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1.
Nat Commun ; 11(1): 133, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919407

RESUMO

Neurons are subjected to strain due to body movement and their location within organs and tissues. However, how they withstand these forces over the lifetime of an organism is still poorly understood. Here, focusing on touch receptor neuron-epidermis interactions using Caenorhabditis elegans as a model system, we show that UNC-70/ß-spectrin and TBC-10, a conserved GTPase-activating protein, function non-cell-autonomously within the epidermis to dynamically maintain attachment of the axon. We reveal that, in response to strain, UNC-70/ß-spectrin and TBC-10 stabilize trans-epidermal hemidesmosome attachment structures which otherwise become lost, causing axonal breakage and degeneration. Furthermore, we show that TBC-10 regulates axonal attachment and maintenance by inactivating RAB-35, and reveal functional conservation of these molecules with their vertebrate orthologs. Finally, we demonstrate that ß-spectrin functions in this context non-cell-autonomously. We propose a model in which mechanically resistant epidermal attachment structures are maintained by UNC-70/ß-spectrin and TBC-10 during movement, preventing axonal detachment and degeneration.


Assuntos
Axônios/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Espectrina/metabolismo , Estresse Fisiológico/fisiologia , Animais , Citoesqueleto/fisiologia , Epiderme/metabolismo , Hemidesmossomos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
2.
Cell Mol Life Sci ; 77(5): 859-874, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31960115

RESUMO

Phosphatidylethanolamine-binding protein 1 (PEBP1), a small 21 kDa protein, is implicated in several key processes of the living cell. The deregulation of PEBP1, especially its downregulation, leads to major diseases such as cancer and Alzheimer's disease. PEBP1 was found to interact with numerous proteins, especially kinases and GTPases, generally inhibiting their activity. To understand the basic functionality of this amazing small protein, we have considered several known processes that it modulates and we have discussed the role of each molecular target in these processes. Here, we propose that cortical actin organization, associated with membrane changes, is involved in the majority of the processes modulated by PEBP1. Furthermore, based on recent data, we summarize some key PEBP1-interacting proteins, and we report their respective functions and focus on their relationships with actin organization. We suggest that, depending on the cell status and environment, PEBP1 is an organizer of the actin-membrane composite material.


Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Sequência de Aminoácidos , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
3.
Toxicol Lett ; 321: 32-43, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31862506

RESUMO

Cadmium (Cd) is an important environmental pollutant. Previous studies have shown that Cd can induce liver cell injury; however, the toxicity mechanisms of Cd have not been fully elucidated. This study aimed to further confirm the hepatotoxic effects of Cd in mouse liver cells by various methods both in vivo and in vitro. In addition, it found that Cd induced autophagy but also caused autophagy blockade, and autophagy blockade intensified Cd-induced injury in liver cells. Subsequently, the study investigated the effects of Cd on lysosomes and found that Cd induced lysosomal acidification, promoted the expression of lysosomal-associated membrane protein 2 and lysosomal hydrolase cathepsin B both in vivo and in vitro, and enhanced the lysosomal degradation capacity. It indicated that Cd triggered lysosomal activation. However, the fusion of autophagosomes with lysosomes was inhibited by Cd both in vivo and in vitro. Next, the expression of Rab7, a key protein that regulates autophagosome-lysosome fusion, was examined. Cd was found to inhibit Rab7 expression both in vivo and in vitro. In conclusion, the results indicated that Cd obstructed the autophagic flux by inhibiting the fusion of autophagosomes with lysosomes, thus exacerbating the Cd-induced hepatotoxicity. Moreover, the molecular mechanism of Cd-induced inhibition of autophagosome-lysosome fusion is probably related to the Cd-induced downregulation of Rab7.


Assuntos
Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cloreto de Cádmio/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fígado/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Animais , Autofagossomos/metabolismo , Autofagossomos/patologia , Proteínas Relacionadas à Autofagia/metabolismo , Catepsina B/metabolismo , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Feminino , Concentração de Íons de Hidrogênio , Fígado/metabolismo , Fígado/patologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos Endogâmicos C57BL , Proteólise , Transdução de Sinais , Proteínas rab de Ligação ao GTP/metabolismo
4.
PLoS Biol ; 17(11): e3000531, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31682603

RESUMO

Recycling endosomes regulate plasma membrane recycling. Recently, recycling endosome-associated proteins have been implicated in the positioning and orientation of the mitotic spindle and cytokinesis. Loss of MYO5B, encoding the recycling endosome-associated myosin Vb, is associated with tumor development and tissue architecture defects in the gastrointestinal tract. Whether loss of MYO5B expression affects mitosis is not known. Here, we demonstrate that loss of MYO5B expression delayed cytokinesis, perturbed mitotic spindle orientation, led to the misorientation of the plane of cell division during the course of mitosis, and resulted in the delamination of epithelial cells. Remarkably, the effects on spindle orientation, but not cytokinesis, were a direct consequence of physical hindrance by giant late endosomes, which were formed in a chloride channel-sensitive manner concomitant with a redistribution of chloride channels from the cell periphery to late endosomes upon loss of MYO5B. Rab7 availability was identified as a limiting factor for the development of giant late endosomes. In accordance, increasing rab7 availability corrected mitotic spindle misorientation and cell delamination in cells lacking MYO5B expression. In conclusion, we identified a novel role for MYO5B in the regulation of late endosome size control and identify the inability to control late endosome size as an unexpected novel mechanism underlying defects in cell division orientation and epithelial architecture.


Assuntos
Endossomos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Fuso Acromático/metabolismo , Animais , Células CACO-2 , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Citocinese/genética , Citocinese/fisiologia , Endossomos/genética , Células Epiteliais/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose/fisiologia , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Proteínas rab de Ligação ao GTP/metabolismo
5.
Nat Commun ; 10(1): 5125, 2019 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-31719531

RESUMO

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, due in part to the propensity of lung cancer to metastasize. Aberrant epithelial-to-mesenchymal transition (EMT) is a proposed model for the initiation of metastasis. During EMT cell-cell adhesion is reduced allowing cells to dissociate and invade. Of the EMT-associated transcription factors, ZEB1 uniquely promotes NSCLC disease progression. Here we apply two independent screens, BioID and an Epigenome shRNA dropout screen, to define ZEB1 interactors that are critical to metastatic NSCLC. We identify the NuRD complex as a ZEB1 co-repressor and the Rab22 GTPase-activating protein TBC1D2b as a ZEB1/NuRD complex target. We find that TBC1D2b suppresses E-cadherin internalization, thus hindering cancer cell invasion and metastasis.


Assuntos
Caderinas/metabolismo , Endocitose , Proteínas Ativadoras de GTPase/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proteínas Correpressoras/metabolismo , Humanos , Camundongos , Modelos Biológicos , Metástase Neoplásica , Ligação Proteica , Proteínas rab de Ligação ao GTP/metabolismo
6.
PLoS Biol ; 17(10): e3000466, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658245

RESUMO

The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The membrane trafficking mechanisms by which neurons control surface polarization of synaptic receptors remain poorly understood. The sorting receptor Sortilin-related CNS expressed 1 (SorCS1) is a critical regulator of trafficking of neuronal receptors, including the presynaptic adhesion molecule neurexin (Nrxn), an essential synaptic organizer. Here, we show that SorCS1 maintains a balance between axonal and dendritic Nrxn surface levels in the same neuron. Newly synthesized Nrxn1α traffics to the dendritic surface, where it is endocytosed. Endosomal SorCS1 interacts with the Rab11 GTPase effector Rab11 family-interacting protein 5 (Rab11FIP5)/Rab11 interacting protein (Rip11) to facilitate the transition of internalized Nrxn1α from early to recycling endosomes and bias Nrxn1α surface polarization towards the axon. In the absence of SorCS1, Nrxn1α accumulates in early endosomes and mispolarizes to the dendritic surface, impairing presynaptic differentiation and function. Thus, SorCS1-mediated sorting in dendritic endosomes controls Nrxn axonal surface polarization required for proper synapse development and function.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Córtex Cerebral/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Neurônios/metabolismo , Receptores de Superfície Celular/genética , Membranas Sinápticas/metabolismo , Transmissão Sináptica/genética , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Polaridade Celular , Córtex Cerebral/citologia , Embrião de Mamíferos , Endocitose , Endossomos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/ultraestrutura , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ratos , Ratos Wistar , Receptores de Superfície Celular/metabolismo , Membranas Sinápticas/ultraestrutura , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
7.
Nat Cell Biol ; 21(10): 1234-1247, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570833

RESUMO

Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.


Assuntos
Retículo Endoplasmático/metabolismo , Monócitos/metabolismo , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/genética , Transdução de Sinais , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Sistemas CRISPR-Cas , Retículo Endoplasmático/ultraestrutura , Edição de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Monócitos/ultraestrutura , Fagocitose , Fagossomos/ultraestrutura , Cultura Primária de Células , Proteólise , Células RAW 264.7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
8.
Zhonghua Xin Xue Guan Bing Za Zhi ; 47(10): 829-835, 2019 Oct 24.
Artigo em Chinês | MEDLINE | ID: mdl-31648466

RESUMO

Objective: To investigate whether CD137-CD137L signaling could affect the secretion of mouse vascular smooth muscle cells (VSMCs) -derived exosomes through autophagy mediated Rab7 pathway. Methods: Primary thoracic aorta VSMCs from C57BL/6J mouse were obtained by tissue block adherence method. VSMCs between the third to fifth passages were used and VSMCs were divided into 4 groups: control group, CD137 agonist group, lentivirus control group, Rab7 lentiviral interference group. VSMCs in CD137 agonist group were treated with recombinant protein of CD137L (10 µg/ml), VSMCs in lentivirus control group were treated with lentiviral followed by recombinant protein of CD137L (10 µg/ml), VSMCs in Rab7 lentiviral interference group were treated with Rab7 lentiviral intervention followed by recombinant protein of CD137L (10 µg/ml). Western blot was used to detect the protein expression of LC3Ⅱ, p62, Rab7, CD9, CD81 and Hsc70. Fluorescence microscopy was used to track the changes of autophagy in cells infected with mRFP-GFP-LC3. Transmission electron microscope was used to observe the morphology and size of VSMCs-derived exosomes. The nanoparticle tracking analysis(NTA) was used to detect the concentration and size of exosomes in each group. Results: (1) The expressions of Rab7, LC3Ⅱ and p62 protein in VSMCs of CD137 activation group were significantly higher than those in control group (all P<0.05). The expressions of Rab7, LC3Ⅱ and p62 protein in Rab7 lentivirus interference group was lower than in CD137 activation group (all P<0.05), while the expressions were similar between the lentivirus control group and the CD137 activation group (all P>0.05). (2) The total number of fluorescent spots and yellow fluorescent spots in the VSMCs of the CD137 activation group were higher than those in the control group (all P<0.05), and the number of yellow fluorescent spots was higher than that of the red fluorescent spots in the VSMCs of the CD137 activation group ((50.3±0.9) vs. (10.3±1.5)/cell). The total numbers of fluorescent spots and yellow fluorescent spots in VSMCs of Rab7 lentivirus interference group were lower than those of CD137 activation group (both P<0.05), and the number of red fluorescent spots in VSMCs was higher than that of yellow fluorescent spots ((40.7±4.0) and (10.7±1.2)/cell) in the Rab7 lentiviral interference group. The total numbers of fluorescent spots and yellow fluorescent spots in the VSMCs were similar between the lentivirus control group and the CD137 activation group (all P>0.05). (3) Under transmission electron microscopy, the size of the VSMCs-derived exosomes was about 30-150 nm. The exosome markers (CD9, CD81) could be detected in vesicles by Western blot. NTA results showed that the concentration of VSMCs-derived exosomes was significantly higher in the CD137-activated group than in the control group (P<0.05), which was significantly lower in the Rab7 lentiviral interference group than in the CD137-activation group (P<0.05) and was similar between the lentivirus control group and the CD137 activation group (P>0.05). The expression of Hsc70 protein in exosomes secreted by CD137 activation group was higher than that in the control group (P<0.05). The expression of Hsc70 protein in exosomes was lower in Rab7 lentivirus interference group than in the CD137 activation group (P<0.05), which was similar between the lentivirus control group and the CD137 activation group (P>0.05). The expression of LC3Ⅱ protein in exosome was higher in CD137 activation group than in control group (P<0.05), which was lower in Rab7 lentivirus interference group than in CD137 activation group (P<0.05), which was similar between the lentivirus control group and the CD137 activation group (P>0.05). Conclusion: The CD137-CD137L signaling may affect the secretion of mouse VSMCs-derived exosomes through modulating the Rab7 pathway mediated autophagy.


Assuntos
Ligante 4-1BB/metabolismo , Exossomos/metabolismo , Músculo Liso Vascular/metabolismo , Transdução de Sinais , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso
9.
PLoS Pathog ; 15(9): e1008030, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31518366

RESUMO

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with multiple human malignancies. EBV drives B-cell proliferation, which contributes to the pathogenesis of multiple lymphomas. Yet, knowledge of how EBV subverts host biosynthetic pathways to transform resting lymphocytes into activated lymphoblasts remains incomplete. Using a temporal proteomic dataset of EBV primary human B-cell infection, we identified that cholesterol and fatty acid biosynthetic pathways were amongst the most highly EBV induced. Epstein-Barr nuclear antigen 2 (EBNA2), sterol response element binding protein (SREBP) and MYC each had important roles in cholesterol and fatty acid pathway induction. Unexpectedly, HMG-CoA reductase inhibitor chemical epistasis experiments revealed that mevalonate pathway production of geranylgeranyl pyrophosphate (GGPP), rather than cholesterol, was necessary for EBV-driven B-cell outgrowth, perhaps because EBV upregulated the low-density lipoprotein receptor in newly infected cells for cholesterol uptake. Chemical and CRISPR genetic analyses highlighted downstream GGPP roles in EBV-infected cell small G protein Rab activation. Rab13 was highly EBV-induced in an EBNA3-dependent manner and served as a chaperone critical for latent membrane protein (LMP) 1 and 2A trafficking and target gene activation in newly infected and in lymphoblastoid B-cells. Collectively, these studies identify highlight multiple potential therapeutic targets for prevention of EBV-transformed B-cell growth and survival.


Assuntos
Linfócitos B/virologia , Ácidos Graxos/biossíntese , Herpesvirus Humano 4/patogenicidade , Ácido Mevalônico/metabolismo , Alquil e Aril Transferases/metabolismo , Linfócitos B/patologia , Proliferação de Células , Sobrevivência Celular , Colesterol/biossíntese , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/patologia , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Redes e Vias Metabólicas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Proteínas Virais/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
10.
Nat Commun ; 10(1): 4392, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31558725

RESUMO

The targeted endocytic recycling of the T cell receptor (TCR) to the immunological synapse is essential for T cell activation. Despite this, the mechanisms that underlie the sorting of internalised receptors into recycling endosomes remain poorly understood. To build a comprehensive picture of TCR recycling during T cell activation, we developed a suite of new imaging and quantification tools centred on photoactivation of fluorescent proteins. We show that the membrane-organising proteins, flotillin-1 and -2, are required for TCR to reach Rab5-positive endosomes immediately after endocytosis and for transfer from Rab5- to Rab11a-positive compartments. We further observe that after sorting into in Rab11a-positive vesicles, TCR recycles to the plasma membrane independent of flotillin expression. Our data suggest a mechanism whereby flotillins delineate a fast Rab5-Rab11a endocytic recycling axis and functionally contribute to regulate the spatial organisation of these endosomes.


Assuntos
Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Proteínas de Membrana , Microscopia Confocal , Transporte Proteico , Receptores de Antígenos de Linfócitos T/genética
11.
Cell Host Microbe ; 26(4): 551-563.e6, 2019 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-31540829

RESUMO

During infection, Legionella pneumophila translocates over 300 effector proteins into the host cytosol, allowing the pathogen to establish an endoplasmic reticulum (ER)-like Legionella-containing vacuole (LCV) that supports bacterial replication. Here, we perform a genome-wide CRISPR-Cas9 screen and secondary targeted screens in U937 human monocyte/macrophage-like cells to systematically identify host factors that regulate killing by L. pneumophila. The screens reveal known host factors hijacked by L. pneumophila, as well as genes spanning diverse trafficking and signaling pathways previously not linked to L. pneumophila pathogenesis. We further characterize C1orf43 and KIAA1109 as regulators of phagocytosis and show that RAB10 and its chaperone RABIF are required for optimal L. pneumophila replication and ER recruitment to the LCV. Finally, we show that Rab10 protein is recruited to the LCV and ubiquitinated by the effectors SidC/SdcA. Collectively, our results provide a wealth of previously undescribed insights into L. pneumophila pathogenesis and mammalian cell function.


Assuntos
Legionella pneumophila/patogenicidade , Doença dos Legionários/patologia , Fagocitose/imunologia , Proteínas/genética , Vacúolos/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Retículo Endoplasmático/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/genética , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Células RAW 264.7 , Células U937 , Fatores de Virulência/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
12.
Int J Mol Sci ; 20(16)2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31408960

RESUMO

Autophagy (particularly macroautophagy) is a bulk degradation process used by eukaryotic cells in order to maintain adequate energy levels and cellular homeostasis through the delivery of long-lived proteins and organelles to the lysosome, resulting in their degradation. It is becoming increasingly clear that many of the molecular requirements to fulfil autophagy intersect with those of conventional and unconventional membrane trafficking pathways. Of particular interest is the dependence of these processes on multiple members of the Rab family of small GTP binding proteins. Rab33b is a protein that localises to the Golgi apparatus and has suggested functions in both membrane trafficking and autophagic processes. Interestingly, mutations in the RAB33B gene have been reported to cause the severe skeletal disorder, Smith-McCort Dysplasia; however, the molecular basis for Rab33b in this disorder remains to be determined. In this review, we focus on the current knowledge of the participation of Rab33b and its interacting partners in membrane trafficking and macroautophagy, and speculate on how its function, and dysfunction, may contribute to human disease.


Assuntos
Autofagia , Mapas de Interação de Proteínas , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Transporte Biológico , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Lisossomos/metabolismo , Osteocondrodisplasias/metabolismo
13.
Results Probl Cell Differ ; 67: 95-123, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31435794

RESUMO

The Golgi apparatus is a central sorting station in the cell. It receives newly synthesized molecules from the endoplasmic reticulum and directs them to different subcellular destinations, such as the plasma membrane or the endocytic pathway. Importantly, in the last few years, it has emerged that the maintenance of Golgi structure is connected to the proper regulation of membrane trafficking. Rab proteins are small GTPases that are considered to be the master regulators of the intracellular membrane trafficking. Several of the over 60 human Rabs are involved in the regulation of transport pathways at the Golgi as well as in the maintenance of its architecture. This chapter will summarize the different roles of Rab GTPases at the Golgi, both as regulators of membrane transport, scaffold, and tethering proteins and in preserving the structure and function of this organelle.


Assuntos
Complexo de Golgi/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Transporte Biológico , Retículo Endoplasmático/metabolismo , Humanos
14.
EMBO J ; 38(8): e100312, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31368593

RESUMO

The small GTPase Rab7 is a key organizer of receptor sorting and lysosomal degradation by recruiting of a variety of effectors depending on its GDP/GTP-bound state. However, molecular mechanisms that trigger Rab7 inactivation remain elusive. Here we find that, among the endosomal pools, Rab7-positive compartments possess the highest level of PI4P, which is primarily produced by PI4K2A kinase. Acute conversion of this endosomal PI4P to PI(4,5)P2 causes Rab7 dissociation from late endosomes and releases a regulator of autophagosome-lysosome fusion, PLEKHM1, from the membrane. Rab7 effectors Vps35 and RILP are not affected by acute PI(4,5)P2 production. Deletion of PI4K2A greatly reduces PIP5Kγ-mediated PI(4,5)P2 production in Rab7-positive endosomes leading to impaired Rab7 inactivation and increased number of LC3-positive structures with defective autophagosome-lysosome fusion. These results reveal a late endosomal PI4P-PI(4,5)P2 -dependent regulatory loop that impacts autophagosome flux by affecting Rab7 cycling and PLEKHM1 association.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagossomos/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Fusão de Membrana , Glicoproteínas de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Endocitose , Células HEK293 , Humanos , Ligação Proteica , Transporte Proteico
15.
Mol Pharmacol ; 96(3): 308-319, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31266815

RESUMO

The dopamine D2 receptor (D2R) mediates ligand-biased signaling with potential therapeutic implications. However, internalization, choice of endocytic routes, and degradation of the D2R in lysosomes may also participate in agonist-directed trafficking. We developed bioluminescence resonance energy transfer (BRET) assays that measure relative distances between Renilla luciferase8-tagged D2R and green fluorescent protein 2 (GFP2)-tagged K-Ras (plasma membrane marker), and between luciferase8-tagged D2R and GFP2-Rab5 (early), GFP2-Rab4 (recycling), or GFP2-Rab7 (late) endosomal markers. The BRET signal between D2R-Luc and GFP2-K-Ras was robustly diminished after receptor internalization induced by dopamine, with subsequent BRET signals increasing when luciferase8-tagged D2R approached GFP2-Rab proteins in endosomal compartments. All BRET signals were blocked by the selective D2R antagonist haloperidol and were decreased by low temperature and high sucrose blocks, two parameters interfering with internalization. Some antipsychotic drugs, such as aripiprazole, are less efficacious in internalizing D2R than most of the antiparkinsonian agents. However, antipsychotics were nearly as efficacious as antiparkinsonians in directing the D2R toward early and recycling endosomes. The Rab7 marker for the late endosome/lysosome route was also capable of discriminating between D2R compounds. We could show that some drugs engaged the D2R either to interact preferentially with arrestin-3 or to internalize. Our study revealed that D2R trafficking in cells was differentially regulated by antipsychotic and antiparkinsonian drugs. Taken together, the BRET assays reported here could further help decipher D2R ligand-induced arrestin-3 recruitment and trafficking, with potentially more selective therapeutic profiles and fewer undesired side effects.


Assuntos
Arrestinas/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Proteínas de Fluorescência Verde/metabolismo , Receptores de Dopamina D2/agonistas , Animais , Células CHO , Cricetulus , Antagonistas de Dopamina/farmacologia , Haloperidol/farmacologia , Lisossomos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais , Proteínas rab de Ligação ao GTP/metabolismo
16.
Invest Ophthalmol Vis Sci ; 60(8): 2861-2874, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31260037

RESUMO

Purpose: Phosphatidylinositol-3-phosphate (PI(3)P), and Vps34, the type III phosphatidylinositol 3-kinase primarily responsible for its production, are important for function and survival of sensory neurons, where they have key roles in membrane processing events, such as autophagy, endosome processing, and fusion of membranes bearing ubiquitinated cargos with lysosomes. We examined their roles in the most abundant class of secondary neurons in the vertebrate retina, the ON-bipolar cells (ON-BCs). Methods: A conditional Vps34 knockout mouse line was generated by crossing Vps34 floxed mice with transgenic mice expressing Cre recombinase in ON-BCs. Structural changes in the retina were determined by immunofluorescence and electron microscopy, and bipolar cell function was determined by electroretinography. Results: Vps34 deletion led to selective death of ON-BCs, a thinning of the inner nuclear layer, and a progressive decline of electroretinogram b-wave amplitudes. There was no evidence for loss of other retinal neurons, or disruption of rod-horizontal cell contacts in the outer plexiform layer. Loss of Vps34 led to aberrant accumulation of membranes positive for autophagy markers LC3, p62, and ubiquitin, accumulation of endosomal membranes positive for Rab7, and accumulation of lysosomes. Similar effects were observed in Purkinje cells of the cerebellum, leading to severe and progressive ataxia. Conclusions: These results support an essential role for PI(3)P in fusion of autophagosomes with lysosomes and in late endosome maturation. The cell death resulting from Vps34 knockout suggests that these processes are essential for the health of ON-BCs.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases/fisiologia , Células Bipolares da Retina/metabolismo , Animais , Autofagossomos , Eletroporação , Eletrorretinografia , Lisossomos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-myc/metabolismo , Células Bipolares da Retina/citologia , Ubiquitina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
17.
Nat Commun ; 10(1): 3106, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308374

RESUMO

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.


Assuntos
Antígeno CTLA-4/metabolismo , Proteínas de Ligação a DNA/deficiência , Fatores de Troca do Nucleotídeo Guanina/deficiência , /genética , Antígeno B7-1/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Técnicas de Inativação de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Homeostase , Humanos , Células Jurkat , Linfócitos T/metabolismo , Linfócitos T/fisiologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
18.
J Pharmacol Sci ; 140(3): 300-304, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31353211

RESUMO

Endocytosis after insulin secretion plays a pivotal role in the regulation of insulin secretion in pancreatic ß-cells. Our recent study suggested that EPI64, a GTPase activating protein for Rab27a, contributes to the regulation of glucose-induced endocytosis, which is mediated by the GDP-bound form of Rab27a. Here, we identified insulin receptor-related receptor (IRR) as an EPI64-interacting protein. Knockdown of IRR inhibited glucose-induced uptake of transferrin, a marker of endocytosis, translocation of the guanine-nucleotide-exchange factor ARNO to the plasma membrane, and generation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). These results suggest that IRR functions upstream of PIP3 generation and controls endocytosis after insulin secretion.


Assuntos
Endocitose/fisiologia , Glucose/metabolismo , Secreção de Insulina/fisiologia , Insulina/metabolismo , Receptor de Insulina/metabolismo , Animais , Transporte Biológico/fisiologia , Membrana Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Células Secretoras de Insulina/metabolismo , Camundongos , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo
19.
Elife ; 82019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31355749

RESUMO

Morphogenesis in plants depends critically on directional (anisotropic) growth. This occurs principally perpendicular to the net orientation of cellulose microfibrils (CMFs), which is in turn controlled by cortical microtubules (CMTs). In young lateral roots of Arabidopsis thaliana, growth anisotropy also depends on RAB-A5c, a plant-specific small GTPase that specifies a membrane trafficking pathway to the geometric edges of cells. Here we investigate the functional relationship between structural anisotropy at faces and RAB-A5c activity at edges during lateral root development. We show that surprisingly, inhibition of RAB-A5c function is associated with increased CMT/CMF anisotropy. We present genetic, pharmacological, and modelling evidence that this increase in CMT/CMF anisotropy partially compensates for loss of an independent RAB-A5c-mediated mechanism that maintains anisotropic growth in meristematic cells. We show that RAB-A5c associates with CMTs at cell edges, indicating that CMTs act as an integration point for both mechanisms controlling cellular growth anisotropy in lateral roots.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Proliferação de Células , Morfogênese , Células Vegetais/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Microtúbulos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
20.
Mediators Inflamm ; 2019: 7538071, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31182932

RESUMO

Rab26 GTPase modulates the trafficking of cell surface receptors, such as G protein-coupled receptors including α2-adrenergic receptors in some cell types. However, the effect of Rab26 on ß2-adrenergic receptor (ß2-AR) trafficking or/and Toll-like receptor 4 (TLR4) expression in human pulmonary microvascular endothelial cells (HPMECs) is still unclear. Here, we investigated the role of Rab26 in regulating the expression of ß2-ARs and TLR4 in HPMECs and the effect of these receptors' imbalance on endothelial cell barrier function. The results showed that there was unbalance expression in these receptors, where ß2-AR expression was remarkably reduced, and TLR4 was increased on the cell membrane after lipopolysaccharide (LPS) treatment. Furthermore, we found that Rab26 overexpression not only upregulated ß2-ARs but also downregulated TLR4 expression on the cell membrane. Subsequently, the TLR4-related inflammatory response was greatly attenuated, and the hyperpermeability of HPMECs also was partially relived. Taken together, these data suggest that basal Rab26 maintains the balance between ß2-ARs and TLR4 on the cell surface, and it might be a potential therapeutic target for diseases involving endothelial barrier dysfunction.


Assuntos
Células Endoteliais/metabolismo , Inflamação/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Citometria de Fluxo , Humanos , Inflamação/imunologia , Microscopia Confocal , Microvasos/citologia , Microvasos/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas rab de Ligação ao GTP/imunologia
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