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      Identification of Proteins Differentially Expressed in the Striatum by Melatonin in a Middle Cerebral Artery Occlusion Rat Model—a Proteomic and in silico Approach

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          Abstract

          Ischemic stroke is characterized by permanent or transient obstruction of blood flow, which initiates a cascading pathological process, starting from acute ATP loss to subsequent membrane depolarization, glutamate excitotoxicity, and calcium overload. Melatonin is a potent antioxidant that exerts protective effects in different experimental stroke models. In this study, melatonin effects were demonstrated by a proteomic and in silico approach. The proteomic study identified differentially expressed proteins by 2D gel electrophoresis in the striatum 24 h after middle cerebral artery occlusion. Proteomic analysis revealed several proteins with aberrant expression and was validated by western blot and immunofluorescence analysis. Homology modeling was performed to build 3D structures for γ-enolase, thioredoxin (TRX), and heat shock 60 (HSP60) by the template crystal structures using a protein data bank as a sequence database. The structure refinement of each model was achieved by energy minimization via molecular dynamic simulation, and the generated models were further assessed for stability by Procheck and ProSA. The models were processed for docking analysis using AutoDock Vina, and post-docking analysis was determined by discovery studio. The proteomic study showed decreased expression of γ-enolase, TRX, and protein phosphatase 2A subunit B and increased expression of collapsin response mediator protein 2 and HSP60 in the striatum after ischemic injury. Treatment with melatonin modulated the expression profiles of these proteins. This study demonstrated the neuroprotective role of melatonin in the ischemic striatum using a proteomic and in silico approach. Collectively, melatonin may act in a multimechanistic way by modulating the expression of several proteins in the ischemic striatum.

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          Most cited references70

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          Multiple sequence alignment with the Clustal series of programs.

          R Chenna (2003)
          The Clustal series of programs are widely used in molecular biology for the multiple alignment of both nucleic acid and protein sequences and for preparing phylogenetic trees. The popularity of the programs depends on a number of factors, including not only the accuracy of the results, but also the robustness, portability and user-friendliness of the programs. New features include NEXUS and FASTA format output, printing range numbers and faster tree calculation. Although, Clustal was originally developed to run on a local computer, numerous Web servers have been set up, notably at the EBI (European Bioinformatics Institute) (http://www.ebi.ac.uk/clustalw/).
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            GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity.

            Neurons are highly polarized and comprised of two structurally and functionally distinct parts, an axon and dendrites. We previously showed that collapsin response mediator protein-2 (CRMP-2) is critical for specifying axon/dendrite fate, possibly by promoting neurite elongation via microtubule assembly. Here, we showed that glycogen synthase kinase-3beta (GSK-3beta) phosphorylated CRMP-2 at Thr-514 and inactivated it. The expression of the nonphosphorylated form of CRMP-2 or inhibition of GSK-3beta induced the formation of multiple axon-like neurites in hippocampal neurons. The expression of constitutively active GSK-3beta impaired neuronal polarization, whereas the nonphosphorylated form of CRMP-2 counteracted the inhibitory effects of GSK-3beta, indicating that GSK-3beta regulates neuronal polarity through the phosphorylation of CRMP-2. Treatment of hippocampal neurons with neurotrophin-3 (NT-3) induced inactivation of GSK-3beta and dephosphorylation of CRMP-2. Knockdown of CRMP-2 inhibited NT-3-induced axon outgrowth. These results suggest that NT-3 decreases phosphorylated CRMP-2 and increases nonphosphorylated active CRMP-2, thereby promoting axon outgrowth.
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              Contributions of protein phosphatases PP1, PP2A, PP2B and PP5 to the regulation of tau phosphorylation.

              Abnormal hyperphosphorylation of tau is believed to lead to neurofibrillary degeneration in Alzheimer's disease (AD) and other tauopathies. Recent studies have shown that protein phosphatases (PPs) PP1, PP2A, PP2B and PP5 dephosphorylate tau in vitro, but the exact role of each of these phosphatases in the regulation of site-specific phosphorylation of tau in the human brain was unknown. Hence, we investigated the contributions of these PPs to the regulation of tau phosphorylation quantitatively. We found that these four phosphatases all dephosphorylated tau at Ser199, Ser202, Thr205, Thr212, Ser214, Ser235, Ser262, Ser396, Ser404 and Ser409, but with different efficiencies toward different sites. The K(m) values of tau dephosphorylation catalysed by PP1, PP2A and PP5 were 8-12 microm, similar to the intraneuronal tau concentration of human brain, whereas the K(m) of PP2B was fivefold higher. PP2A, PP1, PP5 and PP2B accounted for approximately 71%, approximately 11%, approximately 10% and approximately 7%, respectively, of the total tau phosphatase activity of human brain. The total phosphatase activity and the activities of PP2A and PP5 toward tau were significantly decreased, whereas that of PP2B was increased in AD brain. PP2A activity negatively correlated to the level of tau phosphorylation at the most phosphorylation sites in human brains. Our findings indicate that PP2A is the major tau phosphatase that regulates its phosphorylation at multiple sites in human brain. The abnormal hyperphosphorylation of tau is partially due to a downregulation of PP2A activity in AD brain.
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                Author and article information

                Contributors
                Journal
                Front Neurosci
                Front Neurosci
                Front. Neurosci.
                Frontiers in Neuroscience
                Frontiers Media S.A.
                1662-4548
                1662-453X
                10 December 2018
                2018
                : 12
                : 888
                Affiliations
                [1] 1Division of Applied Life Science (BK 21), College of Natural Science, Gyeongsang National University , Jinju, South Korea
                [2] 2Department of Pharmacology, Riphah Institute of Pharmaceutical Sciences, Riphah International University Islamabad , Rawalpindi, Pakistan
                [3] 3Department of Pharmacy, Faculty of Life Science, Sarhad University of Science and Information Technology , Peshawar, Pakistan
                [4] 4Department of Anatomy, College of Veterinary Medicine, Research Institute of Life Science, Gyeongsang National University , Jinju, South Korea
                Author notes

                Edited by: Francisco Lopez-Munoz, Universidad Camilo José Cela, Spain

                Reviewed by: Anwen Shao, Second Affiliated Hospital, Zhejiang University School of Medicine, China; Alejandro Romero, Complutense University of Madrid, Spain

                *Correspondence: Myeong Ok Kim, mokim@ 123456gnu.ac.kr ; mokim@ 123456gsnu.ac.kr

                These authors have contributed equally to this work

                This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neuroscience

                Article
                10.3389/fnins.2018.00888
                6295458
                30618542
                30992f2c-ad6b-40d6-86be-b567cdfc5ab4
                Copyright © 2018 Shah, Zeb, Ali, Muhammad, Faheem, Alam, Saeed, Koh, Lee and Kim.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 28 July 2018
                : 13 November 2018
                Page count
                Figures: 6, Tables: 2, Equations: 0, References: 84, Pages: 15, Words: 0
                Categories
                Neuroscience
                Original Research

                Neurosciences
                melatonin,striatum,ischemic stroke,docking,neuroprotection
                Neurosciences
                melatonin, striatum, ischemic stroke, docking, neuroprotection

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