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1.
J Environ Pathol Toxicol Oncol ; 39(3): 261-279, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32865917

RESUMO

Among the neurodegenerative diseases, Alzheimer's disease (AD) is a predominant public health issue, affecting 16 million people around the world. It is clinically manifested by the presence of amyloid plaques (Aß) and neurofibrillary tangles (NFT) within the brain. Due to intraneuronal processing, Aß interacts with cellular targets such as mitochondria, ER, and Golgi apparatus and hampers their normal functions. Alteration in the mitochondrial function, closely related to the production of reactive oxygen species (ROS), Ca+2 overload, and apoptosis in the brain, is one of the key pathological events studied in AD pathogenesis. It is also an important pivot for the intracellular interaction with ER and Golgi through signal transduction and membrane contact to regulate cell survival and death mechanism. Alteration in mitochondrial function is intimately connected with abnormal ER or Golgi function. Stimuli that enhance perturbation in the normal ER or Golgi organelles function can involve mitochondria mediated apoptotic cell death. In this review, we address the importance of the mitochondria and their cross talk with ER and Golgi in AD pathogenesis and animal models with a therapeutic strategy to improve the mitochondrial functions.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Mitocôndrias/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Apoptose , Encéfalo/patologia , Retículo Endoplasmático/patologia , Complexo de Golgi/patologia , Humanos , Mitocôndrias/patologia , Transdução de Sinais
2.
Nat Commun ; 11(1): 3711, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709891

RESUMO

The skeletal muscle T-tubule is a specialized membrane domain essential for coordinated muscle contraction. However, in the absence of genetically tractable systems the mechanisms involved in T-tubule formation are unknown. Here, we use the optically transparent and genetically tractable zebrafish system to probe T-tubule development in vivo. By combining live imaging of transgenic markers with three-dimensional electron microscopy, we derive a four-dimensional quantitative model for T-tubule formation. To elucidate the mechanisms involved in T-tubule formation in vivo, we develop a quantitative screen for proteins that associate with and modulate early T-tubule formation, including an overexpression screen of the entire zebrafish Rab protein family. We propose an endocytic capture model involving firstly, formation of dynamic endocytic tubules at transient nucleation sites on the sarcolemma, secondly, stabilization by myofibrils/sarcoplasmic reticulum and finally, delivery of membrane from the recycling endosome and Golgi complex.


Assuntos
Contração Muscular/fisiologia , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Sarcolema/fisiologia , Sarcolema/ultraestrutura , Animais , Canais de Cálcio/metabolismo , Canais de Cálcio/ultraestrutura , Canais de Cálcio Tipo L/metabolismo , Proteínas de Transporte/metabolismo , Biologia do Desenvolvimento , Complexo de Golgi/metabolismo , Masculino , Microscopia Eletrônica , Proteínas Musculares/química , Músculo Esquelético/química , Miofibrilas/metabolismo , Sarcolema/química , Retículo Sarcoplasmático/metabolismo , Peixe-Zebra
3.
Cell Signal ; 73: 109706, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32629149

RESUMO

Chloroquine (CQ) and its analogue hydroxychloroquine (HCQ) have been thrust into our everyday vernacular because some believe, based on very limited basic and clinical data, that they might be helpful in preventing and/or lessening the severity of the pandemic coronavirus disease 2019 (COVID-19). However, lacking is a temperance in enthusiasm for their possible use as well as sufficient perspective on their effects and side-effects. CQ and HCQ have well-known properties of being diprotic weak bases that preferentially accumulate in acidic organelles (endolysosomes and Golgi apparatus) and neutralize luminal pH of acidic organelles. These primary actions of CQ and HCQ are responsible for their anti-malarial effects; malaria parasites rely on acidic digestive vacuoles for survival. Similarly, de-acidification of endolysosomes and Golgi by CQ and HCQ may block severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) integration into host cells because SARS-CoV-2 may require an acidic environment for its entry and for its ability to bud and infect bystander cells. Further, de-acidification of endolysosomes and Golgi may underly the immunosuppressive effects of these two drugs. However, modern cell biology studies have shown clearly that de-acidification results in profound changes in the structure, function and cellular positioning of endolysosomes and Golgi, in signaling between these organelles and other subcellular organelles, and in fundamental cellular functions. Thus, studying the possible therapeutic effects of CQ and HCQ against COVID-19 must occur concurrent with studies of the extent to which these drugs affect organellar and cell biology. When comprehensively examined, a better understanding of the Janus sword actions of these and other drugs might yield better decisions and better outcomes.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Cloroquina/farmacologia , Endossomos/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Hidroxicloroquina/farmacologia , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Antivirais/uso terapêutico , Betacoronavirus/enzimologia , Betacoronavirus/metabolismo , Betacoronavirus/patogenicidade , Cloroquina/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Citocinas/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidroxicloroquina/uso terapêutico , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Malária/tratamento farmacológico , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico
5.
Proc Natl Acad Sci U S A ; 117(22): 12341-12351, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32430335

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Currently, there is no effective therapy for PDAC, and a detailed molecular and functional evaluation of PDACs is needed to identify and develop better therapeutic strategies. Here we show that the transcription factor Krüppel-like factor 7 (KLF7) is overexpressed in PDACs, and that inhibition of KLF7 blocks PDAC tumor growth and metastasis in cell culture and in mice. KLF7 expression in PDACs can be up-regulated due to activation of a MAP kinase pathway or inactivation of the tumor suppressor p53, two alterations that occur in a large majority of PDACs. ShRNA-mediated knockdown of KLF7 inhibits the expression of IFN-stimulated genes (ISGs), which are necessary for KLF7-mediated PDAC tumor growth and metastasis. KLF7 knockdown also results in the down-regulation of Discs Large MAGUK Scaffold Protein 3 (DLG3), resulting in Golgi complex fragmentation, and reduced protein glycosylation, leading to reduced secretion of cancer-promoting growth factors, such as chemokines. Genetic or pharmacologic activation of Golgi complex fragmentation blocks PDAC growth and metastasis similar to KLF7 inhibition. Our results demonstrate a therapeutically amenable, KLF7-driven pathway that promotes PDAC growth and metastasis by activating ISGs and maintaining Golgi complex integrity.


Assuntos
Complexo de Golgi/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Complexo de Golgi/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Camundongos Knockout , Metástase Neoplásica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/fisiopatologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Nat Commun ; 11(1): 2170, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358503

RESUMO

Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens.


Assuntos
Arabidopsis/metabolismo , Citocininas/metabolismo , Resistência à Doença/genética , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Raízes de Plantas/metabolismo , Transcriptoma/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Endossomos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/metabolismo , Plasmodioforídeos/patogenicidade , Via Secretória/genética , Solo , Proteínas de Transporte Vesicular/metabolismo
7.
Nat Commun ; 11(1): 1754, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273498

RESUMO

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. Unc51-like kinase 1 (Ulk1) is an essential initiator not only for Atg5-dependent canonical autophagy but also for alternative autophagy. However, the mechanism as to how Ulk1 differentially regulates both types of autophagy has remained unclear. In this study, we identify a phosphorylation site of Ulk1 at Ser746, which is phosphorylated during genotoxic stress-induced alternative autophagy. Phospho-Ulk1746 localizes exclusively on the Golgi and is required for alternative autophagy, but not canonical autophagy. We also identify receptor-interacting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1746 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk1 at Ser746, and loss of RIPK3 abolishes Ulk1746 phosphorylation. These findings indicate that RIPK3-dependent Ulk1746 phosphorylation on the Golgi plays a pivotal role in genotoxic stress-induced alternative autophagy.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Dano ao DNA , Complexo de Golgi/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Animais , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Sítios de Ligação/genética , Células Cultivadas , Embrião de Mamíferos/citologia , Etoposídeo/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos Knockout , Microscopia Confocal , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Homologia de Sequência de Aminoácidos , Serina/genética
8.
Nature ; 580(7804): 530-535, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32322062

RESUMO

Cancer cells increase lipogenesis for their proliferation and the activation of sterol regulatory element-binding proteins (SREBPs) has a central role in this process. SREBPs are inhibited by a complex composed of INSIG proteins, SREBP cleavage-activating protein (SCAP) and sterols in the endoplasmic reticulum. Regulation of the interaction between INSIG proteins and SCAP by sterol levels is critical for the dissociation of the SCAP-SREBP complex from the endoplasmic reticulum and the activation of SREBPs1,2. However, whether this protein interaction is regulated by a mechanism other than the abundance of sterol-and in particular, whether oncogenic signalling has a role-is unclear. Here we show that activated AKT in human hepatocellular carcinoma (HCC) cells phosphorylates cytosolic phosphoenolpyruvate carboxykinase 1 (PCK1), the rate-limiting enzyme in gluconeogenesis, at Ser90. Phosphorylated PCK1 translocates to the endoplasmic reticulum, where it uses GTP as a phosphate donor to phosphorylate INSIG1 at Ser207 and INSIG2 at Ser151. This phosphorylation reduces the binding of sterols to INSIG1 and INSIG2 and disrupts the interaction between INSIG proteins and SCAP, leading to the translocation of the SCAP-SREBP complex to the Golgi apparatus, the activation of SREBP proteins (SREBP1 or SREBP2) and the transcription of downstream lipogenesis-related genes, proliferation of tumour cells, and tumorigenesis in mice. In addition, phosphorylation of PCK1 at Ser90, INSIG1 at Ser207 and INSIG2 at Ser151 is not only positively correlated with the nuclear accumulation of SREBP1 in samples from patients with HCC, but also associated with poor HCC prognosis. Our findings highlight the importance of the protein kinase activity of PCK1 in the activation of SREBPs, lipogenesis and the development of HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Gluconeogênese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipogênese , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Animais , Carcinogênese , Carcinoma Hepatocelular/patologia , Proliferação de Células , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana/química , Camundongos , Camundongos Nus , Oxisteróis/metabolismo , Fosforilação , Prognóstico , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(18): 9884-9895, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32321832

RESUMO

The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo VAC2. We report here on the identification of 17 mtv mutations, corresponding to mutant alleles of MTV2/VSR4, MTV3/PTEN2A MTV7/EREL1, MTV8/ARFC1, MTV9/PUF2, MTV10/VPS3, MTV11/VPS15, MTV12/GRV2, MTV14/GFS10, MTV15/BET11, MTV16/VPS51, MTV17/VPS54, and MTV18/VSR1 Eight of the MTV proteins localize at the interface between the trans-Golgi network (TGN) and the multivesicular bodies (MVBs), supporting that the trafficking step between these compartments is essential for segregating vacuolar proteins from those destined for secretion. Importantly, the GARP tethering complex subunits MTV16/VPS51 and MTV17/VPS54 were found at endoplasmic reticulum (ER)- and microtubule-associated compartments (EMACs). Moreover, MTV16/VPS51 interacts with the motor domain of kinesins, suggesting that, in addition to tethering vesicles, the GARP complex may regulate the motors that transport them. Our findings unveil a previously uncharacterized compartment of the plant vacuolar trafficking pathway and support a role for microtubules and kinesins in GARP-dependent transport of soluble vacuolar cargo in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Transporte Proteico/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética , Alelos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Vesículas Citoplasmáticas/genética , Vesículas Citoplasmáticas/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Cinesina/genética , Cinesina/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Mutação , Vacúolos/genética , Proteínas de Transporte Vesicular/metabolismo
10.
Life Sci ; 253: 117700, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32335164

RESUMO

AIMS: Although previous studies elaborated that selective autophagy was involved in quality control of some organelles, including nucleus, mitochondria, the endoplasmic reticulum and peroxisomes, it remained unclear whether the selective autophagy of the Golgi apparatus (Golgiphagy) existed or not. MAIN METHODS: In this study, H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells were treated with autophagy inducers, Golgi stress inducers and cardiomyocytes hypertrophy stimulators. The Golgiphagy was evaluated by analysing the co-localization of Golgi markers and LC3B. Furthermore, the transmission electron microscope was used to observe the occurrence of Golgiphagy. The co-immunoprecipitation assay was used to evaluate the interaction of GOLPH3 and LC3B. KEY FINDINGS: Results showed that starvation promoted the co-localization of both GM130-positive and TGN46-positive Golgi fragments with LC3B-positive autophagosomes in H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells. Transmission electron microscopy images showed that Golgi apparatus was sequestered into the autophagosomes in the starvation group. Moreover, Golgi stress inducers also facilitated the co-localization of Golgi markers and LC3B in H9c2 cells, HUVECs, HA-VSMCs and HEK293T cells. Furthermore, cardiomyocyte hypertrophy stimulators also triggered the appearance of Golgiphagy in H9c2 cells. Importantly, the co-immunoprecipitation assay indicated endogenous GOLPH3 interacted with LC3B in H9c2 cells, HUVECs, HA-VSMCs. However, knocking down GOLPH3 inhibited the Golgiphagy. SIGNIFICANCE: This study unveiled a new selective autophagy of the Golgi apparatus (Golgiphagy). In addition, GOLPH3 might act as a novel cargo receptor to regulate Golgiphagy. Maintaining homeostasis of the Golgi apparatus via GOLPH3-mediated autophagy was indispensable for cell survival.


Assuntos
Autofagia/fisiologia , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/metabolismo , Ratos
11.
Curr Diab Rep ; 20(6): 20, 2020 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-32306181

RESUMO

PURPOSE OF REVIEW: Impairments in mitochondrial function in patients with insulin resistance and type 2 diabetes have been disputed for decades. This review aims to briefly summarize the current knowledge on mitochondrial dysfunction in metabolic tissues and to particularly focus on addressing a new perspective of mitochondrial dysfunction, the altered capacity of mitochondria to communicate with other organelles within insulin-resistant tissues. RECENT FINDINGS: Organelle interactions are temporally and spatially formed connections essential for normal cell function. Recent studies have shown that mitochondria interact with various cellular organelles, such as the endoplasmic reticulum, lysosomes and lipid droplets, forming inter-organelle junctions. We will discuss the current knowledge on alterations in these mitochondria-organelle interactions in insulin resistance and diabetes, with a focus on changes in mitochondria-lipid droplet communication as a major player in ectopic lipid accumulation, lipotoxicity and insulin resistance.


Assuntos
Comunicação Celular/fisiologia , Diabetes Mellitus Tipo 2/fisiopatologia , Resistência à Insulina/fisiologia , Mitocôndrias/fisiologia , Doenças Mitocondriais/fisiopatologia , Organelas/fisiologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Complexo de Golgi/metabolismo , Complexo de Golgi/fisiologia , Humanos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/fisiologia , Lisossomos/metabolismo , Lisossomos/fisiologia , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Organelas/metabolismo , Sobrepeso/metabolismo , Sobrepeso/fisiopatologia , Peroxissomos/metabolismo , Peroxissomos/fisiologia
12.
Proc Natl Acad Sci U S A ; 117(19): 10565-10574, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32345721

RESUMO

Numerous mutations that impair retrograde membrane trafficking between endosomes and the Golgi apparatus lead to neurodegenerative diseases. For example, mutations in the endosomal retromer complex are implicated in Alzheimer's and Parkinson's diseases, and mutations of the Golgi-associated retrograde protein (GARP) complex cause progressive cerebello-cerebral atrophy type 2 (PCCA2). However, how these mutations cause neurodegeneration is unknown. GARP mutations in yeast, including one causing PCCA2, result in sphingolipid abnormalities and impaired cell growth that are corrected by treatment with myriocin, a sphingolipid synthesis inhibitor, suggesting that alterations in sphingolipid metabolism contribute to cell dysfunction and death. Here we tested this hypothesis in wobbler mice, a murine model with a homozygous partial loss-of-function mutation in Vps54 (GARP protein) that causes motor neuron disease. Cytotoxic sphingoid long-chain bases accumulated in embryonic fibroblasts and spinal cords from wobbler mice. Remarkably, chronic treatment of wobbler mice with myriocin markedly improved their wellness scores, grip strength, neuropathology, and survival. Proteomic analyses of wobbler fibroblasts revealed extensive missorting of lysosomal proteins, including sphingolipid catabolism enzymes, to the Golgi compartment, which may contribute to the sphingolipid abnormalities. Our findings establish that altered sphingolipid metabolism due to GARP mutations contributes to neurodegeneration and suggest that inhibiting sphingolipid synthesis might provide a useful strategy for treating these disorders.


Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Esfingolipídeos/metabolismo , Animais , Modelos Animais de Doenças , Endossomos/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Feminino , Fibroblastos/metabolismo , Complexo de Golgi/metabolismo , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Doença dos Neurônios Motores/genética , Doença dos Neurônios Motores/metabolismo , Doença dos Neurônios Motores/patologia , Neurônios Motores/metabolismo , Células-Tronco Embrionárias Murinas , Mutação , Malformações do Sistema Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Transporte Proteico , Proteômica , Proteínas de Transporte Vesicular/metabolismo
13.
Parasitol Res ; 119(5): 1629-1640, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32219551

RESUMO

Rab proteins constitute the largest group of small GTPases and act as molecular switches in a wide variety of cellular processes, including proliferation, cytoskeleton assembly, and membrane trafficking in all eukaryotic cells. Rab21 has been reported in several eukaryotic cells, and our results suggest that in Entamoeba histolytica, Rab21 is involved in the vesicular traffic associated with the Golgi apparatus, where its function appears to be important to maintain the structure of this organelle. In addition, proteins such as Rab1A and Sec24, identified in this work associated with EhRab21, participate in the traffic of COPII vesicles from the endoplasmic reticulum to the Golgi apparatus and are necessary to maintain the latter's structure in human cells. In addition, EhRab21 probably affects the lysosome biogenesis, as indicated by an increase in the number of lysosomes as a result of the increase in EhRab21 activity. The participation of EhRab21 in the pathogenesis of amebiasis was verified on the amoebic liver abscess formation model using hamsters (Mesocricetus auratus), in which the overexpression of EhRab21Q64L (positive dominant mutant protein) decreased the number of liver abscesses formed.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Entamoeba histolytica/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Amebíase/patologia , Animais , Cricetinae , Retículo Endoplasmático/metabolismo , Humanos , Abscesso Hepático Amebiano/patologia , Lisossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo
14.
Nat Commun ; 11(1): 1304, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161259

RESUMO

The integrated stress response (ISR) converges on eIF2α phosphorylation to regulate protein synthesis. ISR is activated by several stress conditions, including endoplasmic reticulum (ER) stress, executed by protein kinase R-like endoplasmic reticulum kinase (PERK). We report that ER stress combined with ISR inhibition causes an impaired maturation of several tyrosine kinase receptors (RTKs), consistent with a partial block of their trafficking from the ER to the Golgi. Other proteins mature or are secreted normally, indicating selective retention in the ER (sERr). sERr is relieved upon protein synthesis attenuation and is accompanied by the generation of large mixed disulfide bonded complexes, including ERp44. sERr was pharmacologically recapitulated by combining the HIV-protease inhibitor nelfinavir with ISRIB, an experimental drug that inhibits ISR. Nelfinavir/ISRIB combination is highly effective to inhibit the growth of RTK-addicted cell lines and hepatocellular (HCC) cells in vitro and in vivo. Thus, pharmacological sERr can be utilized as a modality for cancer treatment.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , eIF-2 Quinase/metabolismo , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Sistemas CRISPR-Cas/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Cicloexilaminas/farmacologia , Cicloexilaminas/uso terapêutico , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Nelfinavir/farmacologia , Nelfinavir/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , eIF-2 Quinase/genética
15.
Nat Commun ; 11(1): 1368, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170195

RESUMO

MCFD2 and ERGIC-53, which are the products of causative genes of combined factor V and factor VIII deficiency, form a cargo receptor complex responsible for intracellular transport of these coagulation factors in the early secretory pathway. In this study, using an NMR technique, we successfully identified an MCFD2-binding segment from factor VIII composed of a 10 amino acid sequence that enhances its secretion. This prompted us to examine possible effects of attaching this sequence to recombinant glycoproteins on their secretion. We found that the secretion level of recombinant erythropoietin was significantly increased simply by tagging it with the passport sequence. Our findings not only provide molecular basis for the intracellular trafficking of coagulation factors and their genetic deficiency but also offer a potentially useful tool for increasing the production yields of recombinant glycoproteins of biopharmaceutical interest.


Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Retículo Endoplasmático/fisiologia , Eritropoetina/metabolismo , Fator V , Fator VIII/metabolismo , Glicoproteínas/genética , Complexo de Golgi/fisiologia , Humanos , Lectinas de Ligação a Manose/metabolismo , Transporte Proteico , Via Secretória
16.
Nat Commun ; 11(1): 1418, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184397

RESUMO

The Golgi apparatus plays a central role in the intracellular transport of macromolecules. However, molecular mechanisms of Golgi-mediated lipid transport remain poorly understood. Here, we show that genetic inactivation of the Golgi-resident protein GRASP55 in mice reduces whole-body fat mass via impaired intestinal fat absorption and evokes resistance to high-fat diet induced body weight gain. Mechanistic analyses reveal that GRASP55 participates in the Golgi-mediated lipid droplet (LD) targeting of some LD-associated lipases, such as ATGL and MGL, which is required for sustained lipid supply for chylomicron assembly and secretion. Consequently, GRASP55 deficiency leads to reduced chylomicron secretion and abnormally large LD formation in intestinal epithelial cells upon exogenous lipid challenge. Notably, deletion of dGrasp in Drosophila causes similar defects of lipid accumulation in the midgut. These results highlight the importance of the Golgi complex in cellular lipid regulation, which is evolutionary conserved, and uncover potential therapeutic targets for obesity-associated diseases.


Assuntos
Gorduras/metabolismo , Proteínas da Matriz do Complexo de Golgi/genética , Obesidade/genética , Obesidade/prevenção & controle , Animais , Transporte Biológico , Dieta Hiperlipídica , Drosophila , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Obesidade/fisiopatologia , Ganho de Peso
17.
Nat Commun ; 11(1): 1128, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111832

RESUMO

The sterol-regulatory element binding proteins (SREBP) are central transcriptional regulators of lipid metabolism. Using haploid genetic screens we identify the SREBP Regulating Gene (SPRING/C12ORF49) as a determinant of the SREBP pathway. SPRING is a glycosylated Golgi-resident membrane protein and its ablation in Hap1 cells, Hepa1-6 hepatoma cells, and primary murine hepatocytes reduces SREBP signaling. In mice, Spring deletion is embryonic lethal yet silencing of hepatic Spring expression also attenuates the SREBP response. Mechanistically, attenuated SREBP signaling in SPRINGKO cells results from reduced SREBP cleavage-activating protein (SCAP) and its mislocalization to the Golgi irrespective of the cellular sterol status. Consistent with limited functional SCAP in SPRINGKO cells, reintroducing SCAP restores SREBP-dependent signaling and function. Moreover, in line with the role of SREBP in tumor growth, a wide range of tumor cell lines display dependency on SPRING expression. In conclusion, we identify SPRING as a previously unrecognized modulator of SREBP signaling.


Assuntos
Colesterol/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Animais , Linhagem Celular , Desenvolvimento Embrionário/genética , Retículo Endoplasmático/metabolismo , Expressão Gênica , Complexo de Golgi/metabolismo , Haploidia , Hepatócitos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Elemento Regulador de Esterol/genética
18.
Nat Rev Mol Cell Biol ; 21(3): 151-166, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32034394

RESUMO

During division, eukaryotic cells undergo a dramatic, complex and coordinated remodelling of their cytoskeleton and membranes. For cell division to occur, chromosomes must be segregated and new cellular structures, such as the spindle apparatus, must be assembled. Pre-existing organelles, such as the nuclear envelope, endoplasmic reticulum and Golgi apparatus, must be disassembled or remodelled, distributed and reformed. Smaller organelles such as mitochondria as well as cytoplasmic content must also be properly distributed between daughter cells. This mixture of organelles and cytoplasm is bound by a plasma membrane that is itself subject to remodelling as division progresses. The lipids resident in these different membrane compartments play important roles in facilitating the division process. In recent years, we have begun to understand how membrane remodelling is coordinated during division; however, there is still much to learn. In this Review, we discuss recent insights into how these important cellular events are performed and regulated.


Assuntos
Divisão Celular/fisiologia , Membranas/metabolismo , Organelas/fisiologia , Animais , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Células Eucarióticas/citologia , Complexo de Golgi/metabolismo , Humanos , Membranas/fisiologia , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Organelas/metabolismo , Fuso Acromático/metabolismo
19.
Nat Chem Biol ; 16(3): 327-336, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32080624

RESUMO

The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.


Assuntos
Benzamidas/metabolismo , Proteínas Qa-SNARE/metabolismo , Tiofenos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Benzamidas/farmacologia , Transporte Biológico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Transporte Proteico , Ricina/metabolismo , Toxina Shiga/metabolismo , Toxinas Shiga/metabolismo , Tiofenos/farmacologia , Proteínas de Transporte Vesicular/fisiologia
20.
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
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