Publications

• Mouse aminoacylase 3: a metalloenzyme activated by cobalt and nickel.

  Tsirulnikov K., Abuladze N., Newman D., Ryazantsev S., Wolak T., Magilnick N., Koag M.C., Kurtz I., Pushkin A.

  Aminoacylase 3 (AA3) deacetylates N-acetyl-aromatic amino acids and mercapturic acids including N-acetyl-1,2-dichlorovinyl-L-cysteine (Ac-DCVC), a metabolite of a xenobiotic trichloroethylene. Previous studies did not demonstrate metal-dependence of AA3 despite a high homology with a Zn(2+)-metall ... >> More

  Aminoacylase 3 (AA3) deacetylates N-acetyl-aromatic amino acids and mercapturic acids including N-acetyl-1,2-dichlorovinyl-L-cysteine (Ac-DCVC), a metabolite of a xenobiotic trichloroethylene. Previous studies did not demonstrate metal-dependence of AA3 despite a high homology with a Zn(2+)-metalloenzyme aminoacylase 2 (AA2). A 3D model of mouse AA3 was created based on homology with AA2. The model showed a putative metal binding site formed by His21, Glu24 and His116, and Arg63, Asp68, Asn70, Arg71, Glu177 and Tyr287 potentially involved in catalysis/substrate binding. The mutation of each of these residues to alanine inactivated AA3 except Asn70 and Arg71, therefore the corrected 3D model of mouse AA3 was created. Wild type (wt) mouse AA3 expressed in E. coli contained approximately 0.35 zinc atoms per monomer. Incubation with Co(2+) and Ni(2+) activated wt-AA3. In the cobalt-activated AA3 zinc was replaced with cobalt. Metal removal completely inactivated wt-AA3, whereas addition of Zn(2+), Mn(2+) or Fe(2+) restored initial activity. Co(2+) and to a lesser extent Ni(2+) increased activity several times in comparison with intact wt-AA3. Co(2+) drastically increased the rate of deacetylation of Ac-DCVC and significantly increased the toxicity of Ac-DCVC in the HEK293T cells expressing wt-AA3. The results indicate that AA3 is a metalloenzyme significantly activated by Co(2+) and Ni(2+). << Less

  Biochim Biophys Acta 1794:1049-1057(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Specificity of aminoacylase III-mediated deacetylation of mercapturic acids.

  Newman D., Abuladze N., Scholz K., Dekant W., Tsuprun V., Ryazantsev S., Bondar G., Sassani P., Kurtz I., Pushkin A.

  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, m ... >> More

  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 beta-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 beta-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. << Less

  Drug Metab. Dispos. 35:43-50(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structures of aminoacylase 3 in complex with acetylated substrates.

  Hsieh J.M., Tsirulnikov K., Sawaya M.R., Magilnick N., Abuladze N., Kurtz I., Abramson J., Pushkin A.

  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 ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 107:17962-17967(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Pushkin A., Carpenito G., Abuladze N., Newman D., Tsuprun V., Ryazantsev S., Motemoturu S., Sassani P., Solovieva N., Dukkipati R., Kurtz I.

  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 cystei ... >> More

  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 beta-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 approximately 35 kDa. Under nondenaturing conditions, the molecular mass of the enzyme is approximately 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 approximately 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. << Less

  Am. J. Physiol. 286:C848-C856(2004) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Inhibition of aminoacylase 3 protects rat brain cortex neuronal cells from the toxicity of 4-hydroxy-2-nonenal mercapturate and 4-hydroxy-2-nonenal.

  Tsirulnikov K., Abuladze N., Bragin A., Faull K., Cascio D., Damoiseaux R., Schibler M.J., Pushkin A.

  4-Hydroxy-2-nonenal (4HNE) and acrolein (ACR) are highly reactive neurotoxic products of lipid peroxidation that are implicated in the pathogenesis and progression of Alzheimer's and Parkinson's diseases. Conjugation with glutathione (GSH) initiates the 4HNE and ACR detoxification pathway, which g ... >> More

  4-Hydroxy-2-nonenal (4HNE) and acrolein (ACR) are highly reactive neurotoxic products of lipid peroxidation that are implicated in the pathogenesis and progression of Alzheimer's and Parkinson's diseases. Conjugation with glutathione (GSH) initiates the 4HNE and ACR detoxification pathway, which generates the mercapturates of 4HNE and ACR that can be excreted. Prior work has shown that the efficiency of the GSH-dependent renal detoxification of haloalkene derived mercapturates is significantly decreased upon their deacetylation because of rapid transformation of the deacetylated products into toxic compounds mediated by β-lyase. The enzymes of the GSH-conjugation pathway and β-lyases are expressed in the brain, and we hypothesized that a similar toxicity mechanism may be initiated in the brain by the deacetylation of 4HNE- and ACR-mercapturate. The present study was performed to identify an enzyme(s) involved in 4HNE- and ACR-mercapturate deacetylation, characterize the brain expression of this enzyme and determine whether its inhibition decreases 4HNE and 4HNE-mercapturate neurotoxicity. We demonstrated that of two candidate deacetylases, aminoacylases 1 (AA1) and 3 (AA3), only AA3 efficiently deacetylates both 4HNE- and ACR-mercapturate. AA3 was further localized to neurons and blood vessels. Using a small molecule screen we generated high-affinity AA3 inhibitors. Two of them completely protected rat brain cortex neurons expressing AA3 from the toxicity of 4HNE-mercapturate. 4HNE-cysteine (4HNE-Cys) was also neurotoxic and its toxicity was mostly prevented by a β-lyase inhibitor, aminooxyacetate. The results suggest that the AA3 mediated deacetylation of 4HNE-mercapturate may be involved in the neurotoxicity of 4HNE. << Less

  Toxicol Appl Pharmacol 263:303-314(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.