scholarly journals Smoothelin-like Protein 1 Regulates the Thyroid Hor-Mone-Induced Homeostasis and Remodeling of C2C12 Cells via the Modulation of Myosin Phosphatase

2021 ◽  
Vol 22 (19) ◽  
pp. 10293
Author(s):  
Evelin Major ◽  
Ilka Keller ◽  
Dániel Horváth ◽  
István Tamás ◽  
Ferenc Erdődi ◽  
...  
Keyword(s):  
Contractile Function ◽  
C2c12 Cells ◽  
Protein Phosphatase 1 ◽  
Transcriptional Level ◽  
Myosin Phosphatase ◽  
Independent Manner ◽  
Physiological Concentration ◽  
Cytoskeletal Remodeling ◽  
Quantitative Western Blot ◽  
Hyperthyroid Patients

The pathological elevation of the active thyroid hormone (T3) level results in the manifestation of hyperthyroidism, which is associated with alterations in the differentiation and contractile function of skeletal muscle (SKM). Myosin phosphatase (MP) is a major cellular regulator that hydrolyzes the phosphoserine of phosphorylated myosin II light chain. MP consists of an MYPT1/2 regulatory and a protein phosphatase 1 catalytic subunit. Smoothelin-like protein 1 (SMTNL1) is known to inhibit MP by directly binding to MP as well as by suppressing the expression of MYPT1 at the transcriptional level. Supraphysiological vs. physiological concentration of T3 were applied on C2C12 myoblasts and differentiated myotubes in combination with the overexpression of SMTNL1 to assess the role and regulation of MP under these conditions. In non-differentiated myoblasts, MP included MYPT1 in the holoenzyme complex and its expression and activity was regulated by SMTNL1, affecting the phosphorylation level of MLC20 assessed using semi-quantitative Western blot analysis. SMTNL1 negatively influenced the migration and cytoskeletal remodeling of myoblasts measured by high content screening. In contrast, in myotubes, the expression of MYPT2 but not MYPT1 increased in a T3-dependent and SMTNL1-independent manner. T3 treatment combined with SMTNL1 overexpression impeded the activity of MP. In addition, MP interacted with Na+/K+-ATPase and dephosphorylated its inhibitory phosphorylation sites, identifying this protein as a novel MP substrate. These findings may help us gain a better understanding of myopathy, muscle weakness and the disorder of muscle regeneration in hyperthyroid patients.

Abstract WMP39: Protein Phosphatase 1 Regulatory Subunit 12C Contributes to Atrial Myosin Light Chain Dephosphorylation in Atrial Fibrillation

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Srikanth Perike ◽  
Katherina M Alsina ◽  
Arvind Sridhar ◽  
Dawood Darbar ◽  
Xander Wehrens ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain ◽  
Stroke Risk ◽  
Contractile Function ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Western Blots ◽  
Atrial Myosin

Background: Atrial fibrillation (AF) increases stroke risk five-fold. Atrial hypocontractility from atrial myosin light chain (MLC2a) dephosphorylation contributes to stroke risk in AF. Recent proteomic data has shown increased protein phosphatase 1 subunit 12C (PPP1R12C) targeting to MLC2a in AF. However, it is unclear whether PPP1R12C causes MLC2a dephosphorylation in AF. Objective: Determine whether increased PPP1R12C expression causes MLC2a dephosphorylation and increases AF risk. Methods: Western blots and co-IPs were performed to evaluate the relationship among PPP1R12C, PP1c and MLC2a in human atrial tissues (AF vs SR). Mice with either a knockout (KO) or lentiviral (LV) cardiac overexpression of PPP1R12C were evaluated with invasive EP studies for AF inducibility vs WT controls. Results: In human AF, PPP1R12C was increased 4-fold ( P <0.005, n=6) with an 88% reduction in S-19-MLC2a phosphorylation ( P <0.05, n=4). PPP1R12C-PP1c and PPP1R12C-MLC2a binding was increased 2-fold in AF ( P <0.05, n=6). AF burden in LV-12C mice increased nearly tenfold vs. KO and WT mice ( P <0.05, n=6). Conclusion: In human AF, increased PPP1R12C expression is associated with reduced P-MLC2a through enhanced binding with the PP1c catalytic subunit. This dephosphorylation is a likely contributor to atrial hypocontractility and stroke risk in AF. Additionally, increased PPP1R12C expression in mice increases AF risk. Future studies will examine the effects of increased PPP1R12C expression upon atrial contractile function in mice.

scholarly journals Multiple roles of heterogeneous nuclear ribonucleoprotein K during skeletal muscle cell differentiation

2021 ◽  
Author(s):  
Keisuke Hitachi ◽  
Yuri Kiyofuji ◽  
Masashi Nakatani ◽  
Kunihiro Tsuchida
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Myogenic Differentiation ◽  
Cell Physiology ◽  
Transcriptional Level ◽  
Loss Of Function ◽  
Independent Manner ◽  
Multiple Functions

RNA-binding proteins (RBPs) regulate cell physiology via the formation of ribonucleic-protein complexes with coding and non-coding RNAs. RBPs have multiple functions in the same cells; however, the precise mechanism through which their pleiotropic functions are determined remains unknown. In this study, we revealed the multiple inhibitory functions of hnRNPK for myogenic differentiation. We first identified hnRNPK as a lncRNA Myoparr binding protein. Gain- and loss-of-function experiments showed that hnRNPK repressed the expression of myogenin at the transcriptional level via binding to Myoparr. Moreover, hnRNPK repressed the expression of a set of genes coding for aminoacyl-tRNA synthetases in a Myoparr-independent manner. Mechanistically, hnRNPK regulated the eIF2α/Atf4 pathway, one branch of the intrinsic pathways of the endoplasmic reticulum sensors, in differentiating myoblasts. Thus, our findings demonstrate that hnRNPK plays multiple lncRNA-dependent and -independent roles in the inhibition of myogenic differentiation, indicating that the analysis of lncRNA-binding proteins will be useful for elucidating both the physiological functions of lncRNAs and the multiple functions of RBPs.

scholarly journals NFκB1 (p50) suppresses SOD2 expression by inhibiting FoxO3a transactivation in a miR190/PHLPP1/Akt-dependent axis

2013 ◽  
Vol 24 (22) ◽  
pp. 3577-3583 ◽  
Author(s):  
Kejun Du ◽  
Yonghui Yu ◽  
Dongyun Zhang ◽  
Wenjing Luo ◽  
Haishan Huang ◽  
...  
Keyword(s):  
P53 Protein ◽  
Protein Translation ◽  
Protein Phosphatase 1 ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Ph Domain ◽  
Embryonic Fibroblasts ◽  
Insight Into ◽  
Leucine Rich Repeat Protein ◽  
Superoxide Dismutase 2

The biological functions of nuclear factor κB1 (NFκB1; p50) have not been studied as often as those of other members of the NFκB family due to its lack of a transcriptional domain. Our recent studies showed that p50 functions as an apoptotic mediator via its inhibition of GADD45α protein degradation and increase in p53 protein translation. Here we report a novel function of p50 in its regulation of superoxide dismutase 2 (SOD2) transcription via an NFκB-independent pathway. We find that deletion of p50 in mouse embryonic fibroblasts (MEFs; p50−/−) up-regulates SOD2 expression at both protein and mRNA levels. SOD2 promoter–driven luciferase is also up-regulated in p50−/− cells compared with wild-type (WT) MEF (p50+/+) cells, suggesting p50 regulation of SOD2 at the transcriptional level. Our results also show that p50 deficiency specifically results in down-regulation of phosphorylation and increased transactivation of FoxO3a compared with WT cells. Further studies indicate that p50–down-regulated FoxO3a phosphorylation is mediated by activated Akt via up-regulation of microRNA 190 (miR190), in turn inhibiting PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) translation. Together our studies identify a novel p50 function in the regulation of SOD2 transcription by modulating the miR190/PHLPP1/Akt-FoxO3a pathway, which provides significant insight into the physiological function of p50.

scholarly journals Vinculin network–mediated cytoskeletal remodeling regulates contractile function in the aging heart

2015 ◽  
Vol 7 (292) ◽  
pp. 292ra99-292ra99 ◽  
Author(s):  
Gaurav Kaushik ◽  
Alice Spenlehauer ◽  
Ayla O. Sessions ◽  
Adriana S. Trujillo ◽  
Alexander Fuhrmann ◽  
...  
Keyword(s):  
Contractile Function ◽  
Aging Heart ◽  
Cytoskeletal Remodeling

Granzyme B Produced by Human Plasmacytoid Dendritic Cells Suppresses T Cell Expansion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2674-2674
Author(s):  
Dorit Fabricius ◽  
Angelika Vollmer ◽  
Sue Blackwell ◽  
Julia Maier ◽  
Kai Sontheimer ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Granzyme B ◽  
Cd40 Ligand ◽  
Plasmacytoid Dendritic Cells ◽  
Transcriptional Level ◽  
Independent Manner ◽  
Complex Interactions

Abstract Abstract 2674 Poster Board II-650 Human plasmacytoid dendritic cells (pDC) play a central role in regulating adaptive T cell responses in the course of neoplastic, viral and autoimmune disorders. In several of these diseases, elevated extracellular levels of the serine protease granzyme B (GrB) are observed. We found that human pDC can be an abundant source of GrB based on FACS analysis, ELISpot, ELISA, Sensizyme, Western immunoblotting, RT-PCR, and fluorescence microscopy. GrB is actively secreted by pDCs and reaches maximal levels up to two logs higher than those produced by classical GrB producers such as CTL or NK cells. However, pDC GrB production is not accompanied by perforin secretion. Spinning disk confocal microscopy revealed that GrB+ pDC bind to and transfer active GrB to T cells. Importantly, this GrB transfer induces a suppression of T cell proliferation in a GrB-dependent, perforin-independent manner, a process reminiscent of regulatory T cells. GrB expression in pDC is regulated on a transcriptional level by JAK1, STAT3 and STAT5. IL-3 and IL-10 enhance GrB production by pDCs while GrB production is inhibited by toll-like-receptor agonists and CD40 ligand. These findings suggest that GrB production by pDCs is involved in the complex interactions between pDC and T cells and that GrB-secreting pDC may play a regulatory role related to anti-tumor immunity, anti-viral immune responses, and autoimmune processes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification and characterization of a neuroretina-specific enhancer element in the quail Pax-6 (Pax-QNR) gene.

1995 ◽  
Vol 15 (2) ◽  
pp. 892-903 ◽  
Author(s):  
S Plaza ◽  
C Dozier ◽  
M C Langlois ◽  
S Saule
Keyword(s):  
Specific Activity ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelial Cells ◽  
Transcriptional Level ◽  
Regulatory Sequences ◽  
Independent Manner ◽  
Specific Expression ◽  
Enhancer Activity ◽  
Enhancer Element

Using nuclear run-on assays, we showed that the tissue-specific expression of quail Pax-6 (Pax-QNR) P0-initiated mRNAs is due in part to regulation of the gene at the transcriptional level. Regulatory sequences governing neuroretina-specific expression of the P0-initiated mRNAs were investigated. By using reporter-based expression assays, we characterized a region within the Pax-QNR gene, located 7.5 kbp downstream from the P0 promoter, that functions as an enhancer in neuroretina cells but not in nonexpressing P0-initiated mRNA cells (quail embryo cells and quail retinal pigment epithelial cells). This enhancer element functioned in a position- and orientation-independent manner both on the Pax-QNR P0 promoter and the heterologous thymidine kinase promoter. Moreover, this enhancer element exhibited a developmental stage-specific activity during embryonic neuroretina development: in contrast to activity at day E7, the enhancer activity was very weak at day E5. This paralleled the level of expression of P0-initiated mRNAs observed at the same stages. Using footprinting, gel retardation, and Southwestern (DNA-protein) analysis, we demonstrated the existence of four neuroretina-specific nuclear protein-binding sites, involving multiple unknown factors. In addition we showed that the quail enhancer element is structurally and functionally conserved in mice. All of these results strongly suggest that this enhancer element may contribute to the neuroretina-specific transcriptional regulation of the Pax-6 gene in vivo.

scholarly journals Control of Flagellar Gene Regulation in Legionella pneumophila and Its Relation to Growth Phase

2009 ◽  
Vol 192 (2) ◽  
pp. 446-455 ◽  
Author(s):  
Christiane Albert-Weissenberger ◽  
Tobias Sahr ◽  
Odile Sismeiro ◽  
Jörg Hacker ◽  
Klaus Heuner ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Environmental Changes ◽  
Bacterial Pathogen ◽  
Class Ii ◽  
Transcriptional Level ◽  
Class Iii ◽  
Phenotypic Analysis ◽  
Independent Manner ◽  
Genes Encoding ◽  
Class Iii Genes

ABSTRACT The bacterial pathogen Legionella pneumophila responds to environmental changes by differentiation. At least two forms are well described: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Phenotypic analysis, Western blotting, and electron microscopy of mutants of the regulatory genes encoding RpoN, FleQ, FleR, and FliA demonstrated that flagellin expression is strongly repressed and that the mutants are nonflagellated in the transmissive phase. Transcriptome analyses elucidated that RpoN, together with FleQ, enhances transcription of 14 out of 31 flagellar class II genes, which code for the basal body, hook, and regulatory proteins. Unexpectedly, FleQ independent of RpoN enhances the transcription of fliA encoding sigma 28. Expression analysis of a fliA mutant showed that FliA activates three out of the five remaining flagellar class III genes and the flagellar class IV genes. Surprisingly, FleR does not induce but inhibits expression of at least 14 flagellar class III genes on the transcriptional level. Thus, we propose that flagellar class II genes are controlled by FleQ and RpoN, whereas the transcription of the class III gene fliA is controlled in a FleQ-dependent but RpoN-independent manner. However, RpoN and FleR might influence flagellin synthesis on a posttranscriptional level. In contrast to the commonly accepted view that enhancer-binding proteins such as FleQ always interact with RpoN to fullfill their regulatory functions, our results strongly indicate that FleQ regulates gene expression that is RpoN dependent and RpoN independent. Finally, FliA induces expression of flagellar class III and IV genes leading to the complete synthesis of the flagellum.

scholarly journals BCA2/Rabring7 Interferes with HIV-1 Proviral Transcription by Enhancing the SUMOylation of IκBα

2017 ◽  
Vol 91 (8) ◽  
Author(s):  
Marta Colomer-Lluch ◽  
Ruth Serra-Moreno
Keyword(s):  
New Drugs ◽  
Host Cells ◽  
Transcriptional Level ◽  
Lysosomal Degradation ◽  
Virus Propagation ◽  
Independent Manner ◽  
Virion Assembly ◽  
Sumo Ligase ◽  
Additional Barrier ◽  
Hiv 1

ABSTRACT BCA2/Rabring7 is a BST2 cofactor that promotes the lysosomal degradation of trapped HIV-1 virions but also functions as a BST2-independent anti-HIV factor by targeting Gag for lysosomal degradation. Since many antiviral factors regulate the NF-κB innate signaling pathway, we investigated whether BCA2 is also connected to this proinflammatory cascade. Here, we show for the first time that BCA2 is induced by NF-κB-activating proinflammatory cytokines and that upregulation of BCA2 provides regulatory negative feedback on NF-κB. Specifically, BCA2 serves as an E3 SUMO ligase in the SUMOylation of IκBα, which in turn enhances the sequestration of NF-κB components in the cytoplasm. Since HIV-1 utilizes NF-κB to promote proviral transcription, the BCA2-mediated inhibition of NF-κB significantly decreases the transcriptional activity of HIV-1 (up to 4.4-fold in CD4+ T cells). Therefore, our findings indicate that BCA2 poses an additional barrier to HIV-1 infection: not only does BCA2 prevent assembly and release of nascent virions, it also significantly restricts HIV-1 transcription by inhibiting the NF-κB pathway. IMPORTANCE Understanding the interactions between HIV-1 and its host cells is highly relevant to the design of new drugs aimed at eliminating HIV-1 from infected individuals. We have previously shown that BCA2, a cofactor of BST2 in the restriction of HIV-1, also prevents virion assembly in a BST2-independent manner. In this study, we found that BCA2 negatively regulates the NF-κB pathway—a signaling cascade necessary for HIV-1 replication and infectivity—which in turn detrimentally affects proviral transcription and virus propagation. Thus, our results indicate that, besides its previously described functions as an antiviral factor, BCA2 poses an additional barrier to HIV-1 replication at the transcriptional level.

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