scholarly journals PGRMC1 acts as a size-selective cargo receptor to drive ER-phagic clearance of mutant prohormones

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yu-Jie Chen ◽  
Jeffrey Knupp ◽  
Anoop Arunagiri ◽  
Leena Haataja ◽  
Peter Arvan ◽  
...  
Keyword(s):  
Transmembrane Protein ◽  
Dominant Negative ◽  
Insulin Deficiency ◽  
Potential Therapeutic Target ◽  
Binding Partner ◽  
Large Size ◽  
Luminal Side ◽  
Cargo Receptor ◽  
Insulin Storage ◽  
Chemical Perturbation

AbstractThe reticulon-3 (RTN3)-driven targeting complex promotes clearance of misfolded prohormones from the endoplasmic reticulum (ER) for lysosomal destruction by ER-phagy. Because RTN3 resides in the cytosolic leaflet of the ER bilayer, the mechanism of selecting misfolded prohormones as ER-phagy cargo on the luminal side of the ER membrane remains unknown. Here we identify the ER transmembrane protein PGRMC1 as an RTN3-binding partner. Via its luminal domain, PGRMC1 captures misfolded prohormones, targeting them for RTN3-dependent ER-phagy. PGRMC1 selects cargos that are smaller than the large size of other reported ER-phagy substrates. Cargos for PGRMC1 include mutant proinsulins that block secretion of wildtype proinsulin through dominant-negative interactions within the ER, causing insulin-deficiency. Chemical perturbation of PGRMC1 partially restores WT insulin storage by preventing ER-phagic degradation of WT and mutant proinsulin. Thus, PGRMC1 acts as a size-selective cargo receptor during RTN3-dependent ER-phagy, and is a potential therapeutic target for diabetes.

scholarly journals Glioblastoma invasion factor ODZ1 is induced by microenvironmental signals through activation of a Stat3-dependent transcriptional pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Veronica Vidal ◽  
Olga Gutierrez ◽  
Ana Talamillo ◽  
Carlos Velasquez ◽  
Jose L. Fernandez-Luna
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Transmembrane Protein ◽  
Gene Promoter ◽  
Dominant Negative ◽  
Surrounding Tissue ◽  
Extracellular Matrix Protein ◽  
Luciferase Reporter ◽  
Dominant Negative Variant ◽  
Transcriptional Pathway

AbstractWe have previously shown that the transmembrane protein ODZ1 serves for glioblastoma (GBM) cells to invade the surrounding tissue through activation of RhoA/ROCK pathway. However, the transcriptional machinery used by GBM cells to regulate the expression of ODZ1 is unknown. Here we show that interaction with tumor microenvironment elements, mainly activated monocytes through IL-6 secretion, and the extracellular matrix protein fibronectin, induces the Stat3 transcriptional pathway and upregulates ODZ1 which results in GBM cell migration. This signaling route is abrogated by blocking the IL-6 receptor, inhibiting Jak kinases or knocking down Stat3. Furthermore, we have identified a Stat3 responsive element in the ODZ1 gene promoter, about 1 kb from the transcription start site. Luciferase-reporter assays confirmed that the promoter responds to the presence of monocytic cells and this activation is greatly reduced when the Stat3 site is mutated or following treatment with a neutralizing anti-IL-6 receptor antibody or transfecting GBM cells with a dominant negative variant of Stat3. Overall, we show that monocyte-secreted IL-6 and the extracellular matrix protein fibronectin activate the axis Stat3-ODZ1 and promote migration of GBM cells. This is the first described transcriptional mechanism used by tumor cells to promote the expression of the invasion factor ODZ1.

scholarly journals Epiblast integrity requires CLASP and Dystroglycan-mediated microtubule anchoring to the basal cortex

2013 ◽  
Vol 202 (4) ◽  
pp. 637-651 ◽  
Author(s):  
Yukiko Nakaya ◽  
Erike W. Sukowati ◽  
Guojun Sheng
Keyword(s):  
Basement Membrane ◽  
Epithelial To Mesenchymal Transition ◽  
Transmembrane Protein ◽  
Basolateral Membrane ◽  
Cortical Microtubule ◽  
Molecular Regulation ◽  
Mesenchymal Transition ◽  
Binding Partner ◽  
Mesoderm Formation ◽  
Lateral Localization

Amniote epiblast cells differentiate into mesoderm and endoderm lineages during gastrulation through a process called epithelial-to-mesenchymal transition (EMT). Molecular regulation of gastrulation EMT is poorly understood. Here we show that epiblast epithelial status was maintained by anchoring microtubules to the basal cortex via CLIP-associated protein (CLASP), a microtubule plus-end tracking protein, and Dystroglycan, a transmembrane protein that bridges the cytoskeleton and basement membrane (BM). Mesoderm formation required down-regulation of CLASP and Dystroglycan, and reducing CLASP activity in pregastrulation epiblast cells caused ectopic BM breakdown and disrupted epiblast integrity. These effects were mediated through the CLASP-binding partner LL5. Live-imaging using EB1–enhanced GFP (eGFP) revealed that reducing CLASP and LL5 levels in the epiblast destabilized basal microtubules. We further show that Dystroglycan is localized to basolateral membrane in epiblast cells. Basal but not lateral localization of Dystroglycan was regulated by CLASP. We propose that epiblast–BM interaction requires CLASP- and Dystroglycan-mediated cortical microtubule anchoring, the disruption of which initiates gastrulation EMT.

scholarly journals Direct Interactions between Calcitonin-Like Receptor (CLR) and CGRP-Receptor Component Protein (RCP) Regulate CGRP Receptor Signaling

Endocrinology ◽  
2012 ◽  
Vol 153 (4) ◽  
pp. 1850-1860 ◽  
Author(s):  
Sophie C. Egea ◽  
Ian M. Dickerson
Keyword(s):  
Cell Surface ◽  
Transmembrane Protein ◽  
Direct Interaction ◽  
Dominant Negative ◽  
In Vivo Studies ◽  
Cgrp Receptor ◽  
Receptor Component ◽  
Soluble Fusion Protein ◽  
Component Protein

Calcitonin gene-related peptide (CGRP) is a neuropeptide with multiple neuroendocrine roles, including vasodilation, migraine, and pain. The receptor for CGRP is a G protein-coupled receptor (GPCR) that requires three proteins for function. CGRP binds to a heterodimer composed of the GPCR calcitonin-like receptor (CLR) and receptor activity-modifying protein (RAMP1), a single transmembrane protein required for pharmacological specificity and trafficking of the CLR/RAMP1 complex to the cell surface. In addition, the CLR/RAMP1 complex requires a third protein named CGRP-receptor component protein (RCP) for signaling. Previous studies have demonstrated that depletion of RCP from cells inhibits CLR signaling, and in vivo studies have demonstrated that expression of RCP correlates with CLR signaling and CGRP efficacy. It is not known whether RCP interacts directly with CLR to exert its effect. The current studies identified a direct interaction between RCP and an intracellular domain of CLR using yeast two-hybrid analysis and coimmunoprecipitation. When this interacting domain of CLR was expressed as a soluble fusion protein, it coimmunoprecipitated with RCP and inhibited signaling from endogenous CLR. Expression of this dominant-negative domain of CLR did not significantly inhibit trafficking of CLR to the cell surface, and thus RCP may not have a chaperone function for CLR. Instead, RCP may regulate CLR signaling in the cell membrane, and direct interaction between RCP and CLR is required for CLR activation. To date, RCP has been found to interact only with CLR and represents a novel neuroendocrine regulatory step in GPCR signaling.

scholarly journals Functional Myc-Max heterodimer is required for activation-induced apoptosis in T cell hybridomas.

1994 ◽  
Vol 180 (6) ◽  
pp. 2413-2418 ◽  
Author(s):  
R P Bissonnette ◽  
A McGahon ◽  
A Mahboubi ◽  
D R Green
Keyword(s):  
T Cell ◽  
Apoptotic Cell ◽  
Tolerance Induction ◽  
Dominant Negative ◽  
Binding Partner ◽  
Dominant Negative Form ◽  
Dominant Negative Activity ◽  
Induced Apoptosis ◽  
Negative Form

T cell hybridomas respond to activation signals by undergoing apoptotic cell death, and this is likely to represent comparable events related to tolerance induction in immature and mature T cells in vivo. Previous studies using antisense oligonucleotides implicated the c-Myc protein in the phenomenon of activation-induced apoptosis. This role for c-Myc in apoptosis is now confirmed in studies using a dominant negative form of its heterodimeric binding partner, Max, which we show here inhibits activation-induced apoptosis. Further, coexpression of a reciprocally mutant Myc protein capable of forming functional heterodimers with the mutant Max can compensate for the dominant negative activity and restore activation-induced apoptosis. These results imply that Myc promotes activation-induced apoptosis by obligatory heterodimerization with Max, and therefore, by regulating gene transcription.

scholarly journals Effect of 11β-hydroxysteroid dehydrogenase-1 inhibition on hepatic glucose metabolism in the conscious dog

2010 ◽  
Vol 298 (5) ◽  
pp. E1019-E1026 ◽  
Author(s):  
Dale S. Edgerton ◽  
Rita Basu ◽  
Christopher J. Ramnanan ◽  
Tiffany D. Farmer ◽  
Doss Neal ◽  
...  
Keyword(s):  
Hepatic Glucose Production ◽  
Glucose Production ◽  
Hydroxysteroid Dehydrogenase ◽  
Insulin Deficiency ◽  
Potential Therapeutic Target ◽  
Hepatic Glucose Metabolism ◽  
Hepatic Glucose ◽  
Healthy Dogs ◽  
Excess Glucose

Inactive cortisone is converted to active cortisol within the liver by 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1), and impaired regulation of this process may be related to increased hepatic glucose production (HGP) in individuals with type 2 diabetes. The primary aim of this study was to investigate the effect of acute 11β-HSD1 inhibition on HGP and fat metabolism during insulin deficiency. Sixteen conscious, 42-h-fasted, lean, healthy dogs were studied. Somatostatin was infused to create insulin deficiency, and the animals were treated with a specific 11β-HSD1 inhibitor (compound 531) or placebo for 5 h. 11β-HSD1 inhibition completely suppressed hepatic cortisol production, and this attenuated the increase in HGP that occurred during insulin deficiency. PEPCK and glucose-6-phosphatase expression were decreased when 11β-HSD1 was inhibited, but gluconeogenic flux was unchanged, implying an effect on glycogenolysis. Since inhibition of hepatic cortisol production reduces HGP during insulin deficiency, 11β-HSD1 is a potential therapeutic target for the treatment of excess glucose production that occurs in diabetes.

scholarly journals Translocation of TMEM175 Lysosomal Potassium Channel to the Plasma Membrane by Dynasore Compounds

2021 ◽  
Vol 22 (19) ◽  
pp. 10515
Author(s):  
Enikő Pergel ◽  
Irén Veres ◽  
Gergely Imre Csigi ◽  
Gábor Czirják
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Protein ◽  
Intracellular Localization ◽  
Growth Factor Receptor ◽  
Direct Interaction ◽  
Dominant Negative ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Increased Risk ◽  
Inhibitor Compounds

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson’s disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.

scholarly journals A theory for polymicrogyria and brain arteriovenous malformations in HHT

Neurology ◽  
2018 ◽  
Vol 92 (1) ◽  
pp. 34-42 ◽  
Author(s):  
Jesse M. Klostranec ◽  
Long Chen ◽  
Shobhit Mathur ◽  
Jamie McDonald ◽  
Marie E. Faughnan ◽  
...  
Keyword(s):  
Arteriovenous Malformations ◽  
Fluid Shear Stress ◽  
Transmembrane Protein ◽  
Dominant Negative ◽  
Spatial Proximity ◽  
Imaging Data ◽  
Fluid Shear ◽  
Brain Avm ◽  
Close Spatial Proximity ◽  
The Brain

Hereditary hemorrhagic telangiectasia (HHT) is generally considered a disorder of endothelial dysfunction, characterized by the development of multiple systemic arteriovenous malformations (AVMs), including within the brain. However, there have recently been a number of reports correlating HHT with malformations of cortical development, of which polymicrogyria is the most common type. Here we present 7 new cases demonstrating polymicrogyria in HHT, 6 of which demonstrate a brain AVM (bAVM) in close spatial proximity, with the aim of providing a common origin for the association. Upon reviewing patient genetics and imaging data and comparing with previously reported findings, we form 2 new conclusions: (1) polymicrogyria in HHT appears exclusively associated with a subset of mutations in the transmembrane protein endoglin that is involved with blood flow–related mechanotransduction signaling during angiogenesis and (2) the polymicrogyria is characteristically unilateral, typically focal, and correlates with vascular regions experiencing low fluid shear stress during corticogenesis in utero. Integrating these with findings in the literature from genetics and molecular biology experiments, we propose a theory suggesting haploinsufficient endoglin mutations, especially those that are dominant-negative, may predispose focal, aberrant hypersprouting angiogenesis during corticogenesis that leads to the production of polymicrogyria. This hypoxic insult may further serve as the revealing trigger for later development of a spatially coincident bAVM. This hypothesis suggests an essential role for endoglin-mediated hemodynamic mechanotransduction in normal corticogenesis.

Abstract 1473: Identification Of A New NRSF-binding Partner, Zfp90, Which Negatively Regulates The Repression Activity Of NRSF, A Critical Transcriptional Regulator Of Cardiac Fetal Gene Program

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Masao Murakami ◽  
Koichiro Kuwahara ◽  
Masaki Harada ◽  
Yasuaki Nakagawa ◽  
Satoru Usami ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Dominant Negative ◽  
Assay System ◽  
Negative Regulator ◽  
Binding Partner ◽  
Arrhythmic Death ◽  
Dominant Negative Form ◽  
Cardiac Gene ◽  
Hybrid Screening ◽  
Negative Form

Neuron-restrictive silencer factor, NRSF is a Zn-finger transcription factor that specifically binds to DNA sequence called NRSE to repress the transcription. NRSF is involved in various biological processes, such as neuronal differentiation, carcinogenesis, and cardiac homeostasis. We previously reported that NRSF regulates the transcription of multiple fetal cardiac genes. Cardiac-specific overexpression of a dominant-negative form of NRSF in mice induced reactivation of fetal cardiac gene program, cardiac dysfunction, and sudden arrhythmic death, suggesting NRSF plays important roles in normal cardiac homeostasis. However, the molecular mechanisms or signaling pathways that regulate the activity of NRSF have not been well understood. In an attempt to clarify the regulators of NRSF, we performed yeast two-hybrid screening using NRSF as bait and identified Zfp90 as NRSF-binding protein. NRSF and Zfp90 co-localize in the nuclear, and Zn-finger domain of NRSF specifically interacted with KRAB domain of Zfp90. To monitor the activity of NRSF sensitively, we made NRSF-responsive reporters using NRSE sequence of BNP promoter and established assay system for estimating the NRSF activity. In this assay system, expression of NRSF repressed the reporter activity and co-expression of Zfp90 restored it. Furthermore the expression levels of Zfp90 were elevated in the hearts with cardiomyopathy. These results suggest that Zfp90 functions as a negative regulator of NRSF and contributes to the genetic remodeling during the development of cardiomyopathy..

scholarly journals Pref-1 Interacts with Fibronectin To Inhibit Adipocyte Differentiation

2010 ◽  
Vol 30 (14) ◽  
pp. 3480-3492 ◽  
Author(s):  
Yuhui Wang ◽  
Ling Zhao ◽  
Cynthia Smas ◽  
Hei Sook Sul
Keyword(s):  
Adipocyte Differentiation ◽  
Transmembrane Protein ◽  
Dominant Negative ◽  
Biologically Active ◽  
Mitogen Activated Protein Kinase ◽  
Soluble Form ◽  
Necrosis Factor Alpha ◽  
Rgd Peptides ◽  
Downstream Signaling ◽  
Erk Mapk

ABSTRACT Pref-1/Dlk1 is made as an epidermal growth factor (EGF) repeat-containing transmembrane protein but is cleaved by tumor necrosis factor alpha converting enzyme (TACE) to generate a biologically active soluble form. Soluble Pref-1 inhibits adipocyte differentiation through the activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and the subsequent upregulation of Sox9 expression. However, others have implicated Notch in Pref-1 signaling and function. Here, we show that Pref-1 does not interact with, or require, Notch for its function. Instead, we show a direct interaction of Pref-1 and fibronectin via the Pref-1 juxtamembrane domain and fibronectin C-terminal domain. We also show that fibronectin is required for the Pref-1-mediated inhibition of adipocyte differentiation, the activation of ERK/MAPK, and the upregulation of Sox9. Furthermore, disrupting fibronectin binding to integrin by the addition of RGD peptides or by the knockdown of α5 integrin prevents the Pref-1 inhibition of adipocyte differentiation. Pref-1 activates the integrin downstream signaling molecules, FAK and Rac, and ERK activation by Pref-1 is blunted by the knockdown of Rac or by the forced expression of dominant-negative Rac. We conclude that, by interacting with fibronectin, Pref-1 activates integrin downstream signaling to activate MEK/ERK and to inhibit adipocyte differentiation.

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