Structural characterization, tissue distribution, and functional expression of murine aminoacylase III

Many xenobiotics are detoxified through the mercapturate metabolic pathway. The final product of the pathway, mercapturic acids ( N-acetylcysteine S-conjugates), are secreted predominantly by renal proximal tubules. Mercapturic acids may undergo a transformation mediated by aminoacylases and cysteine S-conjugate β-lyases that leads to nephrotoxic reactive thiol formation. The deacetylation of cysteine S-conjugates of N-acyl aromatic amino acids is thought to be mediated by an aminoacylase whose molecular identity has not been determined. In the present study, we cloned aminoacylase III, which likely mediates this process in vivo, and characterized its function and structure. The enzyme consists of 318 amino acids and has a molecular mass (determined by SDS-PAGE) of ∼35 kDa. Under nondenaturing conditions, the molecular mass of the enzyme is ∼140 kDa as determined by size-exclusion chromatography, which suggests that it is a tetramer. In agreement with this hypothesis, transmission electron microscopy and image analysis of aminoacylase III showed that the monomers of the enzyme are arranged with a fourfold rotational symmetry. Northern analysis demonstrated an ∼1.4-kb transcript that was expressed predominantly in kidney and showed less expression in liver, heart, small intestine, brain, lung, testis, and stomach. In kidney, aminoacylase III was immunolocalized predominantly to the apical domain of S1 proximal tubules and the cytoplasm of S2 and S3 proximal tubules. The data suggest that in kidney proximal tubules, aminoacylase III plays an important role in deacetylating mercapturic acids. The predominant cytoplasmic localization of aminoacylase III may explain the greater sensitivity of the proximal straight tubule to the nephrotoxicity of mercapturic acids.

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Effect of carcinogenic metabolites of aromatic amino acids on oxidative processes in lipids in vitro and in vivo

Bulletin of Experimental Biology and Medicine ◽

10.1007/bf00841912 ◽

1987 ◽

Vol 104 (1) ◽

pp. 968-970 ◽

Cited By ~ 3

Author(s):

A. A. Levchuk ◽

N. P. Pal'mina ◽

M. O. Raushenbakh

Keyword(s):

Amino Acids ◽

Aromatic Amino Acids ◽

Oxidative Processes

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In vivo cascade catalysis of aromatic amino acids to the respective mandelic acids using recombinant E. coli cells expressing hydroxymandelate synthase (HMS) from Amycolatopsis mediterranei

Molecular Catalysis ◽

10.1016/j.mcat.2019.110713 ◽

2020 ◽

Vol 483 ◽

pp. 110713 ◽

Cited By ~ 1

Author(s):

Jung-Won Youn ◽

Christoph Albermann ◽

Georg A. Sprenger

Keyword(s):

Amino Acids ◽

Aromatic Amino Acids ◽

Amycolatopsis Mediterranei ◽

E Coli ◽

Cascade Catalysis

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Structure/Function Analysis of the Vaccinia Virus F18 Phosphoprotein, an Abundant Core Component Required for Virion Maturation and Infectivity

Journal of Virology ◽

10.1128/jvi.00399-10 ◽

2010 ◽

Vol 84 (13) ◽

pp. 6846-6860 ◽

Cited By ~ 15

Author(s):

Nadi T. Wickramasekera ◽

Paula Traktman

Keyword(s):

Amino Acids ◽

Structure Function ◽

Protein Interactions ◽

Function Analysis ◽

Aromatic Amino Acids ◽

Protein Protein Interactions ◽

Core Component ◽

Virion Morphogenesis

ABSTRACT Poxvirus virions, whose outer membrane surrounds two lateral bodies and a core, contain at least 70 different proteins. The F18 phosphoprotein is one of the most abundant core components and is essential for the assembly of mature virions. We report here the results of a structure/function analysis in which the role of conserved cysteine residues, clusters of charged amino acids and clusters of hydrophobic/aromatic amino acids have been assessed. Taking advantage of a recombinant virus in which F18 expression is IPTG (isopropyl-β-d-thiogalactopyranoside) dependent, we developed a transient complementation assay to evaluate the ability of mutant alleles of F18 to support virion morphogenesis and/or to restore the production of infectious virus. We have also examined protein-protein interactions, comparing the ability of mutant and WT F18 proteins to interact with WT F18 and to interact with the viral A30 protein, another essential core component. We show that F18 associates with an A30-containing multiprotein complex in vivo in a manner that depends upon clusters of hydrophobic/aromatic residues in the N′ terminus of the F18 protein but that it is not required for the assembly of this complex. Finally, we confirmed that two PSSP motifs within F18 are the sites of phosphorylation by cellular proline-directed kinases in vitro and in vivo. Mutation of both of these phosphorylation sites has no apparent impact on virion morphogenesis but leads to the assembly of virions with significantly reduced infectivity.

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The biosynthesis of GDP-d-arabinopyranose in Crithidia fasciculata: characterization of a d-arabino-1-kinase activity and its use in the synthesis of GDP-[5-3H]d-arabinopyranose

Biochemical Journal ◽

10.1042/bj3110307 ◽

1995 ◽

Vol 311 (1) ◽

pp. 307-315 ◽

Cited By ~ 6

Author(s):

P Schneider ◽

A Nikolaev ◽

M A Ferguson

Keyword(s):

Molecular Mass ◽

Kinase Activity ◽

Substrate Inhibition ◽

Size Exclusion ◽

Crithidia Fasciculata ◽

Optimum Ph ◽

Exclusion Chromatography ◽

Trypanosomatid Parasites

GDP-D-arabinopyranose (GDP-D-Ara) is the precursor of the uncommon D-arabinopyranose residues present in the glycoconjugates of a few trypanosomatid parasites. Biosynthetic labelling experiments with Crithidia fasciculata showed that GDP-D-Ara could be labelled with [3H]D-Ara, [2-3H]D-Glc and [6-3H]D-Glc, but not with [1-3H]D-Glc, suggesting that D-Ara can be either taken up directly by the parasite or derived from D-Glc through a pathway involving the loss of carbon C-1. In vivo pulse-chase experiments indicated that D-Ara was sequentially incorporated into D-Ara-1-PO4 and GDP-D-Ara prior to transfer to the acceptor glycoconjugate, lipoarabinogalactan. An MgATP-dependent D-arabino-1-kinase activity present in soluble extracts of C. fasciculata was purified away from phosphatase activities by size-exclusion chromatography. The D-arabino-1-kinase had an apparent molecular mass of 600 kDa, a neutral optimum pH, and displayed substrate inhibition at D-Ara concentrations above 100 microM. It had a KmATP of 1.7 mM and a KmAra of 24 microM. Competition studies indicated that the orientation of every single hydroxyl residue was important for D-Ara recognition by the enzyme, but that methyl or hydroxymethyl groups could be tolerated as equatorial substituents on C-5 of D-Ara. The partially purified D-arabino-1-kinase activity was used in the chemico-enzymic synthesis of GDP-[5-3H]D-Ara from [6-3H]D-GlcN.

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Fluxes and membrane transport of amino acids in rat liver under different protein diets

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1990.259.5.e614 ◽

1990 ◽

Vol 259 (5) ◽

pp. E614-E625 ◽

Cited By ~ 6

Author(s):

P. Fafournoux ◽

C. Remesy ◽

C. Demigne

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Aromatic Amino Acids ◽

High Protein Diet ◽

High Protein ◽

Protein Diet ◽

Transport Systems ◽

Low Concentrations ◽

Significant Uptake

The aim of the present work was to evaluate in vivo the role of the transport step in hepatic amino acid metabolism. To vary hepatic utilization of amino acids, rats were adapted to diets containing various concentrations of casein (5, 15, and 60%). In rats fed 5 or 15% casein diets, Gln and Glu were released by the liver, and there was a significant uptake of Ala. Hepatic fluxes of amino acids increased considerably after adaptation to high-casein diet (up to 1.55 mumol.min-1.g liver-1 for Ala), because of the rise in afferent concentrations as well as enhanced uptake percentage (peaking at 60–75% for most glucogenic amino acids). Adaptation to a high-protein diet led to induction of not only system A but also of most of the other transport systems (Gly, anionic, T, y+, and to a lesser extent system N); only systems ASC and L were unchanged. The study of amino acid repartition between liver and plasma with different diets indicates that transport could modulate utilization of Ala, Ser, Thr, Gly, Gln, and Asp. For Arg and Asn, present in very low concentrations in liver under any condition, the transport step should be the major locus of control of their metabolism. For amino acids chiefly transported by nonconcentrative systems, such as aromatic amino acids, cellular metabolism could also be limited by the transport process. In conclusion, during adaptation to a high-protein diet, there is apparently a coordinated adaptation of amino acid transport and of their intracellular metabolism. For some amino acids, induction of catabolic enzymes seems greater than that of transport, so that the transport step may play an important role in control of metabolic fluxes. For example, concentration of amino acids such as Thr may be markedly depressed in rats adapted to a high-protein diet.

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A host-gut microbial co-metabolite of aromatic amino acids, p-cresol glucuronide, promotes blood-brain barrier integrity in vivo

10.1101/2022.01.11.475932 ◽

2022 ◽

Author(s):

Andrew V Stachulski ◽

Tobias B-A Knausenberger ◽

Sita N Shah ◽

Lesley Hoyles ◽

Simon McArthur

Keyword(s):

Amino Acids ◽

Blood Brain Barrier ◽

Aromatic Amino Acids ◽

Biologically Active ◽

Brain Barrier ◽

Whole Brain ◽

Blood Brain ◽

Bbb Permeability

Purpose: The sequential activity of gut microbial and host processes can exert a powerful modulatory influence on dietary components, as exemplified by the metabolism of the amino acids tyrosine and phenylalanine to p-cresol by gut microbes, and then to p-cresol glucuronide (pCG) by host enzymes. Although such glucuronide conjugates are classically thought to be biologically inert, there is accumulating evidence that this may not always be the case. We investigated the activity of pCG, studying its interactions with the cerebral vasculature and the brain in vitro and in vivo. Methods: Male C57Bl/6J mice were used to assess blood-brain barrier (BBB) permeability and whole brain transcriptomic changes in response to pCG treatment. Effects were then further explored using the human cerebromicrovascular endothelial cell line hCMEC/D3, assessing paracellular permeability, transendothelial electrical resistance and barrier protein expression. Results: Mice exposed to pCG showed reduced BBB permeability and significant changes in whole brain transcriptome expression. Surprisingly, treatment of hCMEC/D3 cells with pCG had no notable effects until co-administered with bacterial lipopolysaccharide, at which point it was able to prevent the permeabilising effects of endotoxin. Further analysis suggested that pCG acts as an antagonist at the principal lipopolysaccharide receptor TLR4. Conclusion: The amino acid phase II metabolic product pCG is biologically active at the BBB, highlighting the complexity of gut microbe to host communication and the gut-brain axis.

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Solubility Parameters of Amino Acids on Liquid–Liquid Phase Separation and Aggregation of Proteins

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.691052 ◽

2021 ◽

Vol 9 ◽

Author(s):

Akira Nomoto ◽

Suguru Nishinami ◽

Kentaro Shiraki

Keyword(s):

Amino Acids ◽

Phase Separation ◽

Amino Acid ◽

Liquid Phase ◽

Aromatic Amino Acids ◽

Liquid Phase Separation ◽

Solubility Parameters ◽

Liquid Liquid Phase Separation

The solution properties of amino acids determine the folding, aggregation, and liquid–liquid phase separation (LLPS) behaviors of proteins. Various indices of amino acids, such as solubility, hydropathy, and conformational parameter, describe the behaviors of protein folding and solubility both in vitro and in vivo. However, understanding the propensity of LLPS and aggregation is difficult due to the multiple interactions among different amino acids. Here, the solubilities of aromatic amino acids (SAs) were investigated in solution containing 20 types of amino acids as amino acid solvents. The parameters of SAs in amino acid solvents (PSASs) were varied and dependent on the type of the solvent. Specifically, Tyr and Trp had the highest positive values while Glu and Asp had the lowest. The PSAS values represent soluble and insoluble interactions, which collectively are the driving force underlying the formation of droplets and aggregates. Interestingly, the PSAS of a soluble solvent reflected the affinity between amino acids and aromatic rings, while that of an insoluble solvent reflected the affinity between amino acids and water. These findings suggest that the PSAS can distinguish amino acids that contribute to droplet and aggregate formation, and provide a deeper understanding of LLPS and aggregation of proteins.

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Altered Oligomerization Properties of N316 Mutants of Escherichia coli TyrR

Journal of Bacteriology ◽

10.1128/jb.00889-08 ◽

2008 ◽

Vol 190 (24) ◽

pp. 8238-8243 ◽

Cited By ~ 7

Author(s):

Takashi Koyanagi ◽

Takane Katayama ◽

Hideyuki Suzuki ◽

Hidehiko Kumagai

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Amino Acids ◽

Gel Filtration ◽

Aromatic Amino Acids ◽

Transcriptional Regulator ◽

Wild Type ◽

Oligomer Formation ◽

Regulatory Properties

ABSTRACT The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor of Escherichia coli TyrRE274Q, a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for TyrRN316D and TyrRN316R mutants. Gel filtration analyses revealed that the ligand-mediated oligomer formation was enhanced and diminished for TyrRN316D and TyrRN316R, respectively, compared with the wild-type TyrR. ADP was substituted for ATP in the oligomer formation of TyrRN316D.

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