Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex.

Persistent infection of hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Searching for a substance with anti-HCV potential, we examined the effects of a variety of compounds on HCV replication using a HCV subgenomic replicon cell culture system. Consequently, the immunosuppressant cyclosporin A (CsA) was found to have a suppressive effect on the HCV replicon RNA level and HCV protein expression in these cells. CsA also inhibited multiplication of the HCV genome in a cultured human hepatocyte cell line infected with HCV using HCV-positive plasma. This anti-HCV activity of CsA appeared to be independent of its immunosuppressive function. In conclusion, our results suggest that CsA may represent a new approach for the development of anti-HCV therapy. (HEPATOLOGY 2003;38:1282-1288 P ersistent infection with the hepatitis C virus (HCV), identified as the major causative agent of non-A, non-B hepatitis, 1,2 has been closely related to liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. 3 The development of these liver diseases from HCV carriers, an estimated 170 million people throughout the world, is a major public health problem. Effective anti-HCV therapy has been restricted mainly to therapy with interferon (IFN) and a combination of IFN and ribavirin. However, because the virus is not eliminated from approximately one half of HCVinfected patients treated with these agents, 4 alternative approaches to the treatment of HCV infection are needed.Recently, an HCV subgenomic replicon cell culture system has been established in which an HCV subgenomic replicon autonomously replicated in Huh-7 cells (HCV replicon cells). 5 This replicon is composed of the HCV 5Ј-untranslated region containing an internal ribosomal entry site, the neomycin phosphotransferase gene, the encephalomyocarditis virus internal ribosomal entry site, HCV nonstructural proteins (NS) 3 through NS5B; and the HCV 3Ј-untranslated region (Fig. 1A). This system provides a unique tool for studying the mechanisms of HCV replication and screening as well as evaluating anti-HCV compounds. Taking advantage of this feature, we examined the effects of various types of compounds on the replication of HCV using HCV replicon cells established in our laboratory 6 (Miyanari et al., manuscript accepted for publication). Consequently, we found that a well-known immunosuppressant, cyclosporin A (CsA), 7 had a strong suppressive effect on HCV replication in these cells. Moreover, we found suppressive activity of CsA for multiplication of the HCV genome in cultured human hepatocytes infected with HCV. The mechanism of the anti-HCV activity of CsA was also studied. Materials and MethodsCell Culture. Huh-7 and MH-14 cells, HCV replicon cells, were cultured in Dulbecco's modified Eagle medium with 10% fetal bovine serum. PH5CH8 cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle medium and F12 medium supplemented with 100 ng/mL epidermal gro...

Recently, we identified and characterized a novel protein, granuphilin, whose domain structure is similar to that of the Rab3 effector protein rabphilin3 (J. Wang, T. Takeuchi, H. Yokota, and T. Izumi, J. Biol. Chem. 274:28542-28548, 1999). Screening its possible Rab partner by a yeast two-hybrid system revealed that an amino-terminal zinc-finger domain of granuphilin interacts with Rab27a. Granuphilin preferentially bound to the GTP form of Rab27a. Formation of the Rab27a/granuphilin complex was readily detected in the pancreatic beta cell line MIN6. Moreover, the tissue distributions of Rab27a and granuphilin are remarkably similar: both had significant and specific expression in pancreatic islets and in pituitary tissue, but no expression was noted in the brain. Analyses by immunofluorescence, immunoelectron microscopy, and sucrose density gradient subcellular fractionation showed that Rab27a and granuphilin are localized on the membrane of insulin granules. These findings suggest that granuphilin functions as a Rab27a effector protein in beta cells. Overexpression of wild-type Rab27a and its GTPase-deficient mutant significantly enhanced high K ؉ -induced insulin secretion without affecting basal insulin release. Although Rab3a, another exocytotic Rab protein, has some similarities with Rab27a in primary sequence, intracellular distribution, and affinity toward granuphilin, overexpression of Rab3a caused different effects on insulin secretion. These results indicate that Rab27a is involved in the regulated exocytosis of conventional dense-core granules possibly through the interaction with granuphilin, in addition to its recently identified role in lysosome-related organelles.

Iridium complex is a promising organic light-emitting diode material for next generation video displays that emits phosphorescence quenched by oxygen. We used this oxygen-quenching feature for imaging tumor hypoxia. Red light-emitting Ir(btp) 2 (acac) (BTP) presented hypoxia-dependent light emission in culture cell lines, whose intensity was in parallel with hypoxia-inducible factor-1α images. BTP was further applied to imaging five nude mouse transplanted with tumors. All tumors presented a bright BTP-emitting image even 5 minutes after injection. The minimal image recognition size was ∼2 mm in diameter. By morphologic examination and phosphorescence lifetime measurement, BTP appeared to localize to the tumor cells. Because BTP is easily modifiable, we synthesized BTP analogues with a longer excitation/emission wavelength. One of them, BTPHSA, depicted clear imaging from tumors transplanted 6 to 7 mm deep from the skin surface. We suggest that iridium complex materials have a vast potential for imaging hypoxic lesions such as tumor tissues. Cancer Res; 70(11); 4490-8. ©2010 AACR.