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1.
Adv Exp Med Biol ; 1287: 31-46, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33034024

RESUMO

The endosomal pathway plays a pivotal role upon signal transduction in the Notch pathway. Recent work on lethal (2) giant discs (lgd) points to an additional critical role in avoiding uncontrolled ligand-independent signalling during trafficking of the Notch receptor through the endosomal pathway to the lysosome for degradation. In this chapter, we will outline the journey of Notch through the endosomal system and present an overview of the current knowledge about Lgd and its mammalian orthologs Lgd1/CC2D1b and Lgd2/CC2D1a. We will then discuss how Notch is activated in the absence of lgd function in Drosophila and ask whether there is evidence that a similar ligand-independent activation of the Notch pathway can also happen in mammals if the orthologs are inactivated.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Neoplasias/metabolismo , Receptores Notch/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Endossomos/metabolismo , Humanos
2.
Gene ; 760: 145017, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32755655

RESUMO

Cytoplasmic vacuolization usually occurs in cells treated with different agents and substances. We found that LZ-106, an analog of enoxacin, is a potent lysosomotropic agent, contributing to the formation of cytoplasmic vacuoles in cells. Studies of LZ-106-induced vacuolization in H460 cells showed acid environment inside these vacuoles. Further study demonstrated that markers in the late endosomes and lysosomes, like LAMP1 and RAB7, on the surface of the vacuoles, implying that these vacuoles might derive from endosomes and/or lysosomes. By studying the fluorescence intensity of LZ-106, we discovered that LZ-106 tended to locate in acid organelles, and Bafilomycin A1, a V-ATPase inhibitor, was able to suppress its acid organelles localization. Also, we noticed that LZ-106 could induce lysosome stress, involving pH increment and lysosomal membrane damage. Moreover, the expression levels of some lysosome-related proteins, like LAMP1, EEA1, and Cathepsin B, were also altered upon LZ-106 treatment. At last, we confirmed LZ-106 can activate TFEB, a key regulator of lysosomes. Knockdown of TFEB could also reverse LZ-106's effect on vacuolization in H460 cells. Taken together, due to LZ-106's lysosomotropic properties, it is able to accumulate in the acid organelles and induce lysosomal dysfunction in H460 cells, leading to TFEB activation and the following cytoplasmic vacuolization.


Assuntos
Enoxacino/análogos & derivados , Enoxacino/farmacologia , Vacúolos/efeitos dos fármacos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Endossomos/metabolismo , Humanos , Lisossomos/química , Macrolídeos/farmacologia
3.
PLoS One ; 15(8): e0233820, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32804976

RESUMO

Molecular markers derived from cerebrospinal fluid (CSF) represent an accessible means of exploring the pathobiology of Huntington's disease (HD) in vivo. The endo-lysosomal/autophagy system is dysfunctional in HD, potentially contributing to disease pathogenesis and representing a potential target for therapeutic intervention. Several endo-lysosomal proteins have shown promise as biomarkers in other neurodegenerative diseases; however, they have yet to be fully explored in HD. We performed parallel reaction monitoring mass spectrometry analysis (PRM-MS) of multiple endo-lysosomal proteins in the CSF of 60 HD mutation carriers and 20 healthy controls. Using generalised linear models controlling for age and CAG, none of the 18 proteins measured displayed significant differences in concentration between HD patients and controls. This was affirmed by principal component analysis, in which no significant difference across disease stage was found in any of the three components representing lysosomal hydrolases, binding/transfer proteins and innate immune system/peripheral proteins. However, several proteins were associated with measures of disease severity and cognition: most notably amyloid precursor protein, which displayed strong correlations with composite Unified Huntington's Disease Rating Scale, UHDRS Total Functional Capacity, UHDRS Total Motor Score, Symbol Digit Modalities Test and Stroop Word Reading. We conclude that although endo-lysosomal proteins are unlikely to have value as disease state CSF biomarkers for Huntington's disease, several proteins demonstrate associations with clinical severity, thus warranting further, targeted exploration and validation in larger, longitudinal samples.


Assuntos
Proteínas do Líquido Cefalorraquidiano/metabolismo , Doença de Huntington/líquido cefalorraquidiano , Adulto , Idoso , Precursor de Proteína beta-Amiloide/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Cognição , Estudos Transversais , Progressão da Doença , Endossomos/metabolismo , Feminino , Proteína Ativadora de G(M2)/líquido cefalorraquidiano , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/psicologia , Modelos Lineares , Estudos Longitudinais , Proteína 2 de Membrana Associada ao Lisossomo/líquido cefalorraquidiano , Glicoproteínas de Membrana Associadas ao Lisossomo/líquido cefalorraquidiano , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Análise de Componente Principal , Estudos Prospectivos , Proteínas/metabolismo , Expansão das Repetições de Trinucleotídeos
4.
Int J Mol Sci ; 21(16)2020 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-32784543

RESUMO

Gitelman's syndrome (GS) and Bartter's syndrome (BS) are rare inherited salt-losing tubulopathies whose variations in genotype do not correlate well with either clinical course or electrolyte requirements. Using GS/BS patients as nature's experiments, we found them to be a human model of endogenous Ang II antagonism with activated Renin-Angiotensin System (RAS), resulting in high Ang II levels with blunted cardiovascular effects. These patients are also characterized by increased and directly correlated levels of both Angiotensin Converting Enzyme 2 (ACE2) and Ang 1-7. Understanding the myriad of distinctive and frequently overlapping clinical presentations of GS/BS arises remains challenging. Efforts to find a treatment for COVID-19 has fueled a recent surge in interest in chloroquine/hydroxychloroquine and its effects. Of specific interest are chloroquine/hydroxychloroquine's ability to inhibit SARS-CoV infection by impairing ACE2, the SARS-CoV2 entry point, through terminal glycosylation via effects on TGN/post-Golgi pH homeostasis. Several different studies with a GS or a BS phenotype, along with a nonsyndromic form of X-linked intellectual disability linked to a mutated SLC9A7, provide additional evidence that specific gene defects can act via misregulation of TGN/post-Golgi pH homeostasis, which leads to a common mechanistic basis resulting in overlapping phenotypes. We suggest that linkage between the specific gene defects identified in GS and BS and the myriad of distinctive and frequently overlapping clinical findings may be the result of aberrant glycosylation of ACE2 driven by altered TGN/endosome system acidification caused by the metabolic alkalosis brought about by these salt-losing tubulopathies in addition to their altered intracellular calcium signaling due to a blunted second messenger induced intracellular calcium release that is, in turn, amplified by the RAS system.


Assuntos
Síndrome de Bartter/genética , Infecções por Coronavirus/tratamento farmacológico , Síndrome de Gitelman/genética , Peptidil Dipeptidase A/metabolismo , Fenótipo , Pneumonia Viral/tratamento farmacológico , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Síndrome de Bartter/metabolismo , Síndrome de Bartter/patologia , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Síndrome de Gitelman/metabolismo , Síndrome de Gitelman/patologia , Humanos , Hidroxicloroquina/farmacologia , Hidroxicloroquina/uso terapêutico , Pandemias
5.
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
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.
J Transl Med ; 18(1): 261, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32600410

RESUMO

Amino-bisphosphonates such as zoledronic acid (ZA) can possibly ameliorate or prevent severe COVID-19 disease by at least three distinct mechanisms: (1) as immunostimulants which could boost γδ T cell expansion, important in the acute response in the lung; (2) as DC modulators, limiting their ability to only partially activate T cells; and (3) as prenylation inhibitors of small GTPases in the endosomal pathway of the DC to prevent expulsion of lysosomes containing SARS-CoV-2 virions. Use of ZA or other amino-bisphosphonates as modulators of COVID-19 disease should be considered.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Células Dendríticas/virologia , Difosfonatos/uso terapêutico , Endossomos/metabolismo , Fatores Imunológicos/farmacologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Animais , Células Dendríticas/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Humanos , Pandemias
8.
PLoS Biol ; 18(7): e3000778, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678845

RESUMO

The evolution of transformed cancer cells into metastatic tumors is, in part, driven by altered intracellular signaling downstream of receptor tyrosine kinases (RTKs). The surface levels and activity of RTKs are governed mainly through clathrin-mediated endocytosis (CME), endosomal recycling, or degradation. In turn, oncogenic signaling downstream of RTKs can reciprocally regulate endocytic trafficking by creating feedback loops in cells to enhance tumor progression. We previously showed that FCH/F-BAR and Double SH3 Domain-Containing Protein (FCHSD2) has a cancer-cell specific function in regulating CME in non-small-cell lung cancer (NSCLC) cells. Here, we report that FCHSD2 loss impacts recycling of the RTKs, epidermal growth factor receptor (EGFR) and proto-oncogene c-Met (MET), and shunts their trafficking into late endosomes and lysosomal degradation. Notably, FCHSD2 depletion results in the nuclear translocation of active extracellular signal-regulated kinase 1 and 2 (ERK1/2), leading to enhanced transcription and up-regulation of EGFR and MET. The small GTPase, Ras-related protein Rab-7A (Rab7), is essential for the FCHSD2 depletion-induced effects. Correspondingly, FCHSD2 loss correlates to higher tumor grades of NSCLC. Clinically, NSCLC patients expressing high FCHSD2 exhibit elevated survival, whereas patients with high Rab7 expression display decreased survival rates. Our study provides new insight into the molecular nexus for crosstalk between oncogenic signaling and RTK trafficking that controls cancer progression.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/metabolismo , Endocitose , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Oncogenes , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Linhagem Celular Tumoral , Progressão da Doença , Endossomos/metabolismo , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/enzimologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores da Transferrina/metabolismo , Regulação para Cima , Proteínas rab de Ligação ao GTP/metabolismo
9.
PLoS Negl Trop Dis ; 14(7): e0007960, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32687500

RESUMO

Intracerebral microhemorrhages (CMHs) are small foci of hemorrhages in the cerebrum. Acute infections induced by some intracellular pathogens, including rickettsia, can result in CMHs. Annexin a2 (ANXA2) has been documented to play a functional role during intracellular bacterial adhesion. Here we report that ANXA2-knockout (KO) mice are more susceptible to CMHs in response to rickettsia and Ebola virus infections, suggesting an essential role of ANXA2 in protecting vascular integrity during these intracellular pathogen infections. Proteomic analysis via mass spectrometry of whole brain lysates and brain-derived endosomes from ANXA2-KO and wild-type (WT) mice post-infection with R. australis revealed that a variety of significant proteins were differentially expressed, and the follow-up function enrichment analysis had identified several relevant cell-cell junction functions. Immunohistology study confirmed that both infected WT and infected ANXA2-KO mice were subjected to adherens junctional protein (VE-cadherin) damages. However, key blood-brain barrier (BBB) components, tight junctional proteins ZO-1 and occludin, were disorganized in the brains from R. australis-infected ANXA2-KO mice, but not those of infected WT mice. Similar ANXA2-KO dependent CMHs and fragments of ZO-1 and occludin were also observed in Ebola virus-infected ANXA2-KO mice, but not found in infected WT mice. Overall, our study revealed a novel role of ANXA2 in the formation of CMHs during R. australis and Ebola virus infections; and the underlying mechanism is relevant to the role of ANXA2-regulated tight junctions and its role in stabilizing the BBB in these deadly infections.


Assuntos
Anexina A2/metabolismo , Hemorragia Cerebral/metabolismo , Ebolavirus/fisiologia , Doença pelo Vírus Ebola/metabolismo , Infecções por Rickettsia/metabolismo , Rickettsia/fisiologia , Animais , Anexina A2/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Hemorragia Cerebral/genética , Hemorragia Cerebral/microbiologia , Hemorragia Cerebral/virologia , Endossomos/genética , Endossomos/metabolismo , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/virologia , Humanos , Camundongos , Camundongos Knockout , Rickettsia/genética , Infecções por Rickettsia/genética , Infecções por Rickettsia/microbiologia
10.
PLoS Negl Trop Dis ; 14(7): e0008396, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32722702

RESUMO

The parasitophorous vacuoles (PVs) that insulate Leishmania spp. in host macrophages are vacuolar compartments wherein promastigote forms differentiate into amastigote that are the replicative form of the parasite and are also more resistant to host responses. We revisited the biogenesis of tight-fitting PVs that insulate L. infantum in promastigote-infected macrophage-like RAW 264.7 cells by time-dependent confocal laser multidimensional imaging analysis. Pharmacological disassembly of the cellular microtubule network and silencing of the dynein gene led to an impaired interaction of L. infantum-containing phagosomes with late endosomes and lysosomes, resulting in the tight-fitting parasite-containing phagosomes never transforming into mature PVs. Analysis of the shape of the L. infantum parasite within PVs, showed that factors that impair promastigote-amastigote differentiation can also result in PVs whose maturation is arrested. These findings highlight the importance of the MT-dependent interaction of L. infantum-containing phagosomes with the host macrophage endolysosomal pathway to secure the intracellular fate of the parasite.


Assuntos
Leishmania infantum/fisiologia , Leishmaniose Visceral/parasitologia , Macrófagos/parasitologia , Microtúbulos/parasitologia , Animais , Endossomos/metabolismo , Humanos , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/metabolismo , Camundongos , Microtúbulos/metabolismo , Fagossomos/metabolismo , Células RAW 264.7
11.
PLoS Pathog ; 16(7): e1008622, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32634175

RESUMO

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from phagosomes and induces a robust adaptive immune response in mice, while mutants unable to escape phagosomes fail to induce a robust adaptive immune response and suppress the immunity to wildtype bacteria when co-administered. The capacity to suppress immunity can be reversed by blocking IL-10. In this study, we sought to understand the host receptors that lead to secretion of IL-10 in response to phagosome-confined L. monocytogenes (Δhly), with the ultimate goal of generating strains that fail to induce IL-10. We conducted a transposon screen to identify Δhly L. monocytogenes mutants that induced significantly more or less IL-10 secretion in bone marrow-derived macrophages (BMMs). A transposon insertion in lgt, which encodes phosphatidylglycerol-prolipoprotein diacylglyceryl transferase and is essential for the formation of lipoproteins, induced significantly reduced IL-10 secretion. Mutants with transposon insertions in pgdA and oatA, which encode peptidoglycan N-acetylglucosamine deacetylase and O-acetyltransferase, are sensitive to lysozyme and induced enhanced IL-10 secretion. A ΔhlyΔpgdAΔoatA strain was killed in BMMs and induced enhanced IL-10 secretion that was dependent on Unc93b1, a trafficking molecule required for signaling of nucleic acid-sensing TLRs. These data revealed that nucleic acids released by bacteriolysis triggered endosomal TLR-mediated IL-10 secretion. Secretion of IL-10 in response to infection with the parental strain was mostly TLR2-dependent, while IL-10 secretion in response to lysozyme-sensitive strains was dependent on TLR2 and Unc93b1. In mice, the IL-10 response to vacuole-confined L. monocytogenes was also dependent on TLR2 and Unc93b1. Co-administration of Δhly and ΔactA resulted in suppressed immunity in WT mice, but not in mice with mutations in Unc93b1. These data revealed that secretion of IL-10 in response to L. monocytogenes infection in vitro is mostly TLR2-dependent and immune suppression by phagosome-confined bacteria in vivo is mostly dependent on endosomal TLRs.


Assuntos
Tolerância Imunológica/imunologia , Interleucina-10/metabolismo , Listeriose/imunologia , Receptores Toll-Like/imunologia , Animais , Endossomos/imunologia , Endossomos/metabolismo , Interleucina-10/imunologia , Listeria monocytogenes/imunologia , Listeriose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fagossomos/imunologia , Fagossomos/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo
12.
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
13.
Science ; 369(6505): 787-793, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32675289

RESUMO

Although Huntington's disease is a late-manifesting neurodegenerative disorder, both mouse studies and neuroimaging studies of presymptomatic mutation carriers suggest that Huntington's disease might affect neurodevelopment. To determine whether this is actually the case, we examined tissue from human fetuses (13 weeks gestation) that carried the Huntington's disease mutation. These tissues showed clear abnormalities in the developing cortex, including mislocalization of mutant huntingtin and junctional complex proteins, defects in neuroprogenitor cell polarity and differentiation, abnormal ciliogenesis, and changes in mitosis and cell cycle progression. We observed the same phenomena in Huntington's disease mouse embryos, where we linked these abnormalities to defects in interkinetic nuclear migration of progenitor cells. Huntington's disease thus has a neurodevelopmental component and is not solely a degenerative disease.


Assuntos
Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Sistema Nervoso/embriologia , Animais , Ciclo Celular , Endossomos/metabolismo , Feto , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Camundongos , Camundongos Mutantes , Mitose , Mutação , Células Neuroepiteliais/metabolismo , Junções Íntimas/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
14.
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
15.
Proc Natl Acad Sci U S A ; 117(32): 19507-19516, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723814

RESUMO

Previous analysis of postentry events revealed that human cytomegalovirus (HCMV) displays a unique, extended nuclear translocation pattern in monocytes. We determined that c-Src signaling through pentamer engagement of integrins is required upon HCMV entry to avoid sorting of the virus into late endosomes and subsequent degradation. To follow up on this previous study, we designed experiments to investigate how HCMV-induced signaling through the other major axis-the epidermal growth factor receptor (EGFR) kinase-regulates viral postentry events. Here we show that HCMV induces chronic and functional EGFR signaling that is distinct to the virus as compared to the natural EGFR ligand: EGF. This chronic EGFR kinase activity in infected monocytes is required for the proper subcellular localization of the viral particle during trafficking events, as well as for promoting translocation of viral DNA into the host nucleus. Our data indicate that HCMV glycoprotein B (gB) binds to EGFR at the monocyte surface, the virus and EGFR are internalized together, and gB remains bound to EGFR throughout viral postentry events until de-envelopment to promote the chronic EGFR kinase activity required for viral trafficking and nuclear translocation. These data highlight how initial EGFR signaling via viral binding is necessary for entry, but not sufficient to promote each viral trafficking event. HCMV appears to manipulate the EGFR kinase postentry, via gB-EGFR interaction, to be active at the critical points throughout the trafficking process that leads to nuclear translocation and productive infection of peripheral blood monocytes.


Assuntos
Núcleo Celular/metabolismo , Citomegalovirus/fisiologia , Monócitos/virologia , Proteínas do Envelope Viral/metabolismo , Núcleo Celular/virologia , Células Cultivadas , DNA Viral/metabolismo , Endossomos/metabolismo , Endossomos/virologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Monócitos/metabolismo , Ligação Proteica , Transdução de Sinais , Rede trans-Golgi/metabolismo , Rede trans-Golgi/virologia
16.
Proc Natl Acad Sci U S A ; 117(31): 18530-18539, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690699

RESUMO

Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.


Assuntos
Autofagossomos/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Autofagia , Linhagem Celular , Retículo Endoplasmático/genética , Endossomos/genética , Endossomos/metabolismo , Humanos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
17.
Mol Cell Biol ; 40(19)2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32690545

RESUMO

Neuronal ceroid lipofuscinosis (NCL) is one of the most prevalent neurodegenerative disorders of early life, Parkinson's disease (PD) is the most common neurodegenerative disorder of midlife, while Alzheimer's disease (AD) is the most common neurodegenerative disorder of late life. While they are phenotypically distinct, recent studies suggest that they share a biological pathway, retromer-dependent endosomal trafficking. A retromer is a multimodular protein assembly critical for sorting and trafficking cargo out of the endosome. As a lysosomal storage disease, all 13 of NCL's causative genes affect endolysosomal function, and at least four have been directly linked to retromer. PD has several known causative genes, with one directly linked to retromer and others causing endolysosomal dysfunction. AD has over 25 causative genes/risk factors, with several of them linked to retromer or endosomal trafficking dysfunction. In this article, we summarize the emerging evidence on the association of genes causing NCL with retromer function and endosomal trafficking, review the recent evidence linking NCL genes to AD, and discuss how NCL, AD, and PD converge on a shared molecular pathway. We also discuss this pathway's role in microglia and neurons, cell populations which are critical to proper brain homeostasis and whose dysfunction plays a key role in neurodegeneration.


Assuntos
Doença de Alzheimer/metabolismo , Endossomos/metabolismo , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Doença de Parkinson/metabolismo , Doença de Alzheimer/genética , Animais , Transporte Biológico , Humanos , Doença de Parkinson/genética , Presenilina-1/genética , Presenilina-1/metabolismo
18.
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
19.
Nat Commun ; 11(1): 3645, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32686675

RESUMO

Endosomes are compositionally dynamic organelles that regulate signaling, nutrient status and organelle quality by specifying whether material entering the cells will be shuttled back to the cell surface or degraded by the lysosome. Recently, membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and endosomes have emerged as important players in endosomal protein sorting, dynamics and motility. Here, we show that PDZD8, a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain-containing ER transmembrane protein, utilizes distinct domains to interact with Rab7-GTP and the ER transmembrane protein Protrudin and together these components localize to an ER-late endosome MCS. At these ER-late endosome MCSs, mitochondria are also recruited to form a three-way contact. Thus, our data indicate that PDZD8 is a shared component of two distinct MCSs and suggest a role for SMP-mediated lipid transport in the regulation of endosome function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Mitocôndrias/metabolismo , Transporte Biológico , Linhagem Celular , Humanos , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(30): 17510-17512, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32665439

RESUMO

Type I IFN (IFN-I) is thought to be rapidly internalized and degraded following binding to its receptor and initiation of signaling. However, many studies report the persistent effects mediated by IFN-I for days or even weeks, both ex vivo and in vivo. These long-lasting effects are attributed to downstream signaling molecules or induced effectors having a long half-life, particularly in specific cell types. Here, we describe a mechanism explaining the long-term effects of IFN-I. Following receptor binding, IFN-I is siloed into endosomal compartments. These intracellular "IFN silos" persist for days and can be visualized by fluorescence and electron microscopy. However, they are largely dormant functionally, due to IFN-I-induced negative regulators. By contrast, in individuals lacking these negative regulators, such as ISG15 or USP18, this siloed IFN-I can continue to signal from within the endosome. This mechanism may underlie the long-term effects of IFN-I therapy and may contribute to the pathophysiology of type I interferonopathies.


Assuntos
Endossomos/metabolismo , Interferon Tipo I/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Citocinas/metabolismo , Endossomos/ultraestrutura , Humanos , Transporte Proteico , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo
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