PI3K regulates endocytosis after insulin secretion by mediating signaling crosstalk between Arf6 and Rab27a.

In secretory cells, endocytosis is coupled to exocytosis to enable proper secretion. Although endocytosis is crucial to maintain cellular homeostasis before and after secretion, knowledge about secretagogue-induced endocytosis in secretory cells is still limited. Here, we searched for proteins that interacted with the Rab27a GTPase-activating protein (GAP) EPI64 (also known as TBC1D10A) and identified the Arf6 guanine-nucleotide-exchange factor (GEF) ARNO (also known as CYTH2) in pancreatic ß-cells. We found that the insulin secretagogue glucose promotes phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generation through phosphoinositide 3-kinase (PI3K), thereby recruiting ARNO to the intracellular side of the plasma membrane. Peripheral ARNO promotes clathrin assembly through its GEF activity for Arf6 and regulates the early stage of endocytosis. We also found that peripheral ARNO recruits EPI64 to the same area and that the interaction requires glucose-induced endocytosis in pancreatic ß-cells. Given that GTP- and GDP-bound Rab27a regulate exocytosis and the late stage of endocytosis, our results indicate that the glucose-induced activation of PI3K plays a pivotal role in exocytosis-endocytosis coupling, and that ARNO and EPI64 regulate endocytosis at distinct stages.

Involvement of the 3' Untranslated Region in Encapsidation of the Hepatitis C Virus.

Although information regarding morphogenesis of the hepatitis C virus (HCV) is accumulating, the mechanism(s) by which the HCV genome encapsidated remains unknown. In the present study, in cell cultures producing HCV, the molecular ratios of 3' end- to 5' end-regions of the viral RNA population in the culture medium were markedly higher than those in the cells, and the ratio was highest in the virion-rich fraction. The interaction of the 3' untranslated region (UTR) with Core in vitro was stronger than that of the interaction of other stable RNA structure elements across the HCV genome. A foreign gene flanked by the 3' UTR was encapsidated by supplying both viral NS3-NS5B proteins and Core-NS2 in trans. Mutations within the conserved stem-loops of the 3' UTR were observed to dramatically diminish packaging efficiency, suggesting that the conserved apical motifs of the 3´ X region are important for HCV genome packaging. This study provides evidence of selective packaging of the HCV genome into viral particles and identified that the 3' UTR acts as a cis-acting element for encapsidation.

Synthesis of coumarin derivatives and their cytoprotective effects on t-BHP-induced oxidative damage in HepG2 cells.

Coumarins are ubiquitous in higher plants and exhibit various biological actions. The aim of this study was to investigate the structure-activity relationships of coumarin derivatives on tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in human hepatoma HepG2 cells. A series of coumarin derivatives were prepared and assessed for their cytoprotective effects. Among these, a caffeoyl acid-conjugated dihydropyranocoumarin derivative, caffeoyllomatin, efficiently protected against cell damage elicited by t-BHP. Our findings suggest that caffeoyllomatin appears to be a potent cytoprotective agent.

Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

Adenosine 5'-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV), a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET)-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

Endogenous hydrogen sulfide protects pancreatic beta-cells from a high-fat diet-induced glucotoxicity and prevents the development of type 2 diabetes.

Chronic exposure to high glucose induces the expression of cystathionine gamma-lyase (CSE), a hydrogen sulfide-producing enzyme, in pancreatic beta-cells, thereby suppressing apoptosis. The aim of this study was to examine the effects of hydrogen sulfide on the onset and development of type 2 diabetes. Middle-aged (6-month-old) wild-type (WT) and CSE knockout (CSE-KO) mice were fed a high-fat diet (HFD) for 8weeks. We determined the effects of CSE knockout on beta-cell function and mass in islets from these mice. We also analyzed changes in gene expression in the islets. After 8weeks of HFD, blood glucose levels were markedly increased in middle-aged CSE-KO mice, insulin responses were significantly reduced, and DNA fragmentation of the islet cells was increased. Moreover, expression of thioredoxin binding protein-2 (TBP-2, also known as Txnip) was increased. Administration of NaHS, a hydrogen sulfide donor, reduced TBP-2 gene levels in isolated islets from CSE-KO mice. Gene levels were elevated when islets were treated with the CSE inhibitor dl-propargylglycine (PPG). These results provide evidence that CSE-produced hydrogen sulfide protects beta-cells from glucotoxicity via regulation of TBP-2 expression levels and thus prevents the onset/development of type 2 diabetes.

Independent evolution of multi-dominant viral genome species observed in a hepatitis C virus carrier.

The viral genome quasispecies composition of hepatitis C virus (HCV) could have important implications to viral pathogenesis and resistance to anti-viral treatment. The purpose of the present study was to profile the HCV RNA quasispecies. We developed a strategy to determine the full-length HCV genome sequences co-existing within a single patient serum by using next-generation sequencing technologies. The isolated viral clones were divided into the groups that can be distinguished by core amino acid 70 substitution. Subsequently, we determined HCV full-length genome sequences of three independent dominant species co-existing in the sequential serum with a 7-year interval. From phylogenetic analysis, these dominant species evolved independently. Our study demonstrated that multiple dominant species co-existed in patient sera and evolved independently.

Construction of a small-molecule-integrated semisynthetic split intein for in vivo protein ligation.

A new split intein-based protein ligation tool that is synthetically accessible and can be used for protein semisynthesis on the cell surface and potentially inside cells has been constructed.

The host protein CLUH participates in the subnuclear transport of influenza virus ribonucleoprotein complexes.

The nucleus is highly compartmentalized yet dynamic. Subnuclear functions are regulated by controlling the subnuclear localization of the nuclear proteins. Influenza viral ribonucleoprotein (vRNP) is replicated in the nucleus and then exported to the cytoplasm. However, the precise subnuclear localization and transport of vRNPs remain unclear. Here, we show that CLUH, a host protein whose cellular function is not well established, plays a key role in the subnuclear transport of vRNP. Viral PB2 and M1 induced CLUH translocation to the nucleoplasm and SC35-positive speckles, respectively, even though CLUH is usually cytoplasmic. CLUH depletion inhibited the translocation of M1 to SC35-positive speckles, but did not interfere with PB2 localization to the nucleoplasm and disrupted the subnuclear transport of vRNP, abolishing vRNP nuclear export without affecting viral RNA or protein expression. Our findings suggest that CLUH plays a role in the subnuclear transport of progeny vRNP.

Multi-spectral fluorescent reporter influenza viruses (Color-flu) as powerful tools for in vivo studies.

Seasonal influenza A viruses cause annual epidemics of respiratory disease; highly pathogenic avian H5N1 and the recently emerged H7N9 viruses cause severe infections in humans, often with fatal outcomes. Although numerous studies have addressed the pathogenicity of influenza viruses, influenza pathogenesis remains incompletely understood. Here we generate influenza viruses expressing fluorescent proteins of different colours ('Color-flu' viruses) to facilitate the study of viral infection in in vivo models. On adaptation to mice, stable expression of the fluorescent proteins in infected animals allows their detection by different types of microscopy and by flow cytometry. We use this system to analyse the progression of viral spread in mouse lungs, for live imaging of virus-infected cells, and for differential gene expression studies in virus antigen-positive and virus antigen-negative live cells in the lungs of Color-flu-infected mice. Collectively, Color-flu viruses are powerful tools to analyse virus infections at the cellular level in vivo to better understand influenza pathogenesis.

Activated Cdc42-bound IQGAP1 determines the cellular endocytic site.

Recruitment of specific molecules to a specific membrane site is essential for communication between specialized membranous organelles. In the present study, we identified IQGAP1 as a novel GDP-bound-Rab27a-interacting protein. We found that IQGAP1 interacts with GDP-bound Rab27a when it forms a complex with GTP-bound Cdc42. We also found that IQGAP1 regulates the endocytosis of insulin secretory membranes. Silencing of IQGAP1 inhibits both endocytosis and the glucose-induced redistribution of endocytic machinery, including Rab27a and its binding protein coronin 3. These processes can also be inhibited by disruption of the trimeric complex with dominant negative IQGAP1 and Cdc42. These results indicate that activation of Cdc42 in response to the insulin secretagogue glucose recruits endocytic machinery to IQGAP1 at the cell periphery and regulates endocytosis at this membrane site.

Viral interference with host mRNA surveillance, the nonsense-mediated mRNA decay (NMD) pathway, through a new function of HTLV-1 Rex: implications for retroviral replication.

Nonsense-mediated mRNA decay (NMD) is an essential and conserved cellular mRNA quality control mechanism. RNA signals to express viral genes from overlapping open reading frames potentially initiate NMD, nevertheless it is not clear whether viral RNAs are sensitive to NMD or if viruses have evolved mechanisms to evade NMD. Here we demonstrate that the genomic and full-length mRNAs of Human-T-cell Leukemia Virus type-I (HTLV-1), a retrovirus responsible for Adult T-cell Leukemia (ATL), are sensitive to NMD. They exhibit accelerated turnover in NMD-activated cells, while siRNA-mediated knockdown of NMD-master-regulator, UPF1, promotes enhanced stability of them. These effects on RNA stability were recapitulated by a reporter construct encoding the HTLV-1 translational frameshift signal of gag-pol. In agreement with the RNA stability, viral protein expression from the integrated provirus was inversely correlated with cellular NMD activity. We further demonstrated that the viral RNA-binding protein, Rex, approves the stability of viral RNA by inhibiting NMD. Significantly, Rex establishes a general block to NMD, as both NMD-responsive reporter transcripts and natural host-encoded NMD substrates were stabilized in the presence of Rex. Thus, we suggest that Rex not only stabilizes viral transcripts, but also perturbs cellular mRNA metabolism and host cell homeostasis via inhibition of NMD.

Chaperonin TRiC/CCT participates in replication of hepatitis C virus genome via interaction with the viral NS5B protein.

To identify the host factors implicated in the regulation of hepatitis C virus (HCV) genome replication, we performed comparative proteome analyses of HCV replication complex (RC)-rich membrane fractions prepared from cells harboring genome-length bicistronic HCV RNA at the exponential and stationary growth phases. We found that the eukaryotic chaperonin T-complex polypeptide 1 (TCP1)-ring complex/chaperonin-containing TCP1 (TRiC/CCT) plays a role in the replication possibly through an interaction between subunit CCT5 and the viral RNA polymerase NS5B. siRNA-mediated knockdown of CCT5 suppressed RNA replication and production of the infectious virus. Gain-of-function activity was shown following co-transfection with whole eight TRiC/CCT subunits. HCV RNA synthesis was inhibited by an anti-CCT5 antibody in a cell-free assay. These suggest that recruitment of the chaperonin by the viral nonstructural proteins to the RC, which potentially facilitate folding of the RC component(s) into the mature active form, may be important for efficient replication of the HCV genome.

Electrokinetic supercharging preconcentration and microchip gel electrophoretic separation of sodium dodecyl sulfate-protein complexes.

A microchip gel electrophoresis (MCGE) method with electrokinetic supercharging (EKS, electrokinetic injection with transient isotachophoresis) on a single channel chip was developed for high-sensitive detection of a standard mixture of six proteins (phosphorylase b, albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, and alpha-lactalbumin) in the form of sodium dodecyl sulfate (SDS) complexes. An average lower limit of detectable concentration (LLDC) achieved using UV detection at 214 nm was 0.27 microg/mL that is 30 times lower than that of conventional MCGE on a cross geometry chip. The calibration curves for molecular weight and concentration of SDS-protein complexes suggested that the present EKS-MCGE method had a better linear dynamic range and benefited future applications for qualitative and quantitative analysis of unknown protein samples. It was found that an excessive amount of unbound SDS in the sample deteriorated the preconcentration effect and resolution. The developed method appears greatly promising for high-speed and high sensitive analysis of SDS-proteins by MCGE.