Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 11.865
Filtrar
1.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33912962

RESUMO

Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and late endosomes/lysosomes (LE/lys) are emerging as critical hubs for diverse cellular events, and changes in their extents are linked to severe neurological diseases. While recent studies show that the synaptotagmin-like mitochondrial-lipid-binding (SMP) domain-containing protein PDZD8 may mediate the formation of ER-LE/lys MCSs, the cellular functions of PDZD8 remain largely elusive. Here, we attempt to investigate the lipid transfer activities of PDZD8 and the extent to which its cellular functions depend on its lipid transfer activities. In accordance with recent studies, we demonstrate that PDZD8 is a protrudin (ZFYVE27)-interacting protein and that PDZD8 acts as a tether at ER-LE/lys MCSs. Furthermore, we discover that the SMP domain of PDZD8 binds glycerophospholipids and ceramides both in vivo and in vitro, and that the SMP domain can transport lipids between membranes in vitro. Functionally, PDZD8 is required for LE/lys positioning and neurite outgrowth, which is dependent on the lipid transfer activity of the SMP domain.


Assuntos
Retículo Endoplasmático , Endossomos , Lipídeos , Lisossomos , Crescimento Neuronal
2.
Nanoscale ; 13(31): 13294-13300, 2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34477735

RESUMO

Successful delivery of fluorescent nanodiamonds (FNDs) into the cytoplasm is essential to many biological applications. Other applications require FNDs to stay within the endosomes. The diversity of cellular uptake of FNDs and following endosomal escape are less explored. In this article, we quantify particle uptake at a single cell level. We report that FNDs enter into the cells gradually. The number of internalized FNDs per cell differs significantly for the cell lines we investigated at the same incubation time. In HeLa cells we do not see any significant endosomal escape. We also found a wide distribution of FND endosomal escape efficiency within the same cell type. However, compared with HeLa cells, FNDs in HUVECs can easily escape from the endosomes and less than 25% FNDs remained in the vesicles after 4 h incubation time. We believe this work can bring more attention to the diversity of the cells and provide potential guidelines for future studies.


Assuntos
Nanodiamantes , Endossomos , Corantes Fluorescentes , Células HeLa , Humanos
3.
FEBS J ; 288(17): 5071-5088, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34490733

RESUMO

While there is undeniable evidence to link endosomal acid-base homeostasis to viral pathogenesis, the lack of druggable molecular targets has hindered translation from bench to bedside. The recent identification of variants in the interferon-inducible endosomal Na+ /H+ exchanger 9 associated with severe coronavirus disease-19 (COVID-19) has brought a shift in the way we envision aberrant endosomal acidification. Is it linked to an increased susceptibility to viral infection or a propensity to develop critical illness? This review summarizes the genetic and cellular evidence linking endosomal Na+ /H+ exchangers and viral diseases to suggest how they can act as a broad-spectrum modulator of viral infection and downstream pathophysiology. The review also presents novel insights supporting the complex role of endosomal acid-base homeostasis in viral pathogenesis and discusses the potential causes for negative outcomes of clinical trials utilizing alkalinizing drugs as therapies for COVID-19. These findings lead to a pathogenic model of viral disease that predicts that nonspecific targeting of endosomal pH might fail, even if administered early on, and suggests that endosomal Na+ /H+ exchangers may regulate key host antiviral defence mechanisms and mediators that act to drive inflammatory organ injury.


Assuntos
COVID-19/terapia , SARS-CoV-2/patogenicidade , Trocadores de Sódio-Hidrogênio/genética , Viroses/terapia , COVID-19/genética , COVID-19/virologia , Endossomos/genética , Endossomos/virologia , Humanos , Prótons , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Viroses/genética , Viroses/virologia
5.
Free Radic Biol Med ; 174: 225-235, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34407426

RESUMO

Oxidative stress has been implicated in the aging process and the progression of many neurodegenerative disorders. We previously reported that a novel oxindole compound, GIF-0726-r, effectively prevents endogenous oxidative stress, such as oxytosis/ferroptosis, an iron-dependent form of non-apoptotic cell death, in mouse hippocampal cells. In this study, using two hundred compounds that were developed based on the structure-activity relationship of GIF-0726-r, we screened for the most potent compounds that prevent glutamate- and erastin-induced oxytosis and ferroptosis. Using submicromolar concentrations, we identified nine neuroprotective compounds that have N,N-dimethylaniline as a common structure but no longer contain an oxindole ring. The most potent derivatives, GIF-2114 and GIF-2197-r (the racemate of GIF-2115 and GIF-2196), did not affect glutathione levels, had no antioxidant activity in vitro, or ability to activate the Nrf2 pathway, but prevented oxytosis/ferroptosis via reducing reactive oxygen production and decreasing ferrous ions. Furthermore, we developed fluorescent probes of GIF-2114 and GIF-2197-r to image their distribution in live cells and found that they preferentially accumulated in late endosomes/lysosomes, which play a central role in iron metabolism. These results suggest that GIF-2114 and GIF-2197-r protect hippocampal cells from oxytosis/ferroptosis by targeting late endosomes and lysosomes, as well as decreasing ferrous ions.


Assuntos
Ferroptose , Fármacos Neuroprotetores , Compostos de Anilina , Animais , Endossomos , Lisossomos , Camundongos , Fármacos Neuroprotetores/farmacologia
6.
Int J Mol Sci ; 22(16)2021 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-34445155

RESUMO

A main characteristic of sphingolipids is the presence of a very long chain fatty acid (VLCFA) whose function in cellular processes is not yet fully understood. VLCFAs of sphingolipids are involved in the intracellular traffic to the vacuole and the maturation of early endosomes into late endosomes is one of the major pathways for vacuolar traffic. Additionally, the anionic phospholipid phosphatidylinositol-3-phosphate (PtdIns (3)P or PI3P) is involved in protein sorting and recruitment of small GTPase effectors at late endosomes/multivesicular bodies (MVBs) during vacuolar trafficking. In contrast to animal cells, PI3P mainly localizes to late endosomes in plant cells and to a minor extent to a discrete sub-domain of the plant's early endosome (EE)/trans-Golgi network (TGN) where the endosomal maturation occurs. However, the mechanisms that control the relative levels of PI3P between TGN and MVBs are unknown. Using metazachlor, an inhibitor of VLCFA synthesis, we found that VLCFAs are involved in the TGN/MVB distribution of PI3P. This effect is independent from either synthesis of PI3P by PI3-kinase or degradation of PI(3,5)P2 into PI3P by the SUPPRESSOR OF ACTIN1 (SAC1) phosphatase. Using high-resolution live cell imaging microscopy, we detected transient associations between TGNs and MVBs but VLCFAs are not involved in those interactions. Nonetheless, our results suggest that PI3P might be transferable from TGN to MVBs and that VLCFAs act in this process.


Assuntos
Arabidopsis/metabolismo , Endossomos/metabolismo , Ácidos Graxos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Vias Biossintéticas , Esfingolipídeos/metabolismo , Rede trans-Golgi/metabolismo
7.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445480

RESUMO

Multivesicular body (MVB)-mediated endosomal sorting and macroautophagy are the main pathways mediating the transport of cellular components to the vacuole and are essential for maintaining cellular homeostasis. The interplay of these two pathways remains poorly understood in plants. In this study, we show that FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), which was previously identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT), essential for MVB biogenesis and plant growth, can be transported to the vacuole for degradation in response to iron deficiency. The vacuolar transport of ubiquitinated FREE1 protein is mediated by the autophagy pathway. As a consequence, the autophagy deficient mutants, atg5-1 and atg7-2, accumulate more endogenous FREE1 protein and display hypersensitivity to iron deficiency. Furthermore, under iron-deficient growth condition autophagy related genes are upregulated to promote the autophagic degradation of FREE1, thereby possibly relieving the repressive effect of FREE1 on iron absorption. Collectively, our findings demonstrate a unique regulatory mode of protein turnover of the ESCRT machinery through the autophagy pathway to respond to iron deficiency in plants.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteínas Relacionadas à Autofagia/genética , Ferro/metabolismo , Proteínas de Transporte Vesicular/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Autofagia , Transporte Biológico , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Mutação , Proteólise , Ubiquitinação
8.
Adv Exp Med Biol ; 1331: 19-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34453290

RESUMO

During the development of the nervous system, neurons respond to diffusible cues secreted by target cells. Because such target-derived factors regulate development, maturation, and maintenance of axons as well as somatodendritic compartments, signals initiated at distal axons must be retrogradely transmitted toward cell bodies. Neurotrophins, including the nerve growth factor (NGF), provide one of the best-known examples of target-derived growth factors. The cell biological processes of endocytosis and retrograde trafficking of their Trk receptors from growth cones to cell bodies are key mechanisms by which target-derived neurotrophins influence neurons. Evidence accumulated over the past several decades has begun to uncover the molecular mechanisms of formation, transport, and biological functions of these specialized endosomes called "signaling endosomes."


Assuntos
Endossomos , Fator de Crescimento Neural , Axônios , Fator de Crescimento Neural/genética , Neurônios , Transdução de Sinais
9.
J Immunol ; 207(5): 1448-1455, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34362834

RESUMO

Identification of the receptors involved in innate immune recognition of Staphylococcus aureus, a major cause of morbidity and mortality in humans, is essential to develop alternative strategies to treat infections caused by antibiotic-resistant strains. In the current study, we examine the role of endosomal TLRs, which sense the presence of prokaryotic-type nucleic acids, in anti-staphylococcal host defenses using infection models involving genetically defective mice. Single deficiencies in TLR7, 9, or 13 resulted in mild or no decrease in host defenses. However, the simultaneous absence of TLR7, 9, and 13 resulted in markedly increased susceptibility to cutaneous and systemic S. aureus infection concomitantly with decreased production of proinflammatory chemokines and cytokines, neutrophil recruitment to infection sites, and reduced production of reactive oxygen species. This phenotype was significantly more severe than that of mice lacking TLR2, which senses the presence of staphylococcal lipoproteins. Notably, the combined absence of TLR7, 9, and 13 resulted in complete abrogation of IL-12 p70 and IFN-ß responses to staphylococcal stimulation in macrophages. Taken together, our data highlight the presence of a highly integrated endosomal detection system, whereby TLR7, 9, and 13 cooperate in sensing the presence of staphylococcal nucleic acids. We demonstrate that the combined absence of these receptors cannot be compensated for by cell surface-associated TLRs, such as TLR2, or cytosolic receptors. These data may be useful to devise strategies aimed at stimulating innate immune receptors to treat S. aureus infections.


Assuntos
Endossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Neutrófilos/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/metabolismo , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genética , Receptores Toll-Like/genética
10.
Nat Commun ; 12(1): 4739, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362892

RESUMO

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing second messenger which uniquely mobilizes Ca2+ from acidic endolysosomal organelles. However, the molecular identity of the NAADP receptor remains unknown. Given the necessity of the endolysosomal two-pore channel (TPC1 or TPC2) in NAADP signaling, we performed affinity purification and quantitative proteomic analysis of the interacting proteins of NAADP and TPCs. We identified a Sm-like protein Lsm12 complexed with NAADP, TPC1, and TPC2. Lsm12 directly binds to NAADP via its Lsm domain, colocalizes with TPC2, and mediates the apparent association of NAADP to isolated TPC2 or TPC2-containing membranes. Lsm12 is essential and immediately participates in NAADP-evoked TPC activation and Ca2+ mobilization from acidic stores. These findings reveal a putative RNA-binding protein to function as an NAADP receptor and a TPC regulatory protein and provides a molecular basis for understanding the mechanisms of NAADP signaling.


Assuntos
Cálcio/metabolismo , NADP/análogos & derivados , Porinas/metabolismo , Animais , Canais de Cálcio/metabolismo , Linhagem Celular , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/química , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Endossomos/metabolismo , Fibroblastos , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Mutação , NADP/genética , NADP/metabolismo , Proteômica
11.
Int J Mol Sci ; 22(16)2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34445706

RESUMO

Here, we present the main features of human acid sphingomyelinase (ASM), its biosynthesis, processing and intracellular trafficking, its structure, its broad substrate specificity, and the proposed mode of action at the surface of the phospholipid substrate carrying intraendolysosomal luminal vesicles. In addition, we discuss the complex regulation of its phospholipid cleaving activity by membrane lipids and lipid-binding proteins. The majority of the literature implies that ASM hydrolyses solely sphingomyelin to generate ceramide and ignores its ability to degrade further substrates. Indeed, more than twenty different phospholipids are cleaved by ASM in vitro, including some minor but functionally important phospholipids such as the growth factor ceramide-1-phosphate and the unique lysosomal lysolipid bis(monoacylglycero)phosphate. The inherited ASM deficiency, Niemann-Pick disease type A and B, impairs mainly, but not only, cellular sphingomyelin catabolism, causing a progressive sphingomyelin accumulation, which furthermore triggers a secondary accumulation of lipids (cholesterol, glucosylceramide, GM2) by inhibiting their turnover in late endosomes and lysosomes. However, ASM appears to be involved in a variety of major cellular functions with a regulatory significance for an increasing number of metabolic disorders. The biochemical characteristics of ASM, their potential effect on cellular lipid turnover, as well as a potential impact on physiological processes will be discussed.


Assuntos
Fosfolipídeos/biossíntese , Esfingomielina Fosfodiesterase/biossíntese , Esfingomielina Fosfodiesterase/metabolismo , Transporte Biológico , Ceramidas/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Humanos , Lisossomos/metabolismo , Lipídeos de Membrana/metabolismo , Doença de Niemann-Pick Tipo A/metabolismo , Fosfolipídeos/metabolismo , Esfingomielina Fosfodiesterase/fisiologia , Esfingomielinas/metabolismo , Fosfolipases Tipo C/metabolismo , Fosfolipases Tipo C/fisiologia
12.
Nat Commun ; 12(1): 4697, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349123

RESUMO

Polarized epithelial cells can organize into complex structures with a characteristic central lumen. Lumen formation requires that cells coordinately orient their polarity axis so that the basolateral domain is on the outside and apical domain inside epithelial structures. Here we show that the transmembrane aminopeptidase, CD13, is a key determinant of epithelial polarity orientation. CD13 localizes to the apical membrane and associates with an apical complex with Par6. CD13-deficient cells display inverted polarity in which apical proteins are retained on the outer cell periphery and fail to accumulate at an intercellular apical initiation site. Here we show that CD13 is required to couple apical protein cargo to Rab11-endosomes and for capture of endosomes at the apical initiation site. This role in polarity utilizes the short intracellular domain but is independent of CD13 peptidase activity.


Assuntos
Antígenos CD13/metabolismo , Polaridade Celular , Células Epiteliais/citologia , Epitélio/crescimento & desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD13/química , Antígenos CD13/genética , Células CACO-2 , Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Células Epiteliais/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Domínios Proteicos , Proteínas rab de Ligação ao GTP/metabolismo
13.
Nat Commun ; 12(1): 5131, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446736

RESUMO

Protein delivery with cell-penetrating peptide is opening up the possibility of using targets inside cells for therapeutic or biological applications; however, cell-penetrating peptide-mediated protein delivery commonly suffers from ineffective endosomal escape and low tolerance in serum, thereby limiting in vivo efficacy. Here, we present an intracellular protein delivery system consisting of four modules in series: cell-penetrating peptide, pH-dependent membrane active peptide, endosome-specific protease sites and a leucine zipper. This system exhibits enhanced delivery efficiency and serum tolerance, depending on proteolytic cleavage-facilitated endosomal escape and leucine zipper-based dimerisation. Intravenous injection of protein phosphatase 1B fused with this system successfully suppresses the tumour necrosis factor-α-induced systemic inflammatory response and acetaminophen-induced acute liver failure in a mouse model. We believe that the strategy of using multifunctional chimaeric peptides is valuable for the development of cell-penetrating peptide-based protein delivery systems, and facilitate the development of biological macromolecular drugs for use against intracellular targets.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Falência Hepática Aguda/tratamento farmacológico , Peptídeos/química , Proteína Fosfatase 1/administração & dosagem , Animais , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Endossomos/genética , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Falência Hepática Aguda/genética , Falência Hepática Aguda/metabolismo , Camundongos Endogâmicos BALB C , Peptídeos/genética , Peptídeos/metabolismo , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Transporte Proteico
14.
Biomolecules ; 11(7)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34356645

RESUMO

Microphthalmia-associated transcription factor (MITF) is the principal transcription factor regulating pivotal processes in melanoma cell development, growth, survival, proliferation, differentiation and invasion. In recent years, convincing evidence has been provided attesting key roles of endolysosomal cation channels, specifically TPCs and TRPMLs, in cancer, including breast cancer, glioblastoma, bladder cancer, hepatocellular carcinoma and melanoma. In this review, we provide a gene expression profile of these channels in different types of cancers and decipher their roles, in particular the roles of two-pore channel 2 (TPC2) and TRPML1 in melanocytes and melanoma. We specifically discuss the signaling cascades regulating MITF and the relationship between endolysosomal cation channels, MAPK, canonical Wnt/GSK3 pathways and MITF.


Assuntos
Canais Iônicos/metabolismo , Melanócitos/metabolismo , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Endossomos/metabolismo , Expressão Gênica , Humanos , Canais Iônicos/genética , Lisossomos/metabolismo , Melanócitos/patologia , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo
15.
Antiviral Res ; 194: 105167, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34450201

RESUMO

Niemann-Pick type C1 (NPC1) receptor is an endosomal membrane protein that regulates intracellular cholesterol traffic. This protein has been shown to play an important role for several viruses. It has been reported that SARS-CoV-2 enters the cell through plasma membrane fusion and/or endosomal entry upon availability of proteases. However, the whole process is not fully understood yet and additional viral/host factors might be required for viral fusion and subsequent viral replication. Here, we report a novel interaction between the SARS-CoV-2 nucleoprotein (N) and the cholesterol transporter NPC1. Furthermore, we have found that some compounds reported to interact with NPC1, carbazole SC816 and sulfides SC198 and SC073, were able to reduce SARS-CoV-2 viral infection with a good selectivity index in human cell infection models. These findings suggest the importance of NPC1 for SARS-CoV-2 viral infection and a new possible potential therapeutic target to fight against COVID-19.


Assuntos
Transporte Biológico , COVID-19/tratamento farmacológico , Endossomos/virologia , Proteína C1 de Niemann-Pick/análise , SARS-CoV-2/fisiologia , Animais , Carbazóis/farmacologia , Chlorocebus aethiops , Endossomos/química , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fusão de Membrana , Células Vero , Replicação Viral
16.
Molecules ; 26(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200716

RESUMO

In this study, we report pH-responsive metal-based biopolymer nanoparticles (NPs) for tumor-specific chemotherapy. Here, aminated hyaluronic acid (aHA) coupled with 2,3-dimethylmaleic anhydride (DMA, as a pH-responsive moiety) (aHA-DMA) was electrostatically complexed with ferrous chloride tetrahydrate (FeCl2/4H2O, as a chelating metal) and doxorubicin (DOX, as an antitumor drug model), producing DOX-loaded Fe-based hyaluronate nanoparticles (DOX@aHA-DMA/Fe NPs). Importantly, the DOX@aHA-DMA/Fe NPs improved tumor cellular uptake due to HA-mediated endocytosis for tumor cells overexpressing CD44 receptors. As a result, the average fluorescent DOX intensity observed in MDA-MB-231 cells (with CD44 receptors) was ~7.9 × 102 (DOX@HA/Fe NPs, without DMA), ~8.1 × 102 (DOX@aHA-DMA0.36/Fe NPs), and ~9.3 × 102 (DOX@aHA-DMA0.60/Fe NPs). Furthermore, the DOX@aHA-DMA/Fe NPs were destabilized due to ionic repulsion between Fe2+ and DMA-detached aHA (i.e., positively charged free aHA) in the acidic environment of tumor cells. This event accelerated the release of DOX from the destabilized NPs. Our results suggest that these NPs can be promising tumor-targeting drug carriers responding to acidic endosomal pH.


Assuntos
Doxorrubicina/química , Endossomos/química , Compostos Ferrosos/química , Ácido Hialurônico/química , Nanopartículas/química , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Endocitose/efeitos dos fármacos , Feminino , Humanos , Receptores de Hialuronatos/metabolismo , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C
17.
Nat Commun ; 12(1): 4389, 2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34282141

RESUMO

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.


Assuntos
Exossomos/metabolismo , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismo , Comunicação Celular , Membrana Celular/metabolismo , Endossomos/metabolismo , Vesículas Extracelulares/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão , Técnicas de Inativação de Genes , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Transporte Proteico , Proteômica
18.
J Med Microbiol ; 70(7)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34296983

RESUMO

Introduction. Shigella flexneri is an intracellular bacterial pathogen that utilizes a type III secretion apparatus to inject effector proteins into host cells.Hypothesis/Gap Statement. The T3SS effector IpaH4.5 is important for the virulence of Shigella.Aim. This study aimed to elucidate the molecular mechanism and host target of the IpaH4.5 as well as its roles in S. flexneri infection.Methodology. The GAP assay was used to identify substrate Rab GTPases of IpaH4.5. A coimmunoprecipitation assay was applied to identify the interaction of Rab GTPases with IpaH4.5. A confocal microscopy analysis was used to assess the effects of IpaH4.5 on mannose 6-phosphate receptor (MPR) trafficking. To identify the effects of IpaH4.5 GAP activity on the activity of lysosomal cathepsin B, the Magic Red-RR assay was used. Finally, the intracellular persistence assay was used to identify IpaH4.5 GAP activity in S. flexneri intracellular growth.Results. We found that the effector IpaH4.5 disrupts MPR trafficking and lysosomal function, thereby counteracting host lysosomal degradation. IpaH4.5 harbours TBC-like dual-finger motifs and exhibits potent RabGAP activities towards Rab31. IpaH4.5 disrupts the transport of the cation-dependent mannose 6-phosphate receptor (CD-MPR) from the Golgi to the endosome by targeting Rab31, thereby attenuating lysosomal function. As a result, the intracellular persistence of S. flexneri requires IpaH4.5 TBC-like GAP activity to mediate bacterial escape from host lysosome-mediated elimination.Conclusion. We identified an unknown function of IpaH4.5 and its potential role in S. flexneri infection.


Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Lisossomos/metabolismo , Shigella flexneri/patogenicidade , Proteínas rab de Ligação ao GTP/metabolismo , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Catepsina B/metabolismo , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Shigella flexneri/metabolismo , Proteínas rab de Ligação ao GTP/genética
19.
FASEB J ; 35(8): e21681, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34196428

RESUMO

The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.


Assuntos
Proteínas de Membrana/metabolismo , Simportadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Códon sem Sentido , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Sequência Conservada , Cães , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios PDZ/genética , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores/química , Simportadores/genética , Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
20.
J Phys Chem B ; 125(28): 7732-7741, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34255499

RESUMO

During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. The viral spike (S) protein mediates both the initial contact with the host cell and the subsequent membrane fusion. Proteolytic cleavage of S at the S2' site exposes its fusion peptide (FP) as the new N-terminus. By binding to the host membrane, the FP anchors the virus to the host cell. The reorganization of S2 between virus and host then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 µs long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP, we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than 10-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP-membrane binding.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Sequência de Aminoácidos , Membrana Celular , Endossomos , Humanos , Fusão de Membrana , Peptídeos , SARS-CoV-2 , Internalização do Vírus
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...