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[This corrects the article DOI: 10.1371/journal.pone.0253364.].
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Acute lysosomal membrane damage reduces the cellular population of functional lysosomes. However, these damaged lysosomes have a remarkable recovery potential independent of lysosomal biogenesis and remain unaffected in cells depleted in TFEB and TFE3. We combined proximity-labelling-based proteomics, biochemistry and high-resolution microscopy to unravel a lysosomal membrane regeneration pathway that depends on ATG8, the lysosomal membrane protein LIMP2, the RAB7 GTPase-activating protein TBC1D15 and proteins required for autophagic lysosomal reformation, including dynamin-2, kinesin-5B and clathrin. Following lysosomal damage, LIMP2 acts as a lysophagy receptor to bind ATG8, which in turn recruits TBC1D15 to damaged membranes. TBC1D15 interacts with ATG8 proteins on damaged lysosomes and provides a scaffold to assemble and stabilize the autophagic lysosomal reformation machinery. This potentiates the formation of lysosomal tubules and subsequent dynamin-2-dependent scission. TBC1D15-mediated lysosome regeneration was also observed in a cell culture model of oxalate nephropathy.
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Autofagia , Dinamina II , Dinamina II/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Lisosomas/metabolismoRESUMEN
Ubiquitination is a posttranslational modification that regulates a multitude of cellular functions. Pathogens, such as bacteria and viruses, have evolved sophisticated mechanisms that evade or counteract ubiquitin-dependent host responses, or even exploit the ubiquitin system to their own advantage. This is largely done by numerous pathogen virulence factors that encode E3 ligases and deubiquitinases, which are often used as weapons in pathogen-host cell interactions. Moreover, upon pathogen attack, host cellular signaling networks undergo major ubiquitin-dependent changes to protect the host cell, including coordination of innate immunity, remodeling of cellular organelles, reorganization of the cytoskeleton, and reprogramming of metabolic pathways to restrict growth of the pathogen. Here we provide mechanistic insights into ubiquitin regulation of host-pathogen interactions and how it affects bacterial and viral pathogenesis and the organization and response of the host cell.
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Interacciones Huésped-Patógeno , Ubiquitina , Bacterias/metabolismo , Inmunidad Innata , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Factores de Virulencia/metabolismoRESUMEN
SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.
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Aparato de Golgi/patología , Legionella/patogenicidad , Serina/metabolismo , Humanos , UbiquitinaciónRESUMEN
Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.
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Antivirales/farmacología , Inhibidores de Proteasas/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Anticuerpos de Cadena Única/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Células A549 , Complejo Antígeno-Anticuerpo , Humanos , Concentración 50 Inhibidora , ARN Polimerasa Dependiente del ARN/inmunología , ARN Polimerasa Dependiente del ARN/metabolismo , Anticuerpos de Cadena Única/inmunología , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismoRESUMEN
Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.
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Famotidina/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , SARS-CoV-2/efectos de los fármacos , Receptor Toll-Like 3/metabolismo , Células A549 , Sitios de Unión , Células CACO-2 , Quimiocina CCL2/metabolismo , Proteasas 3C de Coronavirus/metabolismo , Células HeLa , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interleucina-6/metabolismo , Simulación del Acoplamiento Molecular , FN-kappa B/metabolismo , Unión Proteica , SARS-CoV-2/fisiología , Transducción de Señal , Receptor Toll-Like 3/química , Replicación ViralRESUMEN
Turnover of cellular organelles, including endoplasmic reticulum (ER) and mitochondria, is orchestrated by an efficient cellular surveillance system. We have identified a mechanism for dual regulation of ER and mitochondria under stress. It is known that AMFR, an ER E3 ligase and ER-associated degradation (ERAD) regulator, degrades outer mitochondrial membrane (OMM) proteins, MFNs (mitofusins), via the proteasome and triggers mitophagy. We show that destabilized mitochondria are almost devoid of the OMM and generate "mitoplasts". This brings the inner mitochondrial membrane (IMM) in the proximity of the ER. When AMFR levels are high and the mitochondria are stressed, the reticulophagy regulatory protein RETREG1 participates in the formation of the mitophagophore by interacting with OPA1. Interestingly, OPA1 and other IMM proteins exhibit similar RETREG1-dependent autophagosomal degradation as AMFR, unlike most of the OMM proteins. The "mitoplasts" generated are degraded by reticulo-mito-phagy - simultaneously affecting dual organelle turnover.Abbreviations: AMFR/GP78: autocrine motility factor receptor; BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; BFP: blue fluorescent protein; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; CNBr: cyanogen bromide; ER: endoplasmic reticulum; ERAD: endoplasmic-reticulum-associated protein degradation; FL: fluorescence, GFP: green fluorescent protein; HA: hemagglutinin; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN: mitofusin, MGRN1: mahogunin ring finger 1; NA: numerical aperature; OMM: outer mitochondrial membrane; OPA1: OPA1 mitochondrial dynamin like GTPase; PRNP/PrP: prion protein; RER: rough endoplasmic reticulum; RETREG1/FAM134B: reticulophagy regulator 1; RFP: red fluorescent protein; RING: really interesting new gene; ROI: region of interest; RTN: reticulon; SEM: standard error of the mean; SER: smooth endoplasmic reticulum; SIM: structured illumination microscopy; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STOML2: stomatin like 2; TOMM20: translocase of outer mitochondrial membrane 20; UPR: unfolded protein response.
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Autofagosomas/metabolismo , GTP Fosfohidrolasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores del Factor Autocrino de Motilidad/metabolismo , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Lisosomas/metabolismo , Microscopía Confocal , Microscopía Electrónica de Transmisión , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.
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COVID-19/inmunología , COVID-19/virología , Proteasas Similares a la Papaína de Coronavirus/química , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Inmunidad Innata , SARS-CoV-2/enzimología , SARS-CoV-2/inmunología , Animales , Proteasas Similares a la Papaína de Coronavirus/antagonistas & inhibidores , Citocinas/química , Citocinas/metabolismo , Enzimas Desubicuitinizantes/antagonistas & inhibidores , Enzimas Desubicuitinizantes/química , Enzimas Desubicuitinizantes/metabolismo , Humanos , Factor 3 Regulador del Interferón/metabolismo , Interferones/inmunología , Interferones/metabolismo , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , FN-kappa B/inmunología , FN-kappa B/metabolismo , Unión Proteica , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Ubiquitinación , Ubiquitinas/química , Ubiquitinas/metabolismo , Tratamiento Farmacológico de COVID-19RESUMEN
The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection.
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Enzimas Desubicuitinizantes/metabolismo , Serina/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Células A549 , Proteínas Bacterianas/metabolismo , Dominio Catalítico/fisiología , Línea Celular , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno/fisiología , Humanos , Legionella pneumophila/patogenicidad , Enfermedad de los Legionarios/metabolismo , Vacuolas/metabolismoRESUMEN
Presence of cytosolic protein aggregates and membrane damage are two common attributes of neurodegenerative diseases. These aggregates delay degradation of non-translocated protein precursors leading to their persistence and accumulation in the cytosol. Here, we find that cells with intracellular protein aggregates (of cytosolic prion protein or huntingtin) destabilize the endoplasmic reticulum (ER) morphology and dynamics when non-translocated protein load is high. This affects trafficking of proteins out from the ER, relative distribution of the rough and smooth ER and three-way junctions that are essential for the structural integrity of the membrane network. The changes in ER membranes may be due to high aggregation tendency of the ER structural proteins-reticulons, and altered distribution of those associated with the three-way ER junctions-Lunapark. Reticulon4 is seen to be enriched in the aggregate fractions in presence of non-translocated protein precursors. This could be mitigated by improving signal sequence efficiencies of the proteins targeted to the ER. These were observed using PrP variants and the seven-pass transmembrane protein (CRFR1) with different signal sequences that led to diverse translocation efficiencies. This identifies a previously unappreciated consequence of cytosolic aggregates on non-translocated precursor proteins-their persistent presence affects ER morphology and dynamics. This may be one of the ways in which cytosolic aggregates can affect endomembranes during neurodegenerative disease.
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Retículo Endoplásmico/metabolismo , Agregado de Proteínas , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Nogo/metabolismo , Proteínas Priónicas/química , Proteínas Priónicas/metabolismo , Unión Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína , Transporte de Proteínas , Receptores de Hormona Liberadora de Corticotropina/química , Receptores de Hormona Liberadora de Corticotropina/metabolismoRESUMEN
The family of bacterial SidE enzymes catalyses phosphoribosyl-linked serine ubiquitination and promotes infectivity of Legionella pneumophila, a pathogenic bacteria that causes Legionnaires' disease1-3. SidE enzymes share the genetic locus with the Legionella effector SidJ that spatiotemporally opposes the toxicity of these enzymes in yeast and mammalian cells, through a mechanism that is currently unknown4-6. Deletion of SidJ leads to a substantial defect in the growth of Legionella in both its natural hosts (amoebae) and in mouse macrophages4,5. Here we demonstrate that SidJ is a glutamylase that modifies the catalytic glutamate in the mono-ADP ribosyl transferase domain of the SdeA, thus blocking the ubiquitin ligase activity of SdeA. The glutamylation activity of SidJ requires interaction with the eukaryotic-specific co-factor calmodulin, and can be regulated by intracellular changes in Ca2+ concentrations. The cryo-electron microscopy structure of SidJ in complex with human apo-calmodulin revealed the architecture of this heterodimeric glutamylase. We show that, in cells infected with L. pneumophila, SidJ mediates the glutamylation of SidE enzymes on the surface of vacuoles that contain Legionella. We used quantitative proteomics to uncover multiple host proteins as putative targets of SidJ-mediated glutamylation. Our study reveals the mechanism by which SidE ligases are inhibited by a SidJ-calmodulin glutamylase, and opens avenues for exploring an understudied protein modification (glutamylation) in eukaryotes.
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Proteínas Bacterianas/metabolismo , Calmodulina/metabolismo , Ácido Glutámico/metabolismo , Legionella pneumophila/enzimología , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina/metabolismo , Factores de Virulencia/metabolismo , ADP-Ribosilación , Apoproteínas/metabolismo , Proteínas Bacterianas/agonistas , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Calmodulina/farmacología , Catálisis , Microscopía por Crioelectrón , Cristalografía por Rayos X , Células HEK293 , Humanos , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidad , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Ubiquitina/química , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Factores de Virulencia/agonistas , Factores de Virulencia/químicaRESUMEN
In healthy cells, dysfunctional mitochondria are removed by selective autophagy (mitophagy), impairment of which causes disease. In this issue of Developmental Cell, Princely Abudu et al. (2019) delineate the function of NIPSNAP1 and NIPSNAP2 in recruiting mitophagy receptors to depolarized mitochondria, highlighting their importance in the zebrafish brain.
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Autofagia , Mitofagia , MitocondriasRESUMEN
The mechanism by which the endoplasmic reticulum (ER) ubiquitin ligases sense stress to potentiate their activity is poorly understood. GP78, an ER E3 ligase, is best known for its role in ER-associated protein degradation, although its activity is also linked to mitophagy, ER-mitochondria junctions, and MAPK signaling, thus highlighting the importance of understanding its regulation. In healthy cells, Mahogunin really interesting new gene (RING) finger 1 (MGRN1) interacts with GP78 and proteasomally degrades it to alleviate mitophagy. Here, we identify calmodulin (CaM) as the adapter protein that senses fluctuating cytosolic Ca2+ levels and modulates the Ca2+-dependent MGRN1-GP78 interactions. When stress elevates cytosolic Ca2+ levels in cultured and primary neuronal cells, CaM binds to both E3 ligases and inhibits their interaction. Molecular docking, simulation, and biophysical studies show that CaM interacts with both proteins with different affinities and binding modes. The physiological impact of this interaction switch manifests in the regulation of ER-associated protein degradation, ER-mitochondria junctions, and relative distribution of smooth ER and rough ER.-Mukherjee, R., Bhattacharya, A., Sau, A., Basu, S., Chakrabarti, S., Chakrabarti, O. Calmodulin regulates MGRN1-GP78 interaction mediated ubiquitin proteasomal degradation system.
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Calmodulina/metabolismo , Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Receptores del Factor Autocrino de Motilidad/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Animales , Señalización del Calcio , Calmodulina/química , Calmodulina/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Ratones , Simulación del Acoplamiento Molecular , Neuronas/citología , Complejo de la Endopetidasa Proteasomal/genética , Receptores del Factor Autocrino de Motilidad/química , Receptores del Factor Autocrino de Motilidad/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
MGRN1-mediated ubiquitination of α-tubulin regulates microtubule stability and mitotic spindle positioning in mitotic cells. This study elucidates the effect of MGRN1-mediated ubiquitination of α-tubulin in interphase cells. Here, we show that MGRN1-mediated ubiquitination regulates dynamics of EB1-labeled plus ends of microtubules. Intracellular transport of mitochondria and endosomes are affected in cultured cells where functional MGRN1 is depleted. Defects in microtubule-dependent organellar transport are evident in cells where noncanonical K6-mediated ubiquitination of α-tubulin by MGRN1 is compromised. Loss of MGRN1 has been previously correlated with late-onset spongiform neurodegeneration. Mislocalised cytosolically exposed PrP (Ctm PrP) interacts with MGRN1 leading to its loss of function. Expression of Ctm PrP generating mutants of PrP[PrP(A117V) and PrP(KHII)] lead to decrease in MGRN1-mediated ubiquitination of α-tubulin and intracellular transport defects. Brain lysates from PrP(A117V) transgenic mice also indicate loss of tubulin polymerization as compared to non-transgenic controls. Depletion of MGRN1 activity may hamper physiologically important processes like mitochondrial movement in neuronal processes and intracellular transport of ligands through the endosomal pathway thereby contributing to the pathogenesis of neurodegeneration in certain types of prion diseases.
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Transporte Biológico/fisiología , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células HeLa , Humanos , Microtúbulos/metabolismo , Mitocondrias/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The ubiquitination status of proteins and intracellular calcium levels are two factors which keep changing inside any living cell. These two events appear to be independent of each other but recent experimental evidences show that ubiquitination of cellular proteins are influenced by calcium, Calmodulin, Calmodulin-dependent kinase II and other proteins of calcium dependent pathways. E3 ligases like Nedd4, SCF complex, APC, GP78 and ITCH are important regulators of calcium mediated processes. A bioinformatics analysis to inspect sequences and interacting partners of 242 candidate E3 ligases show the presence of calcium and/or Calmodulin binding motifs/domains within their sequences. Building a protein-protein interaction (PPI) network of human E3 ligase proteins identifies Ca2+ related proteins as direct interacting partners of E3 ligases. Review of literature, analysis of E3 ligase sequences and their interactome suggests an interconnectivity between calcium signaling and the overall UPS system, especially emphasizing that a subset of E3 ligases have importance in physiological pathways modulated by calcium.
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Señalización del Calcio , Calmodulina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Calmodulina/química , Humanos , Mapas de Interacción de ProteínasRESUMEN
Health and homoeostasis are maintained by a dynamic balance between mitochondrial fission and fusion. Mitochondrial fusion machinery is largely unknown in mammals. Only a few reports have illustrated the role of Fzo1 in mitochondrial fusion known in Saccharomyces cerevisiae. We demonstrate that the ubiquitin ligase Mahogunin Ring Finger-1 (MGRN1) interacts with and constitutively ubiquitinates the mammalian homolog, Mitofusin1 (Mfn1) via K63 linkages. In mice models, loss of Mgrn1 function leads to severe developmental defects and adult-onset spongiform neurodegeneration, similar to prion diseases. The tethering of mitochondria to form the ~180kDa Mfn1 complex is independent of MGRN1-mediated ubiquitination. However, successful mitochondrial fusion requires formation of higher oligomers of Mfn1 which in turn needs GTPase activity, intact heptad repeats of Mfn1 and ubiquitination by MGRN1. Following ubiquitination, proteasomal processing of Mfn1 completes the mitochondrial fusion process. This step requires functional p97 activity. These findings suggest a sequence of events where GTPase activity of Mfn1 and tethering of adjacent mitochondria precedes its MGRN1-mediated ubiquitination and proteasomal degradation culminating in mitochondrial fusion.
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GTP Fosfohidrolasas/genética , Mitocondrias/genética , Dinámicas Mitocondriales/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Neuronas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Animales , Línea Celular Tumoral , Fibroblastos/citología , Fibroblastos/metabolismo , GTP Fosfohidrolasas/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Melanocitos/citología , Melanocitos/metabolismo , Ratones , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Neuronas/citología , Complejo de la Endopetidasa Proteasomal/metabolismo , Multimerización de Proteína , Proteolisis , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMEN
Mahogunin RING Finger 1 (MGRN1) is a ubiquitin E3 ligase known to affect spindle tilt in mitotic cells by regulating α-tubulin ubiquitination and polymerization. In cell culture systems we have found that expressing truncated mutants of MGRN1 leads to various other mitotic anomalies, such as lateral and angular spindle displacements. This seems to be independent of the MGRN1 ligase activity. Our experiments suggest that MGRN1 regulates the balance between the lower molecular weight monomeric Gαi and larger trimeric G-protein complex, along with its abundance in the ternary complex that regulates spindle positioning. The cytosolic isoforms of MGRN1 lead to the enrichment of monomeric Gαi in the cytosol and its subsequent recruitment at the plasma membrane. Excess Gαi at the cell cortex results in an imbalance in the assembly of the ternary complex regulating spindle positioning during mitosis. These observations seem independent of the ligase activity of MGRN1, although we cannot exclude the involvement of an intermediate player that acts as a substrate for MGRN1, and in turn, regulates Gαi.
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Ciclo Celular/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Mitosis/fisiología , Huso Acromático/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Western Blotting , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Células HeLa , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Cellular quality control provides an efficient surveillance system to regulate mitochondrial turnover. This study elucidates a new interaction between the cytosolic E3 ligase mahogunin RING finger 1 (MGRN1) and the endoplasmic reticulum (ER) ubiquitin E3 ligase GP78 (also known as AMFR). Loss of Mgrn1 function has been implicated in late-onset spongiform neurodegeneration and congenital heart defects, among several developmental defects. Here, we show that MGRN1 ubiquitylates GP78 in trans through non-canonical K11 linkages. This helps maintain constitutively low levels of GP78 in healthy cells, in turn downregulating mitophagy. GP78, however, does not regulate MGRN1. When mitochondria are stressed, cytosolic Ca(2+) increases. This leads to a reduced interaction between MGRN1 and GP78 and its compromised ubiquitylation. Chelating Ca(2+) restores association between the two ligases and the in trans ubiquitylation. Catalytic inactivation of MGRN1 results in elevated levels of GP78 and a consequential increase in the initiation of mitophagy. This is important because functional depletion of MGRN1 by the membrane-associated disease-causing prion protein (Ctm)PrP affects polyubiquitylation and degradation of GP78, also leading to an increase in mitophagy events. This suggests that MGRN1 participates in mitochondrial quality control and could contribute to neurodegeneration in a subset of (Ctm)PrP-mediated prion diseases.
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Mitocondrias/metabolismo , Receptores del Factor Autocrino de Motilidad/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Ubiquitina/metabolismo , Ubiquitinación , Animales , Células HeLa , Homeostasis , Humanos , Ratones , Mitofagia , Complejo de la Endopetidasa Proteasomal/metabolismo , ProteolisisRESUMEN
The cell has an intricate quality control system to protect its mitochondria from oxidative stress. This surveillance system is multi-tiered and comprises molecules that are present inside the mitochondria, in the cytosol, and in other organelles like the nucleus and endoplasmic reticulum. These molecules cross talk with each other and protect the mitochondria from oxidative stress. Oxidative stress is a fundamental part of early disease pathogenesis of neurodegenerative diseases. These disorders also damage the cellular quality control machinery that protects the cell against oxidative stress. This exacerbates the oxidative damage and causes extensive neuronal cell death that is characteristic of neurodegeneration.