Shaping tubular carriers for intracellular membrane transport

The particular compositions of the intracellular membrane organelles rely on the proteins and lipids received frequently through membrane trafficking. The delivery of these molecules is driven by the membrane-bound organelles known as transport carriers (TCs). Advanced microscopy approaches have revealed that TC morphology ranges from small vesicles to complex tubular membrane structures. These tubular TCs (TTCs) support effectively both sorting and transport events within the biosynthetic and endocytic pathways, while a coherent picture of the processes that define the formation and further fate of TTCs is still missing. Here, we present an overview of the mechanisms operating during the TTC life cycle, as well as of the emerging role of tubular carriers in different intracellular transport routes.     

Functional Analysis of the Transmembrane Domain in Paramyxovirus F Protein-Mediated Membrane Fusion

To enter cells, enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single-cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the paramyxovirus parainfluenza virus 5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion.     

Role of Individual Domains and Identification of Internal Gap in Human Guanylate Binding Protein-1

Unlike other GTPases, interferon-gamma-induced human guanylate binding protein-1 has the ability to hydrolyze GTP to both GDP and GMP, with GMP being the major product of the reaction. This protein has two domains, an N-terminal globular domain and a C-terminal helical domain. These two domains are connected by a short intermediate region consisting of a two-stranded β-sheet and a helix. As human guanylate binding protein-1 has been shown to undergo stimulated GTPase activity without external GTPase-activating protein, we sought to understand the roles of each of the two individual domains, the intermediate region, a conserved motif (¹⁰³DXEKGD¹⁰⁸), and the mechanism of the stimulation of GTPase activity. The steady-state assays using radiolabeled [α-³²P]GTP on the wild-type protein suggest that the stimulation of activity primarily occurs during the cleavage of the second phosphate of GTP rather than the first, through allosteric interaction. Using several truncated and mutant proteins, we demonstrate for the first time that both the α-helix of the intermediate region and the ¹⁰³DXEKGD¹⁰⁸ motif play critical roles for the hydrolysis to GMP, but they appear to act in different ways: α-helix acts through structural stabilization by allosteric interaction and, thus, acts as an internal GTPase-activating protein, whereas the motif might act by providing necessary catalytic residues. Our data also show that the N-terminal globular domain is able to perform only the first catalysis (GTP to GDP, an activity associated with basal level), but the helical domain in the full-length protein stimulates the hydrolysis of GTP to GMP with higher GMP formation by preventing the dissociation of GDP-bound enzyme dimer.     

Translation of mRNAs from Vesicular Stomatitis Virus and Vaccinia Virus Is Differentially Blocked in Cells with Depletion of eIF4GI and/or eIF4GII

Cytolytic viruses abrogate host protein synthesis to maximize the translation of their own mRNAs. In this study, we analyzed the eukaryotic initiation factor (eIF) 4G requirement for translation of vesicular stomatitis virus (VSV) and vaccinia virus (VV) mRNAs in HeLa cells using two different strategies: eIF4G depletion by small interfering RNAs or cleavage of eIF4G by expression of poliovirus 2A protease. Depletion of eIF4GI or eIF4GII moderately inhibits cellular protein synthesis, whereas silencing of both factors has only a slightly higher effect. Under these conditions, the extent of VSV protein synthesis is similar to that of nondepleted control cells, whereas VV expression is substantially reduced. Similar results were obtained when eIF4E was depleted. On the other hand, eIF4G cleavage by poliovirus 2A protease strongly inhibits translation of VV protein expression, whereas translation directed by VSV mRNAs is not abrogated, even though VSV mRNAs are capped. Therefore, the requirement for eIF4F activity is different for VV and VSV, suggesting that the molecular mechanism by which their mRNAs initiate their translation is also different. Consistent with these findings, eIF4GI does not colocalize with ribosomes in VSV-infected cells, while eIF2α locates at perinuclear sites coincident with ribosomes.     

ROLE OF EVOLVED HOST BREADTH IN THE INITIAL EMERGENCE OF AN RNA VIRUS

Understanding how evolution promotes pathogen emergence would aid disease management, and prediction of future host shifts. Increased pathogen infectiousness of different hosts may occur through direct selection, or fortuitously via indirect selection. However, it is unclear which type of selection tends to produce host breadth promoting pathogen emergence. We predicted that direct selection for host breadth should foster emergence by causing higher population growth on new hosts, lower among‐population variance in growth on new hosts, and lower population variance in growth across new hosts. We tested the predictions using experimentally evolved vesicular stomatitis virus populations, containing groups of host‐use specialists, directly selected generalists, and indirectly selected generalists. In novel‐host challenges, viruses directly selected for generalism showed relatively higher or equivalent host growth, lower among‐population variance in host growth, and lower population variance in growth across hosts. Thus, two of three outcomes supported our prediction that directly selected host breadth should favor host colonization. Also, we observed that indirectly selected generalists were advantaged over specialist viruses, indicating that fortuitous changes in host breadth may also promote emergence. We discuss evolution of phenotypic plasticity versus environmental robustness in viruses, virus avoidance of extinction, and surveillance of pathogen niche breadth to predict future likelihood of emergence.     

A 4-deoxy analogue of N-acetyl-d-glucosamine inhibits heparan sulphate expression and growth factor binding in vitro

Heparan sulphate (HS) is a long, linear polysaccharide, which has a basic backbone of -β1-4GlcA-α1-4GlcNAc- units. The involvement of HS in many steps of tumourigenesis, including growth and angiogenesis, makes it an appealing target for cancer therapy. To target the biosynthesis of HS by interfering with its chain elongation, a 4-deoxy analogue of N-acetyl-d-glucosamine (4-deoxy-GlcNAc) was synthesized. Using immunocytochemistry and agarose gel electrophoresis it was shown that incubation with the 4-deoxysugar resulted in a dose dependent reduction of HS expression of MV3 melanoma cells, 1 mM resulting in an almost nullified HS expression. The parent sugar GlcNAc had no effect. 4-deoxysugar treated cells were viable and proliferated at the same rate as control cells. Other glycan structures appeared to be only mildly affected, as staining by various lectins was generally not or only modestly inhibited. At 1 mM of the 4-deoxysugar, the capacity of cells to bind the HS-dependent pro-angiogenic growth factors FGF-2 and VEGF was greatly compromised. Using an in vitro angiogenesis assay, 4-deoxysugar treated endothelial cells showed a sharp reduction of FGF-2-induced sprout formation. Combined, these data indicate that an inexpensive, easily synthesized, water-soluble monosaccharide analogue can interfere with HS expression and pro-angiogenic growth factor binding.     

Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.     

Association between interleukin 4 gene intron 3 VNTR polymorphism and recurrent aphthous stomatitis in a cohort of Turkish patients

Objective

Recurrent aphthous stomatitis (RAS) is one of the most common oral mucosal diseases, with a multifactorial etiopathogenesis, an interaction between predisposing factors and/or systemic conditions and immunological components in genetically predisposed subjects. Although there is no clear genetic mode of inheritance, there is evidence that inheritance of specific gene polymorphisms may predispose individuals to RAS. The purpose of the present study was to investigate a possible association between the functional interleukin 4 (IL4) VNTR genetic polymorphism and RAS in a sample of Turkish patients.

Methods

The study included 145 unrelated patients with a clinical diagnosis of RAS and 150 unrelated healthy controls. Genomic DNA was isolated and IL4 gene 70 bp VNTR polymorphism determined by using polymerase chain reaction (PCR) with specific primers.

Results

The distribution of genotype and allele frequencies of IL4 gene intron 3 VNTR polymorphism was statistically different between RAS patients and control group (p < 0.0001 and p < 0.0001, respectively) P2P2 genotype and P2 allele were also found to be protective with a lower risk for susceptibility to RAS (p < 0.0001).

Conclusion

The results of this study suggest that intron 3 VNTR polymorphism in the IL4 gene is associated with RAS susceptibility in Turkish population.     

Gene therapy for PIDs: Progress,pitfalls and prospects

Substantial progress has been made in the past decade in treating several primary immunodeficiency disorders (PIDs) with gene therapy. Current approaches are based on ex-vivo transfer of therapeutic transgene via viral vectors to patient-derived autologous hematopoietic stem cells (HSCs) followed by transplantation back to the patient with or without conditioning. The overall outcome from all the clinical trials targeting different PIDs has been extremely encouraging but not without caveats. Malignant outcomes from insertional mutagenesis have featured prominently in the adverse events associated with these trials and have warranted intense pre-clinical investigation into defining the tendencies of different viral vectors for genomic integration. Coupled with issues pertaining to transgene expression, the therapeutic landscape has undergone a paradigm shift in determining safety, stability and efficacy of gene therapy approaches. In this review, we aim to summarize the progress made in the gene therapy trials targeting ADA-SCID, SCID-X1, CGD and WAS, review the pitfalls, and outline the recent advancements which are expected to further enhance favourable risk benefit ratios for gene therapeutic approaches in the future.