The RAB25 small GTPase determines aggressiveness of ovarian and breast cancers

High-density array comparative genomic hybridization (CGH) showed amplification of chromosome 1q22 centered on the RAB25 small GTPase, which is implicated in apical vesicle trafficking, in approximately half of ovarian and breast cancers. RAB25 mRNA levels were selectively increased in stage III and IV serous epithelial ovarian cancers compared to other genes within the amplified region, implicating RAB25 as a driving event in the development of the amplicon. Increased DNA copy number or RNA level of RAB25 was associated with markedly decreased disease-free survival or overall survival in ovarian and breast cancers, respectively. Forced expression of RAB25 markedly increased anchorage-dependent and anchorage-independent cell proliferation, prevented apoptosis and anoikis, including that induced by chemotherapy, and increased aggressiveness of cancer cells in vivo. The inhibition of apoptosis was associated with a decrease in expression of the proapoptotic molecules, BAK and BAX, and activation of the antiapoptotic phosphatidylinositol 3 kinase (PI3K) and AKT pathway, providing potential mechanisms for the effects of RAB25 on tumor aggressiveness. Overall, these studies implicate RAB25, and thus the RAB family of small G proteins, in aggressiveness of epithelial cancers.     

Characterization of RAB-like4, the first identified RAB-like protein from Trypanosoma cruzi with GTPase activity

RAB proteins, which belong to the RAS superfamily, regulate exocytic and endocytic pathways of eukaryotic cells, controlling vesicle docking and fusion. Few RAB proteins have been identified in parasites. Molecular markers for cellular compartments are important to studies concerning about the protein traffic in Trypanosoma cruzi, the causal agent of Chagas disease. In this work, we describe the characterization of TcRABL4, the first RAB-like gene identified in T. cruzi (GenBank Accession No.: ), present as a single-copy gene. TcRABL4 contains all five consensus RAB motifs but lacks cysteine residues at the C terminus, which are essential to isoprenylation, an absolute prerequisite for membrane association of these proteins. TcRABL4 is a functional GTPase that is able to bind and hydrolyze GTP, and its gene is transcribed as a single 1.2 kb mRNA in epimastigotes. TcRABL4 appears to be differentially regulated in the three cell forms of the parasite, and the protein is not associated to membranes, unlike other RAB proteins. It is possible that TcRABL4 may be a member of a novel family of small GTPases.     

The small GTPase RAB37 functions as an organizer for autophagosome biogenesis

Macroautophagy/autophagy is a catabolic process that is essential for cellular homeostasis. How autophagosomal vesicle forms in a spatio-temporally regulated manner remains elusive. Our recent study revealed that small GTPase, RAB37 (RAB37, member RAS oncogene family), functions as a key organizer of autophagosomal membrane biogenesis. RAB37 interacts with ATG5 (autophagy related 5) and promotes autophagosome formation by modulating ATG12–ATG5-ATG16L1 complex assembly. These findings provide new insights into autophagy regulation.     

Plant RABs: Role in Development and in Abiotic and Biotic Stress Responses

Endosomal trafficking plays an integral role in various eukaryotic cellular activities and is vital for higher-order functions in multicellular organisms. RAB GTPases are important proteins that influence various aspects of membrane traffic, which consequently influence many cellular functions and responses. Compared to yeast and mammals, plants have evolved a unique set of plant-specific RABs that play a significant role in their development. RABs form the largest family of small guanosine triphosphate (GTP)-binding proteins, and are divided into eight sub-families named RAB1, RAB2, RAB5, RAB6, RAB7, RAB8, RAB11 and RAB18. Recent studies on different species suggest that RAB proteins play crucial roles in intracellular trafficking and cytokinesis, in autophagy, plant microbe interactions and in biotic and abiotic stress responses. This review recaptures and summarizes the roles of RABs in plant cell functions and in enhancing plant survival under stress conditions.     

Chromosome assignment of four RAS-related RAB genes

Summary The human RAB genes share structural and biochemical properties with the RAS gene superfamily. The encoded RAB proteins show 38 to 75% amino acid identity with the yeast YPT1 and SEC4 gene products. We used four human RAB-cDNAs, RAB3B, RAB4, RAB5 and RAB6, to map the corresponding genes on human chromosomes. These genes were assigned to 1p32-p31, 1q42-q43, 3p24-p22 and 2q14-q21, respectively, by in situ hybridization.     

Cloning and characterization of a novel splice variant of human Rab18 gene (RAB18).

Rab GTPase proteins are a kind of small GTP-binding proteins, which functions mainly focus on regulating interacellular trafficking pathways during vesicular transport. To date, 60 distinct human RAB proteins have been identified. RAB18 gene is discovered from endothelial cells. Its function is considered as endosomes and plasma membrane recycling. Research indicates RAB18 may relate to inflammation and some kinds of tumor. Here we report a splice variant of RAB18, which is 2571 bp in length and has an open reading frame coding a predicted 235 amino-acids protein. RT-PCR shows that the cDNA has different expression pattern with RAB18 and is highly expressed in testis.     

The integration of autophagy and cellular trafficking pathways via RAB GAPs

Macroautophagy is a conserved degradative pathway in which a double-membrane compartment sequesters cytoplasmic cargo and delivers the contents to lysosomes for degradation. Efficient formation and maturation of autophagic vesicles, so-called phagophores that are precursors to autophagosomes, and their subsequent trafficking to lysosomes relies on the activity of small RAB GTPases, which are essential factors of cellular vesicle transport systems. The activity of RAB GTPases is coordinated by upstream factors, which include guanine nucleotide exchange factors (RAB GEFs) and RAB GTPase activating proteins (RAB GAPs). A role in macroautophagy regulation for different TRE2-BUB2-CDC16 (TBC) domain-containing RAB GAPs has been established. Recently, however, a positive modulation of macroautophagy has also been demonstrated for the TBC domain-free RAB3GAP1/2, adding to the family of RAB GAPs that coordinate macroautophagy and additional cellular trafficking pathways.     

RAS and RHO family GTPase mutations in cancer: twin sons of different mothers?

Abstract

The RAS and RHO family comprise two major branches of the RAS superfamily of small GTPases. These proteins function as regulated molecular switches and control cytoplasmic signaling networks that regulate a diversity of cellular processes, including cell proliferation and cell migration. In the early 1980s, mutationally activated RAS genes encoding KRAS, HRAS and NRAS were discovered in human cancer and now comprise the most frequently mutated oncogene family in cancer. Only recently, exome sequencing studies identified cancer-associated alterations in two RHO family GTPases, RAC1 and RHOA. RAS and RHO proteins share significant identity in their amino acid sequences, protein structure and biochemistry. Cancer-associated RAS mutant proteins harbor missense mutations that are found primarily at one of three mutational hotspots (G12, G13 and Q61) and have been identified as gain-of-function oncogenic alterations. Although these residues are conserved in RHO family proteins, the gain-of-function mutations found in RAC1 are found primarily at a distinct hotspot. Unexpectedly, the cancer-associated mutations found with RHOA are located at different hotspots than those found with RAS. Furthermore, since the RHOA mutations suggested a loss-of-function phenotype, it has been unclear whether RHOA functions as an oncogene or tumor suppressor in cancer development. Finally, whereas RAS mutations are found in a broad spectrum of cancer types, RHOA and RAC1 mutations occur in a highly restricted range of cancer types. In this review, we focus on RHOA missense mutations found in cancer and their role in driving tumorigenesis, with comparisons to cancer-associated mutations in RAC1 and RAS GTPases.     

一个与非小细胞肺癌转移相关的基因――RAB5A基因

采用mRNA差异展示技术（mRNA DD）研究具有相同细胞来源,但转移能力高低不同的人肺腺癌细胞系AGZY83-a（低转移）和Anip973(高转移),分析在两个细胞系中基因差异表达的情况,发现在高转移细胞系中有RAB5A基因的表达。该基因为蛋白质入胞信号的调控者,为RAS超家族成员。为进一步证实其转录表达的调控改变情况,以及RAB5A高表达的临床意义,进一步采用RT-PCR和免疫组织化学的方法检测了50例临床非小细胞肺癌的手术标本,结果表明,RAB5A的表达有随转移发生而增强的趋势,而RAB5A的蛋白表达程度在有转移的病例中明显增强(P<0.05)。 Abstract：　Using mRNA differential display (mRNA DD)techniques, we analyzed the differences of gene expression between two human lung adenocarcinoma cell lines,AGZY83-a and Anip973. Anip973 was isolated from AGZY83-a, but manifested much higher metastatic potential than the parent line. The results showed that there were significant differences on gene expression between the two cell lines and that there was over-expression of RAB5Agene in the Anip973 cell line. The product of RAB5Agene was recognized as signal regulators of endocytotic pathway and protein trafficking at the cell surface, and belong to a member of the RAS superfamily. Furthemore, we compared to the expression of RAB5Agene and RAB5Aprotein in clinical samples of 50 cases non-small lung carcinoma and nearby lymph node by means of RT-PCR and immunohistochemistry method. The results indicated that the high expression of RAB5Ain metastatic tumor and the enhancement level of RAB5Ain metastatic tumor and the enhancement level of expression were corresponded with the increase of metastatic degree. And there were significance of difference on the expression degree of RAB5Aprotein between non-small lung carcinoma with metastasis and non- metastasis (P<0.05).     

RIN3 Is a Negative Regulator of Mast Cell Responses to SCF

Stimulation of the receptor tyrosine kinase KIT by Stem Cell Factor (SCF) triggers activation of RAS and its downstream effectors. Proper KIT activation is essential for the maturation, survival and proliferation of mast cells. In addition, SCF activation of KIT is critical for recruiting mast cells to sites of infection or injury, where they release a mix of pro-inflammatory substances. RIN3, a RAS effector and RAB5-directed guanine nucleotide exchange factor (GEF), is highly expressed and enriched in human mast cells. SCF treatment of mast cells increased the amount of GTP-bound RAB5, and the degree of RAB5 activation correlated with the expression level of RIN3. At the same time, SCF caused the dissociation of a pre-formed complex of RIN3 with BIN2, a membrane bending protein implicated in endocytosis. Silencing of RIN3 increased the rate of SCF-induced KIT internalization, while persistent RIN3 over-expression led to KIT down regulation. These observations strongly support a role for RIN3 in coordinating the early steps of KIT endocytosis. Importantly, RIN3 also functioned as an inhibitor of mast cell migration toward SCF. Finally, we demonstrate that elevated RIN3 levels sensitize mastocytosis cells to treatment with a KIT tyrosine kinase inhibitor, suggesting the value of a two-pronged inhibitor approach for this difficult to treat malignancy. These findings directly connect KIT activation with a mast cell-specific RAS effector that regulates the cellular response to SCF and provide new insight for the development of more effective mastocytosis treatments.     

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy

ABSTRACT

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.

Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2     

Global effects of RAB3GAP1 dysexpression on the proteome of mouse cortical neurons

Mounting studies have demonstrated that RAB3GAP1 expression is modified in brain diseases with multiple neurobiological functions and processes and acts as a potentially significant target. However, the cellular and molecular events arising from RAB3GAP1 dysexpression are still incompletely understood. In this work, underexpression and overexpression of RAB3GAP1 were first induced into cultured mouse cortical neurons by transfection with lentivirus plasmids. Then we globally explored the effects of RAB3GAP1 dysexpression on the proteome of the neurons through the use of isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics with bioinformatics. A total of 364 proteins in the RAB3GAP1-underexpression group and 314 proteins in the RAB3GAP1-overexpression group were identified to be differentially expressed. Subsequent bioinformatics analysis indicated that the proteome functional expression profiles induced by RAB3GAP1 underexpression and overexpression were different, suggesting the potential differences in biological processes and cellular effects. Subsequent intergroup cross-comparison revealed some candidate target proteins regulated directly by RAB3GAP1. Further parallel reaction monitoring (PRM) analysis illustrated that Sub1, Ssrp1, and Top1 proteins might serve as new potentially important linkers in the RAB3GAP1-mediated autophagy pathway in the cortical neurons. Collectively, the current proteomics data furnished new valuable insights to better understand the regulatory molecular mechanism of neuronal RAB3GAP1.

     

Evolutionary grouping of the RAS-protein family

Over 50 proteins related to the mammalian H-, K-, and N-RAS GTP binding and hydrolyzing proteins are known. These relatively low molecular weight proteins are usually grouped into four subfamilies, termed true RAS, RAS-like, RHO, and RAB/YPT, based on the presence of shared amino acid sequence motifs in addition to those involved in guanine nucleotide binding. Here, we apply parsimony analysis to the overall amino acid sequences of these proteins to infer possible phylogenetic relationships among them.     

Ras oncogenes and their downstream targets

RAS proteins are small GTPases, which serve as master regulators of a myriad of signaling cascades involved in highly diverse cellular processes. RAS oncogenes have been originally discovered as retroviral oncogenes, and ever since constitutively activating RAS mutations have been identified in human tumors, they are in the focus of intense research. In this review, we summarize the biochemical properties of RAS proteins, trace down the evolution of RAS signaling and present an overview of the spatio-temporal activation of major RAS isoforms. We further discuss RAS effector pathways, their role in normal and transformed cell physiology and summarize ongoing attempts to interfere with aberrant RAS signaling. Finally, we comment on the role of micro RNAs in modulating RAS expression, contribution of RAS to stem cell function and on high-throughput analyses of RAS signaling networks.     

RAB11A is essential for transport of the influenza virus genome to the plasma membrane

Influenza A virus assembly is a complex process that requires the intersection of pathways involved in transporting viral glycoproteins, the matrix protein, and viral genomes, incorporated in the viral ribonucleoprotein (vRNP) complex, to plasma membrane sites of virion formation. Among these virion components, the mechanism of vRNP delivery is the most incompletely understood. Here, we reveal a functional relationship between the cellular Rab11 GTPase isoform, RAB11A, and vRNPs and show that RAB11A is indispensable for proper vRNP transport to the plasma membrane. Using an immunofluorescence-based assay with a monoclonal antibody that recognizes nucleoprotein in the form of vRNP, we demonstrate association between RAB11A and vRNPs at all stages of vRNP cytoplasmic transport. Abrogation of RAB11A expression through small interfering RNA (siRNA) treatment or disruption of RAB11A function by overexpression of dominant negative or constitutively active proteins caused aberrant vRNP intracellular accumulation, retention in the perinuclear region, and lack of accumulation at the plasma membrane. Complex formation between RAB11A and vRNPs was further established biochemically. Our results uncover a critical host factor with an essential contribution to influenza virus genome delivery and reveal a potential role for RAB11A in the transport of ribonucleoprotein cargo.     

The small GTPase RAB10 regulates endosomal recycling of the LDL receptor and transferrin receptor in hepatocytes

The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane. We also show that RAB10 similarly promotes the recycling of the transferrin receptor, which binds the transferrin protein that mediates the transport of iron in the blood, albeit from a distinct RAB4-positive compartment. Taken together, our findings suggest a model in which RAB10 regulates LDL and transferrin uptake by promoting both slow and rapid recycling routes for their respective receptor proteins.