Golgi-Resident Gαo Promotes Protrusive Membrane Dynamics.

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR → Gαo → Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues.

Cryo-EM structure of metazoan TRAPPIII, the multi-subunit complex that activates the GTPase Rab1.

The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo-EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur-TRAPP.

SNX32 is a host restriction factor that degrades African swine fever virus CP204L via the RAB1B-dependent autophagy pathway.

African swine fever virus (ASFV) causes a highly contagious and deadly disease in domestic pigs and European wild boars, posing a severe threat to the global pig industry. ASFV CP204L, a highly immunogenic protein, is produced during the early stages of ASFV infection. However, the impact of CP204L protein-interacting partners on the outcome of ASFV infection is poorly understood. To accomplish this, coimmunoprecipitation and mass spectrometry analysis were conducted in ASFV-infected porcine alveolar macrophages (PAMs). We have demonstrated that sorting nexin 32 (SNX32) is a CP204L-binding protein and that CP204L interacted and colocalized with SNX32 in ASFV-infected PAMs. ASFV growth and replication were promoted by silencing SNX32 and suppressed by overexpressing SNX32. SNX32 degraded CP204L by recruiting the autophagy-related protein Ras-related protein Rab-1b (RAB1B). RAB1B overexpression inhibited ASFV replication, while knockdown of RAB1B had the opposite effect. Additionally, RAB1B, SNX32, and CP204L formed a complex upon ASFV infection. Taken together, this study demonstrates that SNX32 antagonizes ASFV growth and replication by recruiting the autophagy-related protein RAB1B. This finding extends our understanding of the interaction between ASFV CP204L and its host and provides new insights into exploring the relationship between ASFV infection and autophagy.IMPORTANCEAfrican swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with a high mortality near 100% in domestic pigs. ASF virus (ASFV), which is the only member of the family Asfarviridae, is a dsDNA virus of great complexity and size, encoding more than 150 proteins. Currently, there are no available vaccines against ASFV. ASFV CP204L represents the most abundantly expressed viral protein early in infection and plays an important role in regulating ASFV replication. However, the mechanism by which the interaction between ASFV CP204L and host proteins affects ASFV replication remains unclear. In this study, we demonstrated that the cellular protein SNX32 interacted with CP204L and degraded CP204L by upregulating the autophagy-related protein RAB1B. In summary, this study will help us understand the interaction mechanism between CP204L and its host upon infection and provide new insights for the development of vaccines and antiviral drugs.

MicroR-380-3p Reduces Sepsis-Induced Acute Kidney Injury via Regulating RAB1P to Restrain NF-κB Pathway.

Septic acute kidney injury (AKI) is a common complication in critically ill patients with high morbidity and mortality. This study intends to clarify the clinical value and molecular mechanism of microR-380-3p in septic AKI by recruiting patients with septic AKI and establishing septic AKI cell models. Patients with septic AKI were included and human kidney-2 (HK-2) cells were induced by lipopolysaccharide (LPS) to construct the AKI cell model of sepsis. The expression of microR-380-3p was detected by quantitative real-time RT-PCR (qRT-PCR). The expression of Bax, cleaved caspase 3, Bcl-2, p65, and p-p65 was detected by Western blot. The contents of inflammation and oxidation were determined by commercial kits. Bioinformatics predicted the binding target of microR-380-3p and a dual luciferase reporting system was used to verify the regulatory relationship between microR-380-3p and RAP1B. The concentration of microR-380-3p was elevated in patients with septic AKI and appeared to be a biomarker for these patients. Silenced microR-380-3p reversed the damage of LPS on HK-2 cells via promoting viability, inhibiting apoptosis, inflammation, and oxidation. RAP1B was a target of microR-380-3p and microR-380-3p exerted targeted inhibition of RAP1B expression level. Down-regulation of RAP1B reversed the influence of silenced microR-380-3p on HK-2 cells. MicroR-380-3p/RAP1B participated in activating the NF-κB pathway. MicroR-380-3p down-regulated RAP1B to exacerbate septic AKI, providing a potential therapeutic biomarker for septic AKI.

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.

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.

Conformational control of small GTPases by AMPylation.

Posttranslational modifications (PTMs) are important physiological means to regulate the activities and structures of central regulatory proteins in health and disease. Small GTPases have been recognized as important molecules that are targeted by PTMs during infections of mammalian cells by bacterial pathogens. The enzymes DrrA/SidM and AnkX from Legionella pneumophila AMPylate and phosphocholinate Rab1b during infection, respectively. Cdc42 is AMPylated by IbpA from Histophilus somni at tyrosine 32 or by VopS from Vibrio parahaemolyticus at threonine 35. These modifications take place in the important regulatory switch I or switch II regions of the GTPases. Since Rab1b and Cdc42 are central regulators of intracellular vesicular trafficking and of the actin cytoskeleton, their modifications by bacterial pathogens have a profound impact on the course of infection. Here, we addressed the biochemical and structural consequences of GTPase AMPylation and phosphocholination. By combining biochemical experiments and NMR analysis, we demonstrate that AMPylation can overrule the activity state of Rab1b that is commonly dictated by binding to guanosine diphosphate or guanosine triphosphate. Thus, PTMs may exert conformational control over small GTPases and may add another previously unrecognized layer of activity control to this important regulatory protein family.

The fungal RABOME: RAB GTPases acting in the endocytic and exocytic pathways of Aspergillus nidulans (with excursions to other filamentous fungi).

RAB GTPases are major determinants of membrane identity that have been exploited as highly specific reporters to study intracellular traffic in vivo. A score of fungal papers have considered individual RABs, but systematic, integrated studies on the localization and physiological role of these regulators and their effectors have been performed only with Aspergillus nidulans. These studies have influenced the intracellular trafficking field beyond fungal specialists, leading to findings such as the maturation of trans-Golgi (TGN) cisternae into post-Golgi RAB11 secretory vesicles, the concept that these RAB11 secretory carriers are loaded with three molecular nanomotors, the understanding of the role of endocytic recycling mediated by RAB6 and RAB11 in determining the hyphal mode of life, the discovery that early endosome maturation and the ESCRT pathway are essential, the identification of specific adaptors of dynein-dynactin to RAB5 endosomes, the exquisite dependence that autophagy displays on RAB1 activity, the role of TRAPPII as a GEF for RAB11, or the conclusion that the RAB1-to-RAB11 transition is not mediated by TRAPP maturation. A remarkable finding was that the A. nidulans Spitzenkörper contains four RABs: RAB11, Sec4, RAB6, and RAB1. How these RABs cooperate during exocytosis represents an as yet outstanding question.

Rab29 activation of the Parkinson's disease-associated LRRK2 kinase.

Parkinson's disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector-binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson's patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans-Golgi network and greatly stimulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild-type LRRK2. We identify conserved residues within the LRRK2 ankyrin domain that are required for Rab29-mediated Golgi recruitment and kinase activation. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2-mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interacting with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation.

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.

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.

TRAPP complexes in membrane traffic: convergence through a common Rab.

Transport protein particle (TRAPP; also known as trafficking protein particle), a multimeric guanine nucleotide-exchange factor for the yeast GTPase Ypt1 and its mammalian homologue, RAB1, regulates multiple membrane trafficking pathways. TRAPP complexes exist in three forms, each of which activates Ypt1 or RAB1 through a common core of subunits and regulates complex localization through distinct subunits. Whereas TRAPPI and TRAPPII tether coated vesicles during endoplasmic reticulum to Golgi and intra-Golgi traffic, respectively, TRAPPIII has recently been shown to be required for autophagy. These advances illustrate how the TRAPP complexes link Ypt1 and RAB1 activation to distinct membrane-tethering events.

Screening Legionella effectors for antiviral effects reveals Rab1 GTPase as a proviral factor coopted for tombusvirus replication.

Bacterial virulence factors or effectors are proteins targeted into host cells to coopt or interfere with cellular proteins and pathways. Viruses often coopt the same cellular proteins and pathways to support their replication in infected cells. Therefore, we screened the Legionella pneumophila effectors to probe virus-host interactions and identify factors that modulate tomato bushy stunt virus (TBSV) replication in yeast surrogate host. Among 302 Legionella effectors tested, 28 effectors affected TBSV replication. To unravel a coopted cellular pathway in TBSV replication, the identified DrrA effector from Legionella was further exploited. We find that expression of DrrA in yeast or plants blocks TBSV replication through inhibiting the recruitment of Rab1 small GTPase and endoplasmic reticulum-derived COPII vesicles into the viral replication compartment. TBSV hijacks Rab1 and COPII vesicles to create enlarged membrane surfaces and optimal lipid composition within the viral replication compartment. To further validate our Legionella effector screen, we used the Legionella effector LepB lipid kinase to confirm the critical proviral function of PI(3)P phosphoinositide and the early endosomal compartment in TBSV replication. We demonstrate the direct inhibitory activity of LegC8 effector on TBSV replication using a cell-free replicase reconstitution assay. LegC8 inhibits the function of eEF1A, a coopted proviral host factor. Altogether, the identified bacterial effectors with anti-TBSV activity could be powerful reagents in cell biology and virus-host interaction studies. This study provides important proof of concept that bacterial effector proteins can be a useful toolbox to identify host factors and cellular pathways coopted by (+)RNA viruses.

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.

Influence of a Ser111-phosphorylation on Rab1b GTPase conformational dynamics studied by advanced sampling simulations.

Rab GTPases constitute the largest branch of the Ras protein superfamily that regulate intra-cellular membrane trafficking. Their signaling activity is mediated by the transition between an active GTP-bound state and an inactive GDP-bound state. In the inactive state the switch I and II segments adopt largely disordered flexible conformations, whereas in the active state these regions are in well-defined conformations. The switch I and II states are central for recognition of Rab GTPases by interacting partners. Phosphorylation of the Rab1b-GTPase at residue Ser111 (pS111) results in modulation of the signaling activity due to alterations of the protein interaction interface and also due to modulation of the conformational flexibility. We have studied the flexibility of native and pS111-Rab1b in complex with GTP or GDP using extensive Molecular Dynamics (MD) simulations and an advanced sampling method called DIhedral Angle-biasing potential Replica-Exchange Molecular dynamics (DIA-REMD). The DIA-REMD method promotes backbone and side chain dihedral transitions along a series of replica simulations in selected protein segments and through exchanges also improves sampling in an unbiased reference simulation. Application to the Rab1b system results in significantly enhanced sampling of different switch I/II conformational states in the GDP-bound Rab1b state. The pS111 modification is found to reduce the conformational flexibility even in the presence of GDP, which may influence signaling activities. The stabilizing effect can be attributed to the formation of additional surface salt bridges between Arg-residues and pS111 not present in the native structure. The DIA-REMD method could be a valuable approach for studying also other signaling proteins that contain flexible segments.

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.

COPI-TRAPPII activates Rab18 and regulates its lipid droplet association.

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII-specific subunits by various methods including siRNA depletion and CRISPR-Cas9-mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII-deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.

The GTPase Rab1 Is Required for NLRP3 Inflammasome Activation and Inflammatory Lung Injury.

Uncontrolled inflammatory response during sepsis predominantly contributes to the development of multiorgan failure and lethality. However, the cellular and molecular mechanisms for excessive production and release of proinflammatory cytokines are not clearly defined. In this study, we show the crucial role of the GTPase Ras-related protein in brain (Rab)1a in regulating the nucleotide binding domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation and lung inflammatory injury. Expression of dominant negative Rab1 N124I plasmid in bone marrow-derived macrophages prevented the release of IL-1ß and IL-18, NLRP3 inflammasome activation, production of pro-IL-1ß and pro-IL-18, and attenuated TLR4 surface expression and NF-кB activation induced by bacterial LPS and ATP compared with control cells. In alveolar macrophage-depleted mice challenged with cecal ligation and puncture, pulmonary transplantation of Rab1a-inactivated macrophages by expression of Rab1 N124I plasmid dramatically reduced the release of IL-1ß and IL-18, neutrophil count in bronchoalveolar lavage fluid, and inflammatory lung injury. Rab1a activity was elevated in alveolar macrophages from septic patients and positively associated with severity of sepsis and respiratory dysfunction. Thus, inhibition of Rab1a activity in macrophages resulting in the suppression of NLRP3 inflammasome activation may be a promising target for the treatment of patients with sepsis.

RAB1A regulates glioma cellular proliferation and invasion via the mTOR signaling pathway and epithelial-mesenchymal transition.

Aim: We aimed at investigating the mechanism of RAB1A proliferation and invasion in gliomas. Materials & methods: Genome-wide expression profile data and immunohistochemistry were analyzed to assess RAB1A expression in gliomas. The Transwell assay, wound healing assay, brain slice coculture model, cellular fluorescence and intracranial xenograft model of nude mice were used to determine the proliferation and invasion of glioma cells. Results & conclusion: RAB1A was highly expressed in gliomas compared with normal brain tissue. The overall survival time of glioma patients with high RAB1A expression was significantly shortened. RAB1A regulated the activity of RAC1 by inhibiting the mTOR signaling pathway, affecting actin polymerization, cell morphology and cell polarity. RAB1A downregulation inhibited the epithelial-mesenchymal transition, proliferation and invasion of glioma cells.