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1.
BMC Med Genet ; 21(1): 4, 2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906877

RESUMO

BACKGROUND: Chediak-Higashi Syndrome (CHS) is a rare autosomal recessive disease caused by loss of function of the lysosomal trafficking regulator protein. The causative gene LYST/CHS1 was cloned and identified in 1996, which showed significant homology to other species such as bovine and mouse. To date, 74 pathogenic or likely pathogenic mutations had been reported. CASE PRESENTATION: Here we describe a compound heterozygote in LYST gene, which was identified in a 4-year-old female patient. The patient showed skin hypopigmentation, sensitivity to light, mild splenomegaly and reduction of platelets in clinical examination. Giant intracytoplasmic inclusions were observed in the bone marrow examination, suggesting the diagnosis of CHS. Amplicon sequencing was performed to detect pathogenic mutation in LYST gene. The result was confirmed by two-generation pedigree analysis base on sanger sequencing. CONCLUSION: A compound heterozygote in LYST gene, consisting of a missense mutation c.5719A > G and an intron mutation c.4863-4G > A, was identified from the patient by using amplicon sequencing. The missense mutation is reported for the first time. Two-generation pedigree analysis showed these two mutations were inherited from the patient's parents, respectively. Our result demonstrated that amplicon sequencing has great potential for accelerating and improving the diagnosis of rare genetic diseases.


Assuntos
Sequência de Aminoácidos/genética , Síndrome de Chediak-Higashi/genética , Proteínas de Transporte Vesicular/genética , Síndrome de Chediak-Higashi/patologia , Criança , Pré-Escolar , Feminino , Heterozigoto , Humanos , Lisossomos/genética , Mutação de Sentido Incorreto , Linhagem
2.
J Agric Food Chem ; 67(41): 11428-11435, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31589037

RESUMO

Diosgenin and diosgenyl saponins as the major bioactive compounds isolated from dietary fenugreek seeds, yam roots, etc. possessed strong antitumor effects. To understand their detailed antitumor mechanisms, a fluorophore-appended derivative of diosgenin [Glc/CNHphth-diosgenin (GND)] was synthesized, starting from diosgenin and glucosamine hydrochloride in overall yields of 7-12% over 7-10 steps. Co-localization of GND with organelle-specific stains, transmission electron microscopy, and relative protein analyses demonstrated that GND crossed the plasma membrane through organic anion-transporting polypeptide 1B1 and distributed in the endoplasmic reticulum (ER), lysosome, and mitochondria. In this process, GND induced ER swelling, mitochondrial damage, and autophagosome and upregulating IRE-1α to induce autophagy and apoptosis. Furthermore, autophagy inhibitor chloroquine delayed the appearance of cleaved poly(ADP-ribose) polymerase and inhibited cleaved caspase 8, which indicated that GND induced autophagy to activate caspase-8-dependent apoptosis. These observations suggested that diosgenyl saponin was a potent anticancer agent that elicited ER stress and mitochondria-mediated apoptotic pathways in liver cancer.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Hepáticas/fisiopatologia , Extratos Vegetais/farmacologia , Saponinas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo
3.
Emerg Microbes Infect ; 8(1): 1511-1523, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31631785

RESUMO

Interferons (IFNs) control viral infections by inducing expression of IFN-stimulated genes (ISGs) that restrict distinct steps of viral replication. We report herein that gamma-interferon-inducible lysosomal thiol reductase (GILT), a lysosome-associated ISG, restricts the infectious entry of selected enveloped RNA viruses. Specifically, we demonstrated that GILT was constitutively expressed in lung epithelial cells and fibroblasts and its expression could be further induced by type II interferon. While overexpression of GILT inhibited the entry mediated by envelope glycoproteins of SARS coronavirus (SARS-CoV), Ebola virus (EBOV) and Lassa fever virus (LASV), depletion of GILT enhanced the entry mediated by these viral envelope glycoproteins. Furthermore, mutations that impaired the thiol reductase activity or disrupted the N-linked glycosylation, a posttranslational modification essential for its lysosomal localization, largely compromised GILT restriction of viral entry. We also found that the induction of GILT expression reduced the level and activity of cathepsin L, which is required for the entry of these RNA viruses in lysosomes. Our data indicate that GILT is a novel antiviral ISG that specifically inhibits the entry of selected enveloped RNA viruses in lysosomes via disruption of cathepsin L metabolism and function and may play a role in immune control and pathogenesis of these viruses.


Assuntos
Ebolavirus/fisiologia , Doença pelo Vírus Ebola/imunologia , Febre Lassa/imunologia , Vírus Lassa/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/imunologia , Vírus da SARS/fisiologia , Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Catepsina L/genética , Catepsina L/imunologia , Linhagem Celular , Ebolavirus/genética , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/virologia , Humanos , Febre Lassa/genética , Febre Lassa/virologia , Vírus Lassa/genética , Lisossomos/genética , Lisossomos/imunologia , Lisossomos/virologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Vírus da SARS/genética , Síndrome Respiratória Aguda Grave/genética , Síndrome Respiratória Aguda Grave/virologia , Proteínas do Envelope Viral/genética , Replicação Viral
4.
Elife ; 82019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31612854

RESUMO

Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties.


Assuntos
Esclerose Amiotrófica Lateral/diagnóstico , Anticorpos Monoclonais/química , Proteína C9orf72/genética , Demência Frontotemporal/diagnóstico , Imuno-Histoquímica/normas , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/imunologia , Esclerose Amiotrófica Lateral/metabolismo , Animais , Anticorpos Monoclonais/classificação , Anticorpos Monoclonais/imunologia , Biomarcadores/metabolismo , Proteína C9orf72/imunologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Demência Frontotemporal/genética , Demência Frontotemporal/imunologia , Demência Frontotemporal/metabolismo , Edição de Genes , Expressão Gênica , Células HEK293 , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Fagossomos/genética , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Células RAW 264.7
5.
J Biosci ; 44(4)2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31502579

RESUMO

In previous studies, we found interferon-α (IFN-α) could reduce protein levels of p11, 5-hydroxytryptamine receptor 1b (5-HT1b) and 5-hydroxytryptamine receptor 4 (5-HT4), but does not influence their messenger RNA levels in SH-sy5y cells. Thus, we investigated the post-transcriptional modulation of these molecules by IFN-α. SH-sy5y cells were treated with IFN-α, NH4Cl or MG132 alone or in combination, and then the protein levels of p11, 5-HT1b and 5-HT4 were analyzed by western blots. The regulatory effects of p11 on 5-HT1b and 5-HT4 were also determined in p11 knock-down cells. NH4Cl but not MG132 could reverse the protein level of p11 in IFN-α-treated SH-sy5y cells. MG132 could recover the protein levels of 5-HT1b and 5-HT4 in p11 knock-down cells. The down-regulation effects of IFN-α on p11, 5-HT1b and 5-HT4 were associated with the lysosome and ubiquitin-proteasome-mediated pathways. p11 was identified as a potent regulator to modulate the ubiquitination of 5-HT1b and 5-HT4. Therefore, it could be potential target therapies in IFN-ainduced depression.


Assuntos
Depressão/tratamento farmacológico , Interferon-alfa/farmacologia , Receptor 5-HT1B de Serotonina/genética , Cloreto de Amônio/farmacologia , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/genética , Depressão/genética , Depressão/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Leupeptinas/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Proteólise , RNA Mensageiro/genética , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ubiquitina
6.
Int J Mol Sci ; 20(18)2019 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505809

RESUMO

Many neurodegenerative disorders have lysosomal impediments, and the list of proposed treatments targeting lysosomes is growing. We investigated the role of lysosomes in Alzheimer's disease (AD) and other age-related disorders, as well as in a strategy to compensate for lysosomal disturbances. Comprehensive immunostaining was used to analyze brains from wild-type mice vs. amyloid precursor protein/presenilin-1 (APP/PS1) mice that express mutant proteins linked to familial AD. Also, lysosomal modulation was evaluated for inducing synaptic and behavioral improvements in transgenic models of AD and Parkinson's disease, and in models of mild cognitive impairment (MCI). Amyloid plaques were surrounded by swollen organelles positive for the lysosome-associated membrane protein 1 (LAMP1) in the APP/PS1 cortex and hippocampus, regions with robust synaptic deterioration. Within neurons, lysosomes contain the amyloid ß 42 (Aß42) degradation product Aß38, and this indicator of Aß42 detoxification was augmented by Z-Phe-Ala-diazomethylketone (PADK; also known as ZFAD) as it enhanced the lysosomal hydrolase cathepsin B (CatB). PADK promoted Aß42 colocalization with CatB in lysosomes that formed clusters in neurons, while reducing Aß deposits as well. PADK also reduced amyloidogenic peptides and α-synuclein in correspondence with restored synaptic markers, and both synaptic and cognitive measures were improved in the APP/PS1 and MCI models. These findings indicate that lysosomal perturbation contributes to synaptic and cognitive decay, whereas safely enhancing protein clearance through modulated CatB ameliorates the compromised synapses and cognition, thus supporting early CatB upregulation as a disease-modifying therapy that may also slow the MCI to dementia continuum.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Disfunção Cognitiva/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/patologia , Disfunção Cognitiva/genética , Disfunção Cognitiva/patologia , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/genética , Lisossomos/patologia , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Sinapses/metabolismo , Sinapses/patologia
7.
PLoS Genet ; 15(9): e1008376, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31487280

RESUMO

Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme whose function is important for the biosynthesis of purines, pyrimidines, and pyridines. Importantly, while missense mutations of PRPS1 have been identified in neurological disorders such as Arts syndrome, how they contribute to neuropathogenesis is still unclear. We identified the Drosophila ortholog of PRPS (dPRPS) as a direct target of RB/E2F in Drosophila, a vital cell cycle regulator, and engineered dPRPS alleles carrying patient-derived mutations. Interestingly, while they are able to develop normally, dPRPS mutant flies have a shortened lifespan and locomotive defects, common phenotypes associated with neurodegeneration. Careful analysis of the fat body revealed that patient-derived PRPS mutations result in profound defects in lipolysis, macroautophagy, and lysosome function. Significantly, we show evidence that the nervous system of dPRPS mutant flies is affected by these defects. Overall, we uncovered an unexpected link between nucleotide metabolism and autophagy/lysosome function, providing a possible mechanism by which PRPS-dysfunction contributes to neurological disorders.


Assuntos
Autofagia/genética , Lisossomos/genética , Ribose-Fosfato Pirofosfoquinase/metabolismo , Sequência de Aminoácidos , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Pleiotropia Genética/genética , Lisossomos/metabolismo , Mutação , Mutação de Sentido Incorreto , Proteostase/genética , Ribose-Fosfato Pirofosfoquinase/genética , Ribose-Fosfato Pirofosfoquinase/fisiologia
8.
Mol Carcinog ; 58(11): 2149-2160, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31448838

RESUMO

Autophagy is a self-proteolytic process that degrades intracellular material to maintain cellular homeostasis. Transcription factor EB (TFEB) is the master activator that regulates the transcription of genes involved in autophagy and lysosomal biogenesis. However, the cotranscriptional factors of TFEB are rarely identified. Here, we found that Yin Yang 1 (YY1) regulated autophagy and lysosome biogenesis in melanoma cells. YY1 cooperates with TFEB to regulate autophagy through controlling the transcription of autophagy and lysosome biogenesis related genes. Moreover, suppression of YY1 enhanced the antitumor efficiency of vemurafenib both in vitro and in vivo. Collectively, these studies identify YY1 as a novel cotranscription factor of TFEB in regulating autophagy and lysosomal functions and suggest YY1 could be a therapeutic target in cancer treatment.


Assuntos
Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Melanoma/genética , Fator de Transcrição YY1/genética , Animais , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Xenoenxertos , Humanos , Lisossomos/genética , Melanoma/patologia , Camundongos , Plasmídeos/genética
9.
Biomed Res Int ; 2019: 4087160, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31317028

RESUMO

Autophagy is a degradative process in which cellular organelles and proteins are recycled to restore homeostasis and cellular metabolism. Autophagy can be either a prosurvival or a prodeath process and remains one of the most fundamental processes for cell vitality. Thus autophagy modulation is an important approach for reinforcement anticancer therapeutics. Earlier we have demonstrated that recombinant analog of human milk protein lactaptin (RL2) induced apoptosis of various cultured cancer cells and activated lipidation of microtubule-associated protein 1 light chain 3 (LC3). In this study we investigated whether autophagy inhibitors-chloroquine (CQ), Ku55933 (Ku), and 3-methyladenine (3MA)-or inducer-rapamycin (Rap)-can enhance cytotoxic activity of lactaptin analog in cancer cells and its anticancer activity in the mice model. Western Blot analysis revealed that RL2 induced short-term autophagy in MDA-MB-231 and MCF-7 cells at early stages of incubation and that these data were confirmed by the transmission electron microscopy of autophagosome/autophagolysosome formation. RL2 stimulates reactive oxygen species (ROS) production, autophagosomes accumulation, upregulation of ATG5 with processing of LC3I to LC3II, and downregulation of p62/sequestosome 1 (p62). We have shown that autophagy modulators, CQ, Ku, and Rap, synergistically increased cytotoxicity of RL2, and RL2 with CQ induced autophagic cell death. In addition, CQ, Ku, and Rap in combination with RL2 decreased activity of lysosomal protease Cathepsin D. More importantly, combining RL2 with CQ, we improved antitumor effect in mice. Detected synergistic cytotoxic effects of both types of autophagy regulators, inhibitors, and inducers with RL2 against cancer cells allow us to believe that these combinations can be a basis for the new anticancer approach. Finally, we suppose that CQ and Rap promoting of short-term RL2-induced autophagy interlinks with final autophagic cell death.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Caseínas/farmacologia , Neoplasias/tratamento farmacológico , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/genética , Caseínas/genética , Catepsina D/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Células MCF-7 , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Morfolinas/farmacologia , Neoplasias/genética , Pironas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/genética
10.
EBioMedicine ; 45: 393-407, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31303501

RESUMO

BACKGROUND: Recent studies have revealed that vitamin D deficiency may increase the risk of Alzheimer's disease, and vitamin D supplementation may be effective strategy to ameliorate the neurodegenerative process in Alzheimer's disease patients. Paricalcitol (PAL), a low-calcemic vitamin D receptor agonist, is clinically used to treat secondary hyperparathyroidism. However, the potential application of PAL for treating neurodegenerative disorders remains unexplored. METHODS: The APP/PS1 mice were intraperitoneally injected with PAL or vehicle every other day for 15 weeks. The ß-amyloid (Aß) production was confirmed using immunostaining and enzyme linked immunosorbent assay. The underlying mechanism was verified by western blot and immunostaining in vivo and in vitro. FINDINGS: Long-term PAL treatment clearly reduced ß-amyloid (Aß) generation and neuronal loss in APP/PS1 transgenic mouse brains. PAL stimulated the expression of low-density lipoprotein receptor-related protein 1 (LRP1) possibly through inhibiting sterol regulatory element binding protein-2 (SREBP2); PAL also promoted LRP1-mediated ß-site APP cleavage enzyme 1 (BACE1) transport to late endosomes, thus increasing the lysosomal degradation of BACE1. Furthermore, PAL diminished 8-hydroxyguanosine (8-OHdG) generation in neuronal mitochondria via enhancing base excision repair (BER), resulting in the attenuation of calpain-1-mediated neuronal loss. INTERPRETATION: The present data demonstrate that PAL can reduce Aß generation through accelerating BACE1 lysosomal degradation and can inhibit neuronal loss through suppressing mitochondrial 8-OHdG generation. Hence, PAL might be a promising agent for treating Alzheimer's disease. FUND: This study was financially supported by the Natural Science Foundation of China (U1608282).


Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Ergocalciferóis/farmacologia , Neurônios/efeitos dos fármacos , /metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Calpaína/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Neurônios/patologia , Oligopeptídeos/genética , Presenilina-1/genética , Proteólise/efeitos dos fármacos
11.
PLoS One ; 14(5): e0217780, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31150494

RESUMO

Gaucher and Fabry diseases are the most prevalent sphingolipidoses. Chronic inflammation is activated in those disorders, which could play a role in pathogenesis. Significant degrees of amelioration occur in patients upon introduction of specific therapies; however, restoration to complete health status is not always achieved. The idea of an adjunctive therapy that targets inflammation may be a suitable option for patients. PPS is a mixture of semisynthetic sulfated polyanions that have been shown to have anti-inflammatory effects in mucopolysaccharidosis type I and II patients and animal models of type I, IIIA and VI. We hypothesized PPS could be a useful adjunctive therapy to inflammation for Gaucher and Fabry diseases. The objective of this work is to analyze the in vitro effect of PPS on inflammatory cytokines in cellular models of Gaucher and Fabry diseases, and to study its effect in Gaucher disease associated in vitro bone alterations. Cultures of peripheral blood mononuclear cells from Fabry and Gaucher patients were exposed to PPS. The secretion of proinflammatory cytokines was significantly reduced. Peripheral blood cells exposed to PPS from Gaucher patients revealed a reduced tendency to differentiate to osteoclasts. Osteoblasts and osteocytes cell lines were incubated with an inhibitor of glucocerebrosidase, and conditioned media was harvested in order to analyze if those cells secrete factors that induce osteoclastogenesis. Conditioned media from this cell cultures exposed to PPS produced lower numbers of osteoclasts. We could demonstrate PPS is an effective molecule to reduce the production of proinflammatory cytokines in in vitro models of Fabry and Gaucher diseases. Moreover, it was effective at ameliorating bone alterations of in vitro models of Gaucher disease. These results serve as preclinical supportive data to start clinical trials in human patients to analyze the effect of PPS as a potential adjunctive therapy for Fabry and Gaucher diseases.


Assuntos
Doença de Fabry/tratamento farmacológico , Doença de Gaucher/tratamento farmacológico , Inflamação/tratamento farmacológico , Poliéster Sulfúrico de Pentosana/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Doença de Fabry/patologia , Doença de Gaucher/patologia , Humanos , Inflamação/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos
12.
Nat Cell Biol ; 21(5): 614-626, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31036939

RESUMO

Cell growth is controlled by a lysosomal signalling complex containing Rag small GTPases and mammalian target of rapamycin complex 1 (mTORC1) kinase. Here, we carried out a microscopy-based genome-wide human short interfering RNA screen and discovered a lysosome-localized G protein-coupled receptor (GPCR)-like protein, GPR137B, that interacts with Rag GTPases, increases Rag localization and activity, and thereby regulates mTORC1 translocation and activity. High GPR137B expression can recruit and activate mTORC1 in the absence of amino acids. Furthermore, GPR137B also regulates the dissociation of activated Rag from lysosomes, suggesting that GPR137B controls a cycle of Rag activation and dissociation from lysosomes. GPR137B-knockout cells exhibited defective autophagy and an expanded lysosome compartment, similar to Rag-knockout cells. Like zebrafish RagA mutants, GPR137B-mutant zebrafish had upregulated TFEB target gene expression and an expanded lysosome compartment in microglia. Thus, GPR137B is a GPCR-like lysosomal regulatory protein that controls dynamic Rag and mTORC1 localization and activity as well as lysosome morphology.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Genoma Humano/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Receptores Acoplados a Proteínas-G/genética , Animais , Autofagia/genética , Regulação da Expressão Gênica/genética , Humanos , Lisossomos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Microglia/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , RNA Interferente Pequeno/genética , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
13.
Genes Cells ; 24(6): 436-448, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31038803

RESUMO

Lysosomes are acidic organelles responsible for degrading both exogenous and endogenous materials. The small GTPase Arl8 localizes primarily to lysosomes and is involved in lysosomal function. In the present study, using Arl8b gene-trapped mutant (Arl8b-/- ) mice, we show that Arl8b is required for the development of dorsal structures of the neural tube, including the thalamus and hippocampus. In embryonic day (E) 10.5 Arl8b-/- embryos, Sox1 (a neuroepithelium marker) was ectopically expressed in the roof plate, whereas the expression of Gdf7 and Msx1 (roof plate markers) was reduced in the dorsal midline of the midbrain. Ectopic expression of Sox1 in Arl8b-/- embryos was detected also at E9.0 in the neural fold, which gives rise to the roof plate. In addition, the levels of Bmp receptor IA and phosphorylated Smad 1/5/8 (downstream of BMP signaling) were increased in the neural fold of E9.0 Arl8b-/- embryos. These results suggest that Arl8b is involved in the development of the neural fold and the subsequently formed roof plate, possibly via control of BMP signaling.


Assuntos
Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/fisiologia , Crista Neural/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Lisossomos/genética , Lisossomos/fisiologia , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Crista Neural/metabolismo , Tubo Neural/embriologia , Tubo Neural/metabolismo , Fatores de Transcrição SOXB1/fisiologia , Transdução de Sinais
15.
Mol Cell ; 74(5): 891-908.e10, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31006537

RESUMO

Cells respond to nutrient stress by trafficking cytosolic contents to lysosomes for degradation via macroautophagy. The endoplasmic reticulum (ER) serves as an initiation site for autophagosomes and is also remodeled in response to nutrient stress through ER-phagy, a form of selective autophagy. Quantitative proteome analysis during nutrient stress identified an unstudied single-pass transmembrane ER protein, TEX264, as an ER-phagy receptor. TEX264 uses an LC3-interacting region (LIR) to traffic into ATG8-positive puncta that often initiate from three-way ER tubule junctions and subsequently fuse with lysosomes. Interaction and proximity biotinylation proteomics identified a cohort of autophagy regulatory proteins and cargo adaptors located near TEX264 in an LIR-dependent manner. Global proteomics and ER-phagy flux analysis revealed the stabilization of a cohort of ER proteins in TEX264-/- cells during nutrient stress. This work reveals TEX264 as an unrecognized ER-phagy receptor that acts independently of other candidate ER-phagy receptors to remodel the ER during nutrient stress.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Retículo Endoplasmático/genética , Proteínas de Membrana/metabolismo , Animais , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Células COS , Citosol/metabolismo , Estresse do Retículo Endoplasmático/genética , Células HCT116 , Células HEK293 , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Nutrientes/metabolismo , Transporte Proteico/genética , Proteoma/genética
16.
J Agric Food Chem ; 67(16): 4578-4587, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30933511

RESUMO

The objective of this study was to investigate the mechanism underlying lysosome-mediated apoptosis, the cross-talk between the lysosomes and mitochondria, and the effect of the pathway on bovine longissimus muscle tenderness during 7 d post-mortem aging through the observation and analysis of longissimus dorsi (LD) muscles of six crossbred cattle. Results showed that an elevated reactive oxygen species level ( P < 0.05) can damage lysosomal membrane stability ( P < 0.05) through accumulating redox-active iron of bovine muscle during post-mortem aging. In addition, the activities of cathepsins B and D increased with post-mortem aging ( P < 0.05). Moreover, cathepsin B and D activated Bid and Bax in the mitochondria ( P < 0.05). Activated Bid and Bax triggered mitochondrial membrane permeability ( P < 0.05) and further activated caspase-9 and caspase-3 ( P < 0.05), leading to apoptosis. Ultimately, the tenderness of bovine muscle was improved during post-mortem aging ( P < 0.05). Importantly, cathepsin D plays a crucial role in the lysosomal-mitochondrial apoptotic pathway and tenderness in post-mortem muscle. These findings provide new insights into the apoptotic pathway of bovine muscle during post-mortem aging.


Assuntos
Bovinos/metabolismo , Lisossomos/metabolismo , Carne/análise , Mitocôndrias/metabolismo , Músculo Esquelético/química , Animais , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspases/genética , Caspases/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Catepsina D/genética , Catepsina D/metabolismo , Bovinos/genética , Ferro/metabolismo , Lisossomos/genética , Masculino , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
17.
J Biol Chem ; 294(20): 8023-8036, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30936203

RESUMO

G protein-coupled receptor (GPCR) signaling is regulated by members of the protein kinase C (PKC) and GPCR kinase (GRK) families, although the relative contribution of each to GPCR function varies among specific GPCRs. The CXC motif receptor 4 (CXCR4) is a member of the GPCR superfamily that binds the CXC motif chemokine ligand 12 (CXCL12), initiating signaling that is subsequently terminated in part by internalization and lysosomal degradation of CXCR4. The purpose of this study is to define the relative contribution of PKC and GRK to CXCR4 signaling attenuation by studying their effects on CXCR4 lysosomal trafficking and degradation. Our results demonstrate that direct activation of PKC via the phorbol ester phorbol 12-myristate 13-acetate (PMA) mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal trafficking of CXCR4. In agreement, heterologous activation of PKC by stimulating the chemokine receptor CXCR5 with its ligand, CXCL13, also mimics CXCL12-mediated desensitization, internalization, ubiquitination, and lysosomal degradation of CXCR4. Similar to CXCL12, PMA promotes PKC-dependent phosphorylation of serine residues within CXCR4 C-tail that are required for binding and ubiquitination by the E3 ubiquitin ligase AIP4 (atrophin-interacting protein 4). However, inhibition of PKC activity does not alter CXCL12-mediated ubiquitination and degradation of CXCR4, suggesting that other kinases are also required. Accordingly, siRNA-mediated depletion of GRK6 results in decreased degradation and ubiquitination of CXCR4. Overall, these results suggest that PKC and GRK6 contribute to unique aspects of CXCR4 phosphorylation and lysosomal degradation to ensure proper signal propagation and termination.


Assuntos
Lisossomos/metabolismo , Proteólise , Receptores CXCR4/metabolismo , Transdução de Sinais , Ubiquitinação , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL13/genética , Quimiocina CXCL13/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Quinases de Receptores Acoplados a Proteína G/genética , Quinases de Receptores Acoplados a Proteína G/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Receptores CXCR4/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
18.
J Immunol ; 202(8): 2407-2420, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30850476

RESUMO

In mammals, tripartite motif (TRIM) proteins have emerged as pivotal players endowed with, directly, antiviral effects and, indirectly, modulatory capacity of the innate immune response. An unprecedented expansion of TRIM family has occurred in fish; however, the functional role of fish TRIM family members remains largely unknown. In this study, we identify a species-specific TRIM gene from crucian carp Carassius auratus, named FTRCA1, phylogenetically similar to the members of finTRIM, a subfamily of TRIM exclusively in teleost fish. FTRCA1 is induced by IFN and IFN stimuli as a typical IFN-stimulated gene. Overexpression of FTRCA1 negatively regulates IFN antiviral response by inhibition of IRF3 phosphorylation; consistently, knockdown of FTRCA1 results in enhanced levels of IRF3 phosphorylation and also IFN expression following poly(I:C) transfection. Whereas FTRCA1 is associated with several pivotal signaling molecules of RIG-I-like receptor pathway, its association with TBK1 results in autophage-lysosomal degradation of TBK1, thus abrogating the downstream IFN induction. Interestingly, FTRCA1 is phosphorylated by TBK1, but this phosphorylation is not required for downregulation of TBK1 protein. Transfection assays indicate that FTRCA1 is likely an E3 ligase with the requirement of RING finger domain, and deletion of N-terminal RING domain or mutation of seven conservative sites abolishes the negative regulatory function of FTRCA1. Collectively, these results illuminate a novel finTRIM-mediated innate immune modulatory pathway, thus providing insights into species-specific regulation of fish IFN response.


Assuntos
Autofagossomos/imunologia , Proteínas de Peixes/imunologia , Carpa Dourada/imunologia , Interferons/imunologia , Lisossomos/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Proteólise , Proteínas com Motivo Tripartido/imunologia , Animais , Proteínas de Peixes/genética , Carpa Dourada/genética , Células HEK293 , Humanos , Interferons/genética , Lisossomos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas com Motivo Tripartido/genética
19.
J Biol Chem ; 294(16): 6494-6505, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30808710

RESUMO

Missing in metastasis (MIM), an inverse Bin-Amphiphysin-Rvs (I-BAR) domain protein, promotes endocytosis of C-X-C chemokine receptor 4 (CXCR4) in mammalian cells. In response to the CXCR4 ligand stromal cell-derived factor 1 (SDF-1 or CXCL12), MIM associates with RAS-related GTP-binding protein 7 (RAB7) 30 min after stimulation. However, RAB7's role in MIM function remains undefined. Here we show that RNAi-mediated suppression of RAB7 expression in human HeLa cells has little effect on the binding of MIM to RAB5 and on the recruitment of CXCR4 to early endosomes but effectively abolishes MIM-mediated CXCR4 degradation, chemotactic response, and sorting into late endosomes and lysosomes. To determine whether I-BAR domain proteins interact with RAB7, we examined cells expressing insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein bearing an Src homology 3 (SH3) domain. We observed that both MIM and IRTKS interact with RAB5 at an early response to SDF-1 and that IRTKS binds poorly to RAB7 but strongly to RAB11 at a later time point. Moreover, IRTKS overexpression reduced CXCR4 internalization and enhanced the chemotactic response to SDF-1. Interestingly, deletion of the SH3 domain in IRTKS abolished the IRTKS-RAB11 interaction and promoted CXCR4 degradation. Furthermore, the SH3 domain was required for selective targeting of MIM-IRTKS fusion proteins by both RAB7 and RAB11. Hence, to the best of our knowledge, our results provide first evidence that the SH3 domain is critical in the regulation of specific endocytic pathways by I-BAR domain proteins.


Assuntos
Endocitose , Proteínas dos Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteólise , Receptores CXCR4/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Endossomos/genética , Endossomos/metabolismo , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas de Neoplasias/genética , Receptores CXCR4/genética , Proteínas rab de Ligação ao GTP/genética , Domínios de Homologia de src
20.
Proc Natl Acad Sci U S A ; 116(9): 3712-3721, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30733285

RESUMO

Two coding variants in the apolipoprotein L1 (APOL1) gene (termed G1 and G2) are strongly associated with increased risk of nondiabetic kidney disease in people of recent African ancestry. The mechanisms by which the risk variants cause kidney damage, although not well-understood, are believed to involve injury to glomerular podocytes. The intracellular localization and function of APOL1 in podocytes remain unclear, with recent studies suggesting possible roles in the endoplasmic reticulum (ER), mitochondria, endosomes, lysosomes, and autophagosomes. Here, we demonstrate that APOL1 also localizes to intracellular lipid droplets (LDs). While a large fraction of risk variant APOL1 (G1 and G2) localizes to the ER, a significant proportion of wild-type APOL1 (G0) localizes to LDs. APOL1 transiently interacts with numerous organelles, including the ER, mitochondria, and endosomes. Treatment of cells that promote LD formation with oleic acid shifted the localization of G1 and G2 from the ER to LDs, with accompanying reduction of autophagic flux and cytotoxicity. Coexpression of G0 APOL1 with risk variant APOL1 enabled recruitment of G1 and G2 from the ER to LDs, accompanied by reduced cell death. The ability of G0 APOL1 to recruit risk variant APOL1 to LDs may help explain the recessive pattern of kidney disease inheritance. These studies establish APOL1 as a bona fide LD-associated protein, and reveal that recruitment of risk variant APOL1 to LDs reduces cell toxicity, autophagic flux, and cell death. Thus, interventions that divert APOL1 risk variants to LDs may serve as a novel therapeutic strategy to alleviate their cytotoxic effects.


Assuntos
Apolipoproteína L1/genética , Autofagia/genética , Nefropatias/genética , Gotículas Lipídicas/metabolismo , Grupo com Ancestrais do Continente Africano/genética , Retículo Endoplasmático/genética , Endossomos/genética , Variação Genética , Células HEK293 , Humanos , Rim/lesões , Rim/patologia , Nefropatias/fisiopatologia , Gotículas Lipídicas/patologia , Lisossomos/genética , Podócitos/metabolismo , Podócitos/patologia , Fatores de Risco
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