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1.
PLoS One ; 15(1): e0227435, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31961879

RESUMO

Wnt/ß-catenin signalling is crucial for maintaining the balance between cell proliferation and differentiation, both during tissue morphogenesis and in tissue maintenance throughout postnatal life. Whereas the signalling activities of the core Wnt/ß-catenin pathway components are understood in great detail, far less is known about the precise role and regulation of the many different modulators of Wnt/ß-catenin signalling that have been identified to date. Here we describe TMEM98, a putative transmembrane protein of unknown function, as an interaction partner and regulator of the GSK3-binding protein FRAT2. We show that TMEM98 reduces FRAT2 protein levels and, accordingly, inhibits the FRAT2-mediated induction of ß-catenin/TCF signalling. We also characterize the intracellular trafficking of TMEM98 in more detail and show that it is recycled between the plasma membrane and the Golgi. Together, our findings not only reveal a new layer of regulation for Wnt/ß-catenin signalling, but also a new biological activity for TMEM98.


Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Via de Sinalização Wnt , Animais , Proteínas de Transporte/genética , Membrana Celular/genética , Complexo de Golgi/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Camundongos , Transporte Proteico , beta Catenina/genética , beta Catenina/metabolismo
2.
Nucleic Acids Res ; 48(3): 1372-1391, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31840180

RESUMO

Release of phosphorothioate antisense oligonucleotides (PS-ASOs) from late endosomes (LEs) is a rate-limiting step and a poorly defined process for productive intracellular ASO drug delivery. Here, we examined the role of Golgi-endosome transport, specifically M6PR shuttling mediated by GCC2, in PS-ASO trafficking and activity. We found that reduction in cellular levels of GCC2 or M6PR impaired PS-ASO release from endosomes and decreased PS-ASO activity in human cells. GCC2 relocated to LEs upon PS-ASO treatment, and M6PR also co-localized with PS-ASOs in LEs or on LE membranes. These proteins act through the same pathway to influence PS-ASO activity, with GCC2 action preceding that of M6PR. Our data indicate that M6PR binds PS-ASOs and facilitates their vesicular escape. The co-localization of M6PR and of GCC2 with ASOs is influenced by the PS modifications, which have been shown to enhance the affinity of ASOs for proteins, suggesting that localization of these proteins to LEs is mediated by ASO-protein interactions. Reduction of M6PR levels also decreased PS-ASO activity in mouse cells and in livers of mice treated subcutaneously with PS-ASO, indicating a conserved mechanism. Together, these results demonstrate that the transport machinery between LE and Golgi facilitates PS-ASO release.


Assuntos
Endossomos/genética , Proteínas da Matriz do Complexo de Golgi/genética , Oligonucleotídeos Antissenso/genética , Receptor IGF Tipo 2/genética , Animais , Endocitose/genética , Endossomos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Células HeLa , Humanos , Camundongos , Oligonucleotídeos Fosforotioatos/genética , Transporte Proteico/genética , Receptor IGF Tipo 2/metabolismo
3.
ACS Appl Mater Interfaces ; 11(50): 46408-46418, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31729218

RESUMO

Recent studies suggest that cancer cell death accompanied by organelle dysfunction might be a promising approach for cancer therapy. The Golgi apparatus has a key role in cell function and may initiate signaling pathways to mitigate stress and, if irreparable, start apoptosis. It has been shown that Golgi disassembly and fragmentation under oxidative stress act as indicators for stress-mediated cell death pathways through cell cycle arrest in the G2/M phase. The present study shows that UV-induced reactive oxygen species (ROS) generation by Ag@ZnO nanoparticles (NPs) transform the Golgi structures from compressed perinuclear ribbons into detached vesicle-like structures distributed in the entire cytoplasm of melanoma cells. This study also demonstrates that Ag@ZnO NP-induced Golgi fragmentation cooccurs with G2 block of cell cycle progression, preventing cells from entering the mitosis phase. Additionally, the increased intracellular ROS production triggered by Ag@ZnO NPs upon UV exposure promoted autophagy. Taken together, Ag@ZnO NPs induce stress-related Golgi fragmentation and autophagy, finally leading to melanoma cell apoptosis. Intracellular oxidative stress generated by Ag@ZnO NPs upon UV irradiation may thus represent a targeted approach to induce cancer cell death through organelle destruction in melanoma cells, while fibroblast cells remained largely unaffected.


Assuntos
Proliferação de Células/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Melanoma/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Autofagia/efeitos dos fármacos , Autofagia/efeitos da radiação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Complexo de Golgi/genética , Humanos , Melanoma/genética , Melanoma/patologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Mitose/efeitos dos fármacos , Mitose/efeitos da radiação , Espécies Reativas de Oxigênio/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Prata/química , Prata/farmacologia , Raios Ultravioleta , Óxido de Zinco/química , Óxido de Zinco/farmacologia
4.
mBio ; 10(5)2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615964

RESUMO

ADP ribosylation factor (Arf) small GTPase family members are involved in vesicle trafficking and organelle maintenance in organisms ranging from Saccharomyces cerevisiae to humans. A previous study identified Magnaporthe oryzae Arf6 (MoArf6) as one of the Arf proteins that regulates growth and conidiation in the rice blast fungus M. oryzae, but the remaining family proteins remain unknown. Here, we identified six additional Arf proteins, including MoArf1, MoArl1, MoArl3, MoArl8, MoCin4, and MoSar1, as well as their sole adaptor protein, MoGga1, and determined their shared and specific functions. We showed that the majority of these proteins exhibit positive regulatory functions, most notably, in growth. Importantly, MoArl1, MoCin4, and MoGga1 are involved in pathogenicity through the regulation of host penetration and invasive hyphal growth. MoArl1 and MoCin4 also regulate normal vesicle trafficking, and MoCin4 further controls the formation of the biotrophic interfacial complex (BIC). Moreover, we showed that Golgi-cytoplasm cycling of MoArl1 is required for its function. Finally, we demonstrated that interactions between MoArf1 and MoArl1 with MoGga1 are important for Golgi localization and pathogenicity. Collectively, our findings revealed the shared and specific functions of Arf family members in M. oryzae and shed light on how these proteins function through conserved mechanisms to govern growth, transport, and virulence of the blast fungus.IMPORTANCE Magnaporthe oryzae is the causal agent of rice blast, representing the most devastating diseases of rice worldwide, which results in losses of amounts of rice that could feed more than 60 million people each year. Arf (ADP ribosylation factor) small GTPase family proteins are involved in vesicle trafficking and organelle maintenance in eukaryotic cells. To investigate the function of Arf family proteins in M. oryzae, we systematically characterized all seven Arf proteins and found that they have shared and specific functions in governing the growth, development, and pathogenicity of the blast fungus. We have also identified the pathogenicity-related protein MoGga1 as the common adaptor of MoArf1 and MoArl1. Our findings are important because they provide the first comprehensive characterization of the Arf GTPase family proteins and their adaptor protein MoGga1 functioning in a plant-pathogenic fungus, which could help to reveal new fungicide targets to control this devastating disease.


Assuntos
Proteínas Fúngicas/metabolismo , Magnaporthe/patogenicidade , Esporos Fúngicos/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Magnaporthe/genética , Oryza/genética , Oryza/metabolismo , Doenças das Plantas/microbiologia , Esporos Fúngicos/fisiologia , Virulência/genética
5.
Cell Struct Funct ; 44(2): 137-151, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31534067

RESUMO

The Golgi apparatus is an organelle where membrane or secretory proteins receive post-translational modifications such as glycosylation and sulfation, after which the proteins are selectively transported to their final destinations through vesicular transport. When the synthesis of secretory or membrane proteins is increased and overwhelms the capacity of the Golgi (Golgi stress), eukaryotic cells activate a homeostatic mechanism called the Golgi stress response to augment the capacity of the Golgi. Four response pathways of the Golgi stress response have been identified, namely the TFE3, CREB3, HSP47, and proteoglycan pathways, which regulate the general function of the Golgi, apoptosis, cell survival, and proteoglycan glycosylation, respectively. Here, we identified a novel response pathway that augments the expression of glycosylation enzymes for mucins in response to insufficiency in mucin-type glycosylation in the Golgi (mucin-type Golgi stress), and we found that expression of glycosylation enzymes for mucins such as GALNT5, GALNT8, and GALNT18 was increased upon mucin-type-Golgi stress. We named this pathway the mucin pathway. Unexpectedly, mucin-type Golgi stress induced the expression and activation of TFE3, a key transcription factor regulating the TFE3 pathway, suggesting that the activated mucin pathway sends a crosstalk signal to the TFE3 pathway. We identified an enhancer element regulating transcriptional induction of TFE3 upon mucin-type Golgi stress, and named it the mucin-type Golgi stress response element, of which consensus was ACTTCC(N9)TCCCCA. These results suggested that crosstalk from the mucin pathway to the TFE3 pathway has an important role in the regulation of the mammalian Golgi stress response.Key words: Golgi stress, mucin, TFE3, organelle autoregulation, organelle zone.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Complexo de Golgi/metabolismo , Mucinas/metabolismo , Elementos de Resposta/genética , Complexo de Golgi/genética , Células HT29 , Células HeLa , Humanos , Mucinas/genética , Mutação Puntual
6.
J Biosci ; 44(4)2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31502569

RESUMO

Intracellular trafficking is a field that has been intensively studied for years and yet there remains much to be learned. Part of the reason that there is so much obscurity remaining in this field is due to all the pathways and the stages that define cellular trafficking. One of the major steps in cellular trafficking is fusion. Fusion is defined as the terminal step that occurs when a cargo-laden vesicle arrives at the proper destination. There are two types of fusion within a cell: homotypic and heterotypic fusion. Homotypic fusion occurs when the two membranes merging together are of the same type such as vacuole to vacuole fusion. Heterotypic fusion occurs when the two membranes at play are of different types such as when an endosomal membrane fuses with a Golgi membrane. In this review, we will focus on all the protein components - Rabs, Golgins, Multisubunit tethers, GTPases, protein phosphatases and SNAREs - that have been known to function in both of these types of fusion. We hope to develop a model of how all of these constituents function together to achieve membrane fusion. Membrane fusion is a biological process absolutely necessary for proper intracellular trafficking. Due to the degree of importance multiple proteins are required for it to be properly carried through. Whether we are talking about heterotypic or homotypic fusion, any defects in the fusion machinery can result in disease states such as Parkinson's and Alzheimer's disease. Although much research has significantly expanded our knowledge of fusion, there is still much more to be learned.


Assuntos
Citoplasma/genética , GTP Fosfo-Hidrolases/genética , Membranas Intracelulares/metabolismo , Fusão de Membrana/genética , Transporte Biológico/genética , Citoplasma/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Complexo de Golgi/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética
7.
Proc Natl Acad Sci U S A ; 116(28): 14029-14038, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31239341

RESUMO

Endoplasmic reticulum (ER) membrane junctions are formed by the dynamin-like GTPase atlastin (ATL). Deletion of ATL results in long unbranched ER tubules in cells, and mutation of human ATL1 is linked to hereditary spastic paraplegia. Here, we demonstrate that COPII formation is drastically decreased in the periphery of ATL-deleted cells. ER export of cargo proteins becomes defective; ER exit site initiation is not affected, but many of the sites fail to recruit COPII subunits. The efficiency of cargo packaging into COPII vesicles is significantly reduced in cells lacking ATLs, or when the ER is transiently fragmented. Cargo is less mobile in the ER in the absence of ATL, but the cargo mobility and COPII formation can be restored by ATL R77A, which is capable of tethering, but not fusing, ER tubules. These findings suggest that the generation of ER junctions by ATL plays a critical role in maintaining the necessary mobility of ER contents to allow efficient packaging of cargo proteins into COPII vesicles.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Retículo Endoplasmático/genética , Proteínas de Ligação ao GTP/genética , Proteínas de Membrana/genética , Transporte Proteico/genética , Animais , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Complexo de Golgi/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Mutantes/genética , Deleção de Sequência/genética , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/patologia
8.
J Cell Biol ; 218(7): 2215-2231, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31142554

RESUMO

To ensure their homeostasis and sustain differentiated functions, cells continuously transport diverse cargos to various cell compartments and in particular to the cell surface. Secreted proteins are transported along intracellular routes from the endoplasmic reticulum through the Golgi complex before reaching the plasma membrane along microtubule tracks. Using a synchronized secretion assay, we report here that exocytosis does not occur randomly at the cell surface but on localized hotspots juxtaposed to focal adhesions. Although microtubules are involved, the RAB6-dependent machinery plays an essential role. We observed that, irrespective of the transported cargos, most post-Golgi carriers are positive for RAB6 and that its inactivation leads to a broad reduction of protein secretion. RAB6 may thus be a general regulator of post-Golgi secretion.


Assuntos
Adesões Focais/genética , Complexo de Golgi/genética , Microtúbulos/genética , Proteínas rab de Ligação ao GTP/genética , Diferenciação Celular/genética , Retículo Endoplasmático/genética , Exocitose/genética , Complexo de Golgi/metabolismo , Células HeLa , Homeostase/genética , Humanos
9.
Genes (Basel) ; 10(3)2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30934642

RESUMO

The Golgi organelle duplicates its protein and lipid content to segregate evenly between two daughter cells after mitosis. However, how Golgi biogenesis is regulated during interphase remains largely unknown. Here we show that messenger RNA (mRNA) expression of GOLPH3 and GOLGA2, two genes encoding Golgi proteins, is induced specifically in G1 phase, suggesting a link between cell cycle regulation and Golgi growth. We have examined the role of E2F transcription factors, critical regulators of G1 to S progression of the cell cycle, in the expression of Golgi proteins during interphase. We show that promoter activity for GOLPH3, a Golgi protein that is also oncogenic, is induced by E2F1-3 and repressed by E2F7. Mutation of the E2F motifs present in the GOLPH3 promoter region abrogates E2F1-mediated induction of a GOLPH3 luciferase reporter construct. Furthermore, we identify a critical CREB/ATF element in the GOLPH3 promoter that is required for its steady state and ATF2-induced expression. Interestingly, depletion of GOLPH3 with small interfering RNA (siRNA) delays the G1 to S transition in synchronized U2OS cells. Taken together, our results reveal a link between cell cycle regulation and Golgi function, and suggest that E2F-mediated regulation of Golgi genes is required for the timely progression of the cell cycle.


Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Fatores de Transcrição E2F/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Animais , Sítios de Ligação , Ciclo Celular , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Células NIH 3T3 , Fosfoproteínas/genética , Regiões Promotoras Genéticas
10.
PLoS One ; 14(4): e0215215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30986258

RESUMO

The close physical proximity between the Golgi and the centrosome is a unique feature of mammalian cells that has baffled scientists for years. Several knockdown and overexpression studies have linked the spatial relationship between these two organelles to the control of directional protein transport, directional migration, ciliogenesis and mitotic entry. However, most of these conditions have not only separated these two organelles, but also caused extensive fragmentation of the Golgi, making it difficult to dissect the specific contribution of Golgi-centrosome proximity. In this study, we present our results with stable retinal pigment epithelial (RPE-1) cell lines in which GM130 was knocked out using a CRISPR/Cas9 approach. While Golgi and centrosome organization appeared mostly intact in cells lacking GM130, there was a clear separation of these organelles from each other. We show that GM130 may control Golgi-centrosome proximity by anchoring AKAP450 to the Golgi. We also provide evidence that the physical proximity between these two organelles is dispensable for protein transport, cell migration, and ciliogenesis. These results suggest that Golgi-centrosome proximity per se is not necessary for the normal function of RPE-1 cells.


Assuntos
Centrossomo/metabolismo , Células Epiteliais/metabolismo , Complexo de Golgi/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/citologia , Deleção de Genes , Complexo de Golgi/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Epitélio Pigmentado da Retina/citologia
11.
PLoS One ; 14(4): e0215009, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30958856

RESUMO

Interorganelle phospholipid transfer is critical for eukaryotic membrane biogenesis. In the yeast Saccharomyces cerevisiae, phosphatidylserine (PS) synthesized by PS synthase, Pss1, in the endoplasmic reticulum (ER) is decarboxylated to phosphatidylethanolamine (PE) by PS decarboxylase, Psd1, in the ER and mitochondria or by Psd2 in the endosome, Golgi, and/or vacuole, but the mechanism of interorganelle PS transport remains to be elucidated. Here we report that Sfh1, a member of Sec14 family proteins of S. cerevisiae, possesses the ability to enhance PE production by Psd2. Overexpression of SFH1 in the strain defective in Psd1 restored its growth on non-fermentable carbon sources and increased the intracellular and mitochondrial PE levels. Sfh1 was found to bind various phospholipids, including PS, in vivo. Bacterially expressed and purified Sfh1 was suggested to have the ability to transport fluorescently labeled PS between liposomes by fluorescence dequenching assay in vitro. Biochemical subcellular fractionation suggested that a fraction of Sfh1 localizes to the endosome, Golgi, and/or vacuole. We propose a model that Sfh1 promotes PE production by Psd2 by transferring phospholipids between the ER and endosome.


Assuntos
Carboxiliases/deficiência , Proteínas de Ciclo Celular/biossíntese , Proteínas Cromossômicas não Histona/biossíntese , Mitocôndrias/metabolismo , Modelos Biológicos , Consumo de Oxigênio , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Endossomos/genética , Endossomos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Mitocôndrias/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/genética , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo
12.
Dev Cell ; 49(1): 130-144.e6, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30827897

RESUMO

Syntaxin 17 (Stx17) has been implicated in autophagosome-lysosome fusion. Here, we report that Stx17 functions in assembly of protein complexes during autophagy initiation. Stx17 is phosphorylated by TBK1 whereby phospho-Stx17 controls the formation of the ATG13+FIP200+ mammalian pre-autophagosomal structure (mPAS) in response to induction of autophagy. TBK1 phosphorylates Stx17 at S202. During autophagy induction, Stx17pS202 transfers from the Golgi, where its steady-state pools localize, to the ATG13+FIP200+ mPAS. Stx17pS202 was in complexes with ATG13 and FIP200, whereas its non-phosphorylatable mutant Stx17S202A was not. Stx17 or TBK1 knockouts blocked ATG13 and FIP200 puncta formation. Stx17 or TBK1 knockouts reduced the formation of ATG13 protein complexes with FIP200 and ULK1. Endogenous Stx17pS202 colocalized with LC3B following induction of autophagy. Stx17 knockout diminished LC3 response and reduced sequestration of the prototypical bulk autophagy cargo lactate dehydrogenase. We conclude that Stx17 is a TBK1 substrate and that together they orchestrate assembly of mPAS.


Assuntos
Autofagia/genética , Complexos Multiproteicos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Qa-SNARE/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Técnicas de Inativação de Genes , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Fusão de Membrana/genética , Complexos Multiproteicos/metabolismo , Mutação/genética , Fosforilação , Proteínas Tirosina Quinases/genética , Transdução de Sinais/genética
13.
J Biosci ; 44(1)2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30837368

RESUMO

Autophagy is a highly conserved intracellular degradation pathway in eukaryotic cells that responds to environmental changes. Genetic analyses have shown that more than 40 autophagy-related genes (ATG) are directly involved in this process in fungi. In addition to Atg proteins, most vesicle transport regulators are also essential for each step of autophagy. The present study showed that one Endoplasmic Reticulum protein in Saccharomyces cerevisiae, Tip20, which controls Golgi-to-ER retrograde transport, was also required for starvation-induced autophagy under high temperature stress. In tip20 conditional mutant yeast, the transport of Atg8 was impaired during starvation, resulting in multiple Atg8 puncta dispersed outside the vacuole that could not be transported to the pre-autophagosomal structure/phagophore assembly site (PAS). Several Atg8 puncta were trapped in ER exit sites (ERES). Moreover, the GFP-Atg8 protease protection assay indicated that Tip20 functions before autophagosome closure. Furthermore, genetic studies showed that Tip20 functions downstream of Atg5 and upstream of Atg1, Atg9 and Atg14 in the autophagy pathway. The present data show that Tip20, as a vesicle transport regulator, has novel roles in autophagy.


Assuntos
Autofagia/genética , Transporte Proteico/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Proteína 5 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Proteínas de Membrana/genética , Mutação , Fagossomos/genética , Proteínas Quinases/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo
14.
FASEB J ; 33(5): 6442-6455, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30776316

RESUMO

Cellular stress or injury induces release of endogenous danger signals such as ATP, which plays a central role in activating immune cells. ATP is essential for the release of nonclassically secreted cytokines such as IL-1ß but, paradoxically, has been reported to inhibit the release of classically secreted cytokines such as TNF. Here, we reveal that ATP does switch off soluble TNF (17 kDa) release from LPS-treated macrophages, but rather than inhibiting the entire TNF secretion, ATP packages membrane TNF (26 kDa) within microvesicles (MVs). Secretion of membrane TNF within MVs bypasses the conventional endoplasmic reticulum- and Golgi transport-dependent pathway and is mediated by acid sphingomyelinase. These membrane TNF-carrying MVs are biologically more potent than soluble TNF in vivo, producing significant lung inflammation in mice. Thus, ATP critically alters TNF trafficking and secretion from macrophages, inducing novel unconventional membrane TNF signaling via MVs without direct cell-to-cell contact. These data have crucial implications for this key cytokine, particularly when therapeutically targeting TNF in acute inflammatory diseases.-Soni, S., O'Dea, K. P., Tan, Y. Y., Cho, K., Abe, E., Romano, R., Cui, J., Ma, D., Sarathchandra, P., Wilson, M. R., Takata, M. ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles.


Assuntos
Trifosfato de Adenosina/imunologia , Membrana Celular/imunologia , Vesículas Extracelulares/imunologia , Macrófagos/imunologia , Pneumonia/imunologia , Transdução de Sinais/imunologia , Fator de Necrose Tumoral alfa/imunologia , Doença Aguda , Trifosfato de Adenosina/genética , Animais , Comunicação Celular/genética , Comunicação Celular/imunologia , Membrana Celular/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Vesículas Extracelulares/genética , Complexo de Golgi/genética , Complexo de Golgi/imunologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Knockout , Pneumonia/induzido quimicamente , Pneumonia/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Necrose Tumoral alfa/genética
15.
J Cell Biol ; 218(3): 783-797, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30659099

RESUMO

Phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide with key roles in the Golgi complex, is made by Golgi-associated phosphatidylinositol-4 kinases and consumed by the 4-phosphatase Sac1 that, instead, is an ER membrane protein. Here, we show that the contact sites between the ER and the TGN (ERTGoCS) provide a spatial setting suitable for Sac1 to dephosphorylate PI4P at the TGN. The ERTGoCS, though necessary, are not sufficient for the phosphatase activity of Sac1 on TGN PI4P, since this needs the phosphatidyl-four-phosphate-adaptor-protein-1 (FAPP1). FAPP1 localizes at ERTGoCS, interacts with Sac1, and promotes its in-trans phosphatase activity in vitro. We envision that FAPP1, acting as a PI4P detector and adaptor, positions Sac1 close to TGN domains with elevated PI4P concentrations allowing PI4P consumption. Indeed, FAPP1 depletion induces an increase in TGN PI4P that leads to increased secretion of selected cargoes (e.g., ApoB100), indicating that FAPP1, by controlling PI4P levels, acts as a gatekeeper of Golgi exit.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Retículo Endoplasmático/genética , Complexo de Golgi/genética , Células HeLa , Células Hep G2 , Humanos , Proteínas de Membrana/genética , Camundongos , Fosfatos de Fosfatidilinositol/genética
16.
J Cell Biol ; 218(3): 1055-1065, 2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30659100

RESUMO

ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Lipídeos de Membrana/metabolismo , Receptores de Esteroides/metabolismo , Motivos de Aminoácidos , Transporte Biológico Ativo/fisiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/genética , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Lipídeos de Membrana/genética , Microscopia Eletrônica , Receptores de Esteroides/genética
17.
Cell Struct Funct ; 44(1): 1-19, 2019 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-30487368

RESUMO

The Golgi stress response is a homeostatic mechanism that augments the functional capacity of the Golgi apparatus when Golgi function becomes insufficient (Golgi stress). Three response pathways of the Golgi stress response have been identified in mammalian cells, the TFE3, HSP47 and CREB3 pathways, which augment the capacity of specific Golgi functions such as N-glycosylation, anti-apoptotic activity and pro-apoptotic activity, respectively. On the contrary, glycosylation of proteoglycans (PGs) is another important function of the Golgi, although the response pathway upregulating expression of glycosylation enzymes for PGs in response to Golgi stress remains unknown. Here, we found that expression of glycosylation enzymes for PGs was induced upon insufficiency of PG glycosylation capacity in the Golgi (PG-Golgi stress), and that transcriptional induction of genes encoding glycosylation enzymes for PGs was independent of the known Golgi stress response pathways and ER stress response. Promoter analyses of genes encoding these glycosylation enzymes revealed the novel enhancer elements PGSE-A and PGSE-B (the consensus sequences are CCGGGGCGGGGCG and TTTTACAATTGGTC, respectively), which regulate their transcriptional induction upon PG-Golgi stress. From these observations, the response pathway we discovered is a novel Golgi stress response pathway, which we have named the PG pathway.Key words: Golgi stress, proteoglycan, ER stress, organelle zone, organelle autoregulation.


Assuntos
Complexo de Golgi/genética , Proteoglicanas/metabolismo , Elementos de Resposta/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Estresse do Retículo Endoplasmático/genética , Proteínas de Choque Térmico HSP47/metabolismo , Células HeLa , Humanos , Transcrição Genética
18.
J Clin Invest ; 129(1): 230-245, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30352046

RESUMO

Levothyroxine (LT4) is a form of thyroid hormone used to treat hypothyroidism. In the brain, T4 is converted to the active form T3 by type 2 deiodinase (D2). Thus, it is intriguing that carriers of the Thr92Ala polymorphism in the D2 gene (DIO2) exhibit clinical improvement when liothyronine (LT3) is added to LT4 therapy. Here, we report that D2 is a cargo protein in ER Golgi intermediary compartment (ERGIC) vesicles, recycling between ER and Golgi. The Thr92-to-Ala substitution (Ala92-D2) caused ER stress and activated the unfolded protein response (UPR). Ala92-D2 accumulated in the trans-Golgi and generated less T3, which was restored by eliminating ER stress with the chemical chaperone 4-phenyl butyric acid (4-PBA). An Ala92-Dio2 polymorphism-carrying mouse exhibited UPR and hypothyroidism in distinct brain areas. The mouse refrained from physical activity, slept more, and required additional time to memorize objects. Enhancing T3 signaling in the brain with LT3 improved cognition, whereas restoring proteostasis with 4-PBA eliminated the Ala92-Dio2 phenotype. In contrast, primary hypothyroidism intensified the Ala92-Dio2 phenotype, with only partial response to LT4 therapy. Disruption of cellular proteostasis and reduced Ala92-D2 activity may explain the failure of LT4 therapy in carriers of Thr92Ala-DIO2.


Assuntos
Encéfalo , Estresse do Retículo Endoplasmático , Hipotireoidismo , Iodeto Peroxidase , Polimorfismo Genético , Resposta a Proteínas não Dobradas , Substituição de Aminoácidos , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Complexo de Golgi/enzimologia , Complexo de Golgi/genética , Células HEK293 , Humanos , Hipotireoidismo/tratamento farmacológico , Hipotireoidismo/enzimologia , Hipotireoidismo/genética , Hipotireoidismo/patologia , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Tiroxina/uso terapêutico , Tri-Iodotironina/uso terapêutico
19.
Hum Mol Genet ; 28(4): 598-614, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30335141

RESUMO

We report two unrelated families with multigenerational nonsyndromic intellectual disability (ID) segregating with a recurrent de novo missense variant (c.1543C>T:p.Leu515Phe) in the alkali cation/proton exchanger gene SLC9A7 (also commonly referred to as NHE7). SLC9A7 is located on human X chromosome at Xp11.3 and has not yet been associated with a human phenotype. The gene is widely transcribed, but especially abundant in brain, skeletal muscle and various secretory tissues. Within cells, SLC9A7 resides in the Golgi apparatus, with prominent enrichment in the trans-Golgi network (TGN) and post-Golgi vesicles. In transfected Chinese hamster ovary AP-1 cells, the Leu515Phe mutant protein was correctly targeted to the TGN/post-Golgi vesicles, but its N-linked oligosaccharide maturation as well as that of a co-transfected secretory membrane glycoprotein, vesicular stomatitis virus G (VSVG) glycoprotein, was reduced compared to cells co-expressing SLC9A7 wild-type and VSVG. This correlated with alkalinization of the TGN/post-Golgi compartments, suggestive of a gain-of-function. Membrane trafficking of glycosylation-deficient Leu515Phe and co-transfected VSVG to the cell surface, however, was relatively unaffected. Mass spectrometry analysis of patient sera also revealed an abnormal N-glycosylation profile for transferrin, a clinical diagnostic marker for congenital disorders of glycosylation. These data implicate a crucial role for SLC9A7 in the regulation of TGN/post-Golgi pH homeostasis and glycosylation of exported cargo, which may underlie the cellular pathophysiology and neurodevelopmental deficits associated with this particular nonsyndromic form of X-linked ID.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Complexo de Golgi/genética , Deficiência Intelectual/genética , Trocadores de Sódio-Hidrogênio/genética , Ácidos/metabolismo , Animais , Células CHO , Membrana Celular/genética , Cricetinae , Cricetulus , Regulação da Expressão Gênica/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Glicoproteínas de Membrana/genética , Mutação de Sentido Incorreto/genética , Transporte Proteico/genética , Transfecção , Proteínas do Envelope Viral/genética , Rede trans-Golgi/genética
20.
ACS Chem Neurosci ; 10(1): 599-609, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30272946

RESUMO

Manganese (Mn) is an essential metal that can be neurotoxic when elevated exposition occurs leading to parkinsonian-like syndromes. Mutations in the Slc30a10 gene have been identified in new forms of familial parkinsonism. SLC30A10 is a cell surface protein involved in the efflux of Mn and protects the cell against Mn toxicity. Disease-causing mutations block the efflux activity of SLC30A10, resulting in Mn accumulation. Determining the intracellular localization of Mn when disease-causing SLC30A10 mutants are expressed is essential to elucidate the mechanisms of Mn neurotoxicity. Here, using organelle fluorescence microscopy and synchrotron X-ray fluorescence (SXRF) imaging, we found that Mn accumulates in the Golgi apparatus of human cells transfected with the disease-causing SLC30A10-Δ105-107 mutant under physiological conditions and after exposure to Mn. In cells expressing the wild-type SLC30A10 protein, cellular Mn content was low after all exposure conditions, confirming efficient Mn efflux. In nontransfected cells that do not express endogenous SLC30A10 and in mock transfected cells, Mn was located in the Golgi apparatus, similarly to its distribution in cells expressing the mutant protein, confirming deficient Mn efflux. The newly developed SXRF cryogenic nanoimaging (<50 nm resolution) indicated that Mn was trapped in single vesicles within the Golgi apparatus. Our results confirm the role of SLC30A10 in Mn efflux and the accumulation of Mn in cells expressing the disease-causing SLC30A10-Δ105-107 mutation. Moreover, we identified suborganelle Golgi nanovesicles as the main compartment of Mn accumulation in SLC30A10 mutants, suggesting interactions with the vesicular trafficking machinery as a cause of the disease.


Assuntos
Proteínas de Transporte de Cátions/genética , Complexo de Golgi/metabolismo , Mutação/genética , Transtornos Parkinsonianos/genética , Proteínas de Transporte de Cátions/metabolismo , Complexo de Golgi/genética , Humanos , Manganês/metabolismo , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/metabolismo , Transtornos Parkinsonianos/metabolismo , Transporte Proteico/genética , Transporte Proteico/fisiologia
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