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  1. Article ; Online: SUMO-Modification of Human Nrf2 at K

    Walters, Treniqka S / McIntosh, Deneshia J / Ingram, Shalonda M / Tillery, Lakeisha / Motley, Evangeline D / Arinze, Ifeanyi J / Misra, Smita

    Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

    2021  Volume 55, Issue 2, Page(s) 141–159

    Abstract: Background/aims: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element(s) (ARE) in target gene promoters, enabling oxidatively stressed cells to respond in order to restore redox ... ...

    Abstract Background/aims: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element(s) (ARE) in target gene promoters, enabling oxidatively stressed cells to respond in order to restore redox homeostasis. Post-translational modifications (PTMs) that mediate activation of Nrf2, in the cytosol and its release from Keap1, have been extensively studied but PTMs that impact its biology after activation are beginning to emerge. In this regard, PTMs like acetylation, phosphorylation, ubiquitination and sumoylation contribute towards the Nrf2 subcellular localization, and its transactivation function. We previously demonstrated that Nrf2 traffics to the promyelocytic leukemia-nuclear bodies (PML-NB), where it is a target for modification by small ubiquitin-like modifier (SUMO) proteins (sumoylation), but the site(s) for SUMO conjugation have not been determined. In this study, we aim to identify SUMO-2 conjugation site(s) and explore the impact, sumoylation of the site(s) have on Nrf2 stability, nuclear localization and transcriptional activation of its target gene expression upon oxidative stress.
    Methods: The putative SUMO-binding sites in Nrf2 for human isoform1 (NP_006155.2) and mouse homolog (NP_035032.1) were identified using a computer-based SUMO-predictive software (SUMOplot™). Site-directed mutagenesis, immunoblot analysis, and ARE-mediated reporter gene assays were used to assess the impact of sumoylation on these site(s) in vitro. Effect of mutation of these sumoylation sites of Nrf2 on expression of Heme Oxygenase1 (HO-1) was determined in HEK293T cell.
    Results: 
Eight putative sumoylation sites were identified by SUMOplot™ analysis. Out of the eight predicted sites only one
    Conclusion: Our findings indicate that SUMO-2
mediated sumoylation of K
    MeSH term(s) Active Transport, Cell Nucleus/genetics ; Active Transport, Cell Nucleus/physiology ; Binding Sites ; Fluorescent Antibody Technique ; HEK293 Cells ; Humans ; NF-E2-Related Factor 2/genetics ; NF-E2-Related Factor 2/metabolism ; Protein Stability ; Sumoylation
    Chemical Substances NF-E2-Related Factor 2 ; NFE2L2 protein, human
    Language English
    Publishing date 2021-03-24
    Publishing country Germany
    Document type Journal Article
    ZDB-ID 1067572-3
    ISSN 1421-9778 ; 1015-8987
    ISSN (online) 1421-9778
    ISSN 1015-8987
    DOI 10.33594/000000351
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Featured Article: Differential regulation of endothelial nitric oxide synthase phosphorylation by protease-activated receptors in adult human endothelial cells.

    Tillery, Lakeisha C / Epperson, Tenille A / Eguchi, Satoru / Motley, Evangeline D

    Experimental biology and medicine (Maywood, N.J.)

    2016  Volume 241, Issue 6, Page(s) 569–580

    Abstract: Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production ...

    Abstract Protease-activated receptors have been shown to regulate endothelial nitric oxide synthase through the phosphorylation of specific sites on the enzyme. It has been established that PAR-2 activation phosphorylates eNOS-Ser-1177 and leads to the production of the potent vasodilator nitric oxide, while PAR-1 activation phosphorylates eNOS-Thr-495 and decreases nitric oxide production in human umbilical vein endothelial cells. In this study, we hypothesize a differential coupling of protease-activated receptors to the signaling pathways that regulates endothelial nitric oxide synthase and nitric oxide production in primary adult human coronary artery endothelial cells. Using Western Blot analysis, we showed that thrombin and the PAR-1 activating peptide, TFLLR, lead to the phosphorylation of eNOS-Ser-1177 in human coronary artery endothelial cells, which was blocked by SCH-79797 (SCH), a PAR-1 inhibitor. Using the nitrate/nitrite assay, we also demonstrated that the thrombin- and TFLLR-induced production of nitric oxide was inhibited by SCH and L-NAME, a NOS inhibitor. In addition, we observed that TFLLR, unlike thrombin, significantly phosphorylated eNOS-Thr-495, which may explain the observed delay in nitric oxide production in comparison to that of thrombin. Activation of PAR-2 by SLIGRL, a PAR-2 specific ligand, leads to dual phosphorylation of both catalytic sites but primarily regulated eNOS-Thr-495 phosphorylation with no change in nitric oxide production in human coronary artery endothelial cells. PAR-3, known as the non-signaling receptor, was activated by TFRGAP, a PAR-3 mimicking peptide, and significantly induced the phosphorylation of eNOS-Thr-495 with minimal phosphorylation of eNOS-Ser-1177 with no change in nitric oxide production. In addition, we confirmed that PAR-mediated eNOS-Ser-1177 phosphorylation was Ca(2+)-dependent using the Ca(2+) chelator, BAPTA, while eNOS-Thr-495 phosphorylation was mediated via Rho kinase using the ROCK inhibitor, Y-27632, suggesting protease-activated receptor coupling to Gq and G12/13, respectively. These data suggest a vascular bed specific differential coupling of protease-activated receptors to the signaling pathways that regulate endothelial nitric oxide synthase and nitric oxide production that may be responsible for endothelial dysfunction associated with cardiovascular disease.
    MeSH term(s) Adult ; Cells, Cultured ; Endothelial Cells/enzymology ; Endothelial Cells/physiology ; Female ; Gene Expression Regulation ; Humans ; Male ; Middle Aged ; Nitric Oxide/metabolism ; Nitric Oxide Synthase Type III/metabolism ; Phosphorylation ; Protein Processing, Post-Translational ; Receptor, PAR-1/metabolism ; Receptor, PAR-2/metabolism ; Receptors, Thrombin/metabolism ; Young Adult
    Chemical Substances Receptor, PAR-1 ; Receptor, PAR-2 ; Receptors, Thrombin ; protease-activated receptor 3 ; Nitric Oxide (31C4KY9ESH) ; Nitric Oxide Synthase Type III (EC 1.14.13.39)
    Language English
    Publishing date 2016-01-04
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 4015-0
    ISSN 1535-3699 ; 1525-1373 ; 0037-9727
    ISSN (online) 1535-3699 ; 1525-1373
    ISSN 0037-9727
    DOI 10.1177/1535370215622584
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Ua VI Zs.111: Show issues Location:
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