Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63.

The protozoan Leishmania parasitizes macrophages and evades the microbicidal consequences of phagocytosis through the inhibition of phagolysosome biogenesis. In this study, we investigated the impact of this parasite on LC3-associated phagocytosis, a non-canonical autophagic process that enhances phagosome maturation and functions. We show that whereas internalization of L. major promastigotes by macrophages promoted LC3 lipidation, recruitment of LC3 to phagosomes was inhibited through the action of the parasite surface metalloprotease GP63. Reactive oxygen species generated by the NOX2 NADPH oxidase are necessary for LC3-associated phagocytosis. We found that L. major promastigotes prevented, in a GP63-dependent manner, the recruitment of NOX2 to phagosomes through a mechanism that does not involve NOX2 cleavage. Moreover, we found that the SNARE protein VAMP8, which regulates phagosomal assembly of the NADPH oxidase NOX2, was down-modulated by GP63. In the absence of VAMP8, recruitment of LC3 to phagosomes containing GP63-deficient parasites was inhibited, indicating that VAMP8 is involved in the phagosomal recruitment of LC3. These findings reveal a role for VAMP8 in LC3-associated phagocytosis and highlight a novel mechanism exploited by L. major promastigotes to interfere with the host antimicrobial machinery.

Alternative splicing as a biomarker and potential target for drug discovery.

Alternative splicing is a key process of multi-exonic gene expression during pre-mRNA maturation. In this process, particular exons of a gene will be included within or excluded from the final matured mRNA, and the resulting transcripts generate diverse protein isoforms. Recent evidence demonstrates that approximately 95% of human genes with multiple exons undergo alternative splicing during pre-mRNA maturation. Thus, alternative splicing plays a critical role in physiological processes and cell development programs, and.dysregulation of alternative splicing is highly associated with human diseases, such as cancer, diabetes and neurodegenerative diseases. In this review, we discuss the regulation of alternative splicing, examine the relationship between alternative splicing and human diseases, and describe several approaches that modify alternative splicing, which could aid in human disease diagnosis and therapy.

Sorting nexin 3 (SNX3) is a component of a tubular endosomal network induced by Salmonella and involved in maturation of the Salmonella-containing vacuole.

Salmonella enterica serovar Typhimurium is an intracellular pathogen that grows within a modified endomembrane compartment, the Salmonella-containing vacuole (SCV). Maturation of nascent SCVs involves the recruitment of early endosome markers and the remodelling of phosphoinositides at the membrane of the vacuole, in particular the production of phosphatidylinositol 3-phosphate [PI(3)P]. Sorting nexins (SNXs) are a family of proteins characterized by the presence of a phox homology (PX) domain that binds to phosphoinositides and are involved in intracellular trafficking in eukaryotic cells. We therefore studied whether sorting nexins, particularly sorting nexin 3 (SNX3), play a role in Salmonella infection. We found that SNX3 transiently localized to SCVs at early times post invasion (10 min) and presented a striking tubulation phenotype in the vicinity of SCVs at later times (30-60 min). The bacterial effector SopB, which is known to promote PI(3)P production on SCVs, was required for the formation of SNX3 tubules. In addition, RAB5 was also required for the formation of SNX3 tubules. Depletion of SNX3 by siRNA impaired RAB7 and LAMP1 recruitment to the SCV. Moreover, the formation of Salmonella-induced filaments (Sifs) was altered by SNX3 knock-down. Therefore, SNX3 plays a significant role in regulating the maturation of SCVs.

Hippo/MST blocks breast cancer by downregulating WBP2 oncogene expression via miRNA processor Dicer.

WBP2 transcription coactivator is an emerging oncoprotein and a key node of convergence between EGF and Wnt signaling pathways. Understanding how WBP2 is regulated has important implications for cancer therapy. WBP2 is tightly controlled by post-translational modifications, including phosphorylation and ubiquitination, leading to changes in subcellular localization, protein-protein interactions, and protein turnover. As the function of WBP2 is intricately linked to YAP and TAZ, we hypothesize that WBP2 is negatively regulated by the Hippo tumor suppressor pathway. Indeed, MST is demonstrated to negatively regulate WBP2 expression in a kinase-dependent but LATS-independent manner. This was observed in the majority of the breast cancer cell lines tested. The effect of MST was enhanced by SAV and concomitant with the inhibition of the transcription co-activation, in vitro and in vivo tumorigenesis activities of WBP2, resulting in good prognosis in xenografts. Downregulation of WBP2 by MST involved miRNA but not proteasomal or lysosomal degradation. Our data support the existence of a novel MST-Dicer signaling axis, which in turn regulates both WBP2 CDS- and UTR-targeting miRNAs expression, including miR-23a. MiR-23a targets the 3'UTR of WBP2 mRNA directly. Significant inverse relationships between WBP2 and MST or miR23a expression levels in clinical specimens were observed. In conclusion, WBP2 is a target of the Hippo/MST kinase; MST is identified as yet another rheostat in the regulation of WBP2 and its oncogenic function. The findings have implications in targeted therapeutics and precision medicine for breast cancer.

Inhibition of enterovirus VP4 myristoylation is a potential antiviral strategy for hand, foot and mouth disease.

The Hand, Foot and Mouth Disease (HFMD) can result from infections by a plethora of human enteroviruses of the species Enterovirus A and B. These infections are highly contagious, resulting in regular outbreaks especially in the Asia-Pacific Region in the recent decade. Although this disease is generally a childhood affliction which manifests as a mild, febrile illness accompanied by the vesicles on the hands, feet and mouth, permanent morbidity or even fatality can result from severe forms of the disease in a subset of the infected patients. The N-terminal myristoylation signal (MGXXXS) of viral capsid protein VP4, one of the four viral structural proteins, is an extremely well conserved feature of enteroviruses, a potential antiviral target that may yield broad-spectrum inhibitors of HFMD. In this study, we have confirmed through the use of small interfering RNAs, human N-myristoyltransferase 1 plays an integral role in human Enterovirus 71 replication. Subsequent studies by inhibition of myristoylation using different myristic acid analogues elicited differential effects on the virus replication in human rhabdomyosarcoma cells. In particular, 2-hydroxymyristic acid specifically inhibited the cleavage between VP4 and VP2, part of the virion maturation process required to ensure infectivity of progeny virions while 4-oxatetradecanoic acid reduced the synthesis of viral RNA. These findings suggest that the requirement of a myristate moiety in viral structural protein precursor cleavage can serve as a viable antiviral target for further research.

Association between the expression levels of TAZ, AXL and CTGF and clinicopathological parameters in patients with colon cancer.

Colon cancer accounts for a large proportion of all the cancer-associated morbidities worldwide. Genetic analysis and stratification of patients based on survival may identify genetic signatures potentially useful for prognostic or treatment planning purposes. Previous studies have reported that the messenger (m)RNA expression levels of tafazzin (TAZ), AXL receptor tyrosine kinase (AXL) and connective tissue growth factor (CTGF) were able to predict the survival of patients with colon cancer in two independent colon cancer datasets. However, limited clinicopathological data were available from these two datasets. By contrast, a large colon cancer dataset comprising 566 patients has been recently published in the Gene Expression Omnibus database, which contains data regarding tumor stage and location, and genetic status of mismatch repair (MMR), Kirsten rat sarcoma viral oncogene homolog (KRAS), B-Raf proto-oncogene serine/threonine kinase (BRAF) and tumor protein p53 (TP53). In the present study, the mRNA expression levels of TAZ, AXL and CTGF were evaluated, and the TAZ-AXL-CTGF signature was correlated with the available pathological parameters and survival data. Overexpression of TAZ, AXL and CTGF was observed to be associated with severe pathological stage, deficiency in MMR, colon cancer subtype C4 and mutations in the BRAF gene. In addition, overexpression of TAZ-AXL-CTGF was associated with short overall survival in patients with mutations in the TP53 gene, colon cancer subtype C6, proficient MMR and wild-type status of the KRAS and BRAF genes. Furthermore, the prognostic value of TAZ-AXL-CTGF overexpression was observed to be independent of all the clinicopathological parameters and mutational statuses analyzed. The results of the present study confirm the previously reported findings, and suggest that the TAZ-AXL-CTGF mRNA signature is a potential prognostic indicator in colon cancer.

Antiviral activities of peptide-based covalent inhibitors of the Enterovirus 71 3C protease.

Hand, Foot and Mouth Disease is a highly contagious disease caused by a range of human enteroviruses. Outbreaks occur regularly, especially in the Asia-Pacific region, putting a burden on public healthcare systems. Currently, there is no antiviral for treating this infectious disease and the only vaccines are limited to circulation in China, presenting an unmet medical need that needs to be filled urgently. The human enterovirus 3 C protease has been deemed a plausible drug target due to its essential roles in viral replication. In this study, we designed and synthesized 10 analogues of the Rhinovirus 3 C protease inhibitor, Rupintrivir, and tested their 3 C protease inhibitory activities followed by a cellular assay using human enterovirus 71 (EV71)-infected human RD cells. Our results revealed that a peptide-based compound containing a trifluoromethyl moiety to be the most potent analogue, with an EC50 of 65 nM, suggesting its potential as a lead for antiviral drug discovery.

Arl2- and Msps-dependent microtubule growth governs asymmetric division.

Asymmetric division of neural stem cells is a fundamental strategy to balance their self-renewal and differentiation. It is long thought that microtubules are not essential for cell polarity in asymmetrically dividing Drosophila melanogaster neuroblasts (NBs; neural stem cells). Here, we show that Drosophila ADP ribosylation factor like-2 (Arl2) and Msps, a known microtubule-binding protein, control cell polarity and spindle orientation of NBs. Upon arl2 RNA intereference, Arl2-GDP expression, or arl2 deletions, microtubule abnormalities and asymmetric division defects were observed. Conversely, overactivation of Arl2 leads to microtubule overgrowth and depletion of NBs. Arl2 regulates microtubule growth and asymmetric division through localizing Msps to the centrosomes in NBs. Moreover, Arl2 regulates dynein function and in turn centrosomal localization of D-TACC and Msps. Arl2 physically associates with tubulin cofactors C, D, and E. Arl2 functions together with tubulin-binding cofactor D to control microtubule growth, Msps localization, and NB self-renewal. Therefore, Arl2- and Msps-dependent microtubule growth is a new paradigm regulating asymmetric division of neural stem cells.

Sequential and compartmentalized action of Rabs, SNAREs, and MAL in the apical delivery of fusiform vesicles in urothelial umbrella cells.

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV-membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network ∼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking.

Leishmania evades host immunity by inhibiting antigen cross-presentation through direct cleavage of the SNARE VAMP8.

During phagocytosis, microorganisms are taken up by immune cells into phagosomes. Through membrane-trafficking events mediated by SNARE proteins, phagosomes fuse with lysosomes, generating degradative phagolysosomes. Phagolysosomes contribute to host immunity by linking microbial killing within these organelles with antigen processing for presentation on MHC class I or II molecules to T cells. We show that the intracellular parasite Leishmania evades immune recognition by inhibiting phagolysosome biogenesis. The Leishmania cell surface metalloprotease GP63 cleaves a subset of SNAREs, including VAMP8. GP63-mediated VAMP8 inactivation or Vamp8 disruption prevents the NADPH oxidase complex from assembling on phagosomes, thus altering their pH and degradative properties. Consequently, the presentation of exogenous Leishmania antigens on MHC class I molecules, also known as cross-presentation, is inhibited, resulting in reduced T cell activation. These findings indicate that Leishmania subverts immune recognition by altering phagosome function and highlight the importance of VAMP8 in phagosome biogenesis and antigen cross-presentation.

Dual role of VAMP8 in regulating insulin exocytosis and islet ß cell growth.

Optimal insulin secretion required to maintain glucose homeostasis is the summation of total pancreatic islet ß cell mass and intrinsic secretory capacity of individual ß cells, which are regulated by distinct mechanisms that could be amplified by glucagon-like-peptide-1 (GLP-1). Because of these actions of GLP-1 on islet ß cells, GLP-1 has been deployed to treat diabetes. We employed SNARE protein VAMP8-null mice to demonstrate that VAMP8 mediates insulin granule recruitment to the plasma membrane, which partly accounts for GLP-1 potentiation of glucose-stimulated insulin secretion. VAMP8-null mice also exhibited increased islet ß cell mass from increased ß cell mitosis, with ß cell proliferative activity greatly amplified by GLP-1. Thus, despite the ß cell exocytotic defect, VAMP8-null mice have an increased total insulin secretory capacity, which improved glucose homeostasis. We conclude that these VAMP8-mediated events partly underlie the therapeutic actions of GLP-1 on insulin secretion and ß cell growth.

Use of an in vitro model and yeast two-hybrid system to investigate the pathogenesis of hepatitis C.

Fifteen years after the discovery of hepatitis C virus (HCV) in 1989, much remains to be learnt about the cell biology of this virus. Using the serum from a patient containing HCV RNA in high titer as a source, a Singapore strain of genotype 1b was recovered and characterized. This full-length HCV genome was then constructed into a tetracycline-inducible vector using the pSTAR plasmid. Transfection of hepatoma cell lines with this HCV genome under tetracycline induction indicated that chemokines (RANTES and monocyte chemoattractant protein-1) were upregulated, possibly contributing to the induction of immune responses. Using the yeast two-hybrid system to discover protein-protein interactions, nonstructural region NS3 was found to interact with itself, forming a dimer that increased helicase activity but was not essential for its activity, thereby disqualifying it as a suitable target of drug actions. The significance of the interaction between core and NS5A is unclear, and the cleavage of NS5A is related to the development of apoptosis. However, the interaction of p68 and NS56B appears to be important because the knockdown of p68 reduced the viral replication. Finally, a new cell model using chimeric CD81 linked to the cytoplasmic domain of either a low-density lipoprotein receptor or a transferrin receptor led to productive infection of HCV that had been recovered from infected serum. These studies allow us to examine the pathogenesis of HCV infection in more detail.

Inducible system in human hepatoma cell lines for hepatitis C virus production.

We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetracycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 h to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect nai;ve Huh7 and stable Huh7-human CD81 cells.