Inhibition of Rab1B Impairs Trafficking and Maturation of SARS-CoV-2 Spike Protein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) utilizes cellular trafficking pathways to process its structural proteins and move them to the site of assembly. Nevertheless, the exact process of assembly and subcellular trafficking of SARS-CoV-2 proteins remains largely unknown. Here, we have identified and characterized Rab1B as an important host factor for the trafficking and maturation of the spike protein (S) after synthesis at the endoplasmic reticulum (ER). Using confocal microscopy, we showed that S and Rab1B substantially colocalized in compartments of the early secretory pathway. Co-expression of dominant-negative (DN) Rab1B N121I leads to an aberrant distribution of S into perinuclear spots after ectopic expression and in SARS-CoV-2-infected cells caused by either structural rearrangement of the ERGIC or Golgi or missing interaction between Rab1B and S. Western blot analyses revealed a complete loss of the mature, cleaved S2 subunit in cell lysates and culture supernatants upon co-expression of DN Rab1B N121I. In sum, our studies indicate that Rab1B is an important regulator of trafficking and maturation of SARS-CoV-2 S, which not only improves our understanding of the coronavirus replication cycle but also may have implications for the development of antiviral strategies.

Dual function of Rab1A in secretion and autophagy: hypervariable domain dependence.

We currently understand how the different intracellular pathways, secretion, endocytosis, and autophagy are regulated by small GTPases. In contrast, it is unclear how these pathways are coordinated to ensure efficient cellular response to stress. Rab GTPases localize to specific organelles through their hypervariable domain (HVD) to regulate discrete steps of individual pathways. Here, we explored the dual role of Rab1A/B (92% identity) in secretion and autophagy. We show that although either Rab1A or Rab1B is required for secretion, Rab1A, but not Rab1B, localizes to autophagosomes and is required early in stress-induced autophagy. Moreover, replacing the HVD of Rab1B with that of Rab1A enables Rab1B to localize to autophagosomes and regulate autophagy. Therefore, Rab1A-HVD is required for the dual functionality of a single Rab in two different pathways: secretion and autophagy. In addition to this mechanistic insight, these findings are relevant to human health because both the pathways and Rab1A/B were implicated in diseases ranging from cancer to neurodegeneration.

LINC00173 facilitates tumor progression by stimulating RAB1B-mediated PA2G4 and SDF4 secretion in nasopharyngeal carcinoma.

An increasing number of studies have found that long non-coding RNA (lncRNA) play important roles in driving the progression of nasopharyngeal carcinoma (NPC). Our microarray screening revealed that expression of the lncRNA long intergenic non-protein coding RNA 173 (LINC00173) was upregulated in NPC. However, its role and mechanism in NPC have not yet been elucidated. In this study, we demonstrate that high LINC00173 expression indicated a poor prognosis in NPC patients. Knockdown of LINC00173 significantly inhibited NPC cell proliferation, migration and invasion in vitro. Mechanistically, LINC00173 interacted and colocalized with Ras-related protein Rab-1B (RAB1B) in the cytoplasm, but the modulation of LINC00173 expression did not affect the expression of RAB1B at either the mRNA or protein levels. Instead, relying on the stimulation of RAB1B, LINC00173 could facilitate the extracellular secretion of proliferation-associated 2G4 (PA2G4) and stromal cell-derived factor 4 (SDF4; also known as 45-kDa calcium-binding protein) proteins, and knockdown of these proteins could reverse the NPC aggressive phenotype induced by LINC00173 overexpression. Moreover, in vivo LINC00173-knockdown models exhibited a marked slowdown in tumor growth and a significant reduction in lymph node and lung metastases. In summary, LINC00173 serves as a crucial driver for NPC progression, and the LINC00173-RAB1B-PA2G4/SDF4 axis might provide a potential therapeutic target for NPC patients.

Circ_0026123 promotes cisplatin resistance and progression of ovarian cancer by upregulating RAB1A through sequestering miR-543.

BACKGROUND: Circular RNAs can act as critical regulators in the tumorigenesis and chemoresistance of ovarian cancer (OC). Herein, this work aimed to probe the function and mechanism of circ_0026123 in the cisplatin (DDP) resistance and progression of OC and its potential value in the clinic. METHODS: The quantitative real-time PCR and western blotting were used to detect the levels of RNAs and proteins. In vitro experiments were conducted using CCK-8, EdU, transwell, tube formation assays and flow cytometry. Mouse subcutaneous xenograft model was used for in vivo experiments. The interaction between circ_0026123 or RAB1A (Ras-related protein Rab-1A) and miR-543 was confirmed using dual-luciferase reporter and RNA immunoprecipitation assays. RESULTS: Circ_0026123 expression was higher in DDP-resistant OC tissues and cells. Silencing of circ_0026123 dramatically boosted the sensitivity of DDP-resistant OC cells to DDP, as well as inhibited cell growth, angiogenesis, invasion and migration abilities in vitro. Circ_0026123 functionally targeted miR-543, and knockdown of miR-543 reversed the impacts of circ_0026123 deficiency on DDP sensitivity and the malignant behaviors of DDP-resistant OC cells. RAB1A was a target of miR-543, RAB1A overexpression attenuated the inhibitory functions of miR-543 on DDP resistance and the malignant phenotypes of DDP-resistant OC cells. Preclinically, lentivirus-mediated circ_0026123 downregulation also suppressed OC growth and enhanced DDP cytotoxicity in vivo. CONCLUSION: Our study demonstrated that circ_0026123 acted as a sponge for miR-543 to elevate RAB1A expression, thus promoting cisplatin resistance and tumorigenesis in ovarian cancer.

Circ_0061140 Contributes to the Malignant Progression in Ovarian Cancer Cells by Mediating the RAB1A Level Through Sponging miR-361-5p.

Ovarian cancer (OC) progression is related to many functional molecules, including circular RNAs (circRNAs). Hsa_circ_0061140 (circ_0061140) promoted cell growth and metastasis in OC. The aim of this study was to explore a specific functional mechanism of circ_0061140. Reverse transcription-quantitative polymerase chain reaction was performed for expression analysis of circ_0061140, microRNA-361-5p (miR-361-5p), and Ras-like protein in rat brain 1A (RAB1A). Cell proliferation was determined using Cell Counting Kit-8 assay, EdU assay, and colony formation assay. The migration and invasion were assessed through transwell assay. Tube formation assay was used for angiogenesis analysis. Cell apoptosis was evaluated using flow cytometry. The protein levels of epithelial-to-mesenchymal transition (EMT) markers and RAB1A were detected via western blot. Target analysis was performed by dual-luciferase reporter assay and RNA immunoprecipitation assay. In vivo research was conducted using xenograft model. The circ_0061140 level was upregulated in OC samples and cells. Downregulation of circ_0061140 impeded proliferation, migration, invasion, EMT, and angiogenesis of OC cells. Circ_0061140 directly interacted with miR-361-5p to act as a miRNA sponge. The miR-361-5p inhibition reversed the si-circ_0061140-induced anti-tumor function in OC cells. RAB1A was a downstream target of miR-361-5p, and miR-361-5p served as a tumor repressor in OC via inhibiting the level of RAB1A. Circ_0061140 could increase the RAB1A expression by sponging miR-361-5p in OC cells. Circ_0061140 also facilitated tumorigenesis in vivo through targeting the miR-361-5p/RAB1A axis. All results demonstrated that circ_0061140 promoted OC development by inhibiting miR-361-5p to upregulate the expression of RAB1A.

The Small GTPase FgRab1 Plays Indispensable Roles in the Vegetative Growth, Vesicle Fusion, Autophagy and Pathogenicity of Fusarium graminearum.

Rab GTPases are key regulators of membrane and intracellular vesicle transports. However, the biological functions of FgRab1 are still unclear in the devastating wheat pathogen Fusarium graminearum. In this study, we generated constitutively active (CA) and dominant-negative (DN) forms of FgRAB1 from the wild-type PH-1 background for functional analyses. Phenotypic analyses of these mutants showed that FgRab1 is important for vegetative growth, cell wall integrity and hyphal branching. Compared to the PH-1 strain, the number of spores produced by the Fgrab1DN strain was significantly reduced, with obviously abnormal conidial morphology. The number of septa in the conidia of the Fgrab1DN mutant was fewer than that observed in the PH-1 conidia. Fgrab1DN was dramatically reduced in its ability to cause Fusarium head blight symptoms on wheat heads. GFP-FgRab1 was observed to partly localize to the Golgi apparatus, endoplasmic reticulum and Spitzenkörper. Furthermore, we found that FgRab1 inactivation blocks not only the transport of the v-SNARE protein FgSnc1 from the Golgi to the plasma membrane but also the fusion of endocytic vesicles with their target membranes and general autophagy. In summary, our results indicate that FgRab1 plays vital roles in vegetative growth, conidiogenesis, pathogenicity, autophagy, vesicle fusion and trafficking in F. graminearum.

Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

CgRab1 regulates Cgcathepsin L1 expression and participates in the phagocytosis of haemocytes in oyster Crassostrea gigas.

Rab protein plays an important role in a variety of cellular activities, especially the fusion process of the inner membrane during endocytosis. In the present study, a Rab1 protein (CgRab1) was identified from the Pacific oyster Crassostrea gigas. The full-length cDNA sequence of CgRab1 was of 2248 bp with an open reading frame of 618 bp, encoding a polypeptide of 205 amino acids containing a Rab domain. The deduced amino acid sequence of CgRab1 shared 94.2% and 89.3% identity with Rab1 from Pomacea canaliculata and Homo sapiens respectively. In the phylogenetic tree, CgRab1 was firstly clustered with the Rab1s from Aplysia californica and Pomacea canaliculata to form a sister group with Rab1 from invertebrates. The recombinant CgRab1 protein (rCgRab1) was able to bind GTP. The mRNA transcripts of CgRab1 were highly expressed in oyster haemocytes, and its expression level in oyster haemocytes was significantly up-regulated at 24 h after the stimulations with Vibro splendidus, which was 2.43-fold (p < 0.01) of that in the control group. After the expression of CgRab1 was knocked down (0.38-fold of that in EGFP-RNAi experimental group) by RNAi，the protein expression of Cgcathepsin L1 were reduced (0.63-fold, p < 0.01) compared with that in EGFP-RNAi experimental group. The phagocytic rate and phagocytic index of haemocytes in CgRab1-RNAi oysters decreased after V. splendidus stimulation, which was 0.50-fold (p < 0.01) and 0.58-fold (p < 0.01) of that in EGFP-RNAi experimental group. These results indicated that CgRab1 was involved in the process of haemocytes phagocytosis by regulating the expression of Cgcathepsin L1 in oyster C. gigas.

Establishment and analysis of conditional Rab1- and Rab5-knockout cells using the auxin-inducible degron system.

Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. They have recently been found to be essential for cell survival and/or growth in cultured mammalian cells, thereby precluding the establishment of Rab1-knockout (KO) and Rab5-KO cells, making it extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed cell lines with conditional KO (CKO) of either Rab1 (Rab1A and Rab1B) or Rab5 (Rab5A, Rab5B and Rab5C) by using the auxin-inducible protein degradation system. Rab1 CKO and Rab5 CKO led to eventual cell death from 18 h and 48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and endoplasmic reticulum (ER)-to-Golgi trafficking was severely inhibited. Moreover, we discovered a novel Rab1-depletion phenotype: perinuclear clustering of early endosomes and delayed transferrin recycling. In contrast, acute Rab5 protein depletion resulted in loss of early endosomes and late endosomes, but lysosomes appeared to be normal. We also observed a dramatic reduction in the intracellular signals of endocytic cargos via receptor-mediated or fluid-phase endocytosis in Rab5-depleted cells.

Focus on the Small GTPase Rab1: A Key Player in the Pathogenesis of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disease. It is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of large aggregates in the survival neurons called Lewy bodies, which mainly contain α-synuclein (α-syn). The cause of cell death is not known but could be due to mitochondrial dysfunction, protein homeostasis failure, and alterations in the secretory/endolysosomal/autophagic pathways. Survival nigral neurons overexpress the small GTPase Rab1. This protein is considered a housekeeping Rab that is necessary to support the secretory pathway, the maintenance of the Golgi complex structure, and the regulation of macroautophagy from yeast to humans. It is also involved in signaling, carcinogenesis, and infection for some pathogens. It has been shown that it is directly linked to the pathogenesis of PD and other neurodegenerative diseases. It has a protective effect against α-σψν toxicity and has recently been shown to be a substrate of LRRK2, which is the most common cause of familial PD and the risk of sporadic disease. In this review, we analyze the key aspects of Rab1 function in dopamine neurons and its implications in PD neurodegeneration/restauration. The results of the current and former research support the notion that this GTPase is a good candidate for therapeutic strategies.

Rab1A promotes cell proliferation and migration by upregulating Gli1 in colorectal cancer.

Rab1A, as a highly conserved small guanosine triphosphatase (GTPase), plays contentious roles in different types of cancers. The role of Rab1A in colorectal cancer (CRC) has been described in previous studies, but the molecular mechanisms of Rab1A in CRC remain far from being addressed. In the present study, we found that Rab1A expression was significantly upregulated in CRC tissues and increased Rab1A expression correlated with tumor size, lymph node metastasis (LNM) and tumor-node-metastasis (TNM) stage of CRC patients. We also found that Rab1A exerts its promotive effect on CRC cell proliferation, migration and EMT progress. Further mechanistic experiments showed that glioma-associated oncogene-1 (Gli1), as a key transcriptional factor of the Hedgehog pathway, was implicated in Rab1A-mediated regulation of CRC cell proliferation and migration. In addition, Rab1A upregulated Gli1 expression through Smoothened homolog (SMO)-independent pathway. Finally, Rab1A activated mechanistic target of rapamycin (mTOR) signaling in CRC cells. Collectively, our results define Rab1A as a novel regulator of Gli1 to promote CRC cell proliferation and migration, and suggest that the Rab1A/mTOR/Gli1 axis may serve as a promising therapeutic target for the treatment of CRC.

Mycobacterium tuberculosis PE_PGRS20 and PE_PGRS47 Proteins Inhibit Autophagy by Interaction with Rab1A.

Autophagy is a fundamental cellular process that has important roles in innate and adaptive immunity against a broad range of microbes. Many pathogenic microbes have evolved mechanisms to evade or exploit autophagy. It has been previously demonstrated that induction of autophagy can suppress the intracellular survival of mycobacteria, and several PE_PGRS family proteins of Mycobacterium tuberculosis have been proposed to act as inhibitors of autophagy to promote mycobacterial survival. However, the mechanisms by which these effectors inhibit autophagy have not been defined. Here, we report detailed studies of M. tuberculosis deletion mutants of two genes, pe_pgrs20 and pe_pgrs47, that we previously reported as having a role in preventing autophagy of infected host cells. These mutants resulted in increased autophagy and reduced intracellular survival of M. tuberculosis in macrophages. This phenotype was accompanied by increased cytokine production and antigen presentation by infected cells. We further demonstrated that autophagy inhibition by PE_PGRS20 and PE_PGRS47 resulted from canonical autophagy rather than autophagy flux inhibition. Using macrophages transfected to express PE_PGRS20 or PE_PGRS47, we showed that these proteins inhibited autophagy initiation directly by interacting with Ras-related protein Rab1A. Silencing of Rab1A in mammalian cells rescued the survival defects of the pe_pgrs20 and pe_pgrs47 deletion mutant strains and reduced cytokine secretion. To our knowledge, this is the first study to identify mycobacterial effectors that directly interact with host proteins responsible for autophagy initiation. IMPORTANCE Tuberculosis is a significant global infectious disease caused by infection of the lungs with Mycobacterium tuberculosis, which then resides and replicates mainly within host phagocytic cells. Autophagy is a complex host cellular process that helps control intracellular infections and enhance innate and adaptive immune responses. During coevolution with humans, M. tuberculosis has acquired various mechanisms to inhibit host cellular processes, including autophagy. We identified two related M. tuberculosis proteins, PE_PGRS20 and PE_PGRS47, as the first reported examples of specific mycobacterial effectors interfering with the initiation stage of autophagy. Autophagy regulation by these PE_PGRS proteins leads to increased bacterial survival in phagocytic cells and increased autophagic degradation of mycobacterial antigens to stimulate adaptive immune responses. A better understanding of how M. tuberculosis regulates autophagy in host cells could facilitate the design of new and more effective therapeutics or vaccines against tuberculosis.

Arabidopsis RAB8A, RAB8B and RAB8D Proteins Interact with Several RTNLB Proteins and are Involved in the Agrobacterium tumefaciens Infection Process.

Arabidopsis thaliana small GTP-binding proteins, AtRAB8s, associate with the endomembrane system and modulate tubulovesicular trafficking between compartments of the biosynthetic and endocytic pathways. There are five members in Arabidopsis, namely AtRAB8A-8E. Yeast two-hybrid assays, bimolecular fluorescence complementation assays and glutathione-S-transferase pull-down assays showed that RAB8A, 8B and 8D interacted with several membrane-associated reticulon-like (AtRTNLB) proteins in yeast, plant cells and in vitro. Furthermore, RAB8A, 8B and 8D proteins showed interactions with the Agrobacterium tumefaciens virulence protein, VirB2, a component of a type IV secretion system (T4SS). A. tumefaciens uses a T4SS to transfer T-DNA and Virulence proteins to plants, which causes crown gall disease in plants. The Arabidopsis rab8A, rab8B and rab8D single mutants showed decreased levels of Agrobacterium-mediated root and seedling transformation, while the RAB8A, 8B and 8D overexpression transgenic Arabidopsis plants were hypersusceptible to A. tumefaciens and Pseudomonas syringae infections. RAB8A-8E transcripts accumulated differently in roots, rosette leaves, cauline leaves, inflorescence and flowers of wild-type plants. In summary, RAB8A, 8B and 8D interacted with several RTNLB proteins and participated in A. tumefaciens and P. syringae infection processes.

Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes.

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.

Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1.

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle - the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.

Rab1A promotes cancer metastasis and radioresistance through activating GSK-3ß/Wnt/ß-catenin signaling in nasopharyngeal carcinoma.

Many articles have reported that Rab1A was overexpressed in a variety of human cancers and involved in tumor progression and metastasis. However, the biological function and molecular mechanism of Rab1A in nasopharyngeal carcinoma (NPC) remained unknown until now. Here we found that Rab1A overexpression is a common event and was positively associated with distant metastasis and poor prognosis of NPC patients. Functionally, Rab1A depletion inhibited the migration and EMT phenotype of NPC cells, whereas Rab1A overexpression led to the opposite effect. Furthermore, we reveal an important role for Rab1A protein in the induction of radioresistance via regulating homologous recombination (HR) signaling pathway. Mechanistically, Rab1A activated Wnt/ß-catenin signaling by inhibiting the activity of GSK-3ß via phosphorylation at Ser9. Then Wnt/ß-catenin signaling induced NPC cells radioresistance and metastasis through nuclear translocation of ß-catenin and transcription upregulation of HR pathway-related and EMT-related genes expression. In general, this study shows that Rab1A may serve as a potential biomarker for predicting prognosis in NPC patients. Targeting Rab1A and Wnt/ß-catenin signaling may hold promise to overcome NPC radioresistance.

Human Golgi phosphoprotein 3 is an effector of RAB1A and RAB1B.

Golgi phosphoprotein 3 (GOLPH3) is a peripheral membrane protein localized at the trans-Golgi network that is also distributed in a large cytosolic pool. GOLPH3 has been involved in several post-Golgi protein trafficking events, but its precise function at the molecular level is not well understood. GOLPH3 is also considered the first oncoprotein of the Golgi apparatus, with important roles in several types of cancer. Yet, it is unknown how GOLPH3 is regulated to achieve its contribution in the mechanisms that lead to tumorigenesis. Binding of GOLPH3 to Golgi membranes depends on its interaction to phosphatidylinositol-4-phosphate. However, an early finding showed that GTP promotes the binding of GOLPH3 to Golgi membranes and vesicles. Nevertheless, it remains largely unknown whether this response is consequence of the function of GTP-dependent regulatory factors, such as proteins of the RAB family of small GTPases. Interestingly, in Drosophila melanogaster the ortholog of GOLPH3 interacts with- and behaves as effector of the ortholog of RAB1. However, there is no experimental evidence implicating GOLPH3 as a possible RAB1 effector in mammalian cells. Here, we show that human GOLPH3 interacted directly with either RAB1A or RAB1B, the two isoforms of RAB1 in humans. The interaction was nucleotide dependent and it was favored with GTP-locked active state variants of these GTPases, indicating that human GOLPH3 is a bona fide effector of RAB1A and RAB1B. Moreover, the expression in cultured cells of the GTP-locked variants resulted in less distribution of GOLPH3 in the Golgi apparatus, suggesting an intriguing model of GOLPH3 regulation.

Micro and Martsolf syndromes in 34 new patients: Refining the phenotypic spectrum and further molecular insights.

Micro and Martsolf syndromes are rare clinically and genetically overlapping disorders caused by mutations in RAB3GAP1, RAB3GAP2, RAB18 and TBC1D20 genes. We describe 34 new patients, 27 with Micro and seven with Martsolf. Patients presented with the characteristic clinical manifestations of the two syndromes, including postnatal microcephaly, congenital cataracts, microphthalmia, optic atrophy, spasticity and intellectual disability. Brain imaging showed in the majority of cases polymicrogyria, thin corpus callosum, cortical atrophy, and white matter dysmyelination. Unusual additional findings were pectus excavatum (four patients), pectus carinatum (three patients), congenital heart disease (three patients) and bilateral calcification in basal ganglia (one patient). Mutational analysis of RAB3GAP1 and RAB3GAP2 revealed 21 mutations, including 14 novel variants. RAB3GAP1 mutations were identified in 22 patients with Micro, including a deletion of the entire gene in one patient. On the other hand, RAB3GAP2 mutations were identified in two patients with Micro and all Martsolf patients. Moreover, exome sequencing unraveled a TBC1D20 mutation in an additional family with Micro syndrome. Our results expand the phenotypic and mutational spectrum associated with Micro and Martsolf syndromes. Due to the overlapped severities and genetic basis of both syndromes, we suggest to be comprehended as one entity "Micro/Martsolf spectrum" or "RAB18 deficiency."

Rab1b-GBF1-ARFs mediated intracellular trafficking is required for classical swine fever virus replication in swine umbilical vein endothelial cells.

Classical swine fever virus (CSFV), a plus-sense RNA virus, utilizes host intracellular membrane organelles for its replication. Our previous studies have shown that disruption of the intracellular membrane-trafficking events can inhibit CSFV replication. However, the underlying mechanism of this process in CSFV infection has not been elucidated. To determine the role of Golgi-associated anterograde and retrograde trafficking in CSFV replication, we revealed the effect of vesicular transport between Golgi and ER inhibitors Brefeldin A (BFA) and 2,2-methyl-N-(2,4,6,-trimethoxyphenyl) dodecanamide (CI-976), the GBF1 inhibitor golgicide A (GCA) on virus production. Our results showed that disruption of vesicular trafficking by BFA, CI-976, and GCA significantly inhibited CSFV infection. Subsequent experiments revealed that knockdown of Rab1b by lentiviruses and negative-mutant Rab1b-N121I transfection inhibited CSFV infection. Furthermore, we showed that the Rab1b downstream vesicular component effectors GBF1, and class I and class II ADP-ribosylation factors (ARFs) were also involved in virus replication. In addition, confocal microscopy assay showed that CSFV infection disrupted the Golgi apparatus resulting in extended Golgi distribution around the nucleus. We also showed that cell secretory pathway, measured using Gaussia luciferase flash assay, was blocked in CSFV infected cells. Taken together, these findings demonstrate that CSFV utilizes Rab1b-GBF1-ARFs mediated trafficking to promote its own replication. These findings also provide new insights into the intracellular trafficking pathways utilized for CSFV life cycle.