RESUMEN

Previously, we have demonstrated that deletion of a growth-regulating gene (LdCen1) in the Leishmania donovani parasite (LdCen1-/-) attenuated the parasite's intracellular amastigote growth but not the growth of extracellular promastigotes. LdCen1-/- parasites were found to be safe and efficacious against homologous and heterologous Leishmania species as a vaccine candidate in animal models. The reason for the differential growth of LdCen1-/- between the two stages of the parasite needed investigation. Here, we report that LdCen1 interacts with a novel Ras-associated binding protein in L. donovani (LdRab2) to compensate for the growth of LdCen1-/- promastigotes. LdRab2 was isolated by protein pull-down from the parasite lysate, followed by nano-LC-MS/MS identification. The RAB domain sequence and the functional binding partners of the LdRab2 protein were predicted via Search Tool for the Retrieval of Interacting Proteins (STRING) analysis. The closeness of the LdRab2 protein to other reported centrin-binding proteins with different functions in other organisms was analyzed via phylogenetic analysis. Furthermore, in vitro and in silico analyses revealed that LdRab2 also interacts with other L. donovani centrins 3-5. Since centrin is a calcium-binding protein, we further investigated calcium-based interactions and found that the binding of LdRab2 to LdCen1 and LdCen4 is calcium-independent, whereas the interactions with LdCen3 and LdCen5 are calcium-dependent. The colocalization of LdCen1 and LdRab2 at the cellular basal-body region by immunofluorescence supports their possible functional association. The elevated expression of the LdRab2 protein in the mutant promastigotes suggested a probable role in compensating for the promastigote growth of this mutant strain, probably in association with other parasite centrins.

Asunto(s)

Leishmania donovani , Proteínas Protozoarias , Proteína de Unión al GTP rab2 , Leishmania donovani/genética , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteína de Unión al GTP rab2/genética , Proteína de Unión al GTP rab2/metabolismo , Unión Proteica , Filogenia , Animales

RESUMEN

Sperm heads contain not only the nucleus but also the acrosome which is a distinctive cap-like structure located anterior to the nucleus and is derived from the Golgi apparatus. The Golgi Associated RAB2 Interactors (GARINs; also known as FAM71) protein family shows predominant expression in the testis and all possess a RAB2-binding domain which confers binding affinity to RAB2, a small GTPase that is responsible for membrane transport and vesicle trafficking. Our previous study showed that GARIN1A and GARIN1B are important for acrosome biogenesis and that GARIN1B is indispensable for male fertility in mice. Here, we generated KO mice of other Garins, namely Garin2, Garin3, Garin4, Garin5a, and Garin5b (Garin2-5b). Using computer-assisted morphological analysis, we found that the loss of each Garin2-5b resulted in aberrant sperm head morphogenesis. While the fertilities of Garin2-/- and Garin4-/- males are normal, Garin5a-/- and Garin5b-/- males are subfertile, and Garin3-/- males are infertile. Further analysis revealed that Garin3-/- males exhibited abnormal acrosomal morphology, but not as severely as Garin1b-/- males; instead, the amounts of membrane proteins, particularly ADAM family proteins, decreased in Garin3 KO spermatozoa. Moreover, only Garin4 KO mice exhibit vacuoles in the sperm head. These results indicate that GARINs assure correct head morphogenesis and some members of the GARIN family function distinctively in male fertility.

Asunto(s)

Fertilidad , Infertilidad Masculina , Ratones Noqueados , Cabeza del Espermatozoide , Animales , Masculino , Ratones , Acrosoma/metabolismo , Fertilidad/genética , Aparato de Golgi/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Morfogénesis/genética , Proteína de Unión al GTP rab2/metabolismo , Proteína de Unión al GTP rab2/genética , Cabeza del Espermatozoide/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Testículo/crecimiento & desarrollo

RESUMEN

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.

Asunto(s)

Retículo Endoplásmico , Proteínas Protozoarias , Vías Secretoras , Toxoplasma , Proteína de Unión al GTP rab2 , Toxoplasma/metabolismo , Toxoplasma/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Retículo Endoplásmico/metabolismo , Proteína de Unión al GTP rab2/metabolismo , Proteína de Unión al GTP rab2/genética , Dominios Proteicos , Transporte de Proteínas , Gotas Lipídicas/metabolismo , Animales , Humanos

RESUMEN

BACKGROUND: The most fatal form of Visceral leishmaniasis or kala-azar is caused by the intracellular protozoan parasite Leishmania donovani. The life cycle and the infection pathway of the parasite are regulated by the small GTPase family of Rab proteins. The involvement of Rab proteins in neurodegenerative amyloidosis is implicated in protein misfolding, secretion abnormalities and dysregulation. The inter and intra-cellular shuttlings of Rab proteins are proposed to be aggregation-prone. However, the biophysical unfolding and aggregation of protozoan Rab proteins is limited. Understanding the aggregation mechanisms of Rab protein will determine their physical impact on the disease pathogenesis and individual health. OBJECTIVE: This work investigates the acidic pH-induced unfolding and aggregation of a recombinant Rab2 protein from L. donovani (rLdRab2) using multi-spectroscopic probes. METHODS: The acidic unfolding of rLdRab2 is characterised by intrinsic fluorescence and ANS assay, while aggregation is determined by Thioflavin-T and 90° light scattering assay. Circular dichroism determined the secondary structure of monomers and aggregates. The aggregate morphology was imaged by transmission electron microscopy. RESULTS: rLdRab2 was modelled to be a Rab2 isoform with loose globular packing. The acidinduced unfolding of the protein is a plausible non-two-state process. At pH 2.0, a partially folded intermediate (PFI) state characterised by ~ 30% structural loss and exposed hydrophobic core was found to accumulate. The PFI state slowly converted into well-developed protofibrils at high protein concentrations demonstrating its amyloidogenic nature. The native state of the protein was also observed to be aggregation-prone at high protein concentrations. However, it formed amorphous aggregation instead of fibrils. CONCLUSION: To our knowledge, this is the first study to report in vitro amyloid-like behaviour of Rab proteins in L donovani. This study provides a novel opportunity to understand the complete biophysical characteristics of Rab2 protein of the lower eukaryote, L. donovani.

Asunto(s)

Amiloide , Leishmania donovani , Proteínas Recombinantes , Proteína de Unión al GTP rab2 , Leishmania donovani/metabolismo , Leishmania donovani/química , Leishmania donovani/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Amiloide/química , Amiloide/metabolismo , Proteína de Unión al GTP rab2/metabolismo , Proteína de Unión al GTP rab2/química , Proteína de Unión al GTP rab2/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Concentración de Iones de Hidrógeno , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Agregado de Proteínas , Dicroismo Circular

RESUMEN

The acrosome is a cap-shaped, Golgi-derived membranous organelle that is located over the anterior of the sperm nucleus and highly conserved throughout evolution. Although morphological changes during acrosome biogenesis in spermatogenesis have been well described, the molecular mechanism underlying this process is still largely unknown. Family with sequence similarity 71, member F1 and F2 (FAM71F1 and FAM71F2) are testis-enriched proteins that contain a RAB2B-binding domain, a small GTPase involved in vesicle transport and membrane trafficking. Here, by generating mutant mice for each gene, we found that Fam71f1 is essential for male fertility. In Fam71f1-mutant mice, the acrosome was abnormally expanded at the round spermatid stage, likely because of enhanced vesicle trafficking. Mass spectrometry analysis after immunoprecipitation indicated that, in testes, FAM71F1 binds not only RAB2B, but also RAB2A. Further study suggested that FAM71F1 binds to the GTP-bound active form of RAB2A/B, but not the inactive form. These results indicate that a complex of FAM71F1 and active RAB2A/B suppresses excessive vesicle trafficking during acrosome formation.

Asunto(s)

Acrosoma/metabolismo , Fertilidad/fisiología , Proteínas Nucleares/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rab2/metabolismo , Acrosoma/patología , Animales , Genética , Aparato de Golgi/metabolismo , Infertilidad Masculina , Masculino , Ratones , Ratones Transgénicos , Proteínas Nucleares/genética , Unión Proteica , Cabeza del Espermatozoide/metabolismo , Espermatogénesis , Teratozoospermia/metabolismo , Testículo/metabolismo

RESUMEN

Progranulin (PGRN) is a key regulator of lysosomes, and its deficiency has been linked to various lysosomal storage diseases (LSDs), including Gaucher disease (GD), one of the most common LSD. Here, we report that PGRN plays a previously unrecognized role in autophagy within the context of GD. PGRN deficiency is associated with the accumulation of LC3-II and p62 in autophagosomes of GD animal model and patient fibroblasts, resulting from the impaired fusion of autophagosomes and lysosomes. PGRN physically interacted with Rab2, a critical molecule in autophagosome-lysosome fusion. Additionally, a fragment of PGRN containing the Grn E domain was required and sufficient for binding to Rab2. Furthermore, this fragment significantly ameliorated PGRN deficiency-associated impairment of autophagosome-lysosome fusion and autophagic flux. These findings not only demonstrate that PGRN is a crucial mediator of autophagosome-lysosome fusion but also provide new evidence indicating PGRN's candidacy as a molecular target for modulating autophagy in GD and other LSDs in general. KEY MESSAGES : PGRN acts as a crucial factor involved in autophagosome-lysosome fusion in GD. PGRN physically interacts with Rab2, a molecule in autophagosome-lysosome fusion. A 15-kDa C-terminal fragment of PGRN is required and sufficient for binding to Rab2. This PGRN derivative ameliorates PGRN deficiency-associated impairment of autophagy. This study provides new insights into autophagy and may develop novel therapy for GD.

Asunto(s)

Autofagosomas , Enfermedad de Gaucher/metabolismo , Lisosomas , Progranulinas/metabolismo , Proteína de Unión al GTP rab2/metabolismo , Alérgenos/administración & dosificación , Animales , Células Cultivadas , Humanos , Ratones Noqueados , Ovalbúmina/administración & dosificación , Progranulinas/genética , Proteína de Unión al GTP rab2/genética

RESUMEN

Dysregulation of microRNA-425 (miR-425) has been reported in several human cancers. However, the role of miR-425 in human cervical cancer via modulation of RAB2B expression is still unclear. This study was therefore designed to examine the expression and decipher the role of miR-425 in cervical cancer. The qRT-PCR was used for expression analysis. MTT and EdU assays were used for the determination of cell viability and proliferation, respectively. Annexin V/PI staining was used to detect apoptosis. Wound healing and transwell assays were used to monitor cell migration and invasion. Western blotting was used for protein expression analysis. The in vivo study was performed in xenografted mice model. The results of the present study revealed miR-425 to be significantly (P = 0.032) down-regulated in cervical cancer tissues and cell lines. Additionally, low expression of miR-425 was associated with significantly (P = 0.035) lower survival rate of the cervical cancer patients. Overexpression of miR-425 resulted in significant (P = 0.024) decline of cervical cancer cell proliferation via induction of apoptosis. The induction of apoptosis was associated with up-regulation of Bax and down-regulation of Bcl-2. Besides, the migration and invasion of cancer cells significantly (P < 0.01) decreased under miR-425 overexpression. Additionally, miR-425 could inhibit the growth of xenografted tumors in vivo. In silico analysis and dual luciferase assay revealed RAB2B as the direct target of miR-425 in cervical cancer. RAB2B was found to be significantly (P < 0.05) up-regulated in cervical cancer tissues and cell lines and miR-425 overexpression suppressed the expression of RAB2B. Additionally, silencing of RAB2B could suppress the growth of cervical cancer cells but its overexpression could rescue the tumor-suppressive effects of miR-425. Taken together, the results revealed the tumor-suppressive roe of miR-425 and point towards its therapeutic potential in the management of cervical cancer.

Asunto(s)

MicroARNs , Neoplasias del Cuello Uterino , Proteína de Unión al GTP rab2/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/mortalidad , Neoplasias del Cuello Uterino/patología , Proteína de Unión al GTP rab2/genética

RESUMEN

Reliable delivery of presynaptic material, including active zone and synaptic vesicle proteins from neuronal somata to synaptic terminals, is prerequisite for successful synaptogenesis and neurotransmission. However, molecular mechanisms controlling the somatic assembly of presynaptic precursors remain insufficiently understood. We show here that in mutants of the small GTPase Rab2, both active zone and synaptic vesicle proteins accumulated in the neuronal cell body at the trans-Golgi and were, consequently, depleted at synaptic terminals, provoking neurotransmission deficits. Ectopic presynaptic material accumulations consisted of heterogeneous vesicles and short tubules of 40 × 60 nm, segregating in subfractions either positive for active zone or synaptic vesicle proteins and LAMP1, a lysosomal membrane protein. Genetically, Rab2 acts upstream of Arl8, a lysosomal adaptor controlling axonal export of precursors. Collectively, we identified a Golgi-associated assembly sequence of presynaptic precursor biogenesis dependent on a Rab2-regulated protein export and sorting step at the trans-Golgi.

Asunto(s)

Aparato de Golgi/metabolismo , Vesículas Sinápticas/metabolismo , Proteína de Unión al GTP rab2/metabolismo , Animales , Axones/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Terminales Presinápticos/metabolismo , Transporte de Proteínas/fisiología , Transmisión Sináptica/fisiología

RESUMEN

Fast axonal transport of neuropeptide-containing dense core vesicles (DCVs), endolysosomal organelles, and presynaptic components is critical for maintaining neuronal functionality. How the transport of DCVs is orchestrated remains an important unresolved question. The small GTPase Rab2 mediates DCV biogenesis and endosome-lysosome fusion. Here, we use Drosophila to demonstrate that Rab2 also plays a critical role in bidirectional axonal transport of DCVs, endosomes, and lysosomal organelles, most likely by controlling molecular motors. We further show that the lysosomal motility factor Arl8 is required as well for axonal transport of DCVs, but unlike Rab2, it is also critical for DCV exit from cell bodies into axons. We also provide evidence that the upstream regulators of Rab2 and Arl8, Ema and BORC, activate these GTPases during DCV transport. Our results uncover the mechanisms underlying axonal transport of DCVs and reveal surprising parallels between the regulation of DCV and lysosomal motility.

Asunto(s)

Factores de Ribosilacion-ADP/genética , Transporte Axonal/genética , Vesículas de Núcleo Denso/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Neuronas/metabolismo , Proteína de Unión al GTP rab2/genética , Factores de Ribosilacion-ADP/metabolismo , Animales , Vesículas de Núcleo Denso/ultraestructura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endosomas/metabolismo , Endosomas/ultraestructura , Regulación de la Expresión Génica , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lisosomas/metabolismo , Lisosomas/ultraestructura , Fusión de Membrana , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Neuronas/ultraestructura , Biogénesis de Organelos , Unión Proteica , Transducción de Señal , Proteína de Unión al GTP rab2/metabolismo

RESUMEN

PURPOSE: Several RAB family genes have been studied extensively and proven to play pivotal roles in the occurrence and development of certain cancers. Here, we explored commonly expressed RAB family genes in humans and their prognostic significance using bioinformatics, and then identified potential biomarkers of breast invasive carcinoma (BRCA). MATERIALS AND METHODS: The prognostic values (overall survival) of RAB family genes in BRCA were obtained using Gene Expression Profiling Interactive Analysis (GEPIA). The expression patterns of RAB family genes and their relationships with clinicopathological parameters in BRCA were measured using the ONCOMINE and UALCAN databases, respectively. Genetic mutations and survival analysis were investigated using the cBio Cancer Genomics Portal (c-BioPortal). Interacting genes of potential biomarkers were identified using STRING, and functional enrichment analyses were performed using FunRich v3.1.3. RESULTS: In total, 64 RAB genes were identified and analyzed in our study. Results showed that RAB1B, RAB2A, and RAB18 were up-regulated and significantly associated with poor overall survival in BRCA. Furthermore, their higher expression was positively correlated with clinicopathological parameters (e.g. cancer stage and nodal metastasis status). DNA copy number amplifications and mRNA up-regulation were the main genetic mutations, and the altered group showed significantly poorer overall survival compared with the unaltered group. Functional enrichment analysis of RAB1B, RAB2A, and RAB18 indicated they were closely involved in GTPase activity. CONCLUSIONS: RAB1B, RAB2A, and RAB18 were up-regulated and significantly correlated with poor prognosis in BRCA. Thus, they could be applied as novel biomarkers of BRCA in future studies.

Asunto(s)

Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Transcriptoma , Proteínas de Unión al GTP rab/genética , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Biología Computacional , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Invasividad Neoplásica , Fenotipo , Pronóstico , Mapas de Interacción de Proteínas , Proteínas de Unión al GTP rab1/genética , Proteína de Unión al GTP rab2/genética

RESUMEN

Intracellular bacterial pathogens remodel cellular functions during their infectious cycle via the coordinated actions of effector molecules delivered through dedicated secretion systems. While the function of many individual effectors is known, how they interact to promote pathogenesis is rarely understood. The zoonotic bacterium Brucella abortus, the causative agent of brucellosis, delivers effector proteins via its VirB type IV secretion system (T4SS) which mediate biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV). Here, we show that T4SS effectors BspB and RicA display epistatic interactions in Brucella replication. Defects in rBCV biogenesis and Brucella replication caused by deletion of bspB were dependent on the host GTPase Rab2a and suppressed by the deletion of ricA, indicating a role of Rab2-binding effector RicA in these phenotypic defects. Rab2a requirements for rBCV biogenesis and Brucella intracellular replication were abolished upon deletion of both bspB and ricA, demonstrating that the functional interaction of these effectors engages Rab2-dependent transport in the Brucella intracellular cycle. Expression of RicA impaired host secretion and caused Golgi fragmentation. While BspB-mediated changes in ER-to-Golgi transport were independent of RicA and Rab2a, BspB-driven alterations in Golgi vesicular traffic also involved RicA and Rab2a, defining BspB and RicA's functional interplay at the Golgi interface. Altogether, these findings support a model where RicA modulation of Rab2a functions impairs Brucella replication but is compensated by BspB-mediated remodeling of Golgi apparatus-associated vesicular transport, revealing an epistatic interaction between these T4SS effectors.IMPORTANCE Bacterial pathogens with an intracellular lifestyle modulate many host cellular processes to promote their infectious cycle. They do so by delivering effector proteins into host cells via dedicated secretion systems that target specific host functions. While the roles of many individual effectors are known, how their modes of action are coordinated is rarely understood. Here, we show that the zoonotic bacterium Brucella abortus delivers the BspB effector that mitigates the negative effect on bacterial replication that the RicA effector exerts via modulation of the host small GTPase Rab2. These findings provide an example of functional integration between bacterial effectors that promotes proliferation of pathogens.

Asunto(s)

Brucella/fisiología , Brucelosis/metabolismo , Brucelosis/microbiología , Epistasis Genética , Interacciones Huésped-Patógeno , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/metabolismo , Proteína de Unión al GTP rab2/metabolismo , Retículo Endoplásmico/metabolismo , Regulación Bacteriana de la Expresión Génica , Aparato de Golgi/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Unión Proteica , Transporte de Proteínas

RESUMEN

Cervical cancer is one of the leading causes of cancer death in women worldwide. While molecular mechanisms of initiation and cervical carcinogenesis are not well studied. Our data showed that the expression of Methyltransferase-like 3 (METTL3) was upregulated in cervical tumor tissues as compared with normal tissues. Its expression was associated with poor prognosis of cervical cancer. Knockdown of METTL3 can suppress the proliferation of cervical cancer cells. The expression of METTL3 was significantly correlated with the expression of RAB2B, one member of RAS oncogene family. Over expression of RAB2B can significantly attenuate sh-METTL3-suppressed cell proliferation. Mechanistically, METTL3 can increase the mRNA stability of RAB2B via an IGF2BP3-dependent manner. Collectively, METTL3 can trigger growth of cervical cancer cells via upregulation of RAB2B. It indicated that METTL3 might be a potential target for cervical cancer therapy.

Asunto(s)

Metiltransferasas/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Línea Celular Tumoral , Proliferación Celular , Cuello del Útero/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metiltransferasas/genética , Pronóstico , Proteínas de Unión al ARN/genética , Regulación hacia Arriba , Neoplasias del Cuello Uterino/genética , Proteína de Unión al GTP rab2/genética , Proteína de Unión al GTP rab2/metabolismo

RESUMEN

Rap2B, belonging to the Ras superfamily of small guanosine triphosphate-binding proteins, is upregulated and contributes to the progression of several tumors by acting as an oncogene, including hepatocellular carcinoma (HCC). However, the mechanism underlying the functional roles of Rap2B in HCC remains unclear. In this study, the evaluation of Rap2B expression in HCC cells and tissues was achieved by qRT-PCR and western blot assays. The effects of Rap2B on the malignant biological behaviors in HCC were explored by means of MTT assay, flow cytometry analysis, and Transwell invasion assay, respectively. Protein levels of Ki67, matrix metalloproteinase (MMP)-2, MMP-9, and cleaved caspase-3, together with the alternations of the ERK1/2 and PTEN/PI3K/Akt pathways were qualified by western blot assay. Further verification of the Rap2B function on HCC tumorigenesis was attained by performing in vivo assays. We found that Rap2B levels were upregulated in HCC tissues and cells. Rap2B silencing led to a reduction of cell-proliferative and invasive abilities, and an increase of apoptosis in HCC cells. In addition, xenograft tumor assay demonstrated that Rap2B silencing repressed HCC xenograft tumor growth in vivo. In addition, we found that Rap2B knockdown significantly inhibited the ERK1/2 and PTEN/PI3K/Akt cascades in HCC cells and xenograft tumor tissues. Together, Rap2B knockdown inhibited HCC-malignant progression, which was involved in inhibiting the ERK1/2 and PTEN/PI3K/Akt pathways. Our findings contribute to understanding of the molecular mechanism of Rap2B in HCC progression.

Asunto(s)

Carcinoma Hepatocelular/metabolismo , Técnicas de Silenciamiento del Gen , Neoplasias Hepáticas/metabolismo , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína de Unión al GTP rab2/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt , Proteína de Unión al GTP rab2/genética

RESUMEN

Multiple sources contribute membrane and protein machineries to construct functional macroautophagic/autophagic structures. However, the underlying molecular mechanisms remain elusive. Here, we show that RAB2 connects the Golgi network to autophagy pathway by delivering membrane and by sequentially engaging distinct autophagy machineries. In unstressed cells, RAB2 resides primarily in the Golgi apparatus, as evidenced by its interaction and colocalization with GOLGA2/GM130. Importantly, autophagy stimuli dissociate RAB2 from GOLGA2 to interact with ULK1 complex, which facilitates the recruitment of ULK1 complex to form phagophores. Intriguingly, RAB2 appears to modulate ULK1 kinase activity to propagate signals for autophagosome formation. Subsequently, RAB2 switches to interact with autophagosomal RUBCNL/PACER and STX17 to further specify the recruitment of HOPS complex for autolysosome formation. Together, our study reveals a multivalent pathway in bulk autophagy regulation, and provides mechanistic insights into how the Golgi apparatus contributes to the formation of different autophagic structures. Abbreviations: ACTB: actin beta; ATG9: autophagy related 9A; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BCAP31: B cell receptor associated protein 31; BECN1: beclin 1; Ctrl: control; CQ: chloroquine; CTSD: cathepsin D; DMSO: dimethyl sulfoxide; EBSS: Earle's balanced salt solution; EEA1: early endosome antigen 1; GDI: guanine nucleotide dissociation inhibitor; GFP: green fluorescent protein; GOLGA2: golgin A2; HOPS: homotypic fusion and protein sorting complex; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LC3: microtubule-associated protein 1 light chain 3; OE: overexpression; PtdIns3K: class III phosphatidylinositol 3-kinase; SQSTM1/p62: sequestosome 1; RAB2: RAB2A, member RAS oncogene family; RAB7: RAB7A, member RAS oncogene family; RAB11: RAB11A, member RAS oncogene family; RUBCNL/PACER: rubicon like autophagy enhancer; STX17: syntaxin 17; TBC1D14: TBC1 domain family member 14; TFRC: transferrin receptor; TGOLN2: trans-golgi network protein 2; TUBB: tubulin beta class I; ULK1: unc-51 like autophagy activating kinase 1; VPS41: VPS41, HOPS complex subunit; WB: western blot; WT: wild type; YPT1: GTP-binding protein YPT1.

Asunto(s)

Autofagosomas/metabolismo , Autofagia/genética , Lisosomas/metabolismo , Proteína de Unión al GTP rab2/fisiología , Animales , Células Cultivadas , Células Eucariotas/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisosomas/genética , Masculino , Mamíferos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína de Unión al GTP rab2/genética

RESUMEN

ZFYVE26/Spastizin and SPG11/Spatacsin encode 2 large proteins that are mutated in hereditary autosomal-recessive spastic paraplegia/paraparesis (HSP) type 15 (AR-SPG15) and type 11 (AR-SPG11), respectively. We previously have reported that AR-SPG15-related ZFYVE26 mutations lead to autophagy defects with accumulation of immature autophagosomes. ZFYVE26 and SPG11 were found to be part of a complex including the AP5 (adaptor related protein complex 5) and to have a critical role in autophagic lysosomal reformation with identification of autophagic and lysosomal defects in cells with both AR-SPG15- and AR-SPG11-related mutations. In spite of these similarities between the 2 proteins, here we report that ZFYVE26 and SPG11 are differently involved in autophagy and endocytosis. We found that both ZFYVE26 and SPG11 interact with RAB5A and RAB11, 2 proteins regulating endosome trafficking and maturation, but only ZFYVE26 mutations affected RAB protein interactions and activation. ZFYVE26 mutations lead to defects in the fusion between autophagosomes and endosomes, while SPG11 mutations do not affect this step and lead to a milder autophagy defect. We thus demonstrate that ZFYVE26 and SPG11 affect the same cellular physiological processes, albeit at different levels: both proteins have a role in autophagic lysosome reformation, but only ZFYVE26 acts at the intersection between endocytosis and autophagy, thus representing a key player in these 2 processes. Indeed expression of the constitutively active form of RAB5A in cells with AR-SPG15-related mutations partially rescues the autophagy defect. Finally the model we propose demonstrates that autophagy and the endolysosomal pathway are central processes in the pathogenesis of these complicated forms of hereditary spastic paraparesis. Abbreviations: ALR, autophagic lysosome reformation; AP5, adaptor related protein complex 5; AR, autosomal-recessive; HSP, hereditary spastic paraplegia/paraparesis; ATG14, autophagy related 14; BafA, bafilomycin A1; BECN1, beclin 1; EBSS, Earle balanced salt solution; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GDP, guanosine diphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; HSP, hereditary spastic paraplegias; LBPA, lysobisphosphatidic acid; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MVBs, multivesicular bodies; PIK3C3, phosphatidylinositol 3-kinase, catalytic subunit type 3; PIK3R4, phosphoinositide-3-kinase regulatory subunit 4; PtdIns3P, phosphatidylinositol-3-phosphate; RFP, red fluorescent protein; RUBCN, RUN and cysteine rich domain containing beclin 1 interacting protein; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; TCC: thin corpus callosum; TF, transferrin; UVRAG, UV radiation resistance associated.

Asunto(s)

Autofagia/genética , Proteínas Portadoras/genética , Endocitosis/genética , Proteínas/genética , Degeneración Retiniana/genética , Paraplejía Espástica Hereditaria/genética , Proteínas Adaptadoras del Transporte Vesicular/sangre , Autofagosomas/metabolismo , Proteínas Portadoras/sangre , Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Femenino , Células HeLa , Humanos , Lisosomas/metabolismo , Masculino , Mutación , Proteínas/metabolismo , Degeneración Retiniana/sangre , Paraplejía Espástica Hereditaria/sangre , Proteínas de Unión al GTP rab/sangre , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rab2/sangre , Proteínas de Unión al GTP rab5/sangre , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo

RESUMEN

Improvements in survival rates for pancreatic cancer have been slow and the morality rate continues to increase in patients. MicroRNA (miR)­448 is reported to be significantly downregulated in several types of cancer. In this study, Rab2B is target of miR­488 was confirmed by bioinformatics analysis and validated using a luciferase reporter assay. A total of 72 cases of pancreatic cancer in patients diagnosed at The First Affiliated Hospital, School of Medicine, Zhejiang University (Hangzhou, China) were enrolled, and cancer specimens and their adjacent normal tissues were collected for analysis. The expression levels of miR­448 and Rab2B in these tissues and in pancreatic cancer cell lines were quantified using reverse transcription­polymerase chain reaction analysis. miR­448 overexpression was achieved by cell transfection. Protein expression was assessed using western blot analysis. Cell viability, cell cycle and apoptosis were analyzed using CCK­8 assay and flow cytometry, respectively. The results revealed a negative correlation between miR­448 and Rab2B in the pancreatic tissues and cell lines. The results of bioinformatics analysis indicated that miR­448 directly targeted Rab2B. Aberrant miR­448 levels in PANC­1 cells downregulated the expression of Rab2B, and significantly decreased cell proliferation and promoted apoptosis of cancer cells. It was also found that miR­448 mimics resulted in G0/G1 cell cycle arrest and affected the expression of cell cycle regulators, including cyclin D1, p21 and p27. In addition, the miR­448 mimics led to inactivation of the Akt/Mammalian target of rapamycin signaling pathway. The miR­448 mimics induced apoptosis and activated the expression of caspase­3, caspase­9 and poly(ADP­ribose) polymerase. The results suggested that miR­448 was a negative regulator of Rab2B and promoted cell cycle arrest and apoptosis in pancreatic cancer.

Asunto(s)

Biomarcadores de Tumor/metabolismo , MicroARNs/genética , Neoplasias Pancreáticas/patología , Proteína de Unión al GTP rab2/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Proliferación Celular , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Pronóstico , Transducción de Señal , Tasa de Supervivencia , Células Tumorales Cultivadas , Proteína de Unión al GTP rab2/genética

RESUMEN

At the onset of metamorphosis, Drosophila salivary gland cells undergo a burst of glue granule secretion to attach the forming pupa to a solid surface. Here, we show that excess granules evading exocytosis are degraded via direct fusion with lysosomes, a secretory granule-specific autophagic process known as crinophagy. We find that the tethering complex HOPS (homotypic fusion and protein sorting); the small GTPases Rab2, Rab7, and its effector, PLEKHM1; and a SNAP receptor complex consisting of Syntaxin 13, Snap29, and Vamp7 are all required for the fusion of secretory granules with lysosomes. Proper glue degradation within lysosomes also requires the Uvrag-containing Vps34 lipid kinase complex and the v-ATPase proton pump, whereas Atg genes involved in macroautophagy are dispensable for crinophagy. Our work establishes the molecular mechanism of developmentally programmed crinophagy in Drosophila and paves the way for analyzing this process in metazoans.

Asunto(s)

Autofagia/fisiología , Drosophila melanogaster/embriología , Proteínas del Pegamento Salivar de Drosophila/metabolismo , Lisosomas/metabolismo , Fusión de Membrana/fisiología , Vesículas Secretoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Células Cultivadas , Fosfatidilinositol 3-Quinasas Clase III/genética , Proteínas de Drosophila/genética , Proteínas del Pegamento Salivar de Drosophila/genética , Proteínas Qa-SNARE/genética , Proteínas R-SNARE/genética , Proteínas SNARE/genética , Proteínas de Unión al GTP rab/genética , Proteína de Unión al GTP rab2/genética , Proteínas de Unión a GTP rab7

RESUMEN

Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5), in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING), the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis.

Asunto(s)

Citosol/metabolismo , ADN/metabolismo , Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteína de Unión al GTP rab2/metabolismo , Animales , Antivirales/farmacología , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Inmunidad Innata/efectos de los fármacos , Factor 3 Regulador del Interferón/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Nucleotidiltransferasas/metabolismo , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Virus Vaccinia/efectos de los fármacos , Virus Vaccinia/fisiología

RESUMEN

Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes.

Asunto(s)

Autofagosomas/enzimología , Autofagia , Neoplasias de la Mama/enzimología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Endocitosis , Endosomas/enzimología , Lisosomas/enzimología , Proteína de Unión al GTP rab2/metabolismo , Animales , Animales Modificados Genéticamente , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Humanos , Fusión de Membrana , Proteolisis , Interferencia de ARN , Transducción de Señal , Transfección , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rab2/genética , Proteínas de Unión a GTP rab7

RESUMEN

Apoptotic cells generated by programmed cell death are engulfed by phagocytes and enclosed within plasma membrane-derived phagosomes. Maturation of phagosomes involves a series of membrane-remodeling events that are governed by the sequential actions of Rab GTPases and lead to formation of phagolysosomes, where cell corpses are degraded. Here we identified gop-1 as a novel regulator of apoptotic cell clearance in Caenorhabditis elegans Loss of gop-1 affects phagosome maturation through the RAB-5-positive stage, causing defects in phagosome acidification and phagolysosome formation, phenotypes identical to and unaffected by loss of unc-108, the C. elegans Rab2 GOP-1 transiently associates with cell corpse-containing phagosomes, and loss of its function abrogates phagosomal association of UNC-108. GOP-1 interacts with GDP-bound and nucleotide-free UNC-108/Rab2, disrupts GDI-UNC-108 complexes, and promotes activation and membrane recruitment of UNC-108/Rab2 in vitro. Loss of gop-1 also abolishes association of UNC-108 with endosomes, causing defects in endosome and dense core vesicle maturation. Thus, GOP-1 is an activator of UNC-108/Rab2 in multiple processes.

Asunto(s)

Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Lectinas Tipo C/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteína de Unión al GTP rab2/metabolismo , Animales , Animales Modificados Genéticamente , Apoptosis , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Endocitosis , Endosomas/enzimología , Activación Enzimática , Genotipo , Lectinas Tipo C/genética , Microscopía Fluorescente , Microscopía por Video , Proteínas de Transporte de Monosacáridos/genética , Fagosomas/enzimología , Fenotipo , Unión Proteica , Transporte de Proteínas , Vesículas Secretoras/enzimología , Transducción de Señal , Factores de Tiempo , Imagen de Lapso de Tiempo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo