Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.     

A novel membrane-dependent on/off switch mechanism of talin FERM domain at sites of cell adhesion

The activation of heterodimeric (α/β) integrin transmembrane receptors by cytosolic protein talin is crucial for regulating diverse cell-adhesion-dependent processes, including blood coagulation, tissue remodeling, and cancer metastasis. This process is triggered by the coincident binding of N-terminal FERM (four-point-one-protein/ezrin/radixin/moesin) domain of talin (talin-FERM) to the inner membrane surface and integrin β cytoplasmic tail, but how these binding events are spatiotemporally regulated remains obscure. Here we report the crystal structure of a dormant talin, revealing how a C-terminal talin rod segment (talin-RS) self-masks a key integrin-binding site on talin-FERM via a large interface. Unexpectedly, the structure also reveals a distinct negatively charged surface on talin-RS that electrostatically hinders the talin-FERM binding to the membrane. Such a dual inhibitory topology for talin is consistent with the biochemical and functional data, but differs significantly from a previous model. We show that upon enrichment with phosphotidylinositol-4,5-bisphosphate (PIP2) – a known talin activator, membrane strongly attracts a positively charged surface on talin-FERM and simultaneously repels the negatively charged surface on talin-RS. Such an electrostatic “pull-push” process promotes the relief of the dual inhibition of talin-FERM, which differs from the classic “steric clash” model for conventional PIP2-induced FERM domain activation. These data therefore unravel a new type of membrane-dependent FERM domain regulation and illustrate how it mediates the talin on/off switches to regulate integrin transmembrane signaling and cell adhesion.     

Mapping the interaction between the cytoplasmic domains of HIV-1 viral protein U and human CD4 with NMR spectroscopy

Viral protein?U (VpU) of HIV-1 plays an important role in downregulation of the main HIV-1 receptor CD4 from the surface of infected cells. Physical binding of VpU to newly synthesized CD4 in the endoplasmic reticulum is an early step in a pathway leading to proteasomal degradation of CD4. In this study, regions in the cytoplasmic domain of VpU involved in CD4 binding were identified by NMR spectroscopy. Amino acids in both helices found in the cytoplasmic region of VpU in membrane-mimicking detergent micelles experience chemical shift perturbations upon binding to CD4, whereas amino acids between the two helices and at the C-terminus of VpU show no or only small changes, respectively. The topology of the complex was further studied with paramagnetic relaxation enhancement. Paramagnetic spin labels were attached at three sequence positions of a CD4 peptide comprising the transmembrane and cytosolic domains of the receptor. VpU binds to a membrane-proximal region in the cytoplasmic domain of CD4. STRUCTURED DIGITAL ABSTRACT: VpU?and?CD4?bind?by?nuclear magnetic resonance?(View interaction).     

Metagenomic Exploration of Viruses throughout the Indian Ocean

The characterization of global marine microbial taxonomic and functional diversity is a primary goal of the Global Ocean Sampling Expedition. As part of this study, 19 water samples were collected aboard the Sorcerer II sailing vessel from the southern Indian Ocean in an effort to more thoroughly understand the lifestyle strategies of the microbial inhabitants of this ultra-oligotrophic region. No investigations of whole virioplankton assemblages have been conducted on waters collected from the Indian Ocean or across multiple size fractions thus far. Therefore, the goals of this study were to examine the effect of size fractionation on viral consortia structure and function and understand the diversity and functional potential of the Indian Ocean virome. Five samples were selected for comprehensive metagenomic exploration; and sequencing was performed on the microbes captured on 3.0-, 0.8- and 0.1 µm membrane filters as well as the viral fraction (<0.1 µm). Phylogenetic approaches were also used to identify predicted proteins of viral origin in the larger fractions of data from all Indian Ocean samples, which were included in subsequent metagenomic analyses. Taxonomic profiling of viral sequences suggested that size fractionation of marine microbial communities enriches for specific groups of viruses within the different size classes and functional characterization further substantiated this observation. Functional analyses also revealed a relative enrichment for metabolic proteins of viral origin that potentially reflect the physiological condition of host cells in the Indian Ocean including those involved in nitrogen metabolism and oxidative phosphorylation. A novel classification method, MGTAXA, was used to assess virus-host relationships in the Indian Ocean by predicting the taxonomy of putative host genera, with Prochlorococcus, Acanthochlois and members of the SAR86 cluster comprising the most abundant predictions. This is the first study to holistically explore virioplankton dynamics across multiple size classes and provides unprecedented insight into virus diversity, metabolic potential and virus-host interactions.     

The guanine nucleotide exchange factor, C3G regulates differentiation and survival of human neuroblastoma cells

Neuronal differentiation involving neurite growth is dependent on environmental cues which are relayed by signalling pathways to actin cytoskeletal remodelling. C3G, the exchange factor for Rap1, functions in pathways leading to actin reorganization and filopodia formation, processes required during neurite growth. In the present study, we have analyzed the function of C3G, in regulating neuronal cell survival and plasticity. Human neuroblastoma cells, IMR-32 induced to differentiate by serum starvation or by treatment with nerve growth factor (NGF) or forskolin showed enhanced C3G protein levels. Transient over-expression of C3G stimulated neurite growth and also increased responsiveness to NGF and serum deprivation induced differentiation. C3G-induced neurite growth was dependent on both its catalytic and N-terminal regulatory domains, and on the functions of Cdc42 and Rap1. Knockdown of C3G using small hairpin RNA inhibited forskolin and NGF-induced morphological differentiation of IMR-32 cells. Forskolin-induced differentiation was dependent on catalytic activity of C3G. Forskolin and NGF treatment resulted in phosphorylation of C3G at Tyr504 predominantly in the Golgi. C3G expression induced the cell cycle inhibitor p21 and C3G knockdown enhanced cell death in response to serum starvation. These findings demonstrate a novel function for C3G in regulating survival and differentiation of human neuroblastoma cells.     

Dissecting the functional role of polyketide synthases in Dictyostelium discoideum: biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol

Dictyostelium discoideum exhibits the largest repository of polyketide synthase (PKS) proteins of all known genomes. However, the functional relevance of these proteins in the biology of this organism remains largely obscure. On the basis of computational, biochemical, and gene expression studies, we propose that the multifunctional Dictyostelium PKS (DiPKS) protein DiPKS1 could be involved in the biosynthesis of the differentiation regulating factor 4-methyl-5-pentylbenzene-1,3-diol (MPBD). Our cell-free reconstitution studies of a novel acyl carrier protein Type III PKS didomain from DiPKS1 revealed a crucial role of protein-protein interactions in determining the final biosynthetic product. Whereas the Type III PKS domain by itself primarily produces acyl pyrones, the presence of the interacting acyl carrier protein domain modulates the catalytic activity to produce the alkyl resorcinol scaffold of MPBD. Furthermore, we have characterized an O-methyltransferase (OMT12) from Dictyostelium with the capability to modify this resorcinol ring to synthesize a variant of MPBD. We propose that such a modification in vivo could in fact provide subtle variations in biological function and specificity. In addition, we have performed systematic computational analysis of 45 multidomain PKSs, which revealed several unique features in DiPKS proteins. Our studies provide a new perspective in understanding mechanisms by which metabolic diversity could be generated by combining existing functional scaffolds.     

Effect of repeated in vitro sub-culturing on the virulence of Metarhizium anisopliae against Helicoverpa armigera (Lepidoptera: Noctuidae)

The effect of repeated conidial sub-culturing of Metarhizium anisopliae on its virulence against Helicoverpa armigera (Hübner) was studied. The LT₅₀ observed against third instar larvae of H. armigera for the first sub-culture was 3.4 days; it increased to 4.5 and 5.6 days for the 20th and the 40th sub-cultures, respectively. The LT₅₀ values after passage of the 40th sub-culture on H. armigera decreased to 4.4 and 3.7 days for the 40th (first in vivo) and the 40th (fifth in vivo) passages, respectively. Similarly, the LC₅₀ of M. anisopliae towards third instar larvae of H. armigera increased from the first sub-culture (0.17×10⁴) to (3.0×10⁴) for the 40th conidial transfers on potato dextrose agar and again decreased to 0.74×10⁴ and 0.23×10⁴ in the 40th (first in vivo) and the 40th (fifth in vivo) passage, respectively. Similar trends for LC₅₀ and LT₅₀ values were seen when sugarcane woolly aphid, Ceratovacuna lanigera Zehntner was used as a host. Significant variation in appressorium formation and cuticle-degrading enzyme production such as chitinase, chitin deacetylase, chitosanase and protease during subsequent sub-culturing and passage through H. armigera was observed. Though there was no effect on internal transcribed spacer (ITS) sequence pattern, interestingly, in randomly amplified polymorphic DNA (RAPD), significant differences in the band intensities and in the banding pattern for different sub-cultures of M. anisopliae were observed. As stable virulence towards the insect pest is desirable for commercialisation of a mycoinsecticide, such changes in virulence due to repeated in vitro transfer need to be monitored and minimised.