The unfolded protein response is required to maintain the integrity of the endoplasmic reticulum, prevent oxidative stress and preserve differentiation in ß-cells.

Diabetes is an epidemic of worldwide proportions caused by ß-cell failure. Nutrient fluctuations and insulin resistance drive ß-cells to synthesize insulin beyond their capacity for protein folding and secretion and thereby activate the unfolded protein response (UPR), an adaptive signalling pathway to promote cell survival upon accumulation of unfolded protein in the endoplasmic reticulum (ER). Protein kinase-like endoplasmic reticulum kinase (PERK) signals one component of the UPR through phosphorylation of eukaryotic initiation factor 2 on the α-subunit (eIF2α) to attenuate protein synthesis, thereby reducing the biosynthetic burden. ß-Cells uniquely require PERK-mediated phosphorylation of eIF2α to preserve cell function. Unabated protein synthesis in ß-cells is sufficient to initiate a cascade of events, including oxidative stress, that are characteristic of ß-cell failure observed in type 2 diabetes. In contrast to acute adaptive UPR activation, chronic activation increases expression of the proapoptotic transcription factor CAAT/enhancer-binding protein homologous protein (CHOP). Chop deletion in insulin-resistant mice profoundly increases ß-cell mass and prevents ß-cell failure to forestall the progression of diabetes. The findings suggest an unprecedented link by which protein synthesis and/or misfolding in the ER causes oxidative stress and should encourage the development of novel strategies to treat diabetes.

La autoantigen enhances translation of BiP mRNA.

Translational initiation of the human BiP mRNA is directed by an internal ribosomal entry site (IRES) located in the 5'-untranslated region (5'-UTR). In order to understand the mechanism of the IRES-dependent translation of BiP mRNA, cellular proteins interacting with the BiP IRES were investigated. La autoantigen, which augments the translation of polioviral mRNA and hepatitis C viral mRNA, bound specifically to the second half of the 5'-UTR of the BiP IRES and enhanced translation of BiP mRNA in both in vitro and in vivo assays. This finding suggests that cellular and viral IRESs containing very different RNA sequences may share a common mechanism of translation.

Expression and purification of an active, full-length hepatitis C viral NS4A.

The nonstructural protein 3 (NS3) of the hepatitis C virus (HCV) is a bifunctional protein with protease and helicase activities. Nonstructural protein 4A (NS4A) is preceded by NS3 and augments the proteolytic activity of NS3 through protein-protein interaction. The central domain of NS4A has been shown to be sufficient for the enhancement of the NS3 protease activity. However, investigations on the roles of the N-terminal and the C-terminal regions of NS4A have been hampered by the difficulty of purification of full-length NS4A, a polypeptide that contains highly hydrophobic amino acid residues. Here we report a procedure by which one can produce and purify an active, full-length NS4A using maltose-binding protein fusion method. The full-length NS4A fused to the maltose binding protein is soluble and maintains its NS3 protease-enhancing activity.

In vivo determination of substrate specificity of hepatitis C virus NS3 protease: genetic assay for site-specific proteolysis.

Hepatitis C virus (HCV) NS3 protease is responsible for the processing of the viral polyprotein and is considered as a primary target for the development of anti-HCV therapy. We have developed a genetic method in yeast to screen for good substrate sequences of the NS3 protease. A library of fusion proteins was constructed with a transcription factor, GAL4, linked to the intracellular domain of an integral membrane protein, STE2, by a randomized protease substrate sequence. In yeast cells expressing NS3 protease, the substrate sequences in the fusion proteins were specifically recognized and cleaved. This cleavage resulted in the release of GAL4 from the cytoplasmic membrane and the subsequent activation of reporter genes by GAL4, which was detected by the growth of yeast cells on selective media. Based on the analysis of 69 isolated substrate sequences, a consensus sequence was deduced: (Glu/Asp)-X-Val-Val-(Leu/Pro)-Cys / (Ser/Ala), with the scissile bond being located between Cys and Ser or Ala and X not being determined. This is largely consistent with the previous results obtained by biochemical methods. An oligopeptide containing the deduced sequence was highly efficiently cleaved in vitro by the purified NS3 protease. These data demonstrated that the present genetic method could be used as an efficient tool for the in vivo determination of substrate specificity of proteases.

The effect of pelvic floor muscle exercises on genuine stress incontinence among Korean women--focusing on its effects on the quality of life.

This study's purpose was to compare the treatment efficacy and the effects on the patients' quality of life of the pelvic floor muscle (PFM) exercise and the functional electrical stimulation (FES)-biofeedback method. Ninety female incontinence patients were randomly selected and evenly divided into three groups: control, intensive PFM exercise, and FES-biofeedback groups. They were treated for 6 weeks. The subjective changes in the severity of incontinence and discomfort in daily and social life were measured using a translated version of Jackson's Bristol female urinary symptom questionnaire. Objective changes of pelvic muscle contraction force were measured by perineometer. Pre and post-treatment maximal pelvic floor muscle contractile pressure (PMC pressure) among the three groups showed statistically significant differences (p < 0.001). Especially the FES-biofeedback group showed significantly increased maximal PMC pressure compared with other groups (p < 0.001). From the questionnaire, pre and post-treatment changes in the severity of urinary incontinence and discomfort due to incontinence showed significant differences among the three groups (p < 0.001). The level of discomfort in daily life, social activity, physical activity, personal relations and discomfort due to urinary symptoms had largely changed and the FES-biofeedback group, in particular, showed a significant decrease after treatment. In conclusion, when PFM exercise and FES-biofeedback were compared in terms of their effects on the patients' quality of life, FES-biofeedback proved to be more effective than verbal explanation or simple PFM exercise.

Subcellular localization of hepatitis C viral proteins in mammalian cells.

We determined the subcellular localization of hepatitis C viral (HCV) proteins as a first step towards the understanding of the functions of these proteins in the mammalian cell (CHO-K1). We used fluorescence emitted from green fluorescent protein (GFP)-fused to the viral proteins to determine the subcellular localization of the viral proteins. We found that most of the viral proteins were excluded from the nucleus. Core exhibited a globular pattern near the nucleus. NS2 was concentrated in the perinuclear space. NS4A accumulated in the ER and the Golgi regions. NS3 was detected in the nucleus as well as the cytoplasm, when it was expressed by itself. However, NS3 became restricted to the cytoplasm, when it was produced together with NS4A. NS4B showed a spot-like pattern throughout the cytoplasm. NS5A and NS5B were distributed throughout the cytoplasm in a mesh-like pattern. These results can provide a basis for further investigations into the functions of the HCV proteins.

Crystallization and preliminary X-ray crystallographic analysis of the helicase domain of hepatitis C virus NS3 protein.

The NS3 protein of hepatitis C virus (HCV) is thought to be essential for viral replication. The N-terminal domain of the protein contains protease activity and the C-terminal domain contains nucleotide triphosphatase and RNA helicase activity. The RNA helicase domain of HCV NS3 protein was purified by using affinity-column chromatographic methods, and crystallized by using the microbatch crystallization method under oil at 277 K. The crystals belong to primitive trigonal space group P3121 or P3221 with cell dimensions of a = b = 93.3, c = 104.6 A. The asymmetric unit contains one molecule of the helicase domain, with the crystal volume per protein mass (Vm) of 2.50 A3 Da-1 and solvent content of about 50.8% by volume. A native data set to 2.3 A resolution was obtained from a frozen crystal indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.

Crystal structure of RNA helicase from genotype 1b hepatitis C virus. A feasible mechanism of unwinding duplex RNA.

Crystal structure of RNA helicase domain from genotype 1b hepatitis C virus has been determined at 2.3 A resolution by the multiple isomorphous replacement method. The structure consists of three domains that form a Y-shaped molecule. One is a NTPase domain containing two highly conserved NTP binding motifs. Another is an RNA binding domain containing a conserved RNA binding motif. The third is a helical domain that contains no beta-strand. The RNA binding domain of the molecule is distinctively separated from the other two domains forming an interdomain cleft into which single stranded RNA can be modeled. A channel is found between a pair of symmetry-related molecules which exhibit the most extensive crystal packing interactions. A stretch of single stranded RNA can be modeled with electrostatic complementarity into the interdomain cleft and continuously through the channel. These observations suggest that some form of this dimer is likely to be the functional form that unwinds double stranded RNA processively by passing one strand of RNA through the channel and passing the other strand outside of the dimer. A "descending molecular see-saw" model is proposed that is consistent with directionality of unwinding and other physicochemical properties of RNA helicases.

Generation of a novel poliovirus with a requirement of hepatitis C virus protease NS3 activity.

Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B hepatitis. One of the difficulties in developing anti-HCV drugs is the lack of an efficient HCV cultivation system. We have generated an artificial surrogate virus suitable for testing the antiviral effects of drugs affecting HCV protease NS3, an enzyme believed to be essential for HCV proliferation. The surrogate virus genome is composed of most of the poliovirus genome and HCV protease NS3 and an NS3-specific cleavage site. The activity of HCV protease NS3 is required for proliferation of this chimeric virus. The antiviral efficacy of HCV protease inhibitors can, therefore, be evaluated by examining the effects of the drugs on the surrogate virus proliferation.

NS3-4A of hepatitis C virus is a chymotrypsin-like protease.

The polyprotein encoded by a single open reading frame of hepatitis C virus (HCV) is processed by host- and virus-encoded proteases. The viral protease NS3 is responsible for the cleavage of at least four sites (NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions) in the nonstructural protein region. To characterize the protease function of NS3 and NS4 on various target sites, efficient cis- and trans-cleavage assay systems were developed by using in vitro transcription and translation. Deletion of the C-terminal two-thirds from NS3 in an NS3-NS4A-4B polypeptide (NS3 delta C-4A-4B) hampered cleavage of the NS3/4A junction but not that of the NS4A/4B junction. As a consequence, expression of NS3 delta C-4A-4B containing an internal deletion of NS3 results in an NS3 delta C-4A fusion protein. NS3 delta C-4A shows very efficient and specific trans-cleavage activity at NS4A/4B, NS4B/5A, and NS5A/5B junctions. In addition, the biochemical properties of HCV NS3 delta C-4A were further elucidated by adding known protease inhibitors in trans-cleavage reactions. The HCV protease NS3-4A is inhibited by chymotrypsin-specific inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), chymostatin, and Pefabloc SC but not by trypsin-like protease inhibitors antipain, leupeptin, and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by the protease inhibitors E-64, bestatin, pepstatin, and phosphoramidon. This finding strongly suggests that HCV protease NS3-4A is a chymotrypsin-like serine protease.