Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.862
Filtrar
1.
Nat Commun ; 12(1): 2616, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972548

RESUMO

FUN14 domain-containing protein 1 (FUNDC1) is an integral mitochondrial outer-membrane protein, and mediates the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs). This study aims to determine the contributions of FUNDC1-mediated MAMs to angiogenesis in vitro and in vivo. In cultured endothelial cells, VEGF significantly increases the formation of MAMs and MAM-related proteins, including FUNDC1. Endothelial cell-specific deletion of FUNDC1, which disrupts MAM formation in endothelial cells, lowers VEGFR2 expression and reduces tube formation, spheroid-sprouting, and functional blood vessel formation in vitro and in vivo. Conversely, increased MAM formation using MAM linkers mimics the effects of VEGF and promotes endothelial angiogenesis. Mechanistically, increased MAMs formation led to increased levels of Ca2+ in cytosol, promoted the phosphorylation of serum response factor (SRF) and enhanced the binding of SRF to VEGFR2 promoter, resulting in increased VEGFR2 production, with consequent angiogenesis. Moreover, blocking FUNDC1-related MAM formation with a cell-penetrating inhibitory peptide significantly suppresses the expressions of downstream angiogenic genes and inhibits tumor angiogenesis. We conclude that decreased MAMs formation by silencing FUNDC1 can inhibit angiogenesis by decreasing VEGFR2 expression, and targeting FUNDC1-dependent MAMs might be a promising approach for treating human disorders characterized by defective angiogenesis.


Assuntos
Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Animais , Cálcio/metabolismo , Citoplasma/metabolismo , Retículo Endoplasmático/ultraestrutura , Inativação Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Membranas Intracelulares/efeitos dos fármacos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , RNA Interferente Pequeno , Retina/metabolismo , Fator de Resposta Sérica/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Nat Commun ; 12(1): 2034, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795673

RESUMO

COPII mediates Endoplasmic Reticulum to Golgi trafficking of thousands of cargoes. Five essential proteins assemble into a two-layer architecture, with the inner layer thought to regulate coat assembly and cargo recruitment, and the outer coat forming cages assumed to scaffold membrane curvature. Here we visualise the complete, membrane-assembled COPII coat by cryo-electron tomography and subtomogram averaging, revealing the full network of interactions within and between coat layers. We demonstrate the physiological importance of these interactions using genetic and biochemical approaches. Mutagenesis reveals that the inner coat alone can provide membrane remodelling function, with organisational input from the outer coat. These functional roles for the inner and outer coats significantly move away from the current paradigm, which posits membrane curvature derives primarily from the outer coat. We suggest these interactions collectively contribute to coat organisation and membrane curvature, providing a structural framework to understand regulatory mechanisms of COPII trafficking and secretion.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Mapas de Interação de Proteínas , Proteínas de Transporte Vesicular/metabolismo , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Células Sf9 , Spodoptera
3.
Science ; 372(6545): 935-941, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33927055

RESUMO

During infection, intracellular bacterial pathogens translocate a variety of effectors into host cells that modify host membrane trafficking for their benefit. We found a self-organizing system consisting of a bacterial phosphoinositide kinase and its opposing phosphatase that formed spatiotemporal patterns, including traveling waves, to remodel host cellular membranes. The Legionella effector MavQ, a phosphatidylinositol (PI) 3-kinase, was targeted to the endoplasmic reticulum (ER). MavQ and the Legionella PI 3-phosphatase SidP, even in the absence of other bacterial components, drove rapid PI 3-phosphate turnover on the ER and spontaneously formed traveling waves that spread along ER subdomains inducing vesicle and tubule budding. Thus, bacteria can exploit a self-organizing membrane-targeting mechanism to hijack host cellular structures for survival.


Assuntos
Proteínas de Bactérias/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Legionella pneumophila/fisiologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Proteínas de Bactérias/química , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Retroalimentação Fisiológica , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Membranas Intracelulares/ultraestrutura , Legionella pneumophila/enzimologia , Legionella pneumophila/genética , Legionella pneumophila/crescimento & desenvolvimento , Camundongos , Mutação , Fosfatidilinositol 3-Quinase/química , Fosfatos de Fosfatidilinositol/química , Monoéster Fosfórico Hidrolases/metabolismo , Domínios Proteicos , Células RAW 264.7
4.
Oxid Med Cell Longev ; 2021: 8810698, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815661

RESUMO

Programmed necrosis of nucleus pulposus (NP) cells caused by excessive compression is a crucial factor in the etiopathogenesis of intervertebral disc degeneration (IVDD). The endoplasmic reticulum (ER) and mitochondria are crucial regulators of the cell death signaling pathway, and their involvement in IVDD has been reported. However, the specific role of ER stress (ERS) and ER-mitochondria interaction in compression-induced programmed necrosis of NP cells remains unknown. Our studies revealed that compression enhanced ERS and the association between ER and mitochondria in NP cells. Suppression of ERS via 4-phenylbutyrate (4-PBA) or ER-mitochondrial Ca2+ crosstalk by inhibiting the inositol 1,4,5-trisphosphate receptor, glucose-regulated protein 75, voltage-dependent anion-selective channel 1 complex (IP3R-GRP75-VDAC1 complex) protected NP cells against programmed necrosis related to the poly(ADP-ribose) polymerase (PARP) apoptosis-inducing factor (AIF) pathway. Moreover, excessive reactive oxygen species are critical activators of ERS, leading to mitochondrial Ca2+ accumulation and consequent programmed necrosis. These data indicate that ERS and ER-mitochondrial Ca2+ crosstalk may be potential therapeutic targets for the treatment of IVDD-associated disorders. These findings provide new insights into the molecular mechanisms underlying IVDD and may provide novel therapeutic targets.


Assuntos
Cálcio/metabolismo , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Núcleo Pulposo/patologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Fator de Indução de Apoptose/metabolismo , Força Compressiva , Citoproteção , Retículo Endoplasmático/ultraestrutura , Proteínas de Choque Térmico HSP70/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Necrose , Núcleo Pulposo/ultraestrutura , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos Sprague-Dawley , Canal de Ânion 1 Dependente de Voltagem/metabolismo
5.
Nat Commun ; 12(1): 720, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526781

RESUMO

Cellular senescence is induced by stresses and results in a stable proliferation arrest accompanied by a pro-inflammatory secretome. Senescent cells accumulate during aging, promoting various age-related pathologies and limiting lifespan. The endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) calcium-release channel and calcium fluxes from the ER to the mitochondria are drivers of senescence in human cells. Here we show that Itpr2 knockout (KO) mice display improved aging such as increased lifespan, a better response to metabolic stress, less immunosenescence, as well as less liver steatosis and fibrosis. Cellular senescence, which is known to promote these alterations, is decreased in Itpr2 KO mice and Itpr2 KO embryo-derived cells. Interestingly, ablation of ITPR2 in vivo and in vitro decreases the number of contacts between the mitochondria and the ER and their forced contacts induce premature senescence. These findings shed light on the role of contacts and facilitated exchanges between the ER and the mitochondria through ITPR2 in regulating senescence and aging.


Assuntos
Senescência Celular/fisiologia , Retículo Endoplasmático/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Longevidade/fisiologia , Mitocôndrias/metabolismo , Animais , Cálcio/metabolismo , Retículo Endoplasmático/ultraestrutura , Feminino , Fibroblastos , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Camundongos , Camundongos Knockout , Microscopia Confocal , Mitocôndrias/ultraestrutura , RNA Interferente Pequeno , Período Refratário Eletrofisiológico , Análise de Célula Única
6.
Virology ; 556: 9-22, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33524849

RESUMO

Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection.


Assuntos
Infecções por Coronavirus/virologia , Retículo Endoplasmático/metabolismo , Vírus da Hepatite Murina/fisiologia , Compartimentos de Replicação Viral/metabolismo , Animais , Linhagem Celular , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático/virologia , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Lisossomos/virologia , Camundongos , Proteínas Virais/metabolismo , Compartimentos de Replicação Viral/ultraestrutura , Vírion/metabolismo , Montagem de Vírus , Replicação Viral
7.
Plant Sci ; 304: 110825, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33568283

RESUMO

Plants have evolved sophisticated mechanisms to adjust to deficiency or excess of nutrients. Membrane transport proteins play a central role in nutrient uptake from soil. In Arabidopsis thaliana, the COPPER TRANSPORTOR (COPT) family encodes high-affinity copper transporters. COPT2 is transcriptionally regulated in response to changing levels of cellular copper. However, little is known about whether COPT2 activity is subject to multiple levels of regulation. Here, we showed that the plasma membrane-/endoplasmic reticulum-resident COPT2 protein is degraded in response to excess copper. Confocal microscopy analysis together with pharmacological treatment with a vesicle trafficking inhibitor or vacuolar ATPase inhibitor indicated that copper-mediated downregulation of COPT2 is unlikely to be controlled by endosomal recycling and vacuolar system. However, COPT2 protein is stabilized by proteasome inhibition. Through site-directed mutagenesis, we found that COPT2 cannot be ubiquitinated, and lysine residues at the C-terminus is dispensable for copper-induced degradation of COPT2 but required for copper acquisition. Altogether, our findings reveal that unlike many metal transporters in Arabidopsis, COPT2 is a substrate of ubiquitin-independent proteasomal degradation but not of vacuolar proteases. These findings highlight the mechanistic diversity and complexity of plasma membrane transporter degradation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas SLC31/metabolismo , Ubiquitina/metabolismo , Arabidopsis/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Cobre/metabolismo , Proteínas de Transporte de Cobre/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Microscopia Confocal , Reação em Cadeia da Polimerase , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação
8.
Biol Cell ; 113(6): 281-293, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33600624

RESUMO

BACKGROUND INFORMATION: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces an alteration in the endomembrane system of the mammalian cells. In this study, we used transmission electron microscopy and electron tomography to investigate the main structural alterations in the cytoplasm of Vero cells infected with a SARS-CoV-2 isolate from São Paulo state (Brazil). RESULTS: Different membranous structures derived from the zippered endoplasmic reticulum were observed along with virus assembly through membrane budding. Also, we demonstrated the occurrence of annulate lamellae in the cytoplasm of infected cells and the presence of virus particles in the perinuclear space. CONCLUSIONS AND SIGNIFICANCE: This study contributes to a better understanding of the cell biology of SARS-CoV-2 and the mechanisms of the interaction of the virus with the host cell that promote morphological changes, recruitment of organelles and cell components, in a context of a virus-induced membrane remodelling.


Assuntos
Retículo Endoplasmático/virologia , Membranas Intracelulares/virologia , Membrana Nuclear/virologia , SARS-CoV-2 , Animais , COVID-19 , Chlorocebus aethiops , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/ultraestrutura , Humanos , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica de Transmissão , Membrana Nuclear/ultraestrutura , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/ultraestrutura , Células Vero , Montagem de Vírus , Replicação Viral
9.
Nat Commun ; 12(1): 61, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397928

RESUMO

Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.


Assuntos
Retículo Endoplasmático/metabolismo , Homeostase , Proteínas de Membrana/metabolismo , Animais , Brefeldina A/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/ultraestrutura , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Lipoilação , Luciferases/metabolismo , Camundongos , Nucleotidiltransferases/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos
10.
Nat Commun ; 12(1): 614, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504762

RESUMO

Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.


Assuntos
Isocitrato Desidrogenase/genética , Mutação/genética , Organelas/metabolismo , Fosfolipídeos/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Glioblastoma/patologia , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Modelos Biológicos , Oligodendroglioma/patologia , Estearoil-CoA Dessaturase/metabolismo
11.
Nat Commun ; 12(1): 568, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33495454

RESUMO

The endoplasmic reticulum (ER) network consists of tubules with high membrane curvature in cross-section, generated by the reticulons and REEPs. These proteins have two pairs of trans-membrane (TM) segments, followed by an amphipathic helix (APH), but how they induce curvature is poorly understood. Here, we show that REEPs form homodimers by interaction within the membrane. When overexpressed or reconstituted at high concentrations with phospholipids, REEPs cause extreme curvature through their TMs, generating lipoprotein particles instead of vesicles. The APH facilitates curvature generation, as its mutation prevents ER network formation of reconstituted proteoliposomes, and synthetic L- or D-amino acid peptides abolish ER network formation in Xenopus egg extracts. In Schizosaccharomyces japonicus, the APH is required for reticulon's exclusive ER-tubule localization and restricted mobility. Thus, the TMs and APH cooperate to generate high membrane curvature. We propose that the formation of splayed REEP/reticulon dimers is responsible for ER tubule formation.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lipoproteínas/química , Lipoproteínas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia Eletrônica , Mutação , Multimerização Proteica , Schizosaccharomyces , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo
12.
Int J Mol Sci ; 22(2)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445576

RESUMO

Protease Inhibitors (PI e.g., ritonavir (RTV) and lopinavir (LPV)) used to treat pregnant mothers infected by HIV induce prematurity and endocrine dysfunctions. The maintenance of pregnancy relies on placental hormone production (human Chorionic Gonadotrophin (hCG) and progesterone (P4)). Those functions are ensured by the villous trophoblast and are mainly regulated by the Unfolded Protein Response (UPR) pathway and mitochondria. We investigated, in vitro, if PI impair hCG and P4 production and the potential intracellular mechanisms involved. Term villous cytotrophoblast (VCT) were cultured with or without RTV or LPV from 6 to 48 h. VCT differentiation into syncytiotrophoblast (ST) was followed measuring hCG and P4 secretion. We evaluated the expression of P4 synthesis partners (Metastatic Lymph Node 64 (MLN64), cholesterol side-chain cleavage (P450SCC), Hydroxy-delta-5-Steroid Dehydrogenase and 3 Beta-and steroid delta-isomerase 1 (HSD3B1)), of mitochondrial pro-fusion factors (Mitofusin 2 (Mfn2), Optic Atrophy 1 (OPA1)) and of UPR factors (Glucose-Regulated Protein 78 (GRP78), Activating Transcription Factor 4 (ATF4), Activating Transcription Factor 6 (ATF6), spliced X-box Binding Protein 1 (sXBP1)). RTV had no significant effect on hCG and P4 secretion, whereas lopinavir significantly decreased both secretions. LPV also decreased P450SCC and HSD3B1 expression, whereas it increased Mfn2, GRP78 and sXBP1 expression in ST. RTV has no effect on the endocrine placenta. LPV impairs both villous trophoblast differentiation and P4 production. It is likely to act via mitochondrial fusion and UPR pathway activation. These trophoblastic alterations may end in decreased P4 levels in maternal circulation, inducing prematurity.


Assuntos
Células Endócrinas/efeitos dos fármacos , Células Endócrinas/metabolismo , Inibidores da Protease de HIV/efeitos adversos , Lopinavir/efeitos adversos , Placenta/efeitos dos fármacos , Placenta/metabolismo , Biomarcadores , Células Cultivadas , Vilosidades Coriônicas/efeitos dos fármacos , Vilosidades Coriônicas/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Gravidez , Progesterona/metabolismo , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo
13.
Int J Mol Sci ; 22(2)2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33466866

RESUMO

The Ca2+ selective channel ORAI1 and endoplasmic reticulum (ER)-resident STIM proteins form the core of the channel complex mediating store operated Ca2+ entry (SOCE). Using liquid phase electron microscopy (LPEM), the distribution of ORAI1 proteins was examined at rest and after SOCE-activation at nanoscale resolution. The analysis of over seven hundred thousand ORAI1 positions revealed a number of ORAI1 channels had formed STIM-independent distinct supra-molecular clusters. Upon SOCE activation and in the presence of STIM proteins, a fraction of ORAI1 assembled in micron-sized two-dimensional structures, such as the known puncta at the ER plasma membrane contact zones, but also in divergent structures such as strands, and ring-like shapes. Our results thus question the hypothesis that stochastically migrating single ORAI1 channels are trapped at regions containing activated STIM, and we propose instead that supra-molecular ORAI1 clusters fulfill an amplifying function for creating dense ORAI1 accumulations upon SOCE-activation.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteína ORAI1/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Células HEK293 , Humanos , Microscopia Eletrônica , Microscopia de Fluorescência , Tamanho da Partícula , Transporte Proteico , Molécula 1 de Interação Estromal/metabolismo
14.
Aquat Toxicol ; 231: 105721, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33373863

RESUMO

Previous studies illustrated that gibel carp F strain displays better lipid mobilization and antioxidant ability and compared to the A strain. We therefore hypothesized that the F strain would exhibit superior defense to cadmium exposure. Comparative studies were conducted between A and F strains using plasma stress biomarkers, histological observations, and analysis of hepatic molecular events to examine exposure to waterborne Cd (11.9 mg L-1) for 48 h and 96 h. Waterborne Cd exposure stimulated stress response and hepatic metallothionein mRNA induction in both gibel carp strains confirming exposure. Antioxidant responses were stimulated to counteract Cd toxicity, suggested by the upregulation of mRNA levels of genes associated with nuclear factor erythroid 2-related factor 2 (nrf2) signaling. Cd exposure induced endoplasmic reticulum (ER) stress, meanwhile, branches of genes in unfolded protein response (UPR) were activated. Slight time-dependent effects were implied by greater ER stress, UPR, and apoptosis signals with the duration of Cd exposure. Genotype-specific effects were identified, revealing that the F strain showed greater stress at 96 h exposure and higher antioxidant response compared to the A strain, as indicated by the mRNA levels of genes in nrf2 signaling. ER stress and UPR were also stronger in the F strain after Cd exposure. In contrast, the A strain showed higher autophagy and apoptosis response compared to the F strain. Collectively, combined autophagy and apoptosis were triggered under ER stress, which might serve as defense strategies in both gibel carp strains. The F strain showed greater antioxidant detoxification response and UPR to mitigate Cd toxicity, whereas excessive ER stress contributed to higher autophagy and apoptosis in the A strain. The present study uncovered the differential regulation and defense strategies in fish strains exposed to metal exposure.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cádmio/toxicidade , Carpas/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Antioxidantes/metabolismo , Apoptose/genética , Autofagia/genética , Biomarcadores/sangue , Carpas/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metalotioneína/genética , Metalotioneína/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos
15.
PLoS One ; 15(8): e0237930, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841274

RESUMO

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.


Assuntos
Microssomos/metabolismo , Proteômica , Animais , Células CHO , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrifugação , Cricetinae , Cricetulus , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Ontologia Genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microssomos/ultraestrutura , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Software , Frações Subcelulares/metabolismo
16.
Nat Commun ; 11(1): 3298, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620747

RESUMO

Communication between organelles is essential for their cellular homeostasis. Neurodegeneration reflects the declining ability of neurons to maintain cellular homeostasis over a lifetime, where the endolysosomal pathway plays a prominent role by regulating protein and lipid sorting and degradation. Here we report that TMEM16K, an endoplasmic reticulum lipid scramblase causative for spinocerebellar ataxia (SCAR10), is an interorganelle regulator of the endolysosomal pathway. We identify endosomal transport as a major functional cluster of TMEM16K in proximity biotinylation proteomics analyses. TMEM16K forms contact sites with endosomes, reconstituting split-GFP with the small GTPase RAB7. Our study further implicates TMEM16K lipid scrambling activity in endosomal sorting at these sites. Loss of TMEM16K function led to impaired endosomal retrograde transport and neuromuscular function, one of the symptoms of SCAR10. Thus, TMEM16K-containing ER-endosome contact sites represent clinically relevant platforms for regulating endosomal sorting.


Assuntos
Anoctaminas/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Animais , Anoctaminas/genética , Transporte Biológico , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Endossomos/ultraestrutura , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Lisossomos/ultraestrutura , Camundongos Knockout , Microscopia Eletrônica , Mutação , Transporte Proteico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo
17.
PLoS Biol ; 18(6): e3000715, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511245

RESUMO

Zoonotic coronavirus (CoV) infections, such as those responsible for the current severe acute respiratory syndrome-CoV 2 (SARS-CoV-2) pandemic, cause grave international public health concern. In infected cells, the CoV RNA-synthesizing machinery associates with modified endoplasmic reticulum membranes that are transformed into the viral replication organelle (RO). Although double-membrane vesicles (DMVs) appear to be a pan-CoV RO element, studies to date describe an assortment of additional CoV-induced membrane structures. Despite much speculation, it remains unclear which RO element(s) accommodate viral RNA synthesis. Here we provide detailed 2D and 3D analyses of CoV ROs and show that diverse CoVs essentially induce the same membrane modifications, including the small open double-membrane spherules (DMSs) previously thought to be restricted to gamma- and delta-CoV infections and proposed as sites of replication. Metabolic labeling of newly synthesized viral RNA followed by quantitative electron microscopy (EM) autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs Middle East respiratory syndrome-CoV (MERS-CoV) and SARS-CoV and the gamma-CoV infectious bronchitis virus. RNA synthesis could not be linked to DMSs or any other cellular or virus-induced structure. Our results provide a unifying model of the CoV RO and clearly establish DMVs as the central hub for viral RNA synthesis and a potential drug target in CoV infection.


Assuntos
Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Coronavirus/classificação , Coronavirus/fisiologia , Retículo Endoplasmático/patologia , Retículo Endoplasmático/virologia , Replicação Viral , Animais , Betacoronavirus/genética , Betacoronavirus/fisiologia , Linhagem Celular , Chlorocebus aethiops , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/ultraestrutura , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , RNA Viral/metabolismo , Células Vero
18.
Nature ; 584(7821): 475-478, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32494008

RESUMO

The endoplasmic reticulum (ER) membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins1-3. How EMC accomplishes this feat has been unclear. Here we report the first, to our knowledge, cryo-electron microscopy structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1-6, Emc7 and Emc10), has a large lumenal region and a smaller cytosolic region, and has a transmembrane region formed by Emc4, Emc5 and Emc6 plus the transmembrane domains of Emc1 and Emc3. We identified a five-TMH fold centred around Emc3 that resembles the prokaryotic YidC insertase and that delineates a largely hydrophilic client protein pocket. The transmembrane domain of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that the flexibility of Emc4 and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals notable evolutionary conservation with the prokaryotic insertases4,5, suggests that eukaryotic TMH insertion involves a similar mechanism, and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins.


Assuntos
Microscopia Crioeletrônica , Retículo Endoplasmático/enzimologia , Membranas Intracelulares/enzimologia , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae , Sítios de Ligação , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , Evolução Molecular , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
19.
Science ; 369(6502): 433-436, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32439656

RESUMO

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved co- and posttranslational insertase at the ER. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 angstroms by cryo-electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer.


Assuntos
Retículo Endoplasmático/química , Membranas Intracelulares/química , Proteínas de Membrana/química , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura
20.
Nat Commun ; 11(1): 2475, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424134

RESUMO

Autoimmune ß-cell destruction leads to type 1 diabetes, but the pathophysiological mechanisms remain unclear. To help address this void, we created an open-access online repository, unprecedented in its size, composed of large-scale electron microscopy images ('nanotomy') of human pancreas tissue obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD; www.nanotomy.org). Nanotomy allows analyses of complete donor islets with up to macromolecular resolution. Anomalies we found in type 1 diabetes included (i) an increase of 'intermediate cells' containing granules resembling those of exocrine zymogen and endocrine hormone secreting cells; and (ii) elevated presence of innate immune cells. These are our first results of mining the database and support recent findings that suggest that type 1 diabetes includes abnormalities in the exocrine pancreas that may induce endocrine cellular stress as a trigger for autoimmunity.


Assuntos
Bases de Dados como Assunto , Diabetes Mellitus Tipo 1/patologia , Ilhotas Pancreáticas/ultraestrutura , Microscopia Eletrônica , Autoanticorpos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Granulócitos/imunologia , Humanos , Imunidade Inata , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Doadores de Tecidos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...