The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant to the inhibitors. Interestingly, proline 495 lies in a recently identified noncatalytic GTPbinding site, thus validating it as a potential allosteric site that can be targeted by small-molecule inhibitors of HCV polymerase.Hepatitis C virus (HCV) is the causative agent of the majority of chronic liver disease throughout the world. More than 170 million individuals are estimated to be infected with this virus (27). The size of the HCV epidemic and the limited efficacy of current therapy (based on the use of alpha interferon) have stimulated intense research efforts towards the development of antiviral drugs that are both better tolerated and more effective. The most widely established strategy for developing novel anti-HCV therapeutics aims at the identification of low-molecular-weight inhibitors of essential HCV enzymes.RNA-dependent RNA polymerase (RdRP) activity, carried out by the NS5B protein, is essential for virus replication (13) and has no functional equivalent in uninfected mammalian cells. It is thus likely that specific inhibitors of this enzyme can be found that block HCV replication with negligible associated toxicity. The NS5B RdRP has been expressed in a variety of recombinant forms (2, 4). The production of highly soluble forms of the enzyme (12, 24), devoid of the C-terminal membrane anchoring domain (23), has allowed considerable progress toward the determination of the enzyme's three-dimensional structure and mechanism of action. The crystal structure of NS5B revealed a classical "right hand" shape, showing the characteristic fingers, palm, and thumb subdomains (1,7,14). More recently, the three-dimensional structure of the HCV polymerase was solved in complex with RNA (20) as well as in a complex with nucleoside triphosphates (6). Three distinct nucleotide-binding sites were observed in the catalytic center of HCV RdRP whose geometry was remarkably similar to that observed in the initiation complex of the RNA phage ⌽6 RdRP (8), strengthening the proposal that the two enzymes initiate replication de novo by similar ...

In order to find small RNA molecules that are specific and high-affinity ligands of nonstructural 5B (NS5B) polymerase, we screened by SELEX (systematic evolution of ligands by exponential amplification) a structurally constrained RNA library with an NS5B⌬C55 enzyme carrying a C-terminal biotinylation sequence. Among the selected clones, two aptamers appeared to be high-affinity ligands of NS5B, with apparent dissociation constants in the low nanomolar range. They share a sequence that can assume a stem-loop structure. By mutation analysis, this structure has been shown to correspond to the RNA motif responsible for the tight interaction with NS5B. The aptamers appeared to be highly specific for the hepatitis C virus (HCV) polymerase since interaction with the GB virus B (GBV-B) NS5B protein cannot be observed. This is consistent with the observation that the activity of the HCV NS5B polymerase is efficiently inhibited by the selected aptamers, while neither GBV-B nor poliovirus 3D polymerases are affected. The mechanism of inhibition of the NS5B activity turned out to be noncompetitive with respect to template RNA, suggesting that aptamers and template RNA do not bind to the same site. As a matter of fact, mutations introduced in a basic exposed surface of the thumb domain severely impaired both the binding of and activity inhibition by the RNA aptamers.Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family, which affects more than 3% of the world population. About 80% of the infected patients develop liver cirrhosis and, in some cases, hepatocarcinoma (13). Besides interferon-based treatments, effective therapies to counteract this important public health problem are still lacking.The HCV positive-strand RNA viral genome contains a single open reading frame flanked by 5Ј-and 3Ј-untranslated regions. The open reading frame encodes a polyprotein of ca. 3,010 amino acids which is processed into at least 10 mature proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) by both host signal peptidases and viral proteases (10,31). In analogy with other positive-strand RNA viruses, HCV replication is supposed to proceed through the synthesis of negative-strand RNA, which is in turn used as a template for the production of genomic RNA molecules. A virally encoded RNA-dependent RNA polymerase (RdRp) is considered one of the key enzymes involved in both steps of HCV replication and is, therefore, a primary target for the development of antiviral drugs. The HCV RdRp activity has been localized in the 66-kDa nonstructural 5B (NS5B) protein (2). In vitro, purified NS5B has been shown to be a processive enzyme (39) capable of transcribing the full-length HCV genome (28) essentially via a snap-back mechanism. Recent studies indicate that NS5B does direct de novo replication, requiring neither an exogenous primer nor a snap-back priming event on a variety of RNA templates (22,30,37,43,44). The lack of specificity toward the HCV RNA genome suggests that NS5B corresponds to the elongation factor...

Transthyretin (TTR) is a 55 kDa homotetrameric protein. TTR in the cerebral spinal fluid (CSF) is primarily synthesized by the choroid plexus. TTR can bind to the beta-amyloid peptide and a number of familial amyloidosis diseases (familial amyloid polyneuropathy) have been associated with its allele variants. In a transgenic mice model overexpression of TTR was positively correlated with a neuroprotective effect from the pathogenic APPsw mutation. TTR has a free reactive sulphydryl moiety located on the Cys(10) residue which has been implicated to undergo a variety of oxidation reactions. To examine the neuroprotective role of TTR, we investigated the conjugated forms of TTR with cysteine (Cys) and cysteinglycine (CsyGly) in the CSF of 39 probable Alzheimer's disease (AD)-affected subjects and in a cohort of subjects without cognitive impairment (27 subjects). Linear MALDI-TOF MS experiments were employed to obtain high-resolution protein profiling of TTR isoforms. Nano-LC-TANDEM MS combined with reflectron MALDI-TOF-MS was used to unequivocally assign the investigated TTR-conjugate signals. Our results indicate a differential distribution of TTR-Cys and TTR-CysGly adducts. Both oxidized forms of TTR are significantly less abundant in the AD group (p = 0.0001). The investigated population (66 subjects) was then diagnosed using the ratio of conjugated TTR to free TTR in each subject. A sensitivity >90% and a specificity >70% were derived from a receiver operating characteristic curve when the overall cohort is analysed by the TTR-Cys signals. This manuscript is the first report describing the presence of differential post-translational oxidations of TTR in the CSF of AD patients.

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI-TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.