzymes in their catabolic pathway, BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase (BCKD), have been investigated in many nutritional and disease states. The current review focused on the underlying mechanisms of altered BCAA catabolism and its contribution to the pathogenesis of a numerous pathological conditions such as diabetes, heart failure and cancer. In addition, we summarize findings that indicate that the recovery of the dysregulated BCAA catabolism may be associated with an improved outcome and the prevention of serious disease complications.

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa causes a variety of infections in immunocompromised individuals, including individuals with the heritable disease cystic fibrosis. Like the carbon sources metabolized by many disease-causing bacteria, the carbon sources metabolized by P. aeruginosa at the host infection site are unknown. We recently reported that l-alanine is a preferred carbon source for P. aeruginosa and that two genes potentially involved in alanine catabolism (dadA and dadX) are induced during in vivo growth in the rat peritoneum and during in vitro growth in sputum (mucus) collected from the lungs of individuals with cystic fibrosis. The goals of this study were to characterize factors required for alanine catabolism in P. aeruginosa and to assess the importance of these factors for in vivo growth. Our results reveal that dadA and dadX are arranged in an operon and are required for catabolism of l-alanine. The dad operon is inducible by l-alanine, d-alanine, and l-valine, and induction is dependent on the transcriptional regulator Lrp. Finally, we show that a mutant unable to catabolize dl-alanine displays decreased competitiveness in a rat lung model of infection.

Ce travail contribue a l'étude de la régulation de la biosynthèse de la spiramycine, macrolide à 16 membres, chez Streptomyces Ambofaciens, par les sources azotées, carbonées et les phosphates. La valine favorise la production de spiramycine en fournissant des précurseurs de l'aglycone. En excès de valine, les ions ammonium libérés lors de son catabolisme font chuter la production. Ils inhibent et/ou répriment partiellement la valine déshydrogénase (vdh) et la cétoisovalerate déshydrogénase (civdh), deux enzymes du catabolisme de la valine. En présence de valine, les sources carbonées facilement assimilables (glycérol) ont un effet inhibiteur sur la production de spiramycine. Le glycérol interférerait avec le catabolisme de la valine par inhibition et/ou répression des systèmes d'activation ou d'assimilation de l'isobutyrate. Une corrélation entre une teneur intracellulaire élevée en atp, un excès de glycérol ou de phosphates et une suppression de la production de spiramycine a été observée. L'implication des métabolites phosphoryles en tant que médiateur commun aux deux effets négatifs phosphate et carbone a été suggérée. Les phosphates provoquent une réduction des biosynthèses de la vdh et des phosphatases alcalines. Grâce à des cultures semi-continues avec une alimentation azote/carbone contrôlée, la vitesse spécifique de production de spiramycine a été améliorée de douze fois. Suite à la mise en évidence d'une phase critique durant laquelle les différents substrats exercent leurs effets négatifs, une étude de la régulation de l'expression des protéines par les sources carbonée, azotée et les phosphates a été ébauchée par électrophorèse

Post-translational modifications constitute an important arm of the regulatory mechanism in mammalian cells. Therefore, bacterial pathogens, which thrive by mimicking the host cell signaling, are proving to be difficult to be tamed. Mycobacterium tuberculosis, for example, the morbid pathogen manipulates the host cellular pathways such as to block the host phagosomal maturation to avoid its killing as well as creates a favorable environment for its replication. NMPylation (transfer of nucleotide monophosphate; NMP from nucleotide triphosphate; NTP) mediated by Fic domain containing proteins has been an area of immense interest in the recent years and has been established as a stratagem of bacterial effectors to manipulate host cell signaling. This thesis entitled “Studies on NMPylase and valine catabolism enzymes from Mycobacterium” presents a study of Fic domain proteins from Mycobacterium sp. in pathogenesis as well as in bacterial dormancy. The work covers de novo motif identification based on sequence analysis of Fic protein from 22 Mycobacterium sp. and in vitro/ in vivo characterization of Fic domain proteins from Mycobacterium tuberculosis, Mycobacterium smegmatis and Mycobacterium marinum. The thesis begins with the literature survey on Fic mediated AMPylation. The “Chapter 1” commences with a brief introduction on the newly discovered PTM, “AMPylation” and its resemblance with “Adenylylation” which was discovered in 1968 by Stadtman in adenylylation mediate regulation of the reaction catalyzed by Glutamine synthase in E. coli. As the modification is mediated by Fic protein, belonging to Fic/Doc superfamily, the chapter further describes the characterization of Fic protein from E. coli where Fic (Filamentation induced by cAMP) name was coined for the first time. The chapter also describes the effect of Fic domain containing bacterial effectors from Vibrio parahaemolyticus, Histophilus somni, Legionella pneumophila and Bartonella sp. It also discusses in detail the proposed catalytic mechanism of AMPylation and other known PTMs mediated by Fic domain proteins. Further, it summarizes the structural features for substrate recognition and nucleotide binding. This is followed by a description of the regulation of Fic domain containing proteins. As another SxxxEG motif containing domain regulates the activity of Fic proteins, these together represent a new type II toxin-antitoxin module. Therefore, a classification based on the antitoxin motif has been detailed. Apart from having a defined role in perturbing host cell signaling, these proteins also modify targets in the bacteria itself, the biological relevance of the same has also been discussed in this chapter. Fic domain proteins are found not only in bacteria but are distributed ubiquitously in archaea and metazoans as well. Chapter 1 also covers the recent findings about the role of Fic proteins from archaea and metazoans. Chapter 1 further details the characteristic features of Mtb, briefly describing the infection cycle in the host, the pathogen-host interplay in the lung macrophages and the strategies elicited by Mtb to evade the host response with a specific emphasis on the Mtb mediated PTMs in infected macrophages. As set in the objectives, the principal aim was to understand the role of AMPylation in the pathogenesis of Mycobacterium. To this end, Chapter 2 covers a detailed analysis of Fic domain proteins across 22 Mycobacterium sp. where 71% of Fic domain proteins shared non-canonical Fic motif and 14% with canonical Fic motif and 15% with Doc motif. Further, the variability in the protein sequences was utilized for de novo identification of linear motifs. The putative functions these Fic domain proteins disrupt in their mammalian host were deciphered from the motifs they display to localize in a particular cellular compartment. De novo identification was supported by the functional characterization of the localization signal in two Fic domain proteins from M. smegmatis. This chapter identified a new class of TA module in Fic domain proteins from Mycobacterium, i.e, mobile mystery protein B and identified putative small XRE family transcriptional regulator protein, which may be the antitoxin counterpart as mobile mystery protein A. With the identification of a new set of putative regulators; TA system; multifunctional enzymes from mycobacterium, all equipped with a Fic domain, either canonical or non-canonical, we characterized Fic protein from Mtb, which is part of its core genome. Chapter 3 describes the role of AMPylation activity and identifies its cognate partner both in Mycobacterium and mammalian cells. The identified substrate from Mtb includes DNA gyrase subunit B. DNA gyrase is the only type II topoisomerase present in Mtb, which catalyzes negative supercoiling of DNA. AMPylation of GyrB by MtFic decreases its ATPase activity required for introduction of negative supercoiling in closed circular DNA. Further, this chapter identifies that apart from Gyrase B, there are multiple interacting partners of MtFic in the bacterial cell lysate. Further, the Chapter describes transient transfection of MtFic in HeLa and HEK293 cells result in cell rounding which eventually culminates in detachment and cell death. Subsequent investigation of sub-cellular localization of MtFic protein shows nuclear and ER lumen as the major sites of localization, which is corroborated by the identification of its interacting partners in nuclear and ER/membrane enriched fractions. A detailed characterization revealed Rac1, Cdc42, and vimentin as targets for MtFic in mammalian host cells. Altogether identification of multiple interacting partners in bacterial and mammalian host including GyraseB, Rac1, Cdc42 and vimentin, opens new avenues for further exploration of the role of AMPylation activity in Mycobacterium survival, dormancy, and pathogenicity. Fic motif residues bind with phosphates of NTPs, however, the residues from flap region and core helices support interactions with the base of NTPs and place NTP in an orientation suitable for NMPylation. As Fic proteins sequences curated from the Mycobacterium sp. showed variabilities in these regions, nucleotide selectivity exhibited by Fic proteins were explored next, Chapter 4 thus covers identification of a unique motif supporting selectivity for CTP over ATP. MtFic preferred AMPylation whereas Fic from M. smegmatis and M. marinum preferred CMPylation. The auto-modifications viz. auto-AMPylation and auto-CMPylation activity were also shown by the antitoxin mutants, however the M. marinum Fic protein, even with antitoxin domain showed auto-CMPylation. The chapter further explores regulation of Fic activity by antitoxin motif SxxxEG. Presence of arginine/asparagine at glycine position interferes with the inhibitory effect of glutamate, antagonizing the activity of antitoxin motif, thus resulting in enhanced toxin activity even in the presence of antitoxin. Moreover, this chapter also covers preliminary studies on residual pyrophosphatase activity exhibited by some CMPylating Fic enzymes. Chapter 5 covers characterization a Fic protein from M. marinum, with winged helix turn helix domain that showed DNA binding properties and a novel PTM, CMPylation, where CMP molecule gets transferred to the cognate substrates Gyrase A and Gyrase B subunits in bacteria and PCNA and Histone1 in the mammalian host. Additionally, this chapter identifies CMPylation, as a reversible PTM, which can be reversed with the same enzyme. CMPylation and de-CMPylation mediated by MmFic is highly regulated by glutamate present in the anti-toxin domain, thus MmFic protein is well-coordinated toxin-antitoxin system also. This chapter, hence, identifies and characterizes a novel TA system from Mycobacterium and identifies its targets as well. Moreover, this chapter also describes functional significance of auto-AMPylation in class I Fic proteins where auto-modification opens the N-terminus region thus enhancing the accessibility of substrate binding residues from the flap region. While these studies were in progress, functional characterization of some of the enzymes involved in valine catabolism, were also explored. In this context, it has been shown that addition of valine in the culture media inhibits the growth of Mycobacterium. Since the components of valine catabolism are critical for the survival and infection cycle of Mtb, it constitutes a potential drug target. In Chapter 6, detailed structural and functional characterizations of HIBADH enzyme catalyzing the sixth step in valine catabolism are described. MtHIBADH utilizes NAD+ as a cofactor and S-HIBA as the substrate. The locations of substrates in the active site region of HIBA dehydrogenases have been defined for the first time. Using a plethora of structural and biochemical techniques, the entry and active sites of the substrate have been unambiguously characterized. The chapter also describes a plausible reaction mechanism, inferred based on the structures and modeling. Further, studies highlight Cys210 as the critical cysteine residue for maintenance of the conformation of the active site of the enzyme. Further, the chapter identifies a novel FRET pair in NAD+ bound MtHIBADH enzyme, where tryptophan (Trp211) acts as a donor and NAD+ bound ({NAD}* acts as the acceptor. Chapter 7 briefly summarizes all the findings of the research carried out and presents an overview of our present understanding of the mycobacterial Fic proteins and HIBADH enzyme. Appendix A summarizes the preliminary results of MMSA and fadE9 enzymes involved in Mtb valine catabolism.

The acute phase response—trauma, tissue necrosis, infection, inflammation, and malignant neoplasia induce a complex series of nonspecific systemic, physiological, and metabolic responses including fever, leucocytosis, catabolism of muscle proteins, greatly increased de novo synthesis and secretion of a number of ‘acute phase’ plasma proteins, and decreased synthesis of albumin, transthyretin, and high- and low-density lipoproteins. The altered plasma protein concentration profile is called the acute phase response. All endothermic animals mount a similar response, suggesting that it may have survival value, and increased availability of proteinase inhibitors, complement, clotting, and transport proteins presumably enhances host resistance, minimizes tissue injury, and promotes regeneration and repair....

Fruit quality is determined by numerous genetic factors that affect taste, aroma, ‎color, texture, nutritional value and shelf life. To unravel the genetic components ‎involved in the metabolic pathways behind these traits, the major goal of the project was to identify novel genes that are involved in, or that regulate, these pathways using correlation analysis between genotype, metabolite and gene expression data. The original and specific research objectives were: (1) Collection of replicated fruit from a population of 96 RI lines derived from parents distinguished by great diversity in fruit development and quality phenotypes, (2) Phenotypic and metabolic profiling of mature fruit from all 96 RI lines and their parents, (3) 454 pyrosequencing of cDNA representing mRNA of mature fruit from each line to facilitate gene expression analysis based on relative EST abundance, (4) Development of a database modeled after an existing database developed for tomato introgression lines (ILs) to facilitate online data analysis by members of this project and by researchers around the world. The main functions of the database will be to store and present metabolite and gene expression data so that correlations can be drawn between variation in target traits or metabolites across the RI population members and variation in gene expression to identify candidate genes which may impact phenotypic and chemical traits of interest, (5) Selection of RI lines for segregation and/or hybridization (crosses) analysis to ascertain whether or not genes associated with traits through gene expression/metabolite correlation analysis are indeed contributors to said traits. The overall research strategy was to utilize an available recombinant inbred population of melon (Cucumis melo L.) derived from phenotypically diverse parents and for which over 800 molecular markers have been mapped for the association of metabolic trait and gene expression QTLs. Transcriptomic data were obtained by high throughput sequencing using the Illumina platform instead of the originally planned 454 platform. The change was due to the fast advancement and proven advantages of the Illumina platform, as explained in the first annual scientific report. Metabolic data were collected using both targeted (sugars, organic acids, carotenoids) and non-targeted metabolomics analysis methodologies. Genes whose expression patterns were associated with variation of particular metabolites or fruit quality traits represent candidates for the molecular mechanisms that underlie them. Candidate genes that may encode enzymes catalyzingbiosynthetic steps in the production of volatile compounds of interest, downstream catabolic processes of aromatic amino acids and regulatory genes were selected and are in the process of functional analyses. Several of these are genes represent unanticipated effectors of compound accumulation that could not be identified using traditional approaches. According to the original plan, the Cucurbit Genomics Network (http://www.icugi.org/), developed through an earlier BARD project (IS-3333-02), was expanded to serve as a public portal for the extensive metabolomics and transcriptomic data resulting from the current project. Importantly, this database was also expanded to include genomic and metabolomic resources of all the cucurbit crops, including genomes of cucumber and watermelon, EST collections, genetic maps, metabolite data and additional information. In addition, the database provides tools enabling researchers to identify genes, the expression patterns of which correlate with traits of interest. The project has significantly expanded the existing EST resource for melon and provides new molecular tools for marker-assisted selection. This information will be opened to the public by the end of 2013, upon the first publication describing the transcriptomic and metabolomics resources developed through the project. In addition, well-characterized RI lines are available to enable targeted breeding for genes of interest. Segregation of the RI lines for specific metabolites of interest has been shown, demonstrating the utility in these lines and our new molecular and metabolic data as a basis for selection targeting specific flavor, quality, nutritional and/or defensive compounds. To summarize, all the specific goals of the project have been achieved and in many cases exceeded. Large scale trascriptomic and metabolomic resources have been developed for melon and will soon become available to the community. The usefulness of these has been validated. A number of novel genes involved in fruit ripening have been selected and are currently being functionally analyzed. We thus fully addressed our obligations to the project. In our view, however, the potential value of the project outcomes as ultimately manifested may be far greater than originally anticipated. The resources developed and expanded under this project, and the tools created for using them will enable us, and others, to continue to employ resulting data and discoveries in future studies with benefits both in basic and applied agricultural - scientific research.