Calreticulin transacetylase mediates the acetylation of nitric oxide synthase by polyphenolic acetate
article
Our earlier investigations identified acetoxy drug: protein transacetylase (TAase), a unique enzyme in the endoplasmic reticulum (ER) catalyzing the transfer of acetyl groups from polyphenolic acetates (PA) to certain functional proteins. Recently we have established the identity of TAase with ER protein calreticulin (CR) and subsequently transacetylase function of CR was termed calreticulin transacetylase (CRTAase). CRTAase was purified and characterized from human placenta. CRTAase catalyzed the acetylation of a receptor protein nNOS, by a model PA 7, 8-diacetoxy-4-methylcoumarin (DAMC), which was visually confirmed by using antiacetyl lysine. The aim of this report was to provide tacit proof by providing mass spectrometry evidence for CRTAase catalyzed acetylation of purified nNOS by DAMC. For this purpose, purified nNOS was incubated with DAMC and CRTAase, the modified nNOS was analyzed by nanoscale LC-MS/MS, which recorded 11 distinct peptides with a significant score as acetylated on lysine residues. The distribution was in order: lysines-24, -33, -38, -131, and -229 of the PDZ domain, Lys-245 of the oxygenase domain, Lys-754 and -856 of FMN binding domain, Lys-989 of connecting domain and Lys-1300, -1321, and -1371 of the NADPH-binding domain were acetylated. The results documented in this paper highlighted for the first time modification of nNOS by way of acetylation. Our earlier work recorded the profound activation of platelet NADPH cytochrome P-450 reductase and the acetylation of the reductase protein by DAMC, which also remarkably enhanced intracellular levels of nitric oxide. The results reported here coupled with the aforementioned previous observations strongly implicate the possible role of the acetylation of the reductase domain of nitric oxide synthase (NOS) in the NOS activation. In addition, the acetylation of nNOS can be expected to potentiate the interaction with CR, eventually leading to the augmented catalytic activity of NOS and expression of the related biological effects. © 2007 Humana Press Inc.
Topics
CalreticulinNitric oxide synthasePolyphenolic acetatesProtein acetylationTransacetylaseAcetylationBinding sitesCell membranesEnzyme activityProteinsCalreticulinPolyphenolic acetatesProtein acetylationTransacetylaseNitric oxide7,8 diacetoxy 4 methylcoumarinacyltransferasecalreticulincalreticulin transacetylasecoumarin derivativelysinenitric oxide synthasepolyphenol acetatepolyphenol derivativereduced nicotinamide adenine dinucleotide phosphateacetylationamino acid sequencearticlebinding affinitycatalysiscontrolled studyenzyme purificationhumanhuman tissuemass spectrometryprotein domainAcetatesAcetylationAcetyltransferasesAmino Acid SequenceAmino-Acid N-AcetyltransferaseCalcium-Binding Protein, Vitamin D-DependentChromatography, LiquidFemaleFlavonoidsHumansLysineMolecular Sequence DataNanotechnologyNitric Oxide Synthase Type IPeptidesPhenolsPlacentaPregnancyTandem Mass Spectrometry
TNO Identifier
240604
ISSN
02732289
Source
Applied Biochemistry and Biotechnology, 144(1), pp. 37-45.
Pages
37-45
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