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1.
Proc Natl Acad Sci U S A ; 117(18): 9876-9883, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32303654

RESUMO

A massive intronic hexanucleotide repeat (GGGGCC) expansion in C9ORF72 is a genetic origin of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recently, C9ORF72, together with SMCR8 and WDR41, has been shown to regulate autophagy and function as Rab GEF. However, the precise function of C9ORF72 remains unclear. Here, we report the cryogenic electron microscopy (cryo-EM) structure of the human C9ORF72-SMCR8-WDR41 complex at a resolution of 3.2 Å. The structure reveals the dimeric assembly of a heterotrimer of C9ORF72-SMCR8-WDR41. Notably, the C-terminal tail of C9ORF72 and the DENN domain of SMCR8 play critical roles in the dimerization of the two protomers of the C9ORF72-SMCR8-WDR41 complex. In the protomer, C9ORF72 and WDR41 are joined by SMCR8 without direct interaction. WDR41 binds to the DENN domain of SMCR8 by the C-terminal helix. Interestingly, the prominent structural feature of C9ORF72-SMCR8 resembles that of the FLNC-FNIP2 complex, the GTPase activating protein (GAP) of RagC/D. Structural comparison and sequence alignment revealed that Arg147 of SMCR8 is conserved and corresponds to the arginine finger of FLCN, and biochemical analysis indicated that the Arg147 of SMCR8 is critical to the stimulatory effect of the C9ORF72-SMCR8 complex on Rab8a and Rab11a. Our study not only illustrates the basis of C9ORF72-SMCR8-WDR41 complex assembly but also reveals the GAP activity of the C9ORF72-SMCR8 complex.


Assuntos
Proteínas Relacionadas à Autofagia/ultraestrutura , Proteína C9orf72/ultraestrutura , Proteínas de Transporte/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Sequência de Aminoácidos/genética , Esclerose Amiotrófica Lateral/genética , Arginina/genética , Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteína C9orf72/genética , Proteínas de Transporte/genética , Microscopia Crioeletrônica , Filaminas/genética , Filaminas/ultraestrutura , Demência Frontotemporal/genética , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/ultraestrutura , Predisposição Genética para Doença , Humanos , Complexos Multiproteicos/genética , Alinhamento de Sequência , Proteínas rab de Ligação ao GTP/genética
2.
Braz J Med Biol Res ; 53(4): e9220, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267310

RESUMO

Rab7, an important member of the Rab family, is closely related to autophagy, endocytosis, apoptosis, and tumor suppression but few studies have described its association with renal fibrosis. In the early stage, our group studied the effects of Rab7 on production and degradation of extracellular matrix in hypoxic renal tubular epithelial cells. Because cell culture in vitro is different from the environment in vivo, it is urgent to understand the effects in vivo. In our current study, we established a renal fibrosis model in Rab7-knock-in mice (prepared by CRISPR/Cas9 technology) and wild type (WT) C57BL/6 mice using unilateral ureteral obstruction (UUO). Seven and 14 days after UUO, the expression of the Rab7 protein in WT mice, as well as the autophagic activity, renal function, and the degree of renal fibrosis in WT and Rab7-knock-in mice were examined by blood biochemical assay, hematoxylin-eosin and Masson staining, immunohistochemistry, and western blotting. We found that the Rab7 expression in WT mice increased over time. Furthermore, the autophagic activity constantly increased in both groups, although it was higher in the Rab7-knock-in mice than in the WT mice at the same time point. Seven days after UUO, the degree of renal fibrosis was milder in the Rab7-knock-in mice than in the WT mice, but it became more severe 14 days after surgery. Similar results were found for renal function. Therefore, Rab7 suppressed renal fibrosis in mice initially, but eventually it aggravated fibrosis with the activation of autophagy.


Assuntos
Autofagia/fisiologia , Nefropatias/etiologia , Rim/patologia , Obstrução Ureteral/complicações , Proteínas rab de Ligação ao GTP/genética , Animais , Feminino , Fibrose , Masculino , Camundongos , Camundongos Knockout , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Regulação para Cima , Proteínas rab de Ligação ao GTP/metabolismo
3.
Nat Commun ; 11(1): 1127, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111841

RESUMO

Although viruses must navigate the complex host endomembrane system to infect cells, the strategies used to achieve this is unclear. During entry, polyomavirus SV40 is sorted from the late endosome (LE) to the endoplasmic reticulum (ER) to cause infection, yet how this is accomplished remains enigmatic. Here we find that EMC4 and EMC7, two ER membrane protein complex (EMC) subunits, support SV40 infection by promoting LE-to-ER targeting of the virus. They do this by engaging LE-associated Rab7, presumably to stabilize contact between the LE and ER. These EMC subunits also bind to the ER-resident fusion machinery component syntaxin18, which is required for SV40-arrival to the ER. Our data suggest that EMC4 and EMC7 act as molecular tethers, inter-connecting two intracellular compartments to enable efficient transport of a virus between these compartments. As LE-to-ER transport of cellular cargos is unclear, our results have broad implications for illuminating inter-organelle cargo transport.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Internalização do Vírus , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático/virologia , Endossomos/metabolismo , Endossomos/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Membranas Intracelulares/virologia , Proteínas de Membrana/genética , Ligação Proteica , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Vírus 40 dos Símios/fisiologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
4.
Genes Dev ; 34(7-8): 580-597, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32115408

RESUMO

Dysregulation of early neurodevelopment is implicated in macrocephaly/autism disorders. However, the mechanism underlying this dysregulation, particularly in human cells, remains poorly understood. Mutations in the small GTPase gene RAB39b are associated with X-linked macrocephaly, autism spectrum disorder (ASD), and intellectual disability. The in vivo roles of RAB39b in the brain remain unknown. We generated Rab39b knockout (KO) mice and found that they exhibited cortical neurogenesis impairment, macrocephaly, and hallmark ASD behaviors, which resembled patient phenotypes. We also produced mutant human cerebral organoids that were substantially enlarged due to the overproliferation and impaired differentiation of neural progenitor cells (NPCs), which resemble neurodevelopmental deficits in KO mice. Mechanistic studies reveal that RAB39b interacts with PI3K components and its deletion promotes PI3K-AKT-mTOR signaling in NPCs of mouse cortex and cerebral organoids. The mTOR activity is robustly enhanced in mutant outer radial glia cells (oRGs), a subtype of NPCs barely detectable in rodents but abundant in human brains. Inhibition of AKT signaling rescued enlarged organoid sizes and NPC overproliferation caused by RAB39b mutations. Therefore, RAB39b mutation promotes PI3K-AKT-mTOR activity and alters cortical neurogenesis, leading to macrocephaly and autistic-like behaviors. Our studies provide new insights into neurodevelopmental dysregulation and common pathways associated with ASD across species.


Assuntos
Transtorno Autístico/genética , Córtex Cerebral/embriologia , Megalencefalia/genética , Neurogênese/genética , Proteínas rab de Ligação ao GTP/genética , Animais , Transtorno Autístico/fisiopatologia , Comportamento Animal/fisiologia , Diferenciação Celular/genética , Proliferação de Células/genética , Córtex Cerebral/citologia , Deleção de Genes , Humanos , Megalencefalia/fisiopatologia , Camundongos , Camundongos Knockout , Modelos Animais , Organoides/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/genética , Células-Tronco/citologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
5.
Nat Commun ; 11(1): 1269, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32152267

RESUMO

Multicellular rosettes are transient epithelial structures that serve as intermediates during diverse organ formation. We have identified a unique contributor to rosette formation in zebrafish Kupffer's vesicle (KV) that requires cell division, specifically the final stage of mitosis termed abscission. KV utilizes a rosette as a prerequisite before forming a lumen surrounded by ciliated epithelial cells. Our studies identify that KV-destined cells remain interconnected by cytokinetic bridges that position at the rosette's center. These bridges act as a landmark for directed Rab11 vesicle motility to deliver an essential cargo for lumen formation, CFTR (cystic fibrosis transmembrane conductance regulator). Here we report that premature bridge cleavage through laser ablation or inhibiting abscission using optogenetic clustering of Rab11 result in disrupted lumen formation. We present a model in which KV mitotic cells strategically place their cytokinetic bridges at the rosette center, where Rab11-associated vesicles transport CFTR to aid in lumen establishment.


Assuntos
Divisão Celular/fisiologia , Polaridade Celular/fisiologia , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/fisiologia , Macrófagos do Fígado/fisiologia , Organogênese/fisiologia , Peixe-Zebra/embriologia , Animais , Linhagem Celular , Movimento Celular , Cílios/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Macrófagos do Fígado/citologia , Mitose , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(7): 3789-3796, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015134

RESUMO

The facultative intracellular pathogen Listeria monocytogenes uses an actin-based motility process to spread within human tissues. Filamentous actin from the human cell forms a tail behind bacteria, propelling microbes through the cytoplasm. Motile bacteria remodel the host plasma membrane into protrusions that are internalized by neighboring cells. A critical unresolved question is whether generation of protrusions by Listeria involves stimulation of host processes apart from actin polymerization. Here we demonstrate that efficient protrusion formation in polarized epithelial cells involves bacterial subversion of host exocytosis. Confocal microscopy imaging indicated that exocytosis is up-regulated in protrusions of Listeria in a manner that depends on the host exocyst complex. Depletion of components of the exocyst complex by RNA interference inhibited the formation of Listeria protrusions and subsequent cell-to-cell spread of bacteria. Additional genetic studies indicated important roles for the exocyst regulators Rab8 and Rab11 in bacterial protrusion formation and spread. The secreted Listeria virulence factor InlC associated with the exocyst component Exo70 and mediated the recruitment of Exo70 to bacterial protrusions. Depletion of exocyst proteins reduced the length of Listeria protrusions, suggesting that the exocyst complex promotes protrusion elongation. Collectively, these results demonstrate that Listeria exploits host exocytosis to stimulate intercellular spread of bacteria.


Assuntos
Exocitose , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CACO-2 , Quinases do Centro Germinativo/genética , Quinases do Centro Germinativo/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/genética , Listeriose/genética , Listeriose/metabolismo , Listeriose/fisiopatologia , Ligação Proteica , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
7.
Am J Physiol Renal Physiol ; 318(4): F956-F970, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32088968

RESUMO

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for osmotic water reabsorption by kidney collecting ducts. In response to vasopressin, AQP2 traffics from intracellular vesicles to the apical plasma membrane of collecting duct principal cells, where it increases water permeability and, hence, water reabsorption. Despite continuing efforts, gaps remain in our knowledge of vasopressin-regulated AQP2 trafficking. Here, we studied the functions of two retromer complex proteins, small GTPase Rab7 and vacuolar protein sorting 35 (Vps35), in vasopressin-induced AQP2 trafficking in a collecting duct cell model (mpkCCD cells). We showed that upon vasopressin removal, apical AQP2 returned to Rab5-positive early endosomes before joining Rab11-positive recycling endosomes. In response to vasopressin, Rab11-associated AQP2 trafficked to the apical plasma membrane before Rab5-associated AQP2 did so. Rab7 knockdown resulted in AQP2 accumulation in early endosomes and impaired vasopressin-induced apical AQP2 trafficking. In response to vasopressin, Rab7 transiently colocalized with Rab5, indicative of a role of Rab7 in AQP2 sorting in early endosomes before trafficking to the apical membrane. Rab7-mediated apical AQP2 trafficking in response to vasopressin required GTPase activity. When Vps35 was knocked down, AQP2 accumulated in recycling endosomes under vehicle conditions and did not traffic to the apical plasma membrane in response to vasopressin. We conclude that Rab7 and Vps35 participate in AQP2 sorting in early endosomes under vehicle conditions and apical membrane trafficking in response to vasopressin.


Assuntos
Aquaporina 2/metabolismo , Endossomos/enzimologia , Túbulos Renais Coletores/enzimologia , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Aquaporina 2/genética , Endossomos/efeitos dos fármacos , Células HEK293 , Humanos , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Camundongos , Transporte Proteico , Proteólise , Fatores de Tempo , Vasopressinas/farmacologia , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética
8.
Invest Ophthalmol Vis Sci ; 61(2): 29, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32084271

RESUMO

Purpose: Cone-rod dystrophy (CRD) is a rare hereditary eye disorder that causes progressive degeneration of cone and rod photoreceptors. More than 30 genes, including RAB28, have been associated with CRD; however, only a few RAB28 variants have been reported to be associated with CRD. In this study, we describe two brothers with CRD and a homozygous missense variant, c.55G>A (p.Gly19Arg), in RAB28. Methods: The missense variant was identified as part of a study investigating underlying genetic defects in a large patient cohort (n = 667) using targeted next-generation sequencing of 125 genes associated with retinal dystrophy. Cellular localization of RAB28 and ciliogenesis in patient fibroblasts were investigated by immunofluorescence microscopy. The effect of the missense variant on RAB28 expression level was investigated by quantitative real-time PCR. Results: Two brothers of a consanguineous couple presented with CRD, postaxial polydactyly (PAP), and myopia. Both brothers had a homozygous missense RAB28 variant located in the G1 box of the guanosine triphosphate/guanosine diphosphate binding domain of RAB28. This missense variant caused a considerable reduction of RAB28 localized to the cilia, whereas ciliogenesis seemed unaffected. Conclusions: The missense variant in RAB28 is classified as likely pathogenic with functional effect on protein localization. The combination of retinal dystrophy and PAP are well known from ciliopathies; however, more data are needed to finally conclude that the RAB28 variant described here is the cause of PAP in these brothers.


Assuntos
Cílios/metabolismo , Distrofias de Cones e Bastonetes/genética , Dedos/anormalidades , Mutação de Sentido Incorreto , Polidactilia/genética , Dedos do Pé/anormalidades , Proteínas rab de Ligação ao GTP , Criança , Humanos , Masculino , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
9.
PLoS Genet ; 16(2): e1008626, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32059017

RESUMO

Neuronal pruning is a commonly observed phenomenon for the developing nervous systems to ensure precise wiring of neural circuits. The function of Ik2 kinase and its downstream mediator, Spindle-F (Spn-F), are essential for dendrite pruning of Drosophila sensory neurons during development. However, little is known about how Ik2/Spn-F signaling is transduced in neurons and ultimately results in dendrite pruning. Our genetic analyses and rescue experiments demonstrated that the small GTPase Rab11, especially the active GTP-bound form, is required for dendrite pruning. We also found that Rab11 shows genetic interactions with spn-F and ik2 on pruning. Live imaging of single neurons and antibody staining reveal normal Ik2 kinase activation in Rab11 mutant neurons, suggesting that Rab11 could have a functional connection downstream of and/or parallel to the Ik2 kinase signaling. Moreover, we provide biochemical evidence that both the Ik2 kinase activity and the formation of Ik2/Spn-F/Rab11 complexes are central to promote Rab11 activation in cells. Together, our studies reveal that a critical role of Ik2/Spn-F signaling in neuronal pruning is to promote Rab11 activation, which is crucial for dendrite pruning in neurons.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Quinase I-kappa B/metabolismo , Plasticidade Neuronal/genética , Células Receptoras Sensoriais/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Dendritos/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrião não Mamífero , Técnicas de Silenciamento de Genes , Microscopia Intravital , Proteínas Associadas aos Microtúbulos/metabolismo , Células Receptoras Sensoriais/citologia , Transdução de Sinais/fisiologia , Imagem com Lapso de Tempo , Proteínas rab de Ligação ao GTP/genética
10.
Nat Commun ; 11(1): 23, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911620

RESUMO

Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A-containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Here, using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/second, 330 nm isotropic resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection and report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with respiratory syncytial virus alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport of Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection.


Assuntos
Dineínas/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Transporte Biológico , Dineínas/genética , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/virologia , RNA Viral/genética , RNA Viral/metabolismo , Proteínas rab de Ligação ao GTP/genética
11.
Nat Commun ; 11(1): 42, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31896748

RESUMO

DNA damage and metabolic disorders are intimately linked with premature disease onset but the underlying mechanisms remain poorly understood. Here, we show that persistent DNA damage accumulation in tissue-infiltrating macrophages carrying an ERCC1-XPF DNA repair defect (Er1F/-) triggers Golgi dispersal, dilation of endoplasmic reticulum, autophagy and exosome biogenesis leading to the secretion of extracellular vesicles (EVs) in vivo and ex vivo. Macrophage-derived EVs accumulate in Er1F/- animal sera and are secreted in macrophage media after DNA damage. The Er1F/- EV cargo is taken up by recipient cells leading to an increase in insulin-independent glucose transporter levels, enhanced cellular glucose uptake, higher cellular oxygen consumption rate and greater tolerance to glucose challenge in mice. We find that high glucose in EV-targeted cells triggers pro-inflammatory stimuli via mTOR activation. This, in turn, establishes chronic inflammation and tissue pathology in mice with important ramifications for DNA repair-deficient, progeroid syndromes and aging.


Assuntos
Dano ao DNA/fisiologia , Exossomos/metabolismo , Macrófagos/citologia , Animais , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Exossomos/patologia , Regulação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/metabolismo , Masculino , Camundongos Transgênicos , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
12.
J Biotechnol ; 308: 118-123, 2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31846628

RESUMO

Lysosome, an intracellular organelle with an acid interior, contains acidic hydrolases and specific membrane proteins. Saccharomyces cerevisiae contains vacuoles (corresponding to lysosomes) that have similar lipid composition membrane to mammalian cell membrane. However, yeast vacuoles do not cause significant immune stimulation in vivo. Taking advantage of these structural similarities and bio-derived strengths, the present study describes encapsulation of daunorubicin into lysosome derived from S. cerevisiae as drug delivery vehicles for acute myeloid leukemia (AML) treatment. Daunorubicin is a chemotherapy medication used to treat cancer, specifically for AML. In this study, recombinant S. cerevisiae that could keep the small size of lysosomal vacuoles was constructed. Appropriate time and concentration to encapsulate the drug were then identified. In addition, release profile and anticancer effect of the drug in lysosome carriers were confirmed. According to this study, a more accurate encapsulation condition into lysosome can be optimized and potential application of S. cerevisiae derived lysosomes as drug carriers is confirmed.


Assuntos
Daunorrubicina/farmacologia , Lisossomos/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/citologia , Proteínas rab de Ligação ao GTP/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Daunorrubicina/química , Composição de Medicamentos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Lisossomos/genética , Tamanho da Partícula , Saccharomyces cerevisiae/genética
13.
PLoS One ; 14(12): e0220220, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31834921

RESUMO

Mutations in the epithelial polarity gene crumbs (crb) lead to retinal degeneration in Drosophila and in humans. The overall morphology of the retina and its deterioration in Drosophila crb mutants has been well-characterized, but the cell biological origin of the degeneration is not well understood. Degenerative conditions in the retina and elsewhere in the nervous system often involve defects in degradative intracellular trafficking pathways. So far, however, effects of crb on the endolysosomal system, or on the spatial organization of these compartments in photoreceptor cells have not been described. We therefore asked whether photoreceptors in crb mutants exhibit alterations in endolysosomal compartments under pre-degenerative conditions, where the retina is still morphologically intact. Data presented here show that, already well before the onset of degeneration, Arl8, Rab7, and Atg8-carrying endolysosomal and autophagosomal compartments undergo changes in morphology and positioning with respect to each other in crb mutant retinas. We propose that these changes may be early signs of the degeneration-prone condition in crb retinas.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Degeneração Retiniana/genética , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Polaridade Celular/genética , Drosophila melanogaster/genética , Endossomos/metabolismo , Proteínas do Olho/genética , Células Fotorreceptoras de Invertebrados/metabolismo , Transporte Proteico/genética , Retina/metabolismo , Degeneração Retiniana/prevenção & controle , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
14.
BMC Dev Biol ; 19(1): 25, 2019 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-31884948

RESUMO

BACKGROUND: Rab proteins are GTPases responsible for intracellular vesicular trafficking regulation. Rab11 proteins, members of the Rab GTPase family, are known to regulate vesicular recycling during embryonic development. In zebrafish, there are 3 rab11 paralogues, known as rab11a, rab11ba and rab11bb, sharing high identity with each other. However, the expression analysis of rab11 is so far lacking. RESULTS: Here, by phylogeny analysis, we found the three rab11 genes are highly conserved especially for their GTPase domains. We examined the expression patterns of rab11a, rab11ba and rab11bb using RT-PCR and in situ hybridization. We found that all the three genes were highly enriched in the central nervous system, but in different areas of the brain. Apart from brain, rab11a was also expressed in caudal vein, pronephric duct, proctodeum, pharyngeal arches and digestive duct, rab11ba was detected to express in muscle, and rab11bb was expressed in kidney, fin and spinal cord. Different from rab11a and rab11ba, which both have maternal expressions in embryos, rab11bb only expresses during 24hpf to 96hpf. CONCLUSIONS: Our results suggest that rab11 genes play important but distinct roles in the development of the nervous system in zebrafish. The findings could provide new evidences for better understanding the functions of rab11 in the development of zebrafish embryos.


Assuntos
Peixe-Zebra/embriologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Sequência Conservada , Feminino , Trato Gastrointestinal/embriologia , Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Herança Materna , Família Multigênica , Domínios Proteicos , Distribuição Tecidual , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas rab de Ligação ao GTP/química
15.
EBioMedicine ; 49: 118-132, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31707148

RESUMO

BACKGROUND: The klotho (KL) gene is an anti-aging gene that has recently been shown to also function as a general tumor suppressor. However, there is currently only limited information regarding the potential molecular signals for regulation of Klotho without identifying precise molecular mechanisms or interactions. METHODS: We performed a mass spectrometry (MS) assay to screen candidate proteins complexed with Klotho derived from immunoprecipitation in human non-small cell lung cancer (NSCLC) cells, and identified Rab8 to be the protein that most prominently interacts with Klotho. We further investigated whether Rab8 can regulate trafficking of Klotho and which process it would modulate using surface biotinylation assay, immunofluorescence and fluorescence ratio microscopy. Furthermore, we explored whether Rab8 is involved in Klotho-mediated function in NSCLC, and verified the results which we found in vivo using xenograft mouse model. FINDINGS: We report discovery of Rab8 as a Klotho-interacting protein that acts as a critical modulator of Klotho surface expression in human NSCLC. In particular, we report that Rab8 is co-localized and associated with Klotho, and Klotho trafficking is regulated by Rab8. Moreover, we found that Rab8 modulates surface levels of Klotho via a post-biosynthetic pathway, as opposed to an endocytic pathway. Furthermore, we demonstrate that Rab8 is involved in Klotho-mediated regulation of cell proliferation, migration, invasiveness, epithelial-mesenchymal transition (EMT), and Wnt-ß-catenin signaling in NSCLC. Additionally, Rab8 overexpression was also found to increase Klotho-mediated inhibition of NSCLC tumorigenesis in vivo. INTERPRETATION: Overall, our findings suggest that Rab8 GTPase can regulate Klotho-mediated inhibition of Wnt signaling activity by modulating translocation of Klotho onto the cell surface, which in turn affects Klotho-mediated inhibition of cell proliferation, migration and invasiveness in NSCLC. These results have important implications for the development of new therapeutic targets, Klotho-related research in the context of NSCLC as well as other areas, and provide a working model for Rab8 function in the context of cancer and cancer biology.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Glucuronidase/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células , Regulação para Baixo/genética , Endocitose , Transição Epitelial-Mesenquimal/genética , Glucuronidase/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica , Análise de Sobrevida , Via de Sinalização Wnt/genética , Proteínas rab de Ligação ao GTP/genética
16.
Nat Cell Biol ; 21(10): 1234-1247, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31570833

RESUMO

Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.


Assuntos
Retículo Endoplasmático/metabolismo , Monócitos/metabolismo , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/genética , Transdução de Sinais , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Sistemas CRISPR-Cas , Retículo Endoplasmático/ultraestrutura , Edição de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Monócitos/ultraestrutura , Fagocitose , Fagossomos/ultraestrutura , Cultura Primária de Células , Proteólise , Células RAW 264.7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
17.
Oncol Rep ; 42(6): 2788-2796, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31578588

RESUMO

Prostate cancer is closely associated with constitutive transactivation of the androgen receptor (AR) signaling pathway. After treatment with androgen­deprivation therapy (ADT), the majority of patients develop castration­resistant prostate cancer within months or years. In order to investigate potential novel therapeutic targets in addition to ADT, the present study examined the regulatory mechanisms of the AR signaling pathway. In the present study, LNCaP cells were metabolically­labeled with Alk­C16, a palmitate probe. In addition, cells were treated with R1881, an androgen, or DMSO. Subsequently, click­chemistry­based palmitoylome profiling was performed in LNCaP cells and palmitoylated proteins were compared between cells treated with androgen and untreated cells. Androgen treatment was revealed to significantly increase the palmitoylation level of α­tubulin. In addition, the palmitoylation level of Ras­related protein Rab­7a (Rab7a) was enhanced by androgen treatment. Palmitoylation of α­tubulin and Rab7a were essential for cell proliferation. Notably, in the supernatant of LNCaP cells, the palmitoylation level of α­tubulin was also increased following androgen treatment. Palmitoylation of α­tubulin may provide a new potential target for the treatment of prostate cancer. In addition, the high level of α­tubulin palmitoylation in the supernatant may represent a biomarker for early­stage prostate cancer.


Assuntos
Androgênios/genética , Neoplasias da Próstata/genética , Tubulina (Proteína)/genética , Proteínas rab de Ligação ao GTP/genética , Antagonistas de Androgênios/farmacologia , Androgênios/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Lipoilação/genética , Masculino , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteína S/genética , Proteína S/metabolismo , Transdução de Sinais/genética , Tubulina (Proteína)/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
18.
PLoS Biol ; 17(10): e3000466, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658245

RESUMO

The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The membrane trafficking mechanisms by which neurons control surface polarization of synaptic receptors remain poorly understood. The sorting receptor Sortilin-related CNS expressed 1 (SorCS1) is a critical regulator of trafficking of neuronal receptors, including the presynaptic adhesion molecule neurexin (Nrxn), an essential synaptic organizer. Here, we show that SorCS1 maintains a balance between axonal and dendritic Nrxn surface levels in the same neuron. Newly synthesized Nrxn1α traffics to the dendritic surface, where it is endocytosed. Endosomal SorCS1 interacts with the Rab11 GTPase effector Rab11 family-interacting protein 5 (Rab11FIP5)/Rab11 interacting protein (Rip11) to facilitate the transition of internalized Nrxn1α from early to recycling endosomes and bias Nrxn1α surface polarization towards the axon. In the absence of SorCS1, Nrxn1α accumulates in early endosomes and mispolarizes to the dendritic surface, impairing presynaptic differentiation and function. Thus, SorCS1-mediated sorting in dendritic endosomes controls Nrxn axonal surface polarization required for proper synapse development and function.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Córtex Cerebral/metabolismo , Moléculas de Adesão de Célula Nervosa/genética , Neurônios/metabolismo , Receptores de Superfície Celular/genética , Membranas Sinápticas/metabolismo , Transmissão Sináptica/genética , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Polaridade Celular , Córtex Cerebral/citologia , Embrião de Mamíferos , Endocitose , Endossomos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/ultraestrutura , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ratos , Ratos Wistar , Receptores de Superfície Celular/metabolismo , Membranas Sinápticas/ultraestrutura , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
19.
Elife ; 82019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31663853

RESUMO

Mutations that activate LRRK2 protein kinase cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all human protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knockout increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation in human A549 cells. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A "substrate-trapping" PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson's disease.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais , Proteínas rab de Ligação ao GTP/metabolismo , Humanos , Fosforilação , RNA Interferente Pequeno/genética , Proteínas rab de Ligação ao GTP/genética
20.
Proc Natl Acad Sci U S A ; 116(45): 22619-22623, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31636202

RESUMO

Contacts between the endoplasmic reticulum (ER) and other membranes are hot spots for protein-mediated lipid transport between the 2 adjacent bilayers. Compiling a molecular inventory of lipid transport proteins present at these sites is a premise to the elucidation of their function. Here we show that PDZD8, an intrinsic membrane protein of the ER with a lipid transport module of the SMP domain family, concentrates at contacts between the ER and late endosomes/lysosomes, where it interacts with GTP-Rab7. These findings suggest that PDZD8 may cooperate with other proteins that function at the ER-endo/lysosome interface in coordinating endocytic flow with lipid transport between endocytic membranes and the ER.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/genética , Endossomos/genética , Humanos , Lisossomos/genética , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética
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