Reactions of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) with the tyrosine side-chain fragment, 4-ethylphenol

Abstract

The melatonin metabolite N¹-acetyl-5-methoxykynuramine (AMK) has previously been shown to interact with various free radicals. Using the ABTS cation radical [ABTS = 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)] as an electron abstracting reactant, which does not destroy the aromate, we found that the reactive intermediate derived from AMK strongly interacts with the benzene rings of other AMK molecules to form di- and oligomers. Since oligomerization is rather unlikely at physiological concentrations, we investigated reactions with other putative reaction partners. The incubation of tyrosine or several of its structural analogs with AMK in the presence of the ABTS cation radical led to numerous products, amongst which were compounds not detected when one of the educts was incubated with the ABTS cation radical alone. With tyrosine and most of its analogs, the number of products formed in the presence of AMK and ABTS cation radical was relatively high and included numerous oligomers. To optimize the yield of products of interest as well as their separation from other compounds, especially oligomers, we investigated the interaction with 4-ethylphenol, which represents the side chain of tyrosine lacking the carboxyl and amino residues of the amino acid, which otherwise can undergo additional reactions. A prominent product was chromatographically separated and analyzed by mass spectrometry [(+)-ESI-MS, (?)-ESI-MS, (+)-HRESI-MS], ¹H-NMR, and H,H-COSY correlations. The substance was identified as N-{3-[2′-(5″-ethyl-2″-hydroxyphenylamino)-5′-methoxyphenyl]-3-oxopropyl} acetamide. This chemically novel compound represents an adduct in which the amino nitrogen of AMK is attached to the C-2 atom of 4-ethylphenol, which corresponds to the C-3 atom in the benzene ring of tyrosine. This finding suggests that, upon interaction of AMK with an electron-abstracting radical, the kynuric intermediate may modify proteins at superficially accessible tyrosine residues. In fact, protein modification by an unidentified melatonin metabolite has been observed in an earlier study. The possibility of protein AMKylation may be of interest with regard to an eventual interference with tyrosine nitration or, more importantly, with tyrosine phosphorylation.     

Zap70 inhibits Syk‐mediated osteoclast function

The αvβ3 integrin stimulates the resorptive capacity of the differentiated osteoclast (OC) by organizing its cytoskeleton via the tyrosine kinase, Syk. Thus, Syk‐deficient OCs fails to spread or form actin rings, in vitro and in vivo. The Syk family of tyrosine kinases consists of Syk itself and Zap70 which are expressed by different cell types. Because of their structural similarity, and its compensatory properties in other cells, we asked if Zap70 can substitute for absence of Syk in OCs. While expression of Syk, as expected, normalizes the cytoskeletal abnormalities of Syk^?/? OCs, Zap70 fails do so. In keeping with this observation, Syk, but not Zap70, rescues αvβ3 integrin‐induced SLP76 phosphorylation in Syk^?/? OCs. Furthermore the kinase sequence of Syk partially rescues the Syk^?/? phenotype but full normalization also requires its SH2 domains. Surprisingly, expression of Zap70 inhibits WT OC spreading, actin ring formation and bone resorptive activity, but not differentiation. In keeping with arrested cytoskeletal organization, Zap70 blocks integrin‐activated endogenous Syk and Vav3, SLP76 phosphorylation. Such inhibition requires Zap70 kinase activity, as it is abolished by mutation of the Zap70 kinase domain. Thus, while the kinase domain of Syk is uniquely required for OC function that of Zap70 inhibits it. J. Cell. Biochem. 114: 1871–1878, 2013. © 2013 Wiley Periodicals, Inc.     

Methylation of the PTPRO gene in human hepatocellular carcinoma and identification of VCP as its substrate

We have previously reported that the gene encoding protein tyrosine phosphatase receptor type‐O (PTPRO) is suppressed by promoter methylation in a rat model of hepatocellular carcinoma (HCC) and it functions as tumor suppressor in leukemia and lung cancer. Here, we explored the methylation and expression of PTPRO as well as its function in human HCC. MassARRAY analysis of primary human HCC and matching liver samples (n = 24) revealed significantly higher (P = 0.004) methylation density at the promoter CGI in tumors. Combined bisulfite restriction analysis (COBRA) of another set of human HCC samples (n = 17) demonstrated that the CGI was methylated in 29% of tumors where expression of PTPRO was lower than that in corresponding matching livers. A substrate‐trapping mutant of PTPRO that stabilizes the bound substrates was used to identify its novel substrate(s). VCP/p97 was found to be a PTPRO substrate by mass spectrometry of the peptides pulled down by the substrate‐trapping mutant of PTPRO. Tyrosyl dephosphorylation of VCP following ectopic expression of wild‐type PTPRO in H293T and HepG2 cells confirmed that it is a bona fide substrate of PTPRO. Treatment of PTPRO overexpressing HepG2 cells with Doxorubicin, a DNA damaging drug commonly used in therapy of primary HCC, sensitized these cells to this potent anticancer drug that correlated with dephosphorylation of VCP. Taken together, these results demonstrate methylation and downregulation of PTPRO in a subset of primary human HCC and establish VCP as a novel functionally important substrate of this tyrosine phosphatase that could be a potential molecular target for HCC therapy. J. Cell. Biochem. 114: 1810–1818, 2013. © 2013 Wiley Periodicals, Inc.     

ACTH Modulates PTP‐PEST Activity and Promotes Its Interaction With Paxillin

Adrenocorticotropic hormone (ACTH) treatment has been proven to promote paxillin dephosphorylation and increase soluble protein tyrosine phosphatase (PTP) activity in rat adrenal zona fasciculata (ZF). Also, in‐gel PTP assays have shown the activation of a 115‐kDa PTP (PTP115) by ACTH. In this context, the current work presents evidence that PTP115 is PTP‐PEST, a PTP that recognizes paxillin as substrate. PTP115 was partially purified from rat adrenal ZF and PTP‐PEST was detected through Western blot in bioactive samples taken in each purification step. Immunohistochemical and RT‐PCR studies revealed PTP‐PEST expression in rat ZF and Y1 adrenocortical cells. Moreover, a PTP‐PEST siRNA decreased the expression of this phosphatase. PKA phosphorylation of purified PTP115 isolated from non‐ACTH‐treated rats increased K_M and V_M. Finally, in‐gel PTP assays of immunoprecipitated paxillin from control and ACTH‐treated rats suggested a hormone‐mediated increase in paxillin–PTP115 interaction, while PTP‐PEST and paxillin co‐localize in Y1 cells. Taken together, these data demonstrate PTP‐PEST expression in adrenal ZF and its regulation by ACTH/PKA and also suggest an ACTH‐induced PTP–PEST–paxillin interaction. J. Cell. Biochem. 117: 2170–2181, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

The Raf-like MAPKKKs STY8, STY17, and STY46 negatively regulate Botrytis cinerea resistance by limiting MKK7 protein accumulation in Arabidopsis

Plant diseases, which seriously damage crop production, are in most cases caused by fungal pathogens. In this study, we found that the Raf-like MAPKKKs STY8 (SERINE/THREONINE/TYROSINE KINASE 8), STY17, and STY46 negatively regulate resistance to the fungal pathogen Botrytis cinerea through jasmonate response in Arabidopsis. Moreover, STY8/STY17/STY46 homologs negatively contribute to chitin signaling. We further identified MKK7 as the MAPKK component interacting with STY8/STY17/STY46 homologs. MKK7 positively contributes to resistance to B. cinerea and chitin signaling. Furthermore, we found that STY8/STY17/STY46 homologs negatively affect the accumulation of MKK7, in accordance with the opposite roles of MKK7 and STY8/STY17/STY46 homologs in defense against B. cinerea. These results provide new insights into the mechanisms precisely regulating plant immunity via Raf-like MAPKKKs.     

Hydrogen peroxide inhibits activity of the IGF-1 receptor kinase

Abstract

IGF-1 receptor (IGF1R) is a transmembrane tyrosine kinase, which is indispensable for cellular growth and differentiation. Using a recombinant GST-tagged cytosolic fragment of IGF1R (GST-IGFK), we now show that oxidation by low doses (50 μM) of hydrogen peroxide markedly inhibits maximum phosphate incorporation in autophosphorylation and substrate phosphorylation assays. A similar inhibition was observed on the activity of intact IGF1R after treatment of T-47D cells. These results are in sharp contrast to the positive influence of hydrogen peroxide on the highly homologous insulin receptor kinase, which was assayed for comparison. This reciprocal influence of physiologically relevant doses of hydrogen peroxide may have important implications on signal transduction of the closely related receptors for insulin and IGF-1.     

On-line antioxidant activity determination: comparison of hydrophilic and lipophilic antioxidant activity using the ABTS•+ assay

Abstract

The ABTS/H₂O₂/HRP decoloration method is capable of determining both hydrophilic (in buffered media) and lipophilic (in organic media) antioxidant properties in complex samples. Now, we have adapted this method for on-line chromatographic determination. The easy, rapid and controlled generation of the ABTS radical and its great stability in buffered and organic media were important characteristics in the measurement of antioxidant activities. The HPLC-ABTS method used two pumps (one for isocratic eluting-phase and the other for preformed ABTS radical) and an UV-VIS diode array detector. The dual analysis of samples – conventional (with UV-VIS detection) and ABTS-scavenging (at 600 nm) – provided valuable on-line information about the correspondence between the presence of a determined compound and its possible antioxidant activity, and was applicable to both hydrophilic and lipophilic antioxidants (HAA and LAA). A comparison between HAA and LAA determined by the end-point method and by the on-line HPLC method is presented. The application to juices showed that both methods are suitable, sensitive and selective, gave similar values, and the HPLC-ABTS method contributed additional information about the antioxidant activity profile.     

Saitohin,which is nested within the tau gene,interacts with tau and Abl and its human‐specific allele influences Abl phosphorylation

Saitohin (STH) is a gene unique to humans and their closest relatives whose function is not yet known. STH contains a single polymorphism (Q7R); the Q allele is human‐specific and confers susceptibility to several neurodegenerative diseases. In previous work, we discovered that STH interacts with Peroxiredoxin 6 (Prdx6), a unique member of that family which is bifunctional and whose levels increase in Pick's disease. In this study, we report that STH also interacts with tau and the non‐receptor tyrosine kinase c‐Abl (Abl). Furthermore, Abl phosphorylates STH on its single tyrosine residue and STH increases tyrosine phosphorylation by Abl. The effect of Saitohin on Abl‐mediated phosphorylation appears to be allele‐specific, providing evidence for a new cellular function for STH. J. Cell. Biochem. 112: 3482–3488, 2011. © 2011 Wiley Periodicals, Inc.