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1.
Cells ; 9(9)2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32847034

RESUMO

The preservation of cellular homeostasis requires the synthesis of new proteins (proteostasis) and organelles, and the effective removal of misfolded or impaired proteins and cellular debris. This cellular homeostasis involves two key proteostasis mechanisms, the ubiquitin proteasome system and the autophagy-lysosome pathway. These catabolic pathways have been known to be involved in respiratory exacerbations and the pathogenesis of various lung diseases, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), idiopathic pulmonary fibrosis (IPF), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and coronavirus disease-2019 (COVID-19). Briefly, proteostasis and autophagy processes are known to decline over time with age, cigarette or biomass smoke exposure, and/or influenced by underlying genetic factors, resulting in the accumulation of misfolded proteins and cellular debris, elevating apoptosis and cellular senescence, and initiating the pathogenesis of acute or chronic lung disease. Moreover, autophagic dysfunction results in an impaired microbial clearance, post-bacterial and/or viral infection(s) which contribute to the initiation of acute and recurrent respiratory exacerbations as well as the progression of chronic obstructive and restrictive lung diseases. In addition, the autophagic dysfunction-mediated cystic fibrosis transmembrane conductance regulator (CFTR) immune response impairment further exacerbates the lung disease. Recent studies demonstrate the therapeutic potential of novel autophagy augmentation strategies, in alleviating the pathogenesis of chronic obstructive or restrictive lung diseases and exacerbations such as those commonly seen in COPD, CF, ALI/ARDS and COVID-19.


Assuntos
Autofagia/imunologia , Betacoronavirus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Progressão da Doença , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/metabolismo , Infecções por Coronavirus/virologia , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Homeostase , Humanos , Fibrose Pulmonar Idiopática/imunologia , Fibrose Pulmonar Idiopática/metabolismo , Lisossomos/metabolismo , Pandemias , Pneumonia Viral/virologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Síndrome do Desconforto Respiratório do Adulto/imunologia , Síndrome do Desconforto Respiratório do Adulto/metabolismo
2.
Nature ; 585(7824): 251-255, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848248

RESUMO

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a ß-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.


Assuntos
Esclerose Amiotrófica Lateral/genética , Proteínas Relacionadas à Autofagia/química , Proteína C9orf72/química , Proteína C9orf72/genética , Proteínas de Transporte/química , Microscopia Crioeletrônica , Demência Frontotemporal/genética , Haploinsuficiência , Complexos Multiproteicos/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/ultraestrutura , Proteína C9orf72/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/ultraestrutura , Demência Frontotemporal/metabolismo , Humanos , Lisossomos/metabolismo , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Domínios Proteicos
3.
Cells ; 9(9)2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32854430

RESUMO

An outbreak of the novel coronavirus (CoV) SARS-CoV-2, the causative agent of COVID-19 respiratory disease, infected millions of people since the end of 2019, led to high-level morbidity and mortality and caused worldwide social and economic disruption. There are currently no antiviral drugs available with proven efficacy or vaccines for its prevention. An understanding of the underlying cellular mechanisms involved in virus replication is essential for repurposing the existing drugs and/or the discovery of new ones. Endocytosis is the important mechanism of entry of CoVs into host cells. Endosomal maturation followed by the fusion with lysosomes are crucial events in endocytosis. Late endosomes and lysosomes are characterized by their acidic pH, which is generated by a proton transporter V-ATPase and required for virus entry via endocytic pathway. The cytoplasmic cAMP pool produced by soluble adenylyl cyclase (sAC) promotes V-ATPase recruitment to endosomes/lysosomes and thus their acidification. In this review, we discuss targeting the sAC-specific cAMP pool as a potential strategy to impair the endocytic entry of the SARS-CoV-2 into the host cell. Furthermore, we consider the potential impact of sAC inhibition on CoV-induced disease via modulation of autophagy and apoptosis.


Assuntos
Inibidores de Adenilil Ciclases/uso terapêutico , Adenilil Ciclases/metabolismo , Betacoronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , AMP Cíclico/antagonistas & inibidores , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
4.
Int J Nanomedicine ; 15: 5803-5811, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32821102

RESUMO

Introduction: Photodynamic therapy (PDT), which induces tissue damage by exposing tissue to a specific wavelength of light in the presence of a photosensitizer and oxygen, is a promising alternative treatment that could be used as an adjunct to chemotherapy and surgery in oncology. Cell-penetrating peptides (CPPs) with high arginine content, such as protamine, have membrane translocation and lysosome localization activities. They have been used in an extensive range of drug delivery applications. Methods: We conjugated cell-penetrating peptides (CPPs) with methylene blue (MB) and then purification by FPLC. Synthesis structure was characterized by the absorbance spectrum, FPLC, Maldi-TOF, and then evaluated cell viability by cytotoxicity assay after photodynamic therapy (PDT) assay. An uptake imaging assay was used to determine the sites of MB and MB-Pro in subcellular compartments. Results: In vitro assays showed that MB-Pro has more efficient photodynamic activities than MB alone for the colon cancer cells, owing to lysosome rupture causing the rapid necrotic cell death. In this study, we coupled protamine with MB for high efficacy PDT. The conjugates localized in the lysosomes and enhanced the efficiency of PDT by inducing necrotic cell death, whereas PDT with non-coupled MB resulted in only apoptotic processes. Discussion: Our research aimed to enhance PDT by engineering the photosensitizers using CPPs coupled with methylene blue (MB). MB alone permeates through the cell membrane and distributes into the cytoplasm, whereas coupling of MB dye with CPPs localizes the MB through an endocytic mechanism to a specific organelle where the localized conjugates enhance the generation of reactive oxygen species (ROS) and induce cell damage.


Assuntos
Peptídeos Penetradores de Células/farmacologia , Azul de Metileno/farmacologia , Fotoquimioterapia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Células HT29 , Humanos , Imageamento Tridimensional , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Azul de Metileno/química , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo
5.
Front Immunol ; 11: 1337, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32733448

RESUMO

Autophagy is a cellular recycling system found in almost all types of eukaryotic organisms. The system is made up of a variety of proteins which function to deliver intracellular cargo to lysosomes for formation of autophagosomes in which the contents are degraded. The maintenance of cellular homeostasis is key in the survival and function of a variety of human cell populations. The interconnection between metabolism and autophagy is extensive, therefore it has a role in a variety of different cell functions. The disruption or dysfunction of autophagy in these cell types have been implicated in the development of a variety of inflammatory diseases including asthma. The role of autophagy in non-immune and immune cells both lead to the pathogenesis of lung inflammation. Autophagy in pulmonary non-immune cells leads to tissue remodeling which can develop into chronic asthma cases with long term effects. The role autophagy in the lymphoid and myeloid lineages in the pathology of asthma differ in their functions. Impaired autophagy in lymphoid populations have been shown, in general, to decrease inflammation in both asthma and inflammatory disease models. Many lymphoid cells rely on autophagy for effector function and maintained inflammation. In stark contrast, autophagy deficient antigen presenting cells have been shown to have an activated inflammasome. This is largely characterized by a TH17 response that is accompanied with a much worse prognosis including granulocyte mediated inflammation and steroid resistance. The cell specificity associated with changes in autophagic flux complicates its targeting for amelioration of asthmatic symptoms. Differing asthmatic phenotypes between TH2 and TH17 mediated disease may require different autophagic modulations. Therefore, treatments call for a more cell specific and personalized approach when looking at chronic asthma cases. Viral-induced lung inflammation, such as that caused by SARS-CoV-2, also may involve autophagic modulation leading to inflammation mediated by lung resident cells. In this review, we will be discussing the role of autophagy in non-immune cells, myeloid cells, and lymphoid cells for their implications into lung inflammation and asthma. Finally, we will discuss autophagy's role viral pathogenesis, immunometabolism, and asthma with insights into autophagic modulators for amelioration of lung inflammation.


Assuntos
Asma/complicações , Asma/patologia , Autofagia/imunologia , Betacoronavirus , Infecções por Coronavirus/complicações , Infecções por Coronavirus/patologia , Pneumonia Viral/complicações , Pneumonia Viral/patologia , Animais , Asma/imunologia , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Células Dendríticas/metabolismo , Humanos , Linfócitos/metabolismo , Lisossomos/metabolismo , Células Mieloides/metabolismo , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Mucosa Respiratória/metabolismo , Transdução de Sinais/imunologia
6.
PLoS One ; 15(8): e0235551, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833964

RESUMO

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ferro/metabolismo , Neoplasias/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Colesterol/biossíntese , Colesterol/genética , Classe III de Fosfatidilinositol 3-Quinases/genética , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Receptores de LDL/metabolismo , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
7.
Nat Commun ; 11(1): 4286, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855403

RESUMO

Intracellular galectins are carbohydrate-binding proteins capable of sensing and repairing damaged lysosomes. As in the physiological conditions glycosylated moieties are mostly in the lysosomal lumen but not cytosol, it is unclear whether galectins reside in lysosomes, bind to glycosylated proteins, and regulate lysosome functions. Here, we show in gut epithelial cells, galectin-9 is enriched in lysosomes and predominantly binds to lysosome-associated membrane protein 2 (Lamp2) in a Asn(N)-glycan dependent manner. At the steady state, galectin-9 binding to glycosylated Asn175 of Lamp2 is essential for functionality of lysosomes and autophagy. Loss of N-glycan-binding capability of galectin-9 causes its complete depletion from lysosomes and defective autophagy, leading to increased endoplasmic reticulum (ER) stress preferentially in autophagy-active Paneth cells and acinar cells. Unresolved ER stress consequently causes cell degeneration or apoptosis that associates with colitis and pancreatic disorders in mice. Therefore, lysosomal galectins maintain homeostatic function of lysosomes to prevent organ pathogenesis.


Assuntos
Galectinas/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Pâncreas/patologia , Celulas de Paneth/patologia , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Autofagia/fisiologia , Colite/metabolismo , Colite/patologia , Estresse do Retículo Endoplasmático , Galectinas/genética , Células HT29 , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Lisossomos/genética , Lisossomos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pâncreas/metabolismo , Pancreatite/metabolismo , Pancreatite/patologia , Celulas de Paneth/metabolismo
8.
Ecotoxicol Environ Saf ; 204: 111069, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32758696

RESUMO

We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 µM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 µM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 µM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 µM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Glutationa/análogos & derivados , Mucosa Intestinal/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Oncorhynchus mykiss/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Saxitoxina/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Catalase/metabolismo , Dinoflagelados/metabolismo , Enterócitos/efeitos dos fármacos , Enterócitos/metabolismo , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Mucosa Intestinal/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lisossomos/metabolismo , Frutos do Mar
9.
Int J Nanomedicine ; 15: 4049-4062, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606663

RESUMO

Background: Calcium carbonate (CC) nanoparticles have broad biomedical utilizations, owing to their multiple intrinsic merits. However, bare CC nanoparticles do not allow for the development of multifunctional devices suitable for advanced drug delivery in cancer therapy. Methods: Phospholipid-modified phospholipid-CC hybrid nanoparticles were prepared in our study using a combination of vapor-diffusion and solvent-diffusion methods to offer optimized pharmaceutical capabilities. Results: Considering that particle size is a critical parameter that plays an important role in both in vitro and in vivo behaviors of nanoparticles, we here for the first time a present detailed protocol for the size-controlled preparation of hybrid nanoparticles, as well as analysis of the in vitro/in vivo behaviors of differently sized hybrid nanoparticles. Conclusion: Our results might significantly advance the application of this promising material in more varied fields.


Assuntos
Carbonato de Cálcio/química , Nanopartículas/química , Tamanho da Partícula , Fosfolipídeos/química , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Sistemas de Liberação de Medicamentos/métodos , Endocitose , Feminino , Humanos , Lisossomos/metabolismo , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/ultraestrutura , Coelhos , Esferoides Celulares/metabolismo , Eletricidade Estática , Temperatura , Fatores de Tempo , Distribuição Tecidual
10.
PLoS Pathog ; 16(7): e1008220, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658937

RESUMO

The intracellular lifestyle of Salmonella enterica is characterized by the formation of a replication-permissive membrane-bound niche, the Salmonella-containing vacuole (SCV). As a further consequence of the massive remodeling of the host cell endosomal system, intracellular Salmonella establish a unique network of various Salmonella-induced tubules (SIT). The bacterial repertoire of effector proteins required for the establishment for one type of these SIT, the Salmonella-induced filaments (SIF), is rather well-defined. However, the corresponding host cell proteins are still poorly understood. To identify host factors required for the formation of SIF, we performed a sub-genomic RNAi screen. The analyses comprised high-resolution live cell imaging to score effects on SIF induction, dynamics and morphology. The hits of our functional RNAi screen comprise: i) The late endo-/lysosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, consisting of STX7, STX8, VTI1B, and VAMP7 or VAMP8, which is, in conjunction with RAB7 and the homotypic fusion and protein sorting (HOPS) tethering complex, a complete vesicle fusion machinery. ii) Novel interactions with the early secretory GTPases RAB1A and RAB1B, providing a potential link to coat protein complex I (COPI) vesicles and reinforcing recently identified ties to the endoplasmic reticulum. iii) New connections to the late secretory pathway and/or the recycling endosome via the GTPases RAB3A, RAB8A, and RAB8B and the SNAREs VAMP2, VAMP3, and VAMP4. iv) An unprecedented involvement of clathrin-coated structures. The resulting set of hits allowed us to characterize completely new host factor interactions, and to strengthen observations from several previous studies.


Assuntos
Proteínas de Bactérias/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Infecções por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Endossomos/metabolismo , Endossomos/microbiologia , Células HeLa , Humanos , Lisossomos/metabolismo , Lisossomos/microbiologia , RNA Interferente Pequeno
11.
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
12.
Int J Mol Sci ; 21(14)2020 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-32668803

RESUMO

In line with SARS and MERS, the SARS-CoV-2/COVID-19 pandemic is one of the largest challenges in medicine and health care worldwide. SARS-CoV-2 infection/COVID-19 provides numerous therapeutic targets, each of them promising, but not leading to the success of therapy to date. Neither an antiviral nor an immunomodulatory therapy in patients with SARS-CoV-2 infection/COVID-19 or pre-exposure prophylaxis against SARS-CoV-2 has proved to be effective. In this review, we try to close the gap and point out the likely relationships among lysosomotropism, increasing lysosomal pH, SARS-CoV-2 infection, and disease process, and we deduce an approach for the treatment and prophylaxis of COVID-19, and cytokine release syndrome (CRS)/cytokine storm triggered by bacteria or viruses. Lysosomotropic compounds affect prominent inflammatory messengers (e.g., IL-1B, CCL4, CCL20, and IL-6), cathepsin-L-dependent viral entry of host cells, and products of lysosomal enzymes that promote endothelial stress response in systemic inflammation. As supported by recent clinical data, patients who have already taken lysosomotropic drugs for other pre-existing conditions likely benefit from this treatment in the COVID-19 pandemic. The early administration of a combination of antivirals such as remdesivir and lysosomotropic drugs, such as the antibiotics teicoplanin or dalbavancin, seems to be able to prevent SARS-CoV-2 infection and transition to COVID-19.


Assuntos
Infecções por Coronavirus/patologia , Lisossomos/metabolismo , Pneumonia Viral/patologia , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Betacoronavirus/isolamento & purificação , Betacoronavirus/fisiologia , Infecções por Coronavirus/complicações , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Síndrome da Liberação de Citocina/etiologia , Síndrome da Liberação de Citocina/patologia , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/complicações , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Internalização do Vírus/efeitos dos fármacos
13.
Nat Commun ; 11(1): 3298, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620747

RESUMO

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


Assuntos
Anoctaminas/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Animais , Anoctaminas/genética , Transporte Biológico , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Endossomos/ultraestrutura , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Lisossomos/ultraestrutura , Camundongos Knockout , Microscopia Eletrônica , Mutação , Transporte Proteico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo
14.
Proc Natl Acad Sci U S A ; 117(30): 17820-17831, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661174

RESUMO

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (Pam Myh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult Pam Myh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam 0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in Pam Myh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by Pam Myh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by Pam Myh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.


Assuntos
Amidina-Liases/metabolismo , Grânulos Citoplasmáticos/metabolismo , Átrios do Coração/metabolismo , Oxigenases de Função Mista/metabolismo , Vesículas Secretórias/metabolismo , Amidina-Liases/genética , Animais , Fator Natriurético Atrial/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Expressão Gênica , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos , Camundongos Knockout , Oxigenases de Função Mista/genética , Monócitos/metabolismo , Células Musculares/metabolismo , Vesículas Secretórias/ultraestrutura
15.
Nat Cell Biol ; 22(7): 815-827, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601373

RESUMO

Lysosomes serve as cellular degradation and signalling centres that coordinate metabolism in response to intracellular cues and extracellular signals. Lysosomal capacity is adapted to cellular needs by transcription factors, such as TFEB and TFE3, which activate the expression of lysosomal and autophagy genes. Nuclear translocation and activation of TFEB are induced by a variety of conditions such as starvation, lysosome stress and lysosomal storage disorders. How these various cues are integrated remains incompletely understood. Here, we describe a pathway initiated at the plasma membrane that controls lysosome biogenesis via the endocytic regulation of intracellular ion homeostasis. This pathway is based on the exo-endocytosis of NHE7, a Na+/H+ exchanger mutated in X-linked intellectual disability, and serves to control intracellular ion homeostasis and thereby Ca2+/calcineurin-mediated activation of TFEB and downstream lysosome biogenesis in response to osmotic stress to promote the turnover of toxic proteins and cell survival.


Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Membrana Celular/metabolismo , Endocitose , Lisossomos/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Calcineurina/genética , Calcineurina/metabolismo , Cálcio/metabolismo , Clatrina/metabolismo , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico , Trocadores de Sódio-Hidrogênio/genética
16.
Nature ; 584(7820): 291-297, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728216

RESUMO

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.


Assuntos
Espaço Extracelular/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos/química , Anticorpos/metabolismo , Antígenos CD/metabolismo , Apolipoproteína E4/metabolismo , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Receptores ErbB/metabolismo , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Humanos , Ligantes , Proteínas de Membrana/química , Camundongos , Domínios Proteicos , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Solubilidade , Especificidade por Substrato
17.
Life Sci ; 257: 118043, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32621922

RESUMO

BACKGROUND: Alveolar macrophages (AMs) are the primary targets of silicosis. Blockade of autophagy may aggravate the apoptosis of AMs. Trehalose (Tre), a transcription factor EB (TFEB) activator, may impact the autophagy-lysosomal system in AMs during silicosis. However, the mechanism by which Tre acts upon AMs in silicosis is unknown. METHODS: We collected AMs from twenty male workers exposed to silica and divided them into observer and silicosis patient groups. AMs from the two groups were then exposed to Tre. Western blot was used to measure the expression of autophagy-associated proteins. Lysosomal-associated membrane protein 1 (LAMP1) expression was observed using immunofluorescence and western blot. Apoptosis of the AMs was detected by TUNEL assay and western blot. RESULTS: Tre induced localization of TFEB to the nucleus in the AMs of both groups. After Tre exposure, LAMP1 levels increased and LC3 levels decreased in the AMs of both groups, suggesting that Tre may increase the function of the autophagy-lysosomal system. The LC3-II/I ratio in the Tre-exposed AMs was lower than in the AMs not exposed to Tre. The LC3-II/I ratio in AMs subjected to Tre plus Bafilomycin (Baf) was higher than the ratio in cells exposed to Tre or Baf individually. Additionally, p62 levels decreased after Tre stimulation in the AMs of both groups. This indicates that Tre may accelerate the process of autophagic degradation. We also found decreased levels of cleaved caspase-3 after Tre treatment in the AMs of both groups. However, p-mTOR (Ser2448) and p-mTOR (Ser2481) levels did not change significantly after Tre treatment, suggesting that the mTOR signaling pathway was not affected by Tre treatment. CONCLUSION: Our findings suggest that the restoration of autophagy-lysosomal function by Tre may be a potential protective strategy against silicosis.


Assuntos
Silicose/tratamento farmacológico , Trealose/metabolismo , Trealose/farmacologia , Adulto , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Silicose/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição
18.
Proc Natl Acad Sci U S A ; 117(31): 18521-18529, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690708

RESUMO

Animal cells acquire cholesterol from receptor-mediated uptake of low-density lipoprotein (LDL), which releases cholesterol in lysosomes. The cholesterol moves to the endoplasmic reticulum (ER), where it inhibits production of LDL receptors, completing a feedback loop. Here we performed a CRISPR-Cas9 screen in human SV589 cells for genes required for LDL-derived cholesterol to reach the ER. We identified the gene encoding PTDSS1, an enzyme that synthesizes phosphatidylserine (PS), a phospholipid constituent of the inner layer of the plasma membrane (PM). In PTDSS1-deficient cells where PS is low, LDL cholesterol leaves lysosomes but fails to reach the ER, instead accumulating in the PM. The addition of PS restores cholesterol transport to the ER. We conclude that LDL cholesterol normally moves from lysosomes to the PM. When the PM cholesterol exceeds a threshold, excess cholesterol moves to the ER in a process requiring PS. In the ER, excess cholesterol acts to reduce cholesterol uptake, preventing toxic cholesterol accumulation. These studies reveal that one lipid-PS-controls the movement of another lipid-cholesterol-between cell membranes. We relate these findings to recent evidence indicating that PM-to-ER cholesterol transport is mediated by GRAMD1/Aster proteins that bind PS and cholesterol.


Assuntos
Membrana Celular/metabolismo , LDL-Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , Fosfatidilserinas/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Colesterol/metabolismo , Humanos
19.
Proc Natl Acad Sci U S A ; 117(32): 19266-19275, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32703809

RESUMO

Mitochondria and lysosomes are critical for cellular homeostasis, and dysfunction of both organelles has been implicated in numerous diseases. Recently, interorganelle contacts between mitochondria and lysosomes were identified and found to regulate mitochondrial dynamics. However, whether mitochondria-lysosome contacts serve additional functions by facilitating the direct transfer of metabolites or ions between the two organelles has not been elucidated. Here, using high spatial and temporal resolution live-cell microscopy, we identified a role for mitochondria-lysosome contacts in regulating mitochondrial calcium dynamics through the lysosomal calcium efflux channel, transient receptor potential mucolipin 1 (TRPML1). Lysosomal calcium release by TRPML1 promotes calcium transfer to mitochondria, which was mediated by tethering of mitochondria-lysosome contact sites. Moreover, mitochondrial calcium uptake at mitochondria-lysosome contact sites was modulated by the outer and inner mitochondrial membrane channels, voltage-dependent anion channel 1 and the mitochondrial calcium uniporter, respectively. Since loss of TRPML1 function results in the lysosomal storage disorder mucolipidosis type IV (MLIV), we examined MLIV patient fibroblasts and found both altered mitochondria-lysosome contact dynamics and defective contact-dependent mitochondrial calcium uptake. Thus, our work highlights mitochondria-lysosome contacts as key contributors to interorganelle calcium dynamics and their potential role in the pathophysiology of disorders characterized by dysfunctional mitochondria or lysosomes.


Assuntos
Cálcio/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Mucolipidoses/metabolismo , Canais de Receptores Transientes de Potencial/metabolismo , Transporte Biológico , Humanos , Lisossomos/genética , Mitocôndrias/genética , Dinâmica Mitocondrial , Mucolipidoses/genética , Canais de Receptores Transientes de Potencial/genética
20.
Cell Prolif ; 53(7): e12813, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32515860

RESUMO

OBJECTIVES: Accumulating studies have investigated the PM2.5-induced pulmonary toxicity, while gaps still remain in understanding its toxic mechanism. Due to its high specific surface area and adsorption capacity similar to nanoparticles, PM2.5 acts as a significant carrier of metals in air and then leads to altered toxic effects. In this study, we aimed to use CBs and Ni as model materials to investigate the autophagy changes and pulmonary toxic effects at 30 days following intratracheal instillation of CBs-Ni mixture. MATERIALS AND METHODS: Groups of mice were instilled with 100 µL normal saline (NS), 20 µg CBs, and 4 µg Ni or CBs-Ni mixture, respectively. At 7 and 30 days post-instillation, all the mice were weighed and then sacrificed. The evaluation system was composed of the following: (a) autophagy and lysosomal function assessment, (b) trace element biodistribution observation in lungs, (c) pulmonary lavage biomedical analysis, (d) lung histopathological evaluation, (e) coefficient analysis of major organs and (f) CBs-Ni interaction and cell proliferation assessment. RESULTS: We found that after CBs-Ni co-exposure, no obvious autophagy and lysosomal dysfunction or pulmonary toxicity was detected, along with complete clearance of Ni from lung tissues as well as recovery of biochemical indexes to normal range. CONCLUSIONS: We conclude that the damaged autophagy and lysosomal function, as well as physiological function, was repaired at 30 days after exposure of CBs-Ni. Our findings provide a new idea for scientific assessment of the impact of fine particles on environment and human health, and useful information for the comprehensive treatment of air pollution.


Assuntos
Autofagia/efeitos dos fármacos , Carbono/efeitos adversos , Pneumopatias/induzido quimicamente , Pulmão/efeitos dos fármacos , Metais/efeitos adversos , Animais , Linhagem Celular , Pulmão/metabolismo , Pneumopatias/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Células RAW 264.7 , Distribuição Tecidual
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