Expression Of Rab7 Research Articles

Rotaviruses Reach Late Endosomes and Require the Cation-Dependent Mannose-6-Phosphate Receptor and the Activity of Cathepsin Proteases To Enter the Cell

Rotaviruses (RVs) enter cells through different endocytic pathways. Bovine rotavirus (BRV) UK uses clathrin-mediated endocytosis, while rhesus rotavirus (RRV) employs an endocytic process independent of clathrin and caveolin. Given the differences in the cell internalization pathway used by these viruses, we tested if the intracellular trafficking of BRV UK was the same as that of RRV, which is known to reach maturing endosomes (MEs) to infect the cell. We found that BRV UK also reaches MEs, since its infectivity depends on the function of Rab5, the endosomal sorting complex required for transport (ESCRT), and the formation of endosomal intraluminal vesicles (ILVs). However, unlike RRV, the infectivity of BRV UK was inhibited by knocking down the expression of Rab7, indicating that it has to traffic to late endosomes (LEs) to infect the cell. The requirement for Rab7 was also shared by other RV strains of human and porcine origin. Of interest, most RV strains that reach LEs were also found to depend on the activities of Rab9, the cation-dependent mannose-6-phosphate receptor (CD-M6PR), and cathepsins B, L, and S, suggesting that cellular factors from the trans-Golgi network (TGN) need to be transported by the CD-M6PR to LEs to facilitate RV cell infection. Furthermore, using a collection of UK × RRV reassortant viruses, we found that the dependence of BRV UK on Rab7, Rab9, and CD-M6PR is associated with the spike protein VP4. These findings illustrate the elaborate pathway of RV entry and reveal a new process (Rab9/CD-M6PR/cathepsins) that could be targeted for drug intervention. Rotavirus is an important etiological agent of severe gastroenteritis in children. In most instances, viruses enter cells through an endocytic pathway that delivers the viral particle to vesicular organelles known as early endosomes (EEs). Some viruses reach the cytoplasm from EEs, where they start to replicate their genome. However, other viruses go deeper into the cell, trafficking from EEs to late endosomes (LEs) to disassemble and reach the cytoplasm. In this work, we show that most RV strains have to traffic to LEs, and the transport of endolysosomal proteases from the Golgi complex to LEs, mediated by the mannose-6-phosphate receptor, is necessary for the virus to exit the vesicular compartment and efficiently start viral replication. We also show that this deep journey into the cell is associated with the virus spike protein VP4. These findings illustrate the elaborate pathway of RV entry that could be used for drug intervention.

MiR-200b as a prognostic factor targets multiple members of RAB family in glioma

miR-200b is a tumor suppressor in multiple tumors including gastric cancer, breast cancer, ovarian cancer and glioma. In this study, we detected the expression of miR-200b and analyzed its correlation with clinicopathological parameters in glioma tissues. miR-200b was downregulated in glioma tissues. And its downexpression was correlated with poor prognosis in gliomas. Members of RAB family, RAB21, RAB23, RAB18 and RAB3B were predicted to be novel targets of miR-200b. The direct suppression of RAB21, RAB23, RAB18 and RAB3B expressions by miR-200b was revealed by luciferase reporter assay, quantitative RT-PCR analysis and Western blot. Furthermore, the overall survival of patients with different expression of RABs was analyzed. The expression of RAB21, RAB23, RAB18 and RAB3B was related to the prognosis and histopathology of glioma. The patients who had the upregulation of all the four RABs had the worst outcome; those who had the downregulation of all RABs had the best outcome (p<0.001). miR-200b was a potential biomarker for glioma prognosis.

Synergism between upregulation of Rab7 and inhibition of autophagic degradation caused by mycoplasma facilitates intracellular mycoplasma infection

Following fusion of a mycoplasma with a host cell membrane, the inserted components of mycoplasma may then be transported through the endocytic pathway. However, the effects of mycoplasmas on the host cell endomembrane system are largely unknown. In this study, mycoplasma-induced changes in the dynamics of endocytic and autophagic systems were investigated. Endocytosis and autophagy are two major processes involved in the survival of intracellular prokaryotic pathogens. It was found that, immediately following infection, mycoplasmas induce endocytosis in the host cell; however, in the long term the mycoplasmas suppress turnover of the components of the endocytic pathway. Immunofluorescence microscopy revealed that Rab7 and LC3-II are recruited to the intracellular mycoplasma-containing compartments. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qPCR) showed that mycoplasmas increase expression of Rab7 by upregulating transcription, but increase levels of LC3-II and p62 by post-translational regulation. Furthermore, it was demonstrated that mycoplasma infection causes inhibition of autophagic degradation of LC3-II and p62. In addition, it was found that upregulation of Rab7 and inhibition of autophagic degradation synergistically contributes to intracellular mycoplasma accumulation. In conclusion, these findings suggest that mycoplasmas may manipulate host cell endosomal and autophagic systems in order to facilitate intracellular infection.

Characterization of Human Astrovirus Cell Entry

Human astroviruses (HAstV) are a frequent cause of gastroenteritis in young children and immunocompromised patients. To understand the early steps of HAstV infection in the highly permissive Caco-2 cell line, the binding and entry processes of the virus were characterized. The half-time of virus binding to the cell surface was about 10 min, while virus decapsidation took around 130 min. Drugs affecting clathrin-mediated endocytosis, endosome acidification, and actin filament polymerization, as well as those that reduce the presence of cholesterol in the cell membrane, decreased the infectivity of the virus. The infection was also reduced by silencing the expression of the clathrin heavy chain (CHC) by RNA interference or by overexpression of dominant-negative mutants of dynamin 2 and Eps15. Furthermore, the entry of HAstV apparently depends on the maturation of endosomes, since the infection was reduced by silencing the expression of Rab7, a small GTPase involved in the early- to late-endosome maturation. Altogether, our results suggest that HAstV enters Caco-2 cells using a clathrin-dependent pathway and reaches late endosomes to enter cells. Here, we have characterized the mechanism used by human astroviruses, important agents of gastroenteritis in children, to gain entry into their host cells. Using a combination of biochemical and genetic tools, we found that these viruses enter Caco-2 cells using a clathrin-dependent endocytic pathway, where they most likely need to travel to late endosomes to reach the cytoplasm and begin their replication cycle.

Secreted protein acidic and rich in cysteine internalization and its age‐related alterations in skeletal muscle progenitor cells

Aging causes phenotypic changes in skeletal muscle progenitor cells (Skm-PCs), such as reduced myogenesis and increased adipogenesis due to alterations in their environment or niche. Secreted protein acidic and rich in cysteine (SPARC), which is secreted into the niche of Skm-PCs, inhibits adipogenesis and promotes myogenesis. We have previously reported that Skm-PC responsiveness to SPARC declines with age, although the mechanism underlying this decline is unknown. In this study, we found that SPARC is internalized by Skm-PCs and that this uptake increases with age. Internalization is dependent on integrin-α5, a cell surface SPARC-binding molecule, and clathrin-mediated endocytosis. We also demonstrated that internalized SPARC is transported to Rab7-positive endosomes. Skm-PCs from old rats exhibited increased clathrin expression and decreased Rab7 expression exclusively in MyoD-negative cells. In loss-of-function analyses, clathrin knockdown increased the anti-adipogenic effect of SPARC, whereas Rab7 knockdown reduced it, indicating that alterations in SPARC internalization may mediate the age-related decline in its anti-adipogenic effect. These results provide insights into age-related SPARC resistance in Skm-PCs, which may lead to sarcopenia.

MiR-144 and MiR-451 Regulate Human Erythropoiesis By Targeting RAB14

MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by inhibiting translation and/or by degrading of target mRNAs. MiR-144 and miR-451 are expressed from a single precursor transcript, encoded by the miR-144∼451 cluster on human chromosome 17. Expression levels of both miR-144 and miR-451 are upregulated during zebrafish, mouse, and human erythroid differentiation. Several mouse and zebrafish studies have knocked down miR-451 expression and observed decreased late erythroid differentiation. In contrast, miR-144 knockdown had no or smaller effects in zebrafish or mouse erythropoiesis, and there is no study of the role of both members of this cluster in primary human erythropoiesis. Therefore, we investigated the effects of lentiviral-mediated loss-of-function of miR-144 and/or miR-451 in human erythropoiesis. We first utilized the TF1 human erythroleukemia cell line as a model, and then tested primary human CD34+ hematopoietic stem-progenitor cells (HSPCs) from normal human donors. When either TF1 or CD34+ cells are cultured with erythropoietin (EPO), the expression of the CD71 (transferrin receptor) and CD235a (glycophorin A) erythroid markers increases, and the expression of the CD34 HSPC marker decreases. In TF1 or primary CD34+ cells transduced with either a miR-144 trap or a miR-451 trap (using a lentivirus containing GFP and a series of 8 tandem complementary miR binding sequences to knock down function of either miR [Ebert, Nature Method, 2007]), as compared to control vector, erythropoiesis was decreased, as evidenced by lower numbers of erythroid (CD34-CD71hiCD235ahi) cells generated during EPO-induced erythroid differentiation.RAB14 was predicted to be a target of both miR-144 and miR-451 by the TargetScan 6.2 target prediction algorithm, with 2 predicted miR-144 binding sites and 1 predicted miR-451 binding site in its 3'UTR. RAB14 was validated to be a target of miR-451 in human non-small cell lung cancer cells [Wang, Oncogene, 2011]. RAB proteins have been reported to have important roles in vesicle trafficking, signal transduction, and receptor recycling. Recently, RAB12 was implicated in constitutive degradation of the transferrin receptor (CD71) in mouse embryonic fibroblast (MEF) cells [Matsui, Traffic, 2011], but the role of RAB14 in human erythropoiesis is unknown. We found that RAB14 protein expression decreases early during EPO-induced erythroid differentiation of TF1 cells. Then we showed that RAB14 is a direct target of miR-144, using luciferase assays and Western blots. We confirmed that shRNA-mediated RAB14 knockdown increased human erythropoiesis, as compared to control vector-transduced cells. Finally, shRNA-mediated RAB14 knockdown protected cells from miR-144/miR-451 trap-mediated erythropoietic inhibition.In summary, miR-144 and miR-451 regulate human erythropoiesis by decreasing RAB14 expression, which in turn might have a role in regulating function of receptors involved in erythropoiesis, such as the transferrin receptor. Disclosures:No relevant conflicts of interest to declare.

Rab11 as a modulator of synaptic transmission

Many neurodegenerative disorders are characterized by synaptic dysfunction preceding general neuronal loss and subsequent cognitive or behavioral anomalies. Much recent research has been aimed at understanding the early underlying processes leading to dysfunction at the synapse, as this knowledge would likely inform interventions that could potentially slow progression and delay onset of disease. We have recently reported that synaptic dysfunction in a Drosophila melanogaster model of Huntington’s disease (HD) can be prevented by enhanced neuronal expression of Rab11, a Rab family GTPase involved in endosomal recycling, which complements studies that have found disrupted Rab11 activity in several models of this disorder. Indeed, inhibition of Rab11 function in fibroblasts of HD patients has been observed to perturb vesicle formation from recycling endosomes. Therefore, our study investigated a potential role of Rab11 in synaptic dysfunction prior to the onset of HD symptoms, with the aim of finding a possible early intervention to disease progression. We found that Rab11 ameliorates synaptic dysfunction due to expression of mutant huntingtin—the causative protein in HD—by normalizing synaptic vesicle size, which consequently ameliorates locomotor deficits in Drosophila larvae. Here we further consider these results and the implications this work has on potential therapeutic intervention in HD and other neurodegenerative disorders.

Virulence loss and amastigote transformation failure determine host cell responses to Leishmania mexicana

The effect of alterations in virulence and transformation by long-term in vitro culture of Leishmania mexicana promastigotes on infectivity and immune responses was investigated. Fresh parasite cultures harvested from Balb/c mice were passaged 20 times in vitro. Infectivity was decreased and was completely avirulent after 20 passages. The qPCR results showed a down-regulation of GP63, LPG2, CPC, CPB2, CPB2.8, CHT1, LACK and LDCEN3 genes after passage seven concomitant with a reduced and absence of infectivity by passages seven and 20, respectively. Parasites at passages one and 20 are referred to as virulent and avirulent, respectively. The growth of avirulent and virulent parasite was affected by conditioned media derived from macrophages or monocytes infected with parasites for 2 h. Giemsa staining showed the failure of avirulent but not virulent parasites to transform to the amastigote stage in infected host cells with both virulent and avirulent modulating the expression of CCL-22, Tgad51, Cox2, IL-1, IL-10, TGF-β, TNF-α, Rab7, Rab9 and A2 genes; virulent but not avirulent L. mexicana significantly up-regulated Th2-associated cytokines, but down-regulated Rab7 and Rab9 gene expression. In conclusion, a model for L. mexicana is reported, which is of potential value in studying host-parasite interaction.

High RAB25 expression is associated with good clinical outcome in patients with locally advanced head and neck squamous cell carcinoma.

Currently there are no molecular markers able to predict clinical outcome in locally advanced head and neck squamous cell carcinoma (HNSCC). In a previous microarray study, RAB25 was identified as a potential prognostic marker. The aim of this study was to analyze the association between RAB25 expression and clinical outcome in patients with locally advanced HNSCC treated with standard therapy. In a retrospective immunohistochemical study (n = 97), we observed that RAB25-negative tumors had lower survival (log-rank, P = 0.01) than patients bearing positive tumors. In an independent prospective mRNA study (n = 117), low RAB25 mRNA expression was associated with poor prognosis. Using classification and regression tree analysis (CART) we established two groups of patients according to their RAB25 mRNA level and their risk of death. Low mRNA level was associated with poor local recurrence-free (log-rank, P = 0.005), progression-free (log-rank, P = 0.002) and cancer-specific (log-rank, P < 0.001) survival. Multivariate Cox model analysis showed that low expression of RAB25 was an independent poor prognostic factor for survival (hazard ratio: 3.84, 95% confidence interval: 1.93–7.62, P < 0.001). Patients whose tumors showed high RAB25 expression had a low probability of death after treatment. We also found lower RAB25 expression in tumors than in normal tissue (Mann–Whitney U, P < 0.001). Moreover, overexpression of RAB25 in the UM-SCC-74B HNSCC cell line increased cisplatin sensitivity, and reduced cell migration and invasion. Our findings support a tumor suppressor role for RAB25 in HNSCC and its potential use to identify locally advanced patients with a high probability of survival after genotoxic treatment.

Exoenzyme S ADP-Ribosylates Rab5 Effector Sites To Uncouple Intracellular Trafficking

Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylates multiple eukaryotic targets to promote cytopathology and bacterial colonization. ADP-ribosylation of the small GTPase Rab5 has previously been shown to block fluid-phase endocytosis and trafficking of plasma membrane receptors to the early endosomes as well as inhibit phagocytosis of the bacterium. In this study, ExoS is shown to be capable of ADP-ribosylating 6 candidate arginine residues that are located in the effector binding region or in the C terminus of Rab5. Two Rab5 derivatives were engineered, which contained Arg→Ala mutations at four Arg residues within the effector binding region (EF) or two Arg residues within the C-terminal tail (TL). Expression of Rab5(TL) does not affect the ability of ExoS to modify intracellular trafficking, while expression of Rab5(EF) rescued the ability of ExoS to inhibit intracellular trafficking. ADP-ribosylation of effector arginines likely uncouples Rab5 signaling to downstream effectors. This is a different mechanism for inhibition than observed for the ADP-ribosylation of Ras by ExoS, where ADP-ribosylated Ras loses the ability to bind guanine nucleotide exchange factor (GEF). Other experiments showed that expression of dominant negative Rab5(Ser34Asn) does not inhibit ExoS trafficking to the perinuclear region of intoxicated cells. This study provides insight into a mechanism for how ExoS ADP-ribosylation of Rab5 inhibits Rab5 function.

Interleukin 22 Inhibits Intracellular Growth of Mycobacterium tuberculosis by Enhancing Calgranulin A Expression

Previously, we found that interleukin 22 (IL-22) inhibits intracellular growth of Mycobacterium tuberculosis in human monocyte-derived macrophages (MDMs). In the current study, we determined the mechanisms underlying these effects. We found that W7, a phagolysosomal fusion inhibitor, abrogates IL-22-dependent M. tuberculosis growth inhibition in MDMs, suggesting that IL-22 acts through enhanced phagolysosomal fusion. Our microarray analysis indicated that recombinant IL-22 (rIL-22) enhances the expression of an intracellular signaling molecule, calgranulin A. This was confirmed by real-time polymerase chain reaction, Western blot, and confocal microscopy. Calgranulin A small interfering RNA (siRNA) abrogated rIL-22-dependent growth inhibition of M. tuberculosis in MDMs. IL-22 enhanced Rab7 expression and downregulated Rab14 expression of M. tuberculosis-infected MDMs, and these effects were reversed by calgranulin A siRNA. These results suggest that M. tuberculosis growth inhibition by IL-22 depends on calgranulin A and enhanced phagolysosomal fusion, which is associated with increased Rab7 and reduced Rab14 expression.

Overexpression of Rab25 contributes to metastasis of bladder cancer through induction of epithelial–mesenchymal transition and activation of Akt/GSK-3β/Snail signaling

Rab25, an epithelial-specific member of the Rab family of small guanosine triphosphatases, is associated with several human cancers. The goal of this study was to determine its function in bladder cancer (BC). We examined the Rab25 expression pattern in two different cohorts of BC patients treated with radical cystectomy by quantitative PCR, western blotting and immunohistochemical staining. A series of in vitro and in vivo assays were performed to elucidate the function of Rab25 in BC and its underlying mechanisms. Rab25 expression was significantly elevated at both the messenger RNA and protein levels in BCs compared with normal bladder tissues. High Rab25 expression was closely associated with lymph node (LN) metastasis and was an independent predictor for poor disease-free survival in BC patients. Downregulation of Rab25 in BC cells markedly inhibited invasive motility in vitro and metastatic potential in vivo. In addition, downregulation of Rab25 in BC EJ and T24 cells increased the expression levels of epithelial markers (E-cadherin and α-catenin) and decreased the levels of mechamechy markers (vimentin and fibronectin). Simultaneously, downregulation of Rab25 in EJ and T24 cells resulted in the inactivation of downstream phosphorylated protein kinase B (p-Akt), phosphorylated glycogen synthase kinase-β (p-GSK-3β) and snail signaling. This study demonstrates that Rab25 can promote BC metastasis through induction of epithelial-mesenchymal transition process and activation of Akt/GSK-3β/Snail signaling pathway; Rab25 expression level can predict LN metastasis and inferior clinical outcome in BC patients.

Molecular characterization and expression of Rab7 from Clonorchis sinensis and its potential role in autophagy

Accumulating evidences suggest that Rab7 GTPase is important for the normal progression of autophagy. However, the role of Rab7 GTPase in regulation of autophagy in Clonorchis sinensis is not known. In this study, a gene encoding Rab7 was isolated from C. sinensis adult cDNA. Recombinant CsRab7 was expressed and purified from Escherichia coli. CsRab7 transcripts were detected in the cDNA of adult worm, metacercaria, cercaria, and egg of C. sinensis, and were highly expressed in the metacercaria. Immunohistochemical localization results revealed that CsRab7 was specifically deposited on the vitellarium and eggs of adult worm. Furthermore, EGFP signal of CsRab7WT and the active mutant CsRab7Q67L were associated with autophagic vesicles in transiently transfected 293T cells. It is concluded from the present study that CsRab7 GTPase possibly contributes to the development of C. sinensis and that the autophagy pathway could be an important site of action with respect to the developmental role of CsRab7 in C. sinensis.

Abstract 4458: EGFR endocytosis is associated with susceptibility to gefitinib in lung cancer cells with wild-type EGFR.

Abstract Although epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a rational targeted drug with dramatic survival benefit in lung cancer, susceptibility to the drug is restricted in lung cancer with activating mutations of EGFR. Nevertheless, the EGFR-TKI still remains an attractive anticancer drug in lung cancer with wild-type EGFR (wtEGFR). Therefore, in this study, we aimed to investigate unknown mechanism which can contribute to response of the EGFR targeted therapy using gefitinib, a well-known EGFR-TKI, in eight lung cancer cells with wtEGFR. We first examined differential effects of gefitinib on cell survival, morphology, motility, cell cycle and EGFR signaling between the TKI-sensitive and -resistant cells. As a result, although the TKI-resistant cells did not show any changes in cell phenotype by gefitinib treatment, phosphorylation of EGF induced EGFR and downstream effectors was inhibited by the drug treatment as if those in the TKI-sensitive cells. Additionally, we found difference in cellular distribution of EGF induced EGFR after gefitinib treatment between the TKI-sensitive and -resistant cells and it was associated with EGFR endocytosis. Based on our results, we next investigated that regulation of EGFR endocytosis could affect on the response of gefitinib in lung cancer cells with wtEGFR. Inhibition of EGFR endocytosis by combination treatment with gefitinib and two endocytosis inhibitors, dynasore and dynole 34-2, led to significantly decreased cell viability in the TKI-resistant cells compared with treatment of these drugs alone. Moreover, the reduced cell survival after the combination treatment was concerned with increased apoptotic cell death, accompanying by elevated cleavage of poly ADP ribose polymerase (PARP) and decreased myeloid cell leukemia-1 (Mcl-1), X-linked inhibitor of apoptosis protein (XIAP), Survivin and Livin among anti-apoptotic proteins. In addition, we found differentially expressed Rab25, a member of the RAS superfamily of small GTPases, between the TKI-sensitive and-resistant cells through genome-wide gene expression analysis. The expression status of Rab25 was partially associated with response of gefitinib in lung cancer cells with wtEGFR. Furthermore, the silencing of Rab25 in the TKI-sensitive cells with its normal expression reversed sensitivity to gefitinib treatment. In conclusion, our results provided molecular evidences that EGFR endocytosis might contribute to cell survival and response to the EGFR-TKI as a bypass survival mechanism in lung cancer with wtEGFR. Thus, targeting EGFR endocytosis could help us to overcome therapeutic limitation of EGFR-TKI in lung cancer with wtEGFR and Rab25 expression may be an important tool for predicting efficacy of the drug treatment. Citation Format: Ukhyun Jo, Young Mi Whang, Jae Sook Sung, Kyong Hwa Park, Seung Tae Kim, Yeul Hong Kim. EGFR endocytosis is associated with susceptibility to gefitinib in lung cancer cells with wild-type EGFR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4458. doi:10.1158/1538-7445.AM2013-4458

Expression of Rab25 correlates with the invasion and metastasis of gastric cancer.

The objective of this study was to determine the expression of the important vesicle trafficking-regulating factor Rab25 in human gastric cancer tissues, to analyze the correlation between Rab25 protein expression with gastric cancer occurrence and development, and to discuss the correlation of Rab25 protein expression with gastric cancer cell metastasis. The overall aim was to provide experimental evidence that can be used to design future biological treatments of human gastric cancer. Human gastric cancer tissue and the adjacent normal gastric tissue were surgically removed, and immunohistochemistry and Western blotting were used to detect Rab25 protein expression. The correlation between Rab25 protein expression with the development and pathological characteristics of gastric cancer was analyzed. Using RNAi, Rab25 expression was reduced in the gastric cancer cell line MGC80-3, and the changes in MGC80-3 cell invasiveness were then monitored. Immunohistochemistry showed that the Rab25 protein expression rates were 78.21% and 23.08% in gastric carcinoma and the adjacent normal gastric tissue, respectively. Immunohistochemistry and Western blot results showed that Rab25 protein expression in gastric cancer was significantly higher than in adjacent normal gastric tissues (P<0.01). Less differentiated gastric cancer cells had higher expression of Rab25 protein (P<0.01). Gastric carcinomas from patients with a late pathological stage (III-IV) had significantly higher Rab25 protein expression than early stage (I-II) patients (P<0.01). Gastric carcinomas from patients with lymph node metastasis had significantly higher Rab25 protein expression than lymph node metastasis-free patients (P<0.01). Gastric carcinomas from patients with distant metastases had significantly higher Rab25 protein expression than the distant metastasis-negative patients (P<0.01). Rab25 protein expression in gastric cancer was not affected by the patients(,) sex, age, or tumor size (P>0.05). MGC80-3 cells transfected with Rab25 siRNA had significantly lower Rab25 protein expression (P<0.01) and a significantly lower number of cells that passed through a Transwell chamber compared with non-transfected controls and the transfected control group (P<0.01). Rab25 protein expression is associated with the development of gastric cancer. siRNA knockdown of Rab25 protein expression in MGC80-3 gastric cancer cells reduced MGC80-3 cell invasiveness and provided experimental evidence for potential future biological treatment strategies of human gastric cancer.

Rab11-FIP1C and Rab14 Direct Plasma Membrane Sorting and Particle Incorporation of the HIV-1 Envelope Glycoprotein Complex

The incorporation of the envelope glycoprotein complex (Env) onto the developing particle is a crucial step in the HIV-1 lifecycle. The long cytoplasmic tail (CT) of Env is required for the incorporation of Env onto HIV particles in T cells and macrophages. Here we identify the Rab11a-FIP1C/RCP protein as an essential cofactor for HIV-1 Env incorporation onto particles in relevant human cells. Depletion of FIP1C reduced Env incorporation in a cytoplasmic tail-dependent manner, and was rescued by replenishment of FIP1C. FIP1C was redistributed out of the endosomal recycling complex to the plasma membrane by wild type Env protein but not by CT-truncated Env. Rab14 was required for HIV-1 Env incorporation, and FIP1C mutants incapable of binding Rab14 failed to rescue Env incorporation. Expression of FIP1C and Rab14 led to an enhancement of Env incorporation, indicating that these trafficking factors are normally limiting for CT-dependent Env incorporation onto particles. These findings support a model for HIV-1 Env incorporation in which specific targeting to the particle assembly microdomain on the plasma membrane is mediated by FIP1C and Rab14.

Regulation of Hepatitis B Virus Infection by Rab5, Rab7, and the Endolysosomal Compartment

Despite important progress toward deciphering human hepatitis B virus (HBV) entry into host cells, many aspects of the early steps of the life cycle remained completely obscure. Following endocytosis, HBV must travel through the complex network of the endocytic pathway to reach the cell nucleus and initiate replication. In addition to guiding the viral particles to the replication site, the endosomal vesicles may play a crucial role in infection, providing the appropriate environment for virus uncoating and nucleocapsid release. In this work, we investigated the trafficking of HBV particles internalized in permissive cells. Expression of key Rab proteins, involved in specific pathways leading to different intracellular locations, was modulated in HepaRG cells, using a stable and inducible short hairpin RNA (shRNA) expression system. The trafficking properties of the newly developed cells were demonstrated by confocal microscopy and flow cytometry using specific markers. The results showed that HBV infection strongly depends on Rab5 and Rab7 expression, indicating that HBV transport from early to mature endosomes is required for a step in the viral life cycle. This may involve reduction of disulfide bond-linked envelope proteins, as alteration of the redox potential of the endocytic pathway resulted in inhibition of infection. Subcellular fractionation of HBV-infected cells showed that viral particles are further transported to lysosomes. Intriguingly, infection was not dependent on the lysosomal activity, suggesting that trafficking to this compartment is a "dead-end" route. Together, these data add to our understanding of the HBV-host cell interactions controlling the early stages of infection.

Rab25 regulates invasion and metastasis in head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is one of the 10 most common cancers with a 50% five-year survival rate, which has remained unchanged for the past three decades. One of the major reasons for the aggressiveness of this cancer is that HNSCCs readily metastasize to cervical lymph nodes that are abundant in the head and neck region. Hence, discovering new molecules controlling the metastatic process as well as understanding their regulation at the molecular level are essential for effective therapeutic strategies. Rab25 expression level was analyzed in HNSCC tissue microarray. We used a combination of intravital microscopy in live animals and immunofluorescence in an in vitro invasion assay to study the role of Rab25 in tumor cell migration and invasion. In this study, we identified the small GTPase Rab25 as a key regulator of HNSCC metastasis. We observed that Rab25 is downregulated in HNSCC patients. Next, we determined that reexpression of Rab25 in a metastatic cell line is sufficient to block invasion in a three-dimensional collagen matrix and metastasis to cervical lymph nodes in a mouse model for oral cancer. Specifically, Rab25 affects the organization of F-actin at the cell surface, rather than cell proliferation, apoptosis, or tumor angiogenesis. These findings suggest that Rab25 plays an important role in tumor migration and metastasis, and that understanding its function may lead to the development of new strategies to prevent metastasis in oral cancer patients.

The Non-Coding RNA Llme23 Drives the Malignant Property of Human Melanoma Cells

Several lines of evidence support the notion that increased RNA-binding ability of polypyrimidine tract-binding (PTB) protein-associated splicing factor (PSF) and aberrant expression of long non-coding RNAs (lncRNAs) are associated with mouse and human tumors. To identify the PSF-binding lncRNA involved in human oncogenesis, we screened a nuclear RNA repertoire of human melanoma cell line, YUSAC, through RNA-SELEX affinity chromatography. A previously unreported lncRNA, termed as Llme23, was found to bind immobilized PSF resin. The specific binding of Llme23 to both recombinant and native PSF protein was confirmed in vitro and in vivo. The expression of PSF-binding Llme23 is exclusively detected in human melanoma lines. Knocking down Llme23 remarkably suppressed the malignant property of YUSAC cells, accompanied by the repressed expression of proto-oncogene Rab23. These results may indicate that Llme23 can function as an oncogenic RNA and directly associate the PSF-binding lncRNA with human melanoma.

Enhancing autophagy with activated protein C and rapamycin protects against sepsis-induced acute lung injury

Autophagy plays distinct roles in apoptosis and the inflammatory process. Understanding the role of autophagy in sepsis-induced acute lung injury (ALI) may provide new insights into developing novel therapeutic strategies for this group of patients. The aim of this study was to investigate the regulation of autophagy in the septic lung and to use pharmacologic agents to modulate autophagy to study its functional significance. Mice were subjected to cecal ligation and puncture (CLP) or a sham operation. At 1 hour after CLP, mice were treated with vehicle, activated protein C (APC), rapamycin, or bafilomycin A1. Mice were humanely killed at 4 or 24 hours after the operation or were observed for ≤ 7 days. CLP induced a systemic inflammatory response and significantly decreased survival. In lung tissue, increased leukocyte infiltration, inflammation, and apoptosis were observed. In contrast, autophagy was suppressed after CLP such that the expression of LC3II, Atg5, and Rab7 were downregulated. Rapamycin activated autophagy, limited the CLP-induced proinflammatory response, and downregulated apoptotic activity after CLP. The administration of APC after CLP had an effect similar to that of rapamycin. Both medications significantly improved survival 7 days after CLP. The downregulation of autophagy may lead to systemic inflammation and ALI after sepsis. The direct or indirect modification of autophagy using rapamycin or APC, respectively, resulted in improved survival. Enhancing or restoring autophagy early after sepsis seems to be a potential strategy for the treatment of sepsis-induced ALI.