RESUMO
Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.
Assuntos
Repetição de Anquirina , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Proteínas rab de Ligação ao GTP , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/química , Humanos , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Células HEK293 , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Fosforilação , Microscopia Crioeletrônica , Ligação ProteicaRESUMO
To provide a basis for promising exosome-based therapies against intervertebral disc degeneration (IDD), our present research aimed to identify a mechanism underlying the vesicle release from nucleus pulposus cells (NPCs). Scutellarin (SC) is a natural chemotherapeutic agent isolated from Erigeron breviscapus with a variety of biological activities. Here, we observed the significantly elevated autophagy levels in rat NPCs under the stimulation of SC, leading to a concomitant enhancement of intracellular vesicle release, which could be attributed to the inactivation of the phosphoinositide 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/protein kinase B (Akt) pathway. To ensure that exosome release was driven by SC via the autophagic pathway, we implemented gain-of-function and loss-of-function studies by additionally using insulin-like growth factor-1 (IGF-1) and small-interfering RNA of autophagy-related gene 5 (ATG5), and the exosome secretion decreased in the case of attenuated autophagy. Evidently, the treatment with SC exerted the remarkable upregulation of Rab8a through the overexpression of ATG5. After the respective knockdown of ATG5 and Rab8a, the increased release of exosomes induced by SC was reversed, whereas the number of intracellular vesicles was restored. Overall, it can be concluded that SC contributes to the autophagy activation in NPCs by acting on the PI3K/PTEN/Akt pathway, which upregulates the expression of Rab8a and promotes the release of exosomes, inspiring novel therapeutic strategies in preventing IDD that might be fruitfully investigated.
Assuntos
Exossomos , Degeneração do Disco Intervertebral , Núcleo Pulposo , Animais , Apigenina , Apoptose/genética , Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Exossomos/metabolismo , Glucuronatos , Degeneração do Disco Intervertebral/tratamento farmacológico , Degeneração do Disco Intervertebral/metabolismo , Núcleo Pulposo/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RatosRESUMO
Rab family small GTPases are master regulators of distinct steps of intracellular vesicle trafficking in eukaryotic cells. GDP-bound cytoplasmic forms of Rab proteins are prone to aggregation due to the exposure of hydrophobic groups but the machinery that determines the fate of Rab species in the cytosol has not been elucidated in detail. In this study, we find that BAG6 (BAT3/Scythe) predominantly recognizes a cryptic portion of GDP-associated Rab8a, while its major GTP-bound active form is not recognized. The hydrophobic residues of the Switch I region of Rab8a are essential for its interaction with BAG6 and the degradation of GDP-Rab8a via the ubiquitin-proteasome system. BAG6 prevents the excess accumulation of inactive Rab8a, whose accumulation impairs intracellular membrane trafficking. BAG6 binds not only Rab8a but also a functionally distinct set of Rab family proteins, and is also required for the correct distribution of Golgi and endosomal markers. From these observations, we suggest that Rab proteins represent a novel set of substrates for BAG6, and the BAG6-mediated pathway is associated with the regulation of membrane vesicle trafficking events in mammalian cells.
Assuntos
Chaperonas Moleculares/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Deleção de Genes , Complexo de Golgi/metabolismo , Humanos , Modelos Biológicos , Chaperonas Moleculares/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteólise , RNA Interferente Pequeno/genética , Ubiquitina/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Mutations in the gene encoding for leucine-rich repeat kinase 2 (LRRK2) are a common cause of hereditary Parkinson's disease. LRRK2 regulates various intracellular vesicular trafficking pathways, including endolysosomal degradative events such as epidermal growth factor receptor (EGFR) degradation. Recent studies have revealed that a subset of RAB proteins involved in secretory and endocytic recycling are LRRK2 kinase substrates in vivo However, the effects of LRRK2-mediated phosphorylation of these substrates on membrane trafficking remain unknown. Here, using an array of immunofluorescence and pulldown assays, we report that expression of active or phosphodeficient RAB8A variants rescues the G2019S LRRK2-mediated effects on endolysosomal membrane trafficking. Similarly, up-regulation of the RAB11-Rabin8-RAB8A cascade, which activates RAB8A, also reverted these trafficking deficits. Loss of RAB8A mimicked the effects of G2019S LRRK2 on endolysosomal trafficking and decreased RAB7A activity. Expression of pathogenic G2019S LRRK2 or loss of RAB8A interfered with EGFR degradation by causing its accumulation in a RAB4-positive endocytic compartment, which was accompanied by a deficit in EGFR recycling and was rescued upon expression of active RAB7A. Dominant-negative RAB7A expression resulted in similar deficits in EGF degradation, accumulation in a RAB4 compartment, and deficits in EGFR recycling, which were all rescued upon expression of active RAB8A. Taken together, these findings suggest that, by impairing RAB8A function, pathogenic G2019S LRRK2 deregulates endolysosomal transport and endocytic recycling events.
Assuntos
Endossomos/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos/metabolismo , Mutação de Sentido Incorreto , Proteínas rab de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Endossomos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Quinases do Centro Germinativo , Células HEK293 , Células HeLa , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Lisossomos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/genética , Proteólise , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Several studies have shown that Cl- channels regulate the differentiation of some cell types. Thus, we investigated the role of Cl- channels on adipocyte differentiation using adipose tissue-derived stem cells (ASCs) and Cl- channel blocker. We induced rabbit ASCs into adipocytes using Cl- channel blocker. The expression levels of adipocyte markers were no significant difference between the cells treated with a Cl- channel blocker NPPB and untreated cells. However, when the cells were treated with NPPB, lipid droplets (LDs) sizes decreased compared with the untreated control. Interestingly, the expression levels of Rab8a, which is known as a regulator of LD fusion, were also decreased in the cells treated with NPPB. Other Cl- channel blockers, DIDS and IAA-94, also inhibited large LDs formation and Rab8a expression. These results demonstrate that Cl- channels do not regulate the adipocyte differentiation, but do regulate the LDs formation via Rab8a expression.Abbreviations: ASCs: adipose tissue-derived stem cells; LDs: lipid droplets; RUNX2: runt-related transcription factor 2; CFTR: cystic fibrosis transmembrane conductance regulator; TG: triacylglycerol; FA: fatty acid; GLUT4: glucose transporter type 4; ER: endoplasmic reticulum; ADRP: adipose differentiation-related protein; TIP47: tail-interacting protein of 47 kD; HSL: hormone sensitive lipase; PBS: phosphate-buffered saline; DMEM: Dulbecco's modified Eagle Medium; FBS: fetal bovine serum; SMA: smooth muscle actin; FAS: fatty acid synthase; ZONAB: ZO-1 associated nucleic acid binding protein; PPAR-γ: peroxisome proliferator-activated receptor-γ; C/EBPα: CCAAT/enhancer binding protein α; CE: cholesteryl ester; V-ATPase: vacuolar H+ ATPase.
Assuntos
Adipócitos/citologia , Diferenciação Celular/fisiologia , Canais de Cloreto/fisiologia , Gotículas Lipídicas/metabolismo , Células-Tronco/citologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Masculino , CoelhosRESUMO
Skeletal muscles are high-insulin tissues responsible for disposing of glucose via the highly regulated process of facilitated glucose transporter 4 (GLUT4). Impaired insulin action in diabetes, as well as disorders of GLUT4 vesicle trafficking in the muscle, are involved in defects in insulin-stimulated GLUT4 translocation. Since the Rab GTPases are the main regulators of vesicular membrane transport in exo- and endo-cytosis, in the present work, we studied the effect of olive leaf polyphenols (OLPs) on Rab8A, Rab13, and Rab14 proteins of the rat soleus muscle in a model of streptozotocin (SZT)-induced diabetes (DM) in a dose-dependent manner. Glucose, cholesterol, and triglyceride levels were determined in the blood, morphological changes of the muscle tissue were captured by hematoxylin and eosin histological staining, and expression of GLUT4, Rab8A, Rab13, and Rab14 proteins were analyzed in the rat soleus muscle by the immunofluorescence staining and immunoblotting. OLPs significantly reduced blood glucose level in all treated groups. Furthermore, significantly reduced blood triglycerides were found in the groups with the lowest and highest OLPs treatment. The dynamics of activation of Rab8A, Rab13, and Rab14 was OLPs dose-dependent and more effective at higher OLP doses. Thus, these results indicate a beneficial role of phenolic compounds from the olive leaf in the regulation of glucose homeostasis in the skeletal muscle.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Músculo Esquelético/metabolismo , Olea/química , Folhas de Planta/química , Polifenóis/farmacologia , Animais , Diabetes Mellitus Experimental/sangue , Teste de Tolerância a Glucose , Masculino , Membranas , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Transporte Proteico/efeitos dos fármacos , Ratos Wistar , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo-tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed "secretory granules." However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome-edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra-/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo-tubular organelles as Rab11-Rab8-positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium-hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.
Assuntos
Enterócitos/metabolismo , Síndromes de Malabsorção/metabolismo , Microvilosidades/patologia , Mucolipidoses/metabolismo , Vesículas Secretórias/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Células CACO-2 , Membrana Celular/metabolismo , Enterócitos/ultraestrutura , Humanos , Síndromes de Malabsorção/genética , Masculino , Microvilosidades/genética , Microvilosidades/metabolismo , Mucolipidoses/genética , Mutação , Miosina Tipo V/genética , Transporte Proteico , Proteínas Qa-SNARE/genética , Vesículas Secretórias/ultraestruturaRESUMO
Although ATP-sensitive K+ (KATP) channels play an important role in the secretion of insulin by pancreatic beta cells, the mechanisms that regulate the intracellular transport of KATP channel subunit proteins (i.e., Kir6.2 and sulfonylurea receptor 1 (SUR1)) to the plasma membrane remain uncharacterized. We investigated the possibility that an interaction between KATP channel subunit proteins and Rab8a protein, a member of the RAS superfamily, may be involved in the membrane trafficking of KATP channels. Co-immunoprecipitation and immunostaining experiments using co-expression systems with fluorescent protein-tagged Kir6.2 were carried out to identify the coupling of KATP channels and Rab8a proteins in the insulin-secreting cell line, MIN6. Rab8a protein co-localized with Kir6.2 protein, a channel pore subunit (in a granular pattern), and with insulin. Knockdown of the Rab8a gene with RNA interference using small interfering RNA systems caused reductions in the amount of total KATP and plasma membrane surface KATP channels without decreasing the messenger RNA transcription of the KATP channel subunits. Rab8a gene knockdown also enhanced glucose-induced insulin secretion. These results suggest that Rab8a may be involved in membrane trafficking of KATP channels and the maintenance of normal insulin secretion in the MIN6 pancreatic beta cell line.
Assuntos
Secreção de Insulina , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Camundongos Endogâmicos BALB CRESUMO
Misplaced formation of microvilli to basolateral domains and intracellular inclusions in enterocytes are pathognomonic features in congenital enteropathy associated with mutation of the apical plasma membrane receptor syntaxin 3 (STX3). Although the demonstrated binding of Myo5b to the Rab8a and Rab11a small GTPases in vitro implicates cytoskeleton-dependent membrane sorting, the mechanisms underlying the microvillar location defect remain unclear. By selective or combinatory disruption of Rab8a and Rab11a membrane traffic in vivo, we demonstrate that transport of distinct cargo to the apical brush border rely on either individual or both Rab regulators, whereas certain basolateral cargos are redundantly transported by both factors. Enterocyte-specific Rab8a and Rab11a double-knockout mouse neonates showed immediate postnatal lethality and more severe enteropathy than single knockouts, with extensive formation of microvilli along basolateral surfaces. Notably, following an inducible Rab11a deletion from neonatal enterocytes, basolateral microvilli were induced within 3â days. These data identify a potentially important and distinct mechanism for a characteristic microvillus defect exhibited by enterocytes of patients with neonatal enteropathy.
Assuntos
Enterócitos/metabolismo , Doenças Genéticas Inatas/metabolismo , Enteropatias/metabolismo , Microvilosidades/metabolismo , Proteínas rab de Ligação ao GTP/deficiência , Animais , Enterócitos/patologia , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Enteropatias/congênito , Enteropatias/patologia , Camundongos , Camundongos Knockout , Microvilosidades/genética , Microvilosidades/patologia , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Actin filaments are widely involved in multiple cellular processes in oocyte meiosis, such as spindle migration and polar body extrusion. The actin nucleators like Arp2/3 complex and formins are the most recognized molecules for actin assembly in oocytes. In the present study, we report that the vesicle trafficking factor, RAB8A GTPase, is a new regulator critical for actin assembly in meiosis. Our results showed that RAB8A was localized at both the spindle periphery and cortex in mouse oocytes, which was similar to the localization patterns of actin filaments. RAB8A depletion caused spindle migration defects and the failure of polar body extrusion, which could have been due to decreases in both cytoplasmic and cortical actin filaments in oocytes. Based on mass spectrometry analysis, we showed that RAB8A promoted actin assembly through its modulation on the ROCK-LIMK signaling pathway. Moreover, we demonstrated that RAB8A colocalized and interacted with GM130 at the spindle periphery and that RAB8A depletion caused the disruption of GM130-docked Golgi distribution. Taken together, our data indicated that RAB8A was required for Golgi distribution, spindle migration, and polar body extrusion via ROCK-mediated actin assembly in mouse oocyte meiosis.
Assuntos
Actinas/metabolismo , Complexo de Golgi/fisiologia , Oócitos/fisiologia , Fuso Acromático/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Meiose/fisiologia , Camundongos , Corpos Polares/fisiologia , Proteínas rab de Ligação ao GTP/genética , Quinases Associadas a rho/genéticaRESUMO
Communication between stem and niche supporting cells maintains the homeostasis of adult tissues. Wnt signaling is a crucial regulator of the stem cell niche, but the mechanism that governs Wnt ligand delivery in this compartment has not been fully investigated. We identified that Wnt secretion is partly dependent on Rab8a-mediated anterograde transport of Gpr177 (wntless), a Wnt-specific transmembrane transporter. Gpr177 binds to Rab8a, depletion of which compromises Gpr177 traffic, thereby weakening the secretion of multiple Wnts. Analyses of generic Wnt/ß-catenin targets in Rab8a knockout mouse intestinal crypts indicate reduced signaling activities; maturation of Paneth cells - a Wnt-dependent cell type - is severely affected. Rab8a knockout crypts show an expansion of Lgr5(+) and Hopx(+) cells in vivo. However, in vitro, the knockout enteroids exhibit significantly weakened growth that can be partly restored by exogenous Wnts or Gsk3ß inhibitors. Immunogold labeling and surface protein isolation identified decreased plasma membrane localization of Gpr177 in Rab8a knockout Paneth cells and fibroblasts. Upon stimulation by exogenous Wnts, Rab8a-deficient cells show ligand-induced Lrp6 phosphorylation and transcriptional reporter activation. Rab8a thus controls Wnt delivery in producing cells and is crucial for Paneth cell maturation. Our data highlight the profound tissue plasticity that occurs in response to stress induced by depletion of a stem cell niche signal.
Assuntos
Celulas de Paneth/citologia , Nicho de Células-Tronco/fisiologia , Células-Tronco/citologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Ativação Transcricional , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Several mutations in leucine rich repeat kinase 2 (LRRK2) gene have been associated with pathogenesis of Parkinson's disease (PD), a neurodegenerative disorder marked by resting tremors, and rigidity, leading to Postural instability. It has been revealed that mutations that lead to an increase of kinase activity of LRRK2 protein are significantly associated with PD pathogenesis. Recent studies have shown that some Rab GTPases, especially Rab8, serve as substrates of LRRK2 and undergo phosphorylation in its switch II domain upon interaction. Current study was performed in order to find out the effects of the phosphorylation of Rab8 and its mutants on lipid metabolism and lipid droplets growth. METHODS: The phosphorylation status of Rab8a was checked by phos-tag gel. Point mutant construct were generated to investigate the function of Rab8a. 3T3L1 cells were transfected with indicated plasmids and the lipid droplets were stained with Bodipy. Fluorescent microscopy experiments were performed to examine the sizes of lipid droplets. The interactions between Rab8a and Optineurin were determined by immunoprecipitation and western blot. RESULTS: Our assays demonstrated that Rab8a was phosphorylated by mutated LRRK2 that exhibits high kinase activity. Phosphorylation of Rab8a on amino acid residue T72 promoted the formation of large lipid droplets. T72D mutant of Rab8a had higher activity to promote the formation of large lipid droplets compared with wild type Rab8a, with increase in average diameter of lipid droplets from 2.10 µm to 2.46 µm. Moreover, phosphorylation of Rab8a weakened the interaction with its effector Optineurin. CONCLUSIONS: Y1699C mutated LRRK2 was able to phosphorylate Rab8a and phosphorylation of Rab8a on site 72 plays important role in the fusion and enlargement of lipid droplets. Taken together, our study suggests an indirect relationship between enhanced lipid storage capacity and PD pathogenesis.
Assuntos
Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Células 3T3-L1 , Animais , Proteínas de Ciclo Celular , Humanos , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Camundongos , Mutação , Doença de Parkinson/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Tirosina/genética , Tirosina/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The Rab11 family of small GTPases, along with the Rab11-family interacting proteins (Rab11-FIPs), are critical regulators of intracellular vesicle trafficking and recycling. We have identified a point mutation of Threonine-197 site to an Alanine in Rab11-FIP1A, which causes a dramatic dominant negative phenotype when expressed in HeLa cells. The normally perinuclear distribution of GFP-Rab11-FIP1A was condensed into a membranous cisternum with almost no GFP-Rab11-FIP1A(T197A) remaining outside of this central locus. Also, this condensed GFP-FIP1A(T197A) altered the distribution of proteins in the Rab11a recycling pathway including endogenous Rab11a, Rab11-FIP1C, and transferrin receptor (CD71). Furthermore, this condensed GFP-FIP1A(T197A)-containing structure exhibited little movement in live HeLa cells. Expression of GFP-FIP1A(T197A) caused a strong blockade of transferrin recycling. Treatment of cells expressing GFP-FIP1A(T197A) with nocodazole did not disperse the Rab11a-containing recycling system. We also found that Rab5 and EEA1 were accumulated in membranes by GFP-Rab11-FIP1A but Rab4 was unaffected, suggesting that a direct pathway may exist from early endosomes into the Rab11a-containing recycling system. Our study of a potent inhibitory trafficking mutation in Rab11-FIP1A shows that Rab11-FIP1A associates with and regulates trafficking at an early step in the process of membrane recycling.
Assuntos
Endossomos/metabolismo , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica , Transporte ProteicoRESUMO
The small GTP-binding protein Rab8 is known to play an essential role in intracellular transport and cilia formation. We have previously demonstrated that Rab8a is required for localising apical markers in various organisms. Rab8a has a closely related isoform, Rab8b. To determine whether Rab8b can compensate for Rab8a, we generated Rab8b-knockout mice. Although the Rab8b-knockout mice did not display an overt phenotype, Rab8a and Rab8b double-knockout mice exhibited mislocalisation of apical markers and died earlier than Rab8a-knockout mice. The apical markers accumulated in three intracellular patterns in the double-knockout mice. However, the localisation of basolateral and/or dendritic markers of the double-knockout mice seemed normal. The morphology and the length of various primary and/or motile cilia, and the frequency of ciliated cells appeared to be identical in control and double-knockout mice. However, an additional knockdown of Rab10 in double-knockout cells greatly reduced the percentage of ciliated cells. Our results highlight the compensatory effect of Rab8a and Rab8b in apical transport, and the complexity of the apical transport process. In addition, neither Rab8a nor Rab8b are required for basolateral and/or dendritic transport. However, simultaneous loss of Rab8a and Rab8b has little effect on ciliogenesis, whereas additional loss of Rab10 greatly affects ciliogenesis.
Assuntos
Polaridade Celular , Cílios/metabolismo , Organogênese , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Animais Recém-Nascidos , Atrofia , Transporte Biológico , Biomarcadores/metabolismo , Células Cultivadas , Cílios/ultraestrutura , Intestino Delgado/patologia , Intestino Delgado/ultraestrutura , Camundongos , Camundongos Knockout , Microvilosidades/metabolismo , Microvilosidades/patologia , Microvilosidades/ultraestrutura , Fenótipo , Proteínas rab de Ligação ao GTP/deficiênciaRESUMO
Mutations in the LRRK2 gene cause familial Parkinson's disease presenting with pleomorphic neuropathology that can involve α-synuclein or tau accumulation. LRRK2 mutations are thought to converge upon a pathogenic increase in LRRK2 kinase activity. A subset of small RAB GTPases has been identified as LRRK2 substrates, with LRRK2-dependent phosphorylation resulting in RAB inactivation. We used CRISPR-Cas9 genome editing to generate a novel series of isogenic iPSC lines deficient in the two most well-validated LRRK2 substrates, RAB8a and RAB10, from deeply phenotyped healthy control lines. Thorough characterization of NGN2-induced neurons revealed opposing effects of RAB8a and RAB10 deficiency on lysosomal pH and Golgi organization, with isolated effects of RAB8a and RAB10 ablation on α-synuclein and tau, respectively. Our data demonstrate largely antagonistic effects of genetic RAB8a or RAB10 inactivation, which provide discrete insight into the pathologic features of their biochemical inactivation by pathogenic LRRK2 mutation in human disease.
Assuntos
alfa-Sinucleína , Proteínas rab de Ligação ao GTP , Humanos , alfa-Sinucleína/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mutação , Neurônios/metabolismo , Fosforilação , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Energy status is linked to the production of reactive oxygen species (ROS) in macrophages, which is elevated in obesity. However, it is unclear how ROS production is upregulated in macrophages in response to energy overload for mediating the development of obesity. Here, we show that the Rab-GTPase activating protein (RabGAP) TBC1D1, a substrate of the energy sensor AMP-activated protein kinase (AMPK), is a critical regulator of macrophage ROS production and consequent adipose inflammation for obesity development. TBC1D1 deletion decreases, whereas an energy overload-mimetic non-phosphorylatable TBC1D1S231A mutation increases, ROS production and M1-like polarization in macrophages. Mechanistically, TBC1D1 and its downstream target Rab8a form an energy-responsive complex with NOX2 for ROS generation. Transplantation of TBC1D1S231A bone marrow aggravates diet-induced obesity whereas treatment with an ultra-stable TtSOD for removal of ROS selectively in macrophages alleviates both TBC1D1S231A mutation- and diet-induced obesity. Our findings therefore have implications for drug discovery to combat obesity.
RESUMO
Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson's disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.
Assuntos
Neurônios Dopaminérgicos , Proteínas Hedgehog , Células-Tronco Pluripotentes Induzidas , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Receptor Patched-1 , Proteína GLI1 em Dedos de Zinco , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios Dopaminérgicos/metabolismo , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , AMP Cíclico/metabolismo , Caveolina 1/genética , Caveolina 1/metabolismo , Mutação/genética , Cálcio/metabolismo , Diferenciação Celular/genética , Transdução de Sinais/genéticaRESUMO
Introduction: Pathogenic mutations in RPGR ORF15, one of two major human RPGR isoforms, were responsible for most X-linked retinitis pigmentosa cases. Previous studies have shown that RPGR plays a critical role in ciliary protein transport. However, the precise mechanisms of disease triggered by RPGR ORF15 mutations have yet to be clearly defined. There are two homologous genes in zebrafish, rpgra and rpgrb. Zebrafish rpgra has a single transcript homologous to human RPGR ORF15; rpgrb has two major transcripts: rpgrb ex1-17 and rpgrb ORF15, similar to human RPGR ex1-19 and RPGR ORF15, respectively. rpgrb knockdown in zebrafish resulted in both abnormal development and increased cell death in the dysplastic retina. However, the impact of knocking down rpgra in zebrafish remains undetermined. Here, we constructed a rpgra mutant zebrafish model to investigate the retina defect and related molecular mechanism. Methods: we utilized transcription activator-like effector nuclease (TALEN) to generate a rpgra mutant zebrafish. Western blot was used to determine protein expression. RT-PCR was used to quantify gene transcription levels. The visual function of embryonic zebrafish was detected by electroretinography. Immunohistochemistry was used to observe the pathological changes in the retina of mutant zebrafish and transmission electron microscope was employed to view subcellular structure of photoreceptor cells. Results: A homozygous rpgra mutant zebrafish with c.1675_1678delins21 mutation was successfully constructed. Despite the normal morphological development of the retina at 5 days post-fertilization, visual dysfunction was observed in the mutant zebrafish. Further histological and immunofluorescence assays indicated that rpgra mutant zebrafish retina photoreceptors progressively began to degenerate at 3-6 months. Additionally, the mislocalization of cone outer segment proteins (Opn1lw and Gnb3) and the accumulation of vacuole-like structures around the connecting cilium below the OSs were observed in mutant zebrafish. Furthermore, Rab8a, a key regulator of opsin-carrier vesicle trafficking, exhibited decreased expression and evident mislocalization in mutant zebrafish. Discussion: This study generated a novel rpgra mutant zebrafish model, which showed retinal degeneration. our data suggested Rpgra is necessary for the ciliary transport of cone-associated proteins, and further investigation is required to determine its function in rods. The rpgra mutant zebrafish constructed in this study may help us gain a better understanding of the molecular mechanism of retinal degeneration caused by RPGR ORF15 mutation and find some useful treatment in the future.
RESUMO
Dynamic interaction between lipid droplets (LDs) and mitochondria controls the mobilization of long-chain fatty acids (LCFAs) from LDs for mitochondrial ß-oxidation in skeletal muscle in response to energy stress. However, little is known about the composition and regulation of the tethering complex mediating LD-mitochondrion interaction. Here, we identify Rab8a as a mitochondrial receptor for LDs forming the tethering complex with the LD-associated PLIN5 in skeletal muscle. In rat L6 skeletal muscle cells, the energy sensor AMPK increases the GTP-bound active Rab8a that promotes LD-mitochondrion interaction through binding to PLIN5 upon starvation. The assembly of the Rab8a-PLIN5 tethering complex also recruits the adipose triglyceride lipase (ATGL), which couples LCFA mobilization from LDs with its transfer into mitochondria for ß-oxidation. Rab8a deficiency impairs fatty acid utilization and decreases endurance during exercise in a mouse model. These findings may help to elucidate the regulatory mechanisms underlying the beneficial effects of exercise on lipid homeostasis control.
Assuntos
Gotículas Lipídicas , Metabolismo dos Lipídeos , Camundongos , Ratos , Animais , Gotículas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte/metabolismo , Ácidos Graxos/metabolismo , Músculo Esquelético/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Despite their significance in receptor-mediated internalization and continued signal transduction in cells, early/sorting endosomes (EE/SE) remain incompletely characterized, with many outstanding questions that surround the dynamics of their size and number. While several studies have reported increases in EE/SE size and number resulting from endocytic events, few studies have addressed such dynamics in a methodological and quantitative manner. Herein we apply quantitative fluorescence microscopy to measure the size and number of EE/SE upon internalization of two different ligands: transferrin and epidermal growth factor. Additionally, we used siRNA knock-down to determine the involvement of 5 different endosomal RAB proteins (RAB4, RAB5, RAB8A, RAB10 and RAB11A) in EE/SE dynamics. Our study provides new information on the dynamics of endosomes during endocytosis, an important reference for researchers studying receptor-mediated internalization and endocytic events.