RESUMO
Super-resolution microscopy, or nanoscopy, enables the use of fluorescent-based molecular localization tools to study molecular structure at the nanoscale level in the intact cell, bridging the mesoscale gap to classical structural biology methodologies. Analysis of super-resolution data by artificial intelligence (AI), such as machine learning, offers tremendous potential for the discovery of new biology, that, by definition, is not known and lacks ground truth. Herein, we describe the application of weakly supervised paradigms to super-resolution microscopy and its potential to enable the accelerated exploration of the nanoscale architecture of subcellular macromolecules and organelles.
Assuntos
Inteligência Artificial , Microscopia , Animais , Humanos , Processamento de Imagem Assistida por Computador/métodos , Aprendizado de Máquina , Microscopia/métodos , Microscopia de Fluorescência/métodosRESUMO
Caveolin-1 (Cav1) is a 22â kDa intracellular protein that is the main protein constituent of bulb-shaped membrane invaginations known as caveolae. Cav1 can be also found in functional non-caveolar structures at the plasma membrane called scaffolds. Scaffolds were originally described as SDS-resistant oligomers composed of 10-15 Cav1 monomers observable as 8S complexes by sucrose velocity gradient centrifugation. Recently, cryoelectron microscopy (cryoEM) and super-resolution microscopy have shown that 8S complexes are interlocking structures composed of 11 Cav1 monomers each, which further assemble modularly to form higher-order scaffolds and caveolae. In addition, Cav1 can act as a critical signaling regulator capable of direct interactions with multiple client proteins, in particular, the endothelial nitric oxide (NO) synthase (eNOS), a role believed by many to be attributable to the highly conserved and versatile scaffolding domain (CSD). However, as the CSD is a hydrophobic domain located by cryoEM to the periphery of the 8S complex, it is predicted to be enmeshed in membrane lipids. This has led some to challenge its ability to interact directly with client proteins and argue that it impacts signaling only indirectly via local alteration of membrane lipids. Here, based on recent advances in our understanding of higher-order Cav1 structure formation, we discuss how the Cav1 CSD may function through both lipid and protein interaction and propose an alternate view in which structural modifications to Cav1 oligomers may impact exposure of the CSD to cytoplasmic client proteins, such as eNOS.
Assuntos
Caveolina 1 , Transdução de Sinais , Animais , Humanos , Cavéolas/metabolismo , Caveolina 1/metabolismo , Caveolina 1/química , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Óxido Nítrico Sintase Tipo III/metabolismo , Domínios ProteicosRESUMO
Caveolin-1 (CAV1), the main structural component of caveolae, is phosphorylated at tyrosine-14 (pCAV1), regulates signal transduction, mechanotransduction, and mitochondrial function, and plays contrasting roles in cancer progression. We report that CRISPR/Cas9 knockout (KO) of CAV1 increases mitochondrial oxidative phosphorylation, increases mitochondrial potential, and reduces ROS in MDA-MB-231 triple-negative breast cancer cells. Supporting a role for pCAV1, these effects are reversed upon expression of CAV1 phosphomimetic CAV1 Y14D but not non-phosphorylatable CAV1 Y14F. pCAV1 is a known effector of Rho-associated kinase (ROCK) signaling and ROCK1/2 signaling mediates CAV1 promotion of increased mitochondrial potential and decreased ROS production in MDA-MB-231 cells. CAV1/ROCK control of mitochondrial potential and ROS is caveolae-independent as similar results were observed in PC3 prostate cancer cells lacking caveolae. Increased mitochondrial health and reduced ROS in CAV1 KO MDA-MB-231 cells were reversed by knockdown of the autophagy protein ATG5, mitophagy regulator PINK1 or the mitochondrial fission protein Drp1 and therefore due to mitophagy. Use of the mitoKeima mitophagy probe confirmed that CAV1 signaling through ROCK inhibited basal mitophagic flux. Activation of AMPK, a major mitochondrial homeostasis protein inhibited by ROCK, is inhibited by CAV1-ROCK signaling and mediates the increased mitochondrial potential, decreased ROS, and decreased basal mitophagy flux observed in wild-type MDA-MB-231 cells. CAV1 regulation of mitochondrial health and ROS in cancer cells therefore occurs via ROCK-dependent inhibition of AMPK. This study therefore links pCAV1 signaling activity at the plasma membrane with its regulation of mitochondrial activity and cancer cell metabolism through control of mitophagy.
Assuntos
Caveolina 1 , Neoplasias da Próstata , Masculino , Humanos , Caveolina 1/genética , Caveolina 1/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mecanotransdução Celular , Mitocôndrias/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Mitocondriais/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismoRESUMO
Mitochondria are major sources of cytotoxic reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, that when uncontrolled contribute to cancer progression. Maintaining a finely tuned, healthy mitochondrial population is essential for cellular homeostasis and survival. Mitophagy, the selective elimination of mitochondria by autophagy, monitors and maintains mitochondrial health and integrity, eliminating damaged ROS-producing mitochondria. However, mechanisms underlying mitophagic control of mitochondrial homeostasis under basal conditions remain poorly understood. E3 ubiquitin ligase Gp78 is an endoplasmic reticulum membrane protein that induces mitochondrial fission and mitophagy of depolarized mitochondria. Here, we report that CRISPR/Cas9 knockout of Gp78 in HT-1080 fibrosarcoma cells increased mitochondrial volume, elevated ROS production and rendered cells resistant to carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitophagy. These effects were phenocopied by knockdown of the essential autophagy protein ATG5 in wild-type HT-1080 cells. Use of the mito-Keima mitophagy probe confirmed that Gp78 promoted both basal and damage-induced mitophagy. Application of a spot detection algorithm (SPECHT) to GFP-mRFP tandem fluorescent-tagged LC3 (tfLC3)-positive autophagosomes reported elevated autophagosomal maturation in wild-type HT-1080 cells relative to Gp78 knockout cells, predominantly in proximity to mitochondria. Mitophagy inhibition by either Gp78 knockout or ATG5 knockdown reduced mitochondrial potential and increased mitochondrial ROS. Live cell analysis of tfLC3 in HT-1080 cells showed the preferential association of autophagosomes with mitochondria of reduced potential. Xenograft tumors of HT-1080 knockout cells show increased labeling for mitochondria and the cell proliferation marker Ki67 and reduced labeling for the TUNEL cell death reporter. Basal Gp78-dependent mitophagic flux is, therefore, selectively associated with reduced potential mitochondria promoting maintenance of a healthy mitochondrial population, limiting ROS production and tumor cell proliferation.
Assuntos
Mitofagia , Superóxidos , Humanos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Antígeno Ki-67/metabolismo , Superóxidos/metabolismo , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Autofagia/genéticaRESUMO
Drug repurposing can accelerate the identification of effective compounds for clinical use against SARS-CoV-2, with the advantage of pre-existing clinical safety data and an established supply chain. RNA viruses such as SARS-CoV-2 manipulate cellular pathways and induce reorganization of subcellular structures to support their life cycle. These morphological changes can be quantified using bioimaging techniques. In this work, we developed DEEMD: a computational pipeline using deep neural network models within a multiple instance learning framework, to identify putative treatments effective against SARS-CoV-2 based on morphological analysis of the publicly available RxRx19a dataset. This dataset consists of fluorescence microscopy images of SARS-CoV-2 non-infected cells and infected cells, with and without drug treatment. DEEMD first extracts discriminative morphological features to generate cell morphological profiles from the non-infected and infected cells. These morphological profiles are then used in a statistical model to estimate the applied treatment efficacy on infected cells based on similarities to non-infected cells. DEEMD is capable of localizing infected cells via weak supervision without any expensive pixel-level annotations. DEEMD identifies known SARS-CoV-2 inhibitors, such as Remdesivir and Aloxistatin, supporting the validity of our approach. DEEMD can be explored for use on other emerging viruses and datasets to rapidly identify candidate antiviral treatments in the future. Our implementation is available online at https://www.github.com/Sadegh-Saberian/DEEMD.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Antivirais/farmacologia , Antivirais/química , Antivirais/metabolismoRESUMO
Caveolin-1 (CAV1), the caveolae coat protein, also associates with non-caveolar scaffold domains. Single molecule localization microscopy (SMLM) network analysis distinguishes caveolae and three scaffold domains, hemispherical S2 scaffolds and smaller S1B and S1A scaffolds. The caveolin scaffolding domain (CSD) is a highly conserved hydrophobic region that mediates interaction of CAV1 with multiple effector molecules. F92A/V94A mutation disrupts CSD function, however the structural impact of CSD mutation on caveolae or scaffolds remains unknown. Here, SMLM network analysis quantitatively shows that expression of the CAV1 CSD F92A/V94A mutant in CRISPR/Cas CAV1 knockout MDA-MB-231 breast cancer cells reduces the size and volume and enhances the elongation of caveolae and scaffold domains, with more pronounced effects on S2 and S1B scaffolds. Convex hull analysis of the outer surface of the CAV1 point clouds confirms the size reduction of CSD mutant CAV1 blobs and shows that CSD mutation reduces volume variation amongst S2 and S1B CAV1 blobs at increasing shrink values, that may reflect retraction of the CAV1 N-terminus towards the membrane, potentially preventing accessibility of the CSD. Detection of point mutation-induced changes to CAV1 domains highlights the utility of SMLM network analysis for mesoscale structural analysis of oligomers in their native environment.
Assuntos
Caveolina 1/química , Domínios Proteicos/genética , Linhagem Celular , Humanos , Mutação , Conformação ProteicaRESUMO
The endoplasmic reticulum (ER) is a complex subcellular organelle composed of diverse structures such as tubules, sheets and tubular matrices. Flaviviruses such as Zika virus (ZIKV) induce reorganization of ER membranes to facilitate viral replication. Here, using 3D super resolution microscopy, ZIKV infection is shown to induce the formation of dense tubular matrices associated with viral replication in the central ER. Viral non-structural proteins NS4B and NS2B associate with replication complexes within the ZIKV-induced tubular matrix and exhibit distinct ER distributions outside this central ER region. Deep neural networks trained to distinguish ZIKV-infected versus mock-infected cells successfully identified ZIKV-induced central ER tubular matrices as a determinant of viral infection. Super resolution microscopy and deep learning are therefore able to identify and localize morphological features of the ER and allow for better understanding of how ER morphology changes due to viral infection.
Assuntos
Aprendizado Profundo , Retículo Endoplasmático/metabolismo , Microscopia/métodos , Zika virus/fisiologia , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular Tumoral , Retículo Endoplasmático/ultraestrutura , Matriz Extracelular/metabolismo , Humanos , Organoides/metabolismo , Organoides/ultraestrutura , Organoides/virologia , RNA de Cadeia Dupla/metabolismo , Proteínas não Estruturais Virais/metabolismo , Zika virus/ultraestrutura , Infecção por Zika virus/virologiaRESUMO
Caveolin-1 (CAV1) has long been implicated in cancer progression, and while widely accepted as an oncogenic protein, CAV1 also has tumor suppressor activity. CAV1 was first identified in an early study as the primary substrate of Src kinase, a potent oncoprotein, where its phosphorylation correlated with cellular transformation. Indeed, CAV1 phosphorylation on tyrosine-14 (Y14; pCAV1) has been associated with several cancer-associated processes such as focal adhesion dynamics, tumor cell migration and invasion, growth suppression, cancer cell metabolism, and mechanical and oxidative stress. Despite this, a clear understanding of the role of Y14-phosphorylated pCAV1 in cancer progression has not been thoroughly established. Here, we provide an overview of the role of Src-dependent phosphorylation of tumor cell CAV1 in cancer progression, focusing on pCAV1 in tumor cell migration, focal adhesion signaling and metabolism, and in the cancer cell response to stress pathways characteristic of the tumor microenvironment. We also discuss a model for Y14 phosphorylation regulation of CAV1 effector protein interactions via the caveolin scaffolding domain.
Assuntos
Caveolina 1/metabolismo , Neoplasias/metabolismo , Tirosina/metabolismo , Animais , Movimento Celular/fisiologia , Progressão da Doença , Adesões Focais/metabolismo , Humanos , Neoplasias/patologia , Fosforilação , Quinases da Família src/metabolismoRESUMO
Caveolae are an abundant, but enigmatic, plasma membrane feature of vertebrate cells. In this brief commentary, the authors attempt to answer some key questions related to the formation and function of caveolae based on round-table discussions at the first EMBO Workshop on Caveolae held in France in May 2019.
Assuntos
Cavéolas , Caveolinas , Animais , Membrana CelularRESUMO
Caveolin-1 is a transmembrane protein with both tumor promoter and suppressor functions that remain poorly understood. Cav1 phosphorylation by Src kinase on tyrosine 14 is closely associated with focal adhesion dynamics and tumor cell migration, however the role of pCav1 in vivo in tumor progression remains poorly characterized. Herein, we expressed phosphomimetic Y14D, wild type, and non-phosphorylatable Y14F forms of Cav1 in MDA-MB-435 cancer cells. Expression of Cav1Y14D reduced cell proliferation and induced the TP53 tumor suppressor. Ectopic expression in MDA-MB-435 cells of Y14 phosphorylatable Cav1 was required for induction of TP53 in response to oxidative stress. Cav1Y14D promotes an apparent reversal of the Warburg effect and markedly inhibited tumor growth in vivo. However, Cav1 induced pseudopodial recruitment of glycolytic enzymes, and time-lapse intravital imaging showed increased invadopodia protrusion and extravasation into blood vessels for Cav1WT and Y14D but not for Y14F. Our results suggest that Cav1 Y14 phosphorylation levels play a role in the conflicting demands on metabolic resources associated with cancer cell proliferation versus motility.
RESUMO
The endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle composed of smooth peripheral tubules and rough, ribosome-studded central ER sheets whose morphology is determined, in part, by the ER-shaping proteins, reticulon (RTN) and cytoskeleton-linking membrane protein 63 (CLIMP-63), respectively. Here, stimulated emission depletion (STED) super-resolution microscopy shows that reticulon4a (RTN4a) and CLIMP-63 also regulate the organization and dynamics of peripheral ER tubule nanodomains. STED imaging shows that lumenal ER monomeric oxidizing environment-optimized green fluorescent protein (ERmoxGFP), membrane Sec61ßGFP, knock-in calreticulin-GFP, and antibody-labeled ER-resident proteins calnexin and derlin-1 are all localized to periodic puncta along the length of peripheral ER tubules that are not readily observable by diffraction limited confocal microscopy. RTN4a segregates away from and restricts lumenal blob length, while CLIMP-63 associates with and increases lumenal blob length. RTN4a and CLIMP-63 also regulate the nanodomain distribution of ER-resident proteins, being required for the preferential segregation of calnexin and derlin-1 puncta away from lumenal ERmoxGFP blobs. High-speed (40 ms/frame) live cell STED imaging shows that RTN4a and CLIMP-63 regulate dynamic nanoscale lumenal compartmentalization along peripheral ER tubules. RTN4a enhances and CLIMP-63 disrupts the local accumulation of lumenal ERmoxGFP at spatially defined sites along ER tubules. The ER-shaping proteins RTN and CLIMP-63 therefore regulate lumenal ER nanodomain heterogeneity, interaction with ER-resident proteins, and dynamics in peripheral ER tubules.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Nogo/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Citoesqueleto/metabolismo , Humanos , Membranas/metabolismo , Microtúbulos/metabolismo , Microscopia Óptica não Linear/métodosRESUMO
Gp78/autocrine motility factor receptor (Gp78/AMFR) is a cancer-associated endoplasmic reticulum-localized E3 ubiquitin ligase and also the cell surface receptor for autocrine motility factor (AMF). The study objective was to determine the association between Gp78/AMFR and AMF endocytosis in thyroid cancer cells. Gp78/AMFR expression and AMF internalization were measured in differentiated thyroid cancer (DTC) and anaplastic thyroid cancer (ATC) cell lines and in freshly resected human papillary thyroid cancers (PTC) relative to benign thyroid tissue. Spheroid-like aggregates generated from explants of cancer, goiter, and collateral thyroid tissue were assessed for expression of cancer stem cell markers, surface Gp78/AMFR and AMF endocytosis. DTC cell lines showed elevated total and surface Gp78/AMFR and AMF internalization relative to ATC lines. Gp78/AMFR, Oct-4 and Sox-2 protein expression, Gp78/AMFR surface expression and AMF internalization were elevated in PTC-derived aggregates relative to fibroblasts. Elevated levels of Gp78/AMFR expression and AMF internalization in PTC were associated with expression of cancer stem cell markers. Gp78/AMFR expression and AMF uptake are more closely associated with DTC compared to benign thyroid lesions or ATC and with PTC-derived cancer stem-like cells.
RESUMO
Caveolin-1 (Cav1), a major Src kinase substrate phosphorylated on tyrosine-14 (Y14), contains the highly conserved membrane-proximal caveolin scaffolding domain (CSD; amino acids 82-101). Here we show, using CSD mutants (F92A/V94A) and membrane-permeable CSD-competing peptides, that Src kinase-dependent pY14Cav1 regulation of focal adhesion protein stabilization, focal adhesion tension, and cancer cell migration is CSD dependent. Quantitative proteomic analysis of Cav1-GST (amino acids 1-101) pull downs showed sixfold-increased binding of vinculin and, to a lesser extent, α-actinin, talin, and filamin, to phosphomimetic Cav1Y14D relative to nonphosphorylatable Cav1Y14F. Consistently, pY14Cav1 enhanced CSD-dependent vinculin tension in focal adhesions, dampening force fluctuation and synchronously stabilizing cellular focal adhesions in a high-tension mode, paralleling effects of actin stabilization. This identifies pY14Cav1 as a molecular regulator of focal adhesion tension and suggests that functional interaction between Cav1 Y14 phosphorylation and the CSD promotes focal adhesion traction and, thereby, cancer cell motility.
Assuntos
Caveolina 1/metabolismo , Movimento Celular/fisiologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Actinina/metabolismo , Actinas/metabolismo , Caveolina 1/genética , Adesão Celular/fisiologia , Células Cultivadas , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/genética , Adesões Focais/metabolismo , Humanos , Masculino , Mutação , Fosforilação , Domínios Proteicos , Talina/metabolismo , Tirosina/metabolismo , Vinculina/metabolismo , Quinases da Família src/metabolismoRESUMO
Viral hijacking and manipulation of host-cell biosynthetic pathways by human enveloped viruses are shared molecular events essential for the viral lifecycle. For Flaviviridae members such as hepatitis C virus and dengue virus (DENV), one of the key subsets of cellular pathways that undergo manipulation is the lipid metabolic pathways, underlining the importance of cellular lipids and, in particular, lipid droplets (LDs) in viral infection. Here, we hypothesize that targeting cellular enzymes that act as key regulators of lipid homeostasis and LD formation could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with all DENV serotypes (1-4) circulating around the world. Using PF-429242, an active-site-directed inhibitor of SKI-1/S1P, we demonstrate that inhibition of SKI-1/S1P enzymatic activity in human hepatoma Huh-7.5.1 cells results in a robust reduction of the LD numbers and LD-positive areas and provides a means of effectively inhibiting infection by DENV (1-4). Pre-treatment of Huh-7.5.1 cells with PF-429242 results in a dose-dependent inhibition of DENV infection [median inhibitory dose (EC50) = 1.2 microM; median cytotoxic dose (CC50) = 81 microM; selectivity index (SI) = 68)] and a ~3-log decrease in DENV-2 titer with 20 microM of PF-429242. Post-treatment of DENV-2 infected Huh-7.5.1 cells with PF-429242 does not affect viral RNA abundance, but it does compromise the assembly and/or release of infectious virus particles. PF-429242 antiviral activity is reversed by exogenous oleic acid, which acts as an inducer of LD formation in PF-429242-treated and non-treated control cells. Collectively, our results demonstrate that human SKI-1/S1P is a potential target for indirect-acting pan-serotypic anti-DENV agents and reveal new therapeutic opportunities associated with the use of lipid-modulating drugs for controlling DENV infection.
Assuntos
Antivirais/uso terapêutico , Citoplasma/metabolismo , Dengue/tratamento farmacológico , Gotículas Lipídicas/metabolismo , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Linhagem Celular Tumoral , Citoplasma/efeitos dos fármacos , Dengue/metabolismo , Dengue/virologia , Vírus da Dengue/efeitos dos fármacos , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Pirrolidinas/farmacologia , Replicação Viral/efeitos dos fármacosRESUMO
The reperfusion of ischemic brain tissue following a cerebral stroke causes oxidative stress, and leads to the generation of reactive oxygen species (ROS). Apart from inflicting oxidative damage, the latter may also trigger the upregulation of aquaporin 4 (AQP4), a water-permeable channel expressed by astroglial cells of the blood-brain barrier (BBB), and contribute to edema formation, the severity of which is known to be the primary determinant of mortality and morbidity. The mechanism through which this occurs remains unknown. In the present study, we have attempted to address this question using primary astrocyte cultures treated with hydrogen peroxide (H2O2) as a model system. First, we showed that H2O2 induces a significant increase in AQP4 protein levels and that this is inhibited by the antioxidant N-acetylcysteine (NAC). Second, we demonstrated using cell surface biotinylation that H2O2 increases AQP4 cell-surface expression independently of it's increased synthesis. In parallel, we found that caveolin-1 (Cav1) is phosphorylated in response to H2O2 and that this is reversed by the Src kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). PP2 also abrogated the H2O2-induced increase in AQP4 surface levels, suggesting thatâ the phosphorylation of tyrosine-14 of Cav1 regulatesâ thisâ process. Weâ furtherâ showedâ that dominant-negative Y14F and phosphomimetic Y14D mutants caused a decrease and increase in AQP4 membrane expression respectively, and that the knockdown of Cav1 inhibits the increase in AQP4 cell-surface, expression following H2O2 treatment. Together, these findings suggest that oxidative stress-induced Cav1 phosphorylation modulates AQP4 subcellular distribution and therefore may indirectly regulate AQP4-mediated water transport.
RESUMO
OBJECTIVE: The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells. METHODS: We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies. RESULTS: Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact. CONCLUSIONS: We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation.
RESUMO
Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER; however, the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an 8-amino acid motif (533-541) in mouse Gp78 isoform 2 that forms part of a highly conserved 41-amino acid region containing 14-3-3- and WW-binding domains and a p38 MAP kinase (p38 MAPK) consensus site on Ser-538 (S538). 3F3A binds selectively to nonphosphorylated S538 Gp78. Using 3F3A as a reporter, we induced Gp78 S538 phosphorylation by serum starvation and showed it to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fission and degrade MFN1 and MFN2 but did not affect in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER-mitochondria interaction, and SB203580 inhibition of p38 MAPK increased ER-mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER-mitochondria association and mitochondria motility.
Assuntos
Mitocôndrias/metabolismo , Receptores do Fator Autócrino de Motilidade/metabolismo , Animais , Linhagem Celular , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático/genética , Humanos , Camundongos , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Fosforilação , Receptores do Fator Autócrino de Motilidade/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0119954.].
RESUMO
Gp78 (also known as AMFR), an endoplasmic-reticulum (ER)-associated protein degradation (ERAD) E3 ubiquitin ligase, localizes to mitochondria-associated ER and targets the mitofusin (Mfn1 and Mfn2) mitochondrial fusion proteins for degradation. Gp78 is also the cell surface receptor for autocrine motility factor (AMF), which prevents Gp78-dependent mitofusin degradation. Gp78 ubiquitin ligase activity promotes ER-mitochondria association and ER-mitochondria Ca(2+) coupling, processes that are reversed by AMF. Electron microscopy of HT-1080 fibrosarcoma cancer cells identified both smooth ER (SER; â¼8â nm) and wider (â¼50-60â nm) rough ER (RER)-mitochondria contacts. Both short hairpin RNA (shRNA)-mediated knockdown of Gp78 (shGp78) and AMF treatment selectively reduced the extent of RER-mitochondria contacts without impacting on SER--mitochondria contacts. Concomitant small interfering RNA (siRNA)-mediated knockdown of Mfn1 increased SER-mitochondria contacts in both control and shGp78 cells, whereas knockdown of Mfn2 increased RER-mitochondria contacts selectively in shGp78 HT-1080 cells. The mitofusins therefore inhibit ER-mitochondria interaction. Regulation of close SER-mitochondria contacts by Mfn1 and of RER-mitochondria contacts by AMF-sensitive Gp78-mediated degradation of Mfn2 define new mechanisms that regulate ER-mitochondria interactions.