RESUMO
Rab35 (Ras-associated binding protein) is a small GTPase that regulates endosomal membrane trafficking and functions in cell polarity, cytokinesis, and growth factor signaling. Altered Rab35 function contributes to progression of glioblastoma, defects in primary cilia formation, and altered cytokinesis. Here, we report a pediatric patient with global developmental delay, hydrocephalus, a Dandy-Walker malformation, axial hypotonia with peripheral hypertonia, visual problems, and conductive hearing impairment. Exome sequencing identified a homozygous missense variant in the GTPase fold of RAB35 (c.80G>A; p.R27H) as the most likely candidate. Functional analysis of the R27H-Rab35 variant protein revealed enhanced interaction with its guanine-nucleotide exchange factor, DENND1A and decreased interaction with a known effector, MICAL1, indicating that the protein is in an inactive conformation. Cellular expression of the variant drives the activation of Arf6, a small GTPase under negative regulatory control of Rab35. Importantly, variant expression leads to delayed cytokinesis and altered length, number, and Arl13b composition of primary cilia, known factors in neurodevelopmental disease. Our findings provide evidence of altered Rab35 function as a causative factor of a neurodevelopmental disorder.
Assuntos
Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento , Proteínas rab de Ligação ao GTP , Feminino , Humanos , Masculino , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Linhagem Celular , Cílios/metabolismo , Cílios/genética , Cílios/patologia , Citocinese/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mutação com Perda de Função , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Linhagem , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Epidermal growth factor (EGF) activates the EGF receptor (EGFR) and stimulates its internalization and trafficking to lysosomes for degradation. However, a percentage of EGFR undergoes ligand-independent endocytosis and is rapidly recycled back to the plasma membrane. Importantly, alterations in EGFR recycling are a common hallmark of cancer, and yet, our understanding of the machineries controlling the fate of endocytosed EGFR is incomplete. Intersectin-s is a multi-domain adaptor protein that is required for internalization of EGFR Here, we discover that intersectin-s binds DENND2B, a guanine nucleotide exchange factor for the exocytic GTPase Rab13, and this interaction promotes recycling of ligand-free EGFR to the cell surface. Intriguingly, upon EGF treatment, DENND2B is phosphorylated by protein kinase D and dissociates from intersectin-s, allowing for receptor targeting to degradation. Our study thus reveals a novel mechanism controlling the fate of internalized EGFR with important implications for cancer.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Membrana Celular/metabolismo , Endocitose , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Neoplasias/fisiopatologia , Fosforilação , Ligação Proteica , Proteína Quinase C/metabolismo , Transporte Proteico , Proteínas Supressoras de Tumor/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction.
Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas 14-3-3/metabolismo , Células 3T3-L1 , Animais , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/química , Fatores de Troca do Nucleotídeo Guanina/química , Células HEK293 , Humanos , Insulina/metabolismo , Camundongos , Fosforilação , Mapas de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Oxidative stress and mitochondrial dysfunction are implicated in neuronal apoptosis associated with Huntington's disease. Naringin is the flavanone present in grapefruit and related citrus species possess diverse pharmacological and therapeutic properties including antioxidant, anti-apoptotic, and neuroprotective properties. The aim of this study was to investigate the protective effect of naringin on 3-nitropropionic acid (3-NP)-induced neurotoxicity in pheochromocytoma cells (PC12) cells and to explore its mechanism of action. Naringin protects PC12 cells from 3-NP neurotoxicity, as evaluated the by cell viability assays. The lactate dehydrogenase release was decreased upon naringin treatment in 3-NP-induced PC12 cells. Naringin treatment enhances the antioxidant defense by increasing the activities of enzymatic antioxidants and the level of reduced glutathione. The increase in levels of reactive oxygen species and lipid peroxidation induced by 3-NP were significantly decreased by naringin. PC12 cells induced with 3-NP showed decrease in the mitochondrial membrane potential and mitochondrial respiratory complex enzymes, succinate dehydrogenase and cytochrome c oxidase activities, and it was significantly altered to near normal upon naringin treatment. Naringin reduced the 3-NP-induced apoptosis through the modulation in expressions of B-cell lymphoma 2 and Bcl-2-associated X protein. Further, naringin enhances the nuclear translocation of Nrf2 and induces the NAD(P)H: quinone oxidoreductase-1 and Heme oxygenase-1 expressions through the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. Taken together, the above findings suggest that naringin augments cellular antioxidant defense capacity and reduces the 3-NP-induced neurotoxicity in PC12 cells through the PI-3K/Akt-dependent Nrf2 activation in PC12 cells.
Assuntos
Flavanonas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Fármacos Neuroprotetores/farmacologia , Nitrocompostos/toxicidade , Propionatos/toxicidade , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glutationa/metabolismo , Heme Oxigenase-1/biossíntese , L-Lactato Desidrogenase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/biossíntese , Síndromes Neurotóxicas/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Ratos , Espécies Reativas de Oxigênio/metabolismo , Succinato Desidrogenase/metabolismo , Proteína X Associada a bcl-2/biossínteseRESUMO
Lysosomes help maintain cellular proteostasis, and defects in lysosomal positioning and function can cause disease, including neurodegenerative disorders. The spatiotemporal distribution of lysosomes is regulated by small GTPases including Rabs, which are activated by guanine nucleotide exchange factors (GEFs). DENN domain proteins are the largest family of Rab GEFs. Using a cell-based assay, we screened DENND6A, a member of the DENN domain protein family against all known Rabs and identified it as a potential GEF for 20 Rabs, including Rab34. Here, we demonstrate that DENND6A activates Rab34, which recruits a RILP/dynein complex to lysosomes, promoting lysosome retrograde transport. Further, we identify DENND6A as an effector of Arl8b, a major regulatory GTPase on lysosomes. We demonstrate that Arl8b recruits DENND6A to peripheral lysosomes to activate Rab34 and initiate retrograde transport, regulating nutrient-dependent lysosomal juxtanuclear repositioning. Loss of DENND6A impairs autophagic flux. Our findings support a model whereby Arl8b/DENND6A/Rab34-dependent lysosomal retrograde trafficking controls autophagy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ligação Proteica , Proteínas rab de Ligação ao GTP/metabolismo , Lisossomos/metabolismo , Autofagia , Dineínas/metabolismoRESUMO
Developmental and epileptic encephalopathies (DEEs) are a heterogenous group of epilepsies in which altered brain development leads to developmental delay and seizures, with the epileptic activity further negatively impacting neurodevelopment. Identifying the underlying cause of DEEs is essential for progress toward precision therapies. Here we describe a group of individuals with biallelic variants in DENND5A and determine that variant type is correlated with disease severity. We demonstrate that DENND5A interacts with MUPP1 and PALS1, components of the Crumbs apical polarity complex, which is required for both neural progenitor cell identity and the ability of these stem cells to divide symmetrically. Induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division during neural induction and have an inherent propensity to differentiate into neurons, and transgenic DENND5A mice, with phenotypes like the human syndrome, have an increased number of neurons in the adult subventricular zone. Disruption of symmetric cell division following loss of DENND5A results from misalignment of the mitotic spindle in apical neural progenitors. A subset of DENND5A is localized to centrosomes, which define the spindle poles during mitosis. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state and ultimately shortening the period of neurogenesis. This study provides a mechanism behind DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.
RESUMO
Developmental and epileptic encephalopathies (DEEs) feature altered brain development, developmental delay and seizures, with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A, encoding a membrane trafficking protein, and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1, components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state, ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.
Assuntos
Divisão Celular , Células-Tronco Pluripotentes Induzidas , Células-Tronco Neurais , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Humanos , Animais , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Neurogênese/genética , Masculino , Feminino , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Modelos Animais de Doenças , Polaridade CelularRESUMO
Cytokinesis relies on membrane trafficking pathways regulated by Rabs and guanine nucleotide exchange factors (GEFs). During cytokinesis, the intercellular cytokinetic bridge (ICB) connecting daughter cells undergoes abscission, which requires actin depolymerization. Rab35 recruits MICAL1 to oxidize and depolymerize actin filaments. We show that DENND2B, a protein linked to cancer and congenital disorders, functions as a Rab35 GEF, recruiting and activating Rab35 at the ICB. DENND2B's N-terminal region also interacts with an active form of Rab35, suggesting that DENND2B is both a Rab35 GEF and effector. Knockdown of DENND2B delays abscission, leading to multinucleated cells and filamentous actin (F-actin) accumulation at the ICB, impairing recruitment of ESCRT-III at the abscission site. Additionally, F-actin accumulation triggers the formation of a chromatin bridge, activating the NoCut/abscission checkpoint, and DENND2B knockdown activates Aurora B kinase, a hallmark of checkpoint activation. Thus, our study identifies DENND2B as a crucial player in cytokinetic abscission.
Assuntos
Actinas , Citocinese , Proteínas de Ligação a DNA , Proteínas rab de Ligação ao GTP , Humanos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citocinese/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Proteínas dos Microfilamentos/metabolismo , Oxigenases de Função Mista/metabolismo , Tetraploidia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas de Ligação a DNA/metabolismoRESUMO
Primary cilia are sensory antennae crucial for cell and organism development, and defects in their biogenesis cause ciliopathies. Ciliogenesis involves membrane trafficking mediated by small guanosine triphosphatases (GTPases) including Rabs, molecular switches activated by guanine nucleotide exchange factors (GEFs). The largest family of Rab GEFs is the DENN domain-bearing proteins. Here, we screen all 60 Rabs against two major DENN domain families using a cellular GEF assay, uncovering 19 novel DENN/Rab pairs. The screen reveals Rab10 as a substrate for DENND2B, a protein previously implicated in cancer and severe mental retardation. Through activation of Rab10, DENND2B represses the formation of primary cilia. Through a second pathway, DENND2B functions as a GEF for RhoA to control the length of primary cilia. This work thus identifies an unexpected diversity in DENN domain-mediated activation of Rabs, a previously unidentified non-Rab substrate for a DENN domain, and a new regulatory protein in primary ciliogenesis.
RESUMO
Glioblastoma is the most common and deadly malignant brain cancer. We now demonstrate that loss of function of the endosomal GTPase Rab35 in human brain tumor initiating cells (BTICs) increases glioblastoma growth and decreases animal survival following BTIC implantation in mouse brains. Mechanistically, we identify that the GTPase Arf5 interacts with the guanine nucleotide exchange factor (GEF) for Rab35, DENND1/connecdenn, and allosterically enhances its GEF activity toward Rab35. Knockdown of either Rab35 or Arf5 increases cell migration, invasiveness, and self-renewal in culture and enhances the growth and invasiveness of BTIC-initiated brain tumors in mice. RNAseq of the tumors reveals up-regulation of the tumor-promoting transcription factor SPOCD1, and disruption of the Arf5/Rab35 axis in glioblastoma cells leads to strong activation of the epidermal growth factor receptor, with resulting enhancement of SPOCD1 levels. These discoveries reveal an unexpected cascade between an Arf and a Rab and indicate a role for the cascade, and thus endosomal trafficking, in brain tumors.