Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 630
Filtrar
1.
Dev Cell ; 56(1): 52-66.e7, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33434526

RESUMO

ER tubules form and maintain membrane contact sites (MCSs) with endosomes. How and why these ER-endosome MCSs persist as endosomes traffic and mature is poorly understood. Here we find that a member of the reticulon protein family, Reticulon-3L (Rtn3L), enriches at ER-endosome MCSs as endosomes mature. We show that this localization is due to the long divergent N-terminal cytoplasmic domain of Rtn3L. We found that Rtn3L is recruited to ER-endosome MCSs by endosomal protein Rab9a, which marks a transition stage between early and late endosomes. Rab9a utilizes an FSV region to recruit Rtn3L via its six LC3-interacting region motifs. Consistent with our localization results, depletion or deletion of RTN3 from cells results in endosome maturation and cargo sorting defects, similar to RAB9A depletion. Together our data identify a tubular ER protein that promotes endosome maturation at ER MCSs.


Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico/genética , Motivos de Aminoácidos , Autofagossomos/genética , Autofagossomos/metabolismo , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Endossomos/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nogo/genética , Proteínas Nogo/metabolismo , RNA Interferente Pequeno , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
2.
Methods Mol Biol ; 2233: 53-70, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33222127

RESUMO

Endocytosis and intracellular retrograde trafficking from endosomes to the Golgi apparatus are key cellular processes. Endocytosis is directly or indirectly involved in many if not all cellular functions ranging from nutrient uptake and receptor signaling to mitosis, cell division, and migration (Scita, Di Fiore. Nature 463(7280):464-473, 2010; McMahon, Boucrot. Nat Rev Mol Cell Biol 12(8):517-533, 2011). Retrograde trafficking is emerging as a key driver for cell polarity. Robust methods are needed to quantify these processes. At the example of the bacterial Shiga toxin and the endogenous α5ß1 integrin, we here describe generic methods to differentiate (1) internalized from cell surface-accessible cargo proteins and (2) endocytic cargo proteins that have reached the Golgi apparatus via the retrograde route from those that have not. The choice of antibodies or natural ligands allows to adjust these methods to virtually any chosen biological system.


Assuntos
Endocitose/genética , Endossomos/genética , Complexo de Golgi/genética , Biologia Molecular/métodos , Transporte Biológico/genética , Movimento Celular/efeitos dos fármacos , Polaridade Celular/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Redes e Vias Metabólicas/efeitos dos fármacos , Toxina Shiga/química , Toxina Shiga/farmacologia , Rede trans-Golgi
3.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118857, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32949647

RESUMO

Intracellular organelle cross-talk is a new and important research area. Under stress conditions, the coordinated action of the autophagy and endosomal systems in tumor cells is essential for maintaining cellular homeostasis and survival. The activation of the IκB kinase (IKK) complex is also involved in the regulation of stress and homeostasis in tumor cells. Here, we try to explore the effects of constitutively active IKKß subunits (CA-IKKß) on autophagy and endosomal system interactions. We confirm that CA-IKKß induces accumulation of autophagosomes and their fusion with MVBs to form amphisomes in cancer cells, and also drives the release of EVs containing autophagy components through an amphisome-dependent mechanism. We further demonstrate that CA-IKKß inhibits the expression of RAB7, thereby weakening the lysosomal-dependent degradation pathway. CA-IKKß also induces phosphorylation of SNAP23 at Ser95 instead of Ser110, which further promotes amphisome-plasma membrane fusion and sEV secretion. These results indicate that CA-IKKß drives the formation and transport of amphisomes, thereby regulating tumor cell homeostasis, which may illuminate a special survival mechanism in tumor cells under stress.


Assuntos
Autofagia/genética , Quinase I-kappa B/genética , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Proteínas rab de Ligação ao GTP/genética , Autofagossomos/genética , Linhagem Celular Tumoral , Endossomos/genética , Exocitose/genética , Vesículas Extracelulares/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Lisossomos/genética , Fusão de Membrana/genética , Neoplasias/genética , Neoplasias/patologia , Fosforilação/genética , Transdução de Sinais/genética
4.
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
5.
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
6.
PLoS One ; 15(6): e0234180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511278

RESUMO

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1H46R;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.


Assuntos
Autofagia/fisiologia , Endossomos/metabolismo , Proteínas Luminescentes/genética , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Animais , Células Cultivadas , Meios de Cultura/farmacologia , Endossomos/genética , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/metabolismo , Lisossomos/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inanição , Superóxido Dismutase-1/genética , Imagem com Lapso de Tempo
7.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376619

RESUMO

HIV-1 assembly occurs principally at the plasma membrane (PM) of infected cells. Gag polyprotein precursors (Pr55Gag) are targeted to the PM, and their binding is mediated by the interaction of myristoylated matrix domain and a PM-specific phosphoinositide, the phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The major synthesis pathway of PI(4,5)P2 involves the activity of phosphatidylinositol-4-phosphate 5-kinase family type 1 composed of three isoforms (PIP5K1α, PIP5K1ß, and PIP5K1γ). To examine whether the activity of a specific PIP5K1 isoform determines proper Pr55Gag localization at the PM, we compared the cellular behavior of Pr55Gag in the context of PIP5K1 inhibition using siRNAs that individually targeted each of the three isoforms in TZM-bl HeLa cells. We found that downregulation of PIP5K1α and PIP5K1γ strongly impaired the targeting of Pr55Gag to the PM with a rerouting of the polyprotein within intracellular compartments. The efficiency of Pr55Gag release was thus impaired through the silencing of these two isoforms, while PIP5K1ß is dispensable for Pr55Gag targeting to the PM. The PM mistargeting due to the silencing of PIP5K1α leads to Pr55Gag hydrolysis through lysosome and proteasome pathways, while the silencing of PIP5K1γ leads to Pr55Gag accumulation in late endosomes. Our findings demonstrated that, within the PIP5K1 family, only the PI(4,5)P2 pools produced by PIP5K1α and PIP5K1γ are involved in the Pr55Gag PM targeting process.IMPORTANCE PM specificity of Pr55Gag membrane binding is mediated through the interaction of PI(4,5)P2 with the matrix (MA) basic residues. It was shown that overexpression of a PI(4,5)P2-depleting enzyme strongly impaired PM localization of Pr55Gag However, cellular factors that control PI(4,5)P2 production required for Pr55Gag-PM targeting have not yet been characterized. In this study, by individually inhibiting PIP5K1 isoforms, we elucidated a correlation between PI(4,5)P2 metabolism pathways mediated by PIP5K1 isoforms and the targeting of Pr55Gag to the PM of TZM-bl HeLa cells. Confocal microscopy analyses of cells depleted from PIP5K1α and PIP5K1γ show a rerouting of Pr55Gag to various intracellular compartments. Notably, Pr55Gag is degraded by the proteasome and/or by the lysosomes in PIP5K1α-depleted cells, while Pr55Gag is targeted to endosomal vesicles in PIP5K1γ-depleted cells. Thus, our results highlight, for the first time, the roles of PIP5K1α and PIP5K1γ as determinants of Pr55Gag targeting to the PM.


Assuntos
Membrana Celular/metabolismo , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , HIV-1/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Precursores de Proteínas/metabolismo , Membrana Celular/genética , Membrana Celular/virologia , Endossomos/genética , Endossomos/metabolismo , Endossomos/virologia , HIV-1/genética , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/virologia , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Precursores de Proteínas/genética , Proteólise
8.
Nat Commun ; 11(1): 1941, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321914

RESUMO

Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Organelas/metabolismo , Sindecana-4/metabolismo , Sinteninas/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Membrana Celular/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Endossomos/metabolismo , Células HeLa , Humanos , Organelas/genética , Ligação Proteica , Sindecana-4/genética , Sinteninas/genética
9.
Nat Struct Mol Biol ; 27(4): 392-399, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251413

RESUMO

The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.


Assuntos
Membrana Celular/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Proteínas Oncogênicas/ultraestrutura , Membrana Celular/química , Membrana Celular/genética , Citocinese/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/química , Endossomos/genética , Endossomos/ultraestrutura , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Polimerização , Conformação Proteica
10.
Genes Cells ; 25(6): 391-401, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32167217

RESUMO

Vesicular transport serves as an important mechanism for cell shape regulation during development. Although the semaphorin signaling molecule, a well-known regulator of axon guidance, induces endocytosis in the growth cone and the axonal transport of vertebrate neurons, the underlying molecular mechanisms remain largely unclear. Here, we show that the Caenorhabditis elegans SNT-1/synaptotagmin-UNC-41/stonin2 system, whose role in synaptic vesicle recycling in neurons has been studied extensively, is involved in semaphorin-regulated vesicular transport in larval epidermal cells. Mutations in the snt-1/unc-41 genes strongly suppressed the cell shape defects of semaphorin mutants. The null mutation in the semaphorin receptor gene, plx-1, altered the expression and localization pattern of endocytic and exocytic markers in the epidermal cells while repressing the transport of SNT-1-containing vesicles toward late endosome/lysosome pathways. Our findings suggest that the nematode semaphorins regulate the vesicular transport in epidermal cells in a manner distinct from that of vertebrate semaphorins in neurons.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Células Epidérmicas/metabolismo , Semaforinas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagminas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Transporte Biológico Ativo/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Endocitose/genética , Endossomos/genética , Endossomos/metabolismo , Exocitose/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Interferência de RNA , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Semaforinas/genética , Transdução de Sinais/genética , Sinaptotagminas/genética , Proteínas de Transporte Vesicular/genética
11.
PLoS Biol ; 18(3): e3000647, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163403

RESUMO

Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, we found that canonical Wnt signaling proteins regulate localization of the core nucleation protein γTubulin (γTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Iγ, G proteins, and Axin reduced γTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic γTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with fz, dsh, Axin, and Rab5. We propose that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points.


Assuntos
Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Microtúbulos/metabolismo , Proteínas Wnt/metabolismo , Animais , Complexo de Sinalização da Axina/genética , Complexo de Sinalização da Axina/metabolismo , Axônios/metabolismo , Polaridade Celular , Dendritos/genética , Drosophila , Proteínas de Drosophila/genética , Endossomos/genética , Microtúbulos/genética , Mutação , Receptores Wnt/genética , Receptores Wnt/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
12.
Am J Physiol Cell Physiol ; 318(5): C870-C878, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32186931

RESUMO

Pituitary adenylate cyclase activating polypeptide (PACAP; ADCYAP1) is a pleiotropic neuropeptide widely distributed in both the peripheral and central nervous systems. PACAP and its specific cognate PAC1 receptor (ADCYAP1R1) play critical roles in the homeostatic maintenance of multiple physiological and behavioral systems. Notably, maladaptations in the PACAPergic system have been associated with several psychopathologies related to fear and anxiety. PAC1 receptor transcripts are highly expressed in granule cells of the dentate gyrus (DG). Here, we examined the direct effects of PACAP on DG granule cells in brain slices using whole cell patch recordings in current clamp mode. PACAP significantly increased the intrinsic excitability of DG granule cells via PAC1 receptor activation. This increased excitability was not mediated by adenylyl cyclase/cAMP or phospholipase C/PKC activation, but instead via activation of an extracellular signal-regulated kinase (ERK) signaling pathway initiated through PAC1 receptor endocytosis/endosomal signaling. PACAP failed to increase excitability in DG granule cells pretreated with the persistent sodium current blocker riluzole, suggesting that the observed PACAP effects required this component of the inward sodium current.


Assuntos
Ansiedade/genética , Encéfalo/metabolismo , Giro Denteado/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Animais , Ansiedade/patologia , Encéfalo/patologia , Proliferação de Células/efeitos dos fármacos , Giro Denteado/patologia , Endocitose/genética , Endossomos/genética , Medo/psicologia , Regulação da Expressão Gênica/genética , Humanos , Sistema de Sinalização das MAP Quinases/genética , Neurônios/metabolismo , Neurônios/patologia , Técnicas de Patch-Clamp , Ratos , Riluzol/farmacologia
13.
Curr Opin Cell Biol ; 63: 102-113, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32036294

RESUMO

Autosomal dominant missense mutations that hyperactivate the leucine-rich repeat protein kinase-2 (LRRK2) are a common cause of inherited Parkinson's disease and therapeutic efficacy of LRRK2 inhibitors is being tested in clinical trials. In this review, we discuss the nuts and bolts of our current understanding of how the LRRK2 is misregulated by mutations and how pathway activity is affected by LRRK2 binding to membrane, microtubule filaments, and 14-3-3, as well as by upstream components such as Rab29 and VPS35. We discuss recent work that points toward a subset of Rab proteins comprising key physiological substrates that bind new sets of effectors, such as RILPL1/2, JIP3 and JIP4 after phosphorylation by LRRK2. We explore what is known about how LRRK2 regulates ciliogenesis, the endosomal-lysosomal system, immune responses and interplay with alpha-synuclein and tau and how this might be linked to Parkinson's' disease.


Assuntos
Fenômenos Fisiológicos Celulares , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/fisiologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Animais , Fenômenos Fisiológicos Celulares/genética , Endossomos/genética , Endossomos/metabolismo , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Mutação , Doença de Parkinson/metabolismo
14.
Cell Mol Life Sci ; 77(24): 5223-5242, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32065241

RESUMO

Endocytosis of the amyloid precursor protein (APP) is critical for generation of ß-amyloid, aggregating in Alzheimer's disease. APP endocytosis depending on the intracellular NPTY motif is well investigated, whereas involvement of the YTSI (also termed BaSS) motif remains controversial. Here, we show that APP lacking the YTSI motif (ΔYTSI) displays reduced localization to early endosomes and decreased internalization rates, similar to APP ΔNPTY. Additionally, we show that the YTSI-binding protein, PAT1a interacts with the Rab5 activator RME-6, as shown by several independent assays. Interestingly, knockdown of RME-6 decreased APP endocytosis, whereas overexpression increased the same. Similarly, APP ΔNPTY endocytosis was affected by PAT1a and RME-6 overexpression, whereas APP ΔYTSI internalization remained unchanged. Moreover, we could show that RME-6 mediated increase of APP endocytosis can be diminished upon knocking down PAT1a. Together, our data identify RME-6 as a novel player in APP endocytosis, involving the YTSI-binding protein PAT1a.


Assuntos
Doença de Alzheimer/genética , Motivos de Aminoácidos/genética , Precursor de Proteína beta-Amiloide/genética , Proteínas rab5 de Ligação ao GTP/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Proteínas de Transporte/genética , Endocitose/genética , Endossomos/genética , Humanos , Camundongos , Transporte Proteico/genética , Vesículas Transportadoras/genética
15.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32045479

RESUMO

Regulated secretion is a fundamental cellular process in which biologically active molecules stored in long-lasting secretory granules (SGs) are secreted in response to external stimuli. Many studies have described mechanisms responsible for biogenesis and secretion of SGs, but how SGs mature remains poorly understood. In a genetic screen, we discovered a large number of endolysosomal trafficking genes required for proper SG maturation, indicating that maturation of SGs might occur in a manner similar to lysosome-related organelles (LROs). CD63, a tetraspanin known to decorate LROs, also decorates SG membranes and facilitates SG maturation. Moreover, CD63-mediated SG maturation requires type II phosphatidylinositol 4 kinase (PI4KII)-dependent early endosomal sorting and accumulation of phosphatidylinositol 4-phosphate (PI4P) on SG membranes. In addition, the PI4P effector Past1 is needed for formation of stable PI4KII-containing endosomal tubules associated with this process. Our results reveal that maturation of post-Golgi-derived SGs requires trafficking via the endosomal system, similar to mechanisms employed by LROs.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Endossomos/genética , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transporte Proteico , Glândulas Salivares/embriologia , Vesículas Secretórias/genética , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Fatores de Tempo
16.
Fish Shellfish Immunol ; 99: 119-129, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32014587

RESUMO

Rab GTPases control trafficking of intracellular vesicles and are key regulators of endocytic and secretory pathways. Due to their specific distribution, they may serve as markers for different endolysosomal compartments. Since Rab GTPases are involved in uptake and trafficking of endocytosed ligands and cell receptors, as well as secretion of immune mediators, they have been implicated in diverse immunological processes and their functions are often exploited by intracellular pathogens such as viruses. While Rab proteins have been studied extensively in mammals, their functions in vesicle trafficking in teleosts are not well known. In the present work, Atlantic salmon Rab5c, Rab7a and Rab27a homologs were studied in terms of intracellular distribution and gene expression. Structured illumination microscopy demonstrated that transgenic, GFP-tagged salmon Rab5c and Rab7a are, predominantly, located within early endosomes and late endosomes/lysosomes, respectively. In contrast, Rab27a showed a broader distribution, which indicates that it associates with diverse intracellular vesicles and organelles. Infection of salmon with Salmonid alphavirus subtype 3 (SAV3) enhanced the mRNA levels of all of the studied Rab isoforms in heart and head kidney and most of them were upregulated in spleen. This may reflect the capacity of the virus to exploit the functions of these rab proteins. It is also possible that the transcriptional regulation of Rab proteins in SAV3-infected organs may play a role in the antiviral immune response. The latter was further supported by in vitro experiments with adherent head kidney leukocytes. The expression of Rab5c and Rab27a was upregulated in these cells following stimulation with TLR ligands including CpG oligonucleotides and polyI:C. The expression of most of the analyzed Rab isoforms in the primary leukocytes was also enhanced by stimulation with type I IFN. Interestingly, IFN-gamma had a negative effect on Rab7a expression which may be linked to the priming activity of this cytokine on monocytes and macrophages. Overall, these data demonstrate that the intracellular distribution of Rab5c, Rab7a and Rab27a is phylogenetically conserved within vertebrates and that these molecules might be implicated in viral infections and the regulation of the antiviral immune response in Atlantic salmon.


Assuntos
Infecções por Alphavirus/veterinária , Proteínas de Peixes/genética , Salmo salar/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/genética , Alphavirus , Infecções por Alphavirus/imunologia , Animais , Células Cultivadas , Endossomos/genética , Proteínas de Peixes/imunologia , Expressão Gênica , Regulação da Expressão Gênica , Rim Cefálico/citologia , Rim Cefálico/imunologia , Leucócitos/imunologia , Lisossomos/genética , Salmo salar/imunologia , Homologia de Sequência , Proteínas rab de Ligação ao GTP/imunologia , Proteínas rab27 de Ligação ao GTP/imunologia , Proteínas rab5 de Ligação ao GTP/imunologia
17.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32049272

RESUMO

The intraluminal vesicles (ILVs) of endosomes mediate the delivery of activated signaling receptors and other proteins to lysosomes for degradation, but they also modulate intercellular communication when secreted as exosomes. The formation of ILVs requires four complexes, ESCRT-0, -I, -II, and -III, with ESCRT-0, -I, and -II presumably involved in cargo sorting and ESCRT-III in membrane deformation and fission. Here, we report that an active form of the ESCRT-associated protein ALIX efficiently recruits ESCRT-III proteins to endosomes. This recruitment occurs independently of other ESCRTs but requires lysobisphosphatidic acid (LBPA) in vivo, and can be reconstituted on supported bilayers in vitro. Our data indicate that this ALIX- and ESCRT-III-dependent pathway promotes the sorting and delivery of tetraspanins to exosomes. We conclude that ALIX provides an additional pathway of ILV formation, secondary to the canonical pathway, and that this pathway controls the targeting of exosomal proteins.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Exossomos/metabolismo , Tetraspaninas/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Exossomos/genética , Células HeLa , Humanos , Lisofosfolipídeos/metabolismo , Monoglicerídeos/metabolismo , Transporte Proteico , Tetraspanina 28/metabolismo , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismo , Tetraspaninas/genética , Fatores de Tempo
18.
J Biol Chem ; 295(7): 2068-2083, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31915245

RESUMO

Many secretory proteins are activated by cleavage at specific sites. The proprotein convertases (PCs) form a family of nine secretory subtilisin-like serine proteases, seven of which cleave at specific basic residues within the trans-Golgi network, granules, or at the cell surface/endosomes. The seventh member, PC7, is a type-I transmembrane (TM) protein with a 97-residue-long cytosolic tail (CT). PC7 sheds human transferrin receptor 1 (hTfR1) into soluble shTfR1 in endosomes. To better understand the physiological roles of PC7, here we focused on the relationship between the CT-regulated trafficking of PC7 and its ability to shed hTfR1. Deletion of the TMCT resulted in soluble PC7 and loss of its hTfR1 shedding activity. Extensive CT deletions and mutagenesis analyses helped us zoom in on three residues in the CT, namely Glu-719, Glu-721, and Leu-725, that are part of a novel motif, EXEXXXL725, critical for PC7 activity on hTfR1. NMR studies of two 14-mer peptides mimicking this region of the CT and its Ala variants revealed that the three exposed residues are on the same side of the molecule. This led to the identification of adaptor protein 2 (AP-2) as a protein that recognizes the EXEXXXL725 motif, thus representing a potentially new regulator of PC7 trafficking and cleavage activity. Immunocytochemistry of the subcellular localization of PC7 and its Ala variants of Leu-725 and Glu-719 and Glu-721 revealed that Leu-725 enhances PC7 localization to early endosomes and that, together with Glu-719 and Glu-721, it increases the endosomal activity of PC7 on hTfR1.


Assuntos
Antígenos CD/genética , Citosol/metabolismo , Transporte Proteico/genética , Receptores da Transferrina/genética , Subtilisinas/genética , Fator de Transcrição AP-2/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Antígenos CD/química , Membrana Celular/genética , Movimento Celular/genética , Citosol/química , Endossomos/genética , Células HEK293 , Humanos , Receptores da Transferrina/química , Subtilisinas/química , Rede trans-Golgi/genética
19.
Sci Rep ; 10(1): 1358, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992816

RESUMO

We previously demonstrated that cyclic guanosine monophosphate (cGMP) stimulates amyloid precursor protein (APP) and beta-secretase (BACE1) approximation in neuronal endo-lysosomal compartments, thus boosting the production of amyloid-ß (Aß) peptides and enhancing synaptic plasticity and memory. Here, we further investigated the mechanism by which cGMP regulates the subcellular localization of APP and BACE1, finding that the cyclic nucleotide inhibits the activity of Rab5, a small GTPase associated with the plasma membrane and early endosomes. Accordingly, we also found that expression of a dominant-negative Rab5 mutant increases both APP-BACE1 approximation and Aß extracellular levels, therefore mimicking the effects induced by cGMP. These results reveal a functional correlation between the cGMP/Aß pathway and the activity of Rab5 that may contribute to the understanding of Alzheimer's disease pathophysiology.


Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Membrana Celular/metabolismo , Sistemas do Segundo Mensageiro , Proteínas rab5 de Ligação ao GTP/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Linhagem Celular , Membrana Celular/genética , Membrana Celular/patologia , GMP Cíclico , Endossomos/genética , Endossomos/metabolismo , Endossomos/patologia , Camundongos , Proteínas rab5 de Ligação ao GTP/genética
20.
ACS Appl Mater Interfaces ; 12(4): 4308-4322, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31939276

RESUMO

Combination therapies consisting of multiple short therapeutic RNAs, such as small interfering RNA (siRNA) and microRNA (miRNA), have enormous potential in cancer treatment as they can precisely silence a specific set of oncogenes and target multiple disease-related pathways. However, clinical use of siRNA/miRNA combinations is limited by the availability of safe and efficient systemic delivery systems with sufficient tumor penetrating and endosomal escaping capabilities. This study reports on the development of multifunctional tumor-penetrating mesoporous silica nanoparticles (iMSNs) for simultaneous delivery of siRNA (siPlk1) and miRNA (miR-200c), using encapsulation of a photosensitizer indocyanine green (ICG) to facilitate endosomal escape and surface conjugation of the iRGD peptide to enable deep tumor penetration. Increased cell uptake of the nanoparticles was observed in both 3D tumor spheroids in vitro and in orthotopic MDA-MB-231 breast tumors in vivo. Using a galectin-8 recruitment assay, we showed that reactive oxygen species generated by ICG upon light irradiation functioned as an endosomolytic stimulus that caused release of the siRNA/miRNA combination from endosomes. Co-delivery of the therapeutic RNAs displayed combined cell killing activity in cancer cells. Systemic intravenous treatment of metastatic breast cancer with the iMSNs loaded with siPlk1 and miR-200c resulted in a significant suppression of the primary tumor growth and in marked reduction of metastasis upon short light irradiation of the primary tumor. This work demonstrates that siRNA-miRNA combination assisted by the photodynamic effect and tumor penetrating delivery system may provide a promising approach for metastatic cancer treatment.


Assuntos
Neoplasias da Mama/terapia , Terapia Genética , MicroRNAs/química , Nanopartículas/química , RNA Interferente Pequeno/química , Dióxido de Silício/química , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Sistemas de Liberação de Medicamentos , Endossomos/genética , Endossomos/metabolismo , Feminino , Humanos , Camundongos , MicroRNAs/administração & dosagem , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...