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

Pushkin, Alexander; Carpenito, Gerardo; Abuladze, Natalia; Newman, Debra K.; Tsuprun, Vladimir; Ryazantsev, Sergey; Motemoturu, Srilakshmi; Sassani, Patrick; Solovieva, N.A.; Dukkipati, Ramnath; Kurtz, Ira

doi:10.1152/ajpcell.00192.2003

Cited by 65 publications

(73 citation statements)

References 39 publications

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“…Mouse AAIII was expressed in human embryonic kidney HEK293T cells as a His 6 -tagged fusion protein using a pcDNA3.1/His vector (Invitrogen, Carlsbad, CA). The AAIII purification was performed as we described before (Pushkin et al, 2004), with minor modifications: ion-exchange chromatography on DEAE-cellulose DE52 (Whatman, Maidstone, Kent, UK) was used before Ni-Superflow resin (Novagen, Madison, WI) chromatography. The purified His 6 -tagged AAIII protein was homogeneous upon polyacrylamide gel electrophoresis (PAGE) in the presence of SDS, and Western blotting with both a mouse kidney AAIIIspecific MR-C1 antibody (Pushkin et al, 2004) and a penta-His antibody (QIAGEN, Valencia, CA).…”

Section: Methodsmentioning

confidence: 99%

“…The molecular mass of this enzyme was ϳ55 kDa (Endo, 1978;Uttamsingh and Anders, 1999), whereas a similar enzyme from rat liver was a ϳ145-kDa protein (Suzuki and Tateishi, 1981). The molecular nature of AAIII was not completely characterized until the recent cloning of mouse AAIII (Pushkin et al, 2004). The molecular mass of mouse AAIII expressed in HEK293T cells was ϳ140 kDa, and the enzyme was shown to be homotetramer (Pushkin et al, 2004).…”

Section: S-alkyl Derivatives Of N-acetyl-l-cysteine Including N-acetymentioning

confidence: 99%

“…The molecular nature of AAIII was not completely characterized until the recent cloning of mouse AAIII (Pushkin et al, 2004). The molecular mass of mouse AAIII expressed in HEK293T cells was ϳ140 kDa, and the enzyme was shown to be homotetramer (Pushkin et al, 2004). It is not known whether rat kidney AAIII is a dimer, or whether the AAIII dimer and AAIII tetramer kinetics are different.…”

Section: S-alkyl Derivatives Of N-acetyl-l-cysteine Including N-acetymentioning

confidence: 99%

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Specificity of Aminoacylase III-Mediated Deacetylation of Mercapturic Acids

et al. 2006

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ABSTRACT:Trichloroethylene (TCE) and other halogenated alkenes are known environmental contaminants with cytotoxic and nephrotoxic effects, and are potential carcinogens. Their metabolism via the mercapturate metabolic pathway was shown to lead to their detoxification. The final products of this pathway, mercapturic acids or N-acetyl-L-cysteine S-conjugates, are secreted into the lumen in the renal proximal tubule. The proximal tubule may also deacetylate mercapturic acids, and the resulting cysteine S-conjugates are transformed by cysteine S-conjugate ␤-lyases to nephrotoxic reactive thiols. The specificity and rate of mercapturic acid deacetylation may determine the toxicity of certain mercapturic acids; however, the exact enzymologic processes involved are not known in detail. In the present study we characterized the kinetics of the recently cloned mouse aminoacylase III (AAIII) toward a wide spectrum of halogenated mercapturic acids and N-acetylated amino acids. In general, the V max value of AAIII was significantly larger with chlorinated and brominated mercapturic acids, whereas fluorination significantly decreased it. The enzyme deacetylated mercapturic acids derived from the TCE metabolism including N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NA-1,2-DCVC) and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine (NA-2,2-DCVC). Both mercapturic acids induced cytotoxicity in mouse proximal tubule mPCT cells expressing AAIII, which was decreased by an inhibitor of ␤-lyase, aminooxyacetate. The toxic effect of NA-2,2-DCVC was smaller than that of NA-1,2-DCVC, indicating that factors other than the intracellular activity of AAIII mediate the cytotoxicity of these mercapturic acids. Our results indicate that in proximal tubule cells, AAIII plays an important role in deacetylating several halogenated mercapturic acids, and this process may be involved in their cyto-and nephrotoxicity.Trichloroethylene (TCE) and other industrial solvents are shown to cause nephrotoxicity and hepatotoxicity, and are probable human carcinogens (Maltoni et al., 1988;Cummings and Lash, 2000). One of the TCE toxicity mechanisms involves the conjugation with glutathione, which is transformed by ␥-glutamyltransferase and dipeptidase to cysteine S-conjugates Berrnauer et al., 1996;Anders and Dekant, 1998). The formation of cysteine S-conjugates takes place mainly in liver and, to some extent, also in kidney. Cysteine S-conjugates may be acetylated in the liver and kidney into corresponding N-acetyl S-conjugates (mercapturic acids) and transported from the liver into the kidney. In the kidney proximal tubules, mercapturic acids may be secreted or deacetylated by aminoacylases into cysteine S-conjugates Dekant, 1994, 1998). The deacetylation reaction may play an important role in the renal nephrotoxicity since cysteine S-conjugates, but not mercapturates, are transformed by cysteine conjugate ␤-lyases into toxic metabolites (Hayden and Stevens, 1990;Commandeur et al., 1995). Cysteine S-conjugates also may be transformed by flavoprotein monooxygenase...

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Section: Methodsmentioning

confidence: 99%

Section: S-alkyl Derivatives Of N-acetyl-l-cysteine Including N-acetymentioning

confidence: 99%

Section: S-alkyl Derivatives Of N-acetyl-l-cysteine Including N-acetymentioning

confidence: 99%

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Specificity of Aminoacylase III-Mediated Deacetylation of Mercapturic Acids

et al. 2006

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“…There are three types of aminoacylases: (i) aminoacylase 1 (AA1) deacetylates neutral aliphatic N-acyl-α-amino acids and mercapturic acids; (ii) aminoacylase 2 or aspartoacylase (AA2) has a strict specificity for N α -acetyl-L-aspartate (NAD); and (iii) aminoacylase 3 (AA3) preferentially deacetylates N α -acetylated aromatic amino acids and mercapturic acids that are usually not deacetylated by AA1 (1)(2)(3)(4)(5)(6). Despite different substrate specificities, AA2 and AA3 have a high degree of sequence (42% identity) and structure homology but are both substantially different from AA1 (∼10% of sequence identity) (2,(6)(7)(8)(9)(10).…”

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confidence: 99%

Structures of aminoacylase 3 in complex with acetylated substrates

Hsieh

Tsirulnikov²,

Sawaya³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Trichloroethylene (TCE) is one of the most widespread environmental contaminants, which is metabolized to N-acetyl-S-1,2-dichlorovinyl-L-cysteine (NA-DCVC) before being excreted in the urine. Alternatively, NA-DCVC can be deacetylated by aminoacylase 3 (AA3), an enzyme that is highly expressed in the kidney, liver, and brain. NA-DCVC deacetylation initiates the transformation into toxic products that ultimately causes acute renal failure. AA3 inhibition is therefore a target of interest to prevent TCE induced nephrotoxicity. Here we report the crystal structure of recombinant mouse AA3 (mAA3) in the presence of its acetate byproduct and two substrates: N α -acetyl-L-tyrosine and NA-DCVC. These structures, in conjunction with biochemical data, indicated that AA3 mediates substrate specificity through van der Waals interactions providing a dynamic interaction interface, which facilitates a diverse range of substrates.mercapturates | metalloprotein | X-ray structure A minoacylase 3 (AA3) is a member of the aminoacylase family of enzymes that deacylates a broad range of substrates including both N α -acetylated amino acids and S-cysteine conjugates of N-acetyl-L-cysteine (mercapturic acids) ( Fig. 1) (1). There are three types of aminoacylases: (i) aminoacylase 1 (AA1) deacetylates neutral aliphatic N-acyl-α-amino acids and mercapturic acids; (ii) aminoacylase 2 or aspartoacylase (AA2) has a strict specificity for N α -acetyl-L-aspartate (NAD); and (iii) aminoacylase 3 (AA3) preferentially deacetylates N α -acetylated aromatic amino acids and mercapturic acids that are usually not deacetylated by AA1 (1-6). Despite different substrate specificities, AA2 and AA3 have a high degree of sequence (42% identity) and structure homology but are both substantially different from AA1 (∼10% of sequence identity) (2, 6-10).AA3 is of particular interest for human health because it participates in mediating toxicity of the xenobiotic trichloroethylene (TCE). The United States produces in excess of 130,000 tons of TCE per year (11), making it the most widespread chemical contaminant in both soil and ground water. TCE is readily absorbed into the body where it can enter the glutathione conjugation detoxification pathway producing the mercapturic acid N-acetyl-S-1,2-dichlorovinyl-L-cysteine (NA-DCVC) for subsequent urinary excretion (12, 13). However, NA-DCVC can be deacetylated by AA3-which is highly expressed in the renal proximal tubule, liver, and brain-to generate S-1,2-dichlorovinyl-L-cysteine (DCVC) (2) and further transformed via β-lyases or flavin monooxygenases into lethal products capable of causing acute renal failure and toxicity to the liver and brain. (4, 13-22). Thus, inhibition of AA3 can decrease DCVC formation and ameliorate TCE toxicity, presenting a potential target for drug discovery.Generating specific inhibitors for AA3 would be greatly aided by high-resolution structural data, but structural studies of aminoacylases have been limited to AA1 (7) and AA2 (8, 9), which differ in both overall architecture and ac...

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“…27 This study further propose and support the hypothesis that b-lactamase production is an important in vivo resistance mechanism in infected patients which could subsequently lead to more severe inflammation and scaring formation. Aspartoacylase-3 reported to localize to the cytoplasm of S2 and S3 proximal tubules and to the apical domain of S1 proximal tubules 28 and may function as an hepatitis C virus (HCV) core binding protein which may play a role in the development of HCV-associated diseases. 29 To our interest, aspartoacylase-3 was identified in the kidney tissues from proteomics investigations on aristolochic acid nephropathy (AAN): a case study on rat kidney tissues.…”

Section: Discussionmentioning

confidence: 99%

Potential biomarkers associated with diabetic glomerulopathy through proteomics

Hsu

Lei

et al. 2015

Renal Failure

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Diabetic nephropathy (DN) is characterized by the development of progressive glomerulosclerotic lesions gradually leading to an increasing loss of functioning kidney parenchyma. Relatively little proteomic research of isolated glomeruli of experimental animal models has been done so far. Isolated glomerular proteomics is an innovative tool that potentially detects simultaneous expressions of glomeruli in diabetic pathological contexts. We compared the isolated glomerular profiles of rats with and without diabetes. The proteins in the aliquots of glomeruli were subjected to two-dimensional gel electrophoresis. The protein spots were matched and quantified using an imaging analysis system. The peptide mass fingerprints were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and a bioinformation search. We found that diabetes increased collagen type I and collagen type IV levels in diabetic glomeruli when compared to normal control group using Dynabeads. We found that rats with diabetes had significantly higher abundance of the Protein disulfide isomerase associated 3, Aspartoacylase-3,3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase, Lactamase beta 2 and Agmat protein. However, diabetic glomeruli in rats had significantly lower levels of the Regucalcin, rCG52140, Aldo-keto reductase family 1, Peroxiredoxin 1, and L-arginine: glycine amidinotransferase. These proteins of interest were reported to modulate disturbances in the homeostasis of endoplasmic reticulum stress, disturbance of inflammatory and fibrinogenic activities, impairing endothelial function, and dysregulation in the antioxidation capacity/oxidative stress in several tissue types under pathological contexts. Taken together, our high-throughput isolated glomerular proteomic findings indicated that multiple pathological reactions presumably occurred in DN.

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

Cited by 65 publications

References 39 publications

Specificity of Aminoacylase III-Mediated Deacetylation of Mercapturic Acids

Specificity of Aminoacylase III-Mediated Deacetylation of Mercapturic Acids

Structures of aminoacylase 3 in complex with acetylated substrates

Potential biomarkers associated with diabetic glomerulopathy through proteomics

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