Anthony F. Mannarino

Bio: Anthony F. Mannarino is an academic researcher from Schering-Plough. The author has contributed to research in topics: Allosteric regulation & NS5B. The author has an hindex of 6, co-authored 7 publications receiving 961 citations.

Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site

Abstract: Various classes of nucleotidyl polymerases with different transcriptional roles contain a conserved core structure. Less is known, however, about the distinguishing features of these enzymes, particularly those of the RNA-dependent RNA polymerase class. The 1. 9 A resolution crystal structure of hepatitis C virus (HCV) nonstructural protein 5B (NS5B) presented here provides the first complete and detailed view of an RNA-dependent RNA polymerase. While canonical polymerase features exist in the structure, NS5B adopts a unique shape due to extensive interactions between the fingers and thumb polymerase subdomains that serve to encircle the enzyme active site. Several insertions in the fingers subdomain account for intersubdomain linkages that include two extended loops and a pair of antiparallel alpha-helices. The HCV NS5B apoenzyme structure reported here can accommodate a template:primer duplex without global conformational changes, supporting the hypothesis that this structure is essentially preserved during the reaction pathway. This NS5B template:primer model also allows identification of a new structural motif involved in stabilizing the nascent base pair.

Crystal structures of MEK1 binary and ternary complexes with nucleotides and inhibitors.

Abstract: MEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. We have determined that the kinase domain spans residues 35−382 by proteolytic cleavage. The complete kinase domain has been crystallized and its X-ray crystal structure as a complex with magnesium and ATP-γS determined at 2.1 A. Unlike crystals of a truncated kinase domain previously published, the crystals of the intact domain can be grown either as a binary complex with a nucleotide or as a ternary complex with a nucleotide and one of a multitude of allosteric inhibitors. Further, the crystals allow for the determination of costructures with ATP competitive inhibitors. We describe the structures of nonphosphorylated MEK1 (npMEK1) binary complexes with ADP and K252a, an ATP-competitive inhibitor (see Table 1), at 1.9 and 2.7 A resolution, respectively. Ternary complexes have also been solved between npMEK1, a nucleotide, and an allosteric non-ATP competitive inhibitor: ATP-γS with compound 1 and ADP w...

Thermodynamics of nucleotide and inhibitor binding to wild-type and ispinesib-resistant forms of human kinesin spindle protein.

Abstract: Current antimitotic cancer chemotherapy based on vinca alkaloids and taxanes target tubulin, a protein required not only for mitotic spindle formation but also for the overall structural integrity of terminally differentiated cells. Among many innovations targeting specific mitotic events, inhibition of motor enzymes including KSP (or Eg5) has been validated as a highly productive approach. Many reported KSP inhibitors bind to an induced allosteric site near the site of ATP hydrolysis, and some have been tested in clinical trials with varying degrees of success. This allosteric site was defined in detail by X-ray crystallography of inhibitor complexes, yet complementary information on binding thermodynamics is still lacking. Using two model ATP-uncompetitive inhibitors, monastrol and ispinesib, we report here the results of thermal denaturation and isothermal titration calorimetric studies. These binding studies were conducted with the wild-type KSP motor domain as well as two ispinesib mutants (D130V and...

Probing the relationship between RNA-stimulated ATPase and helicase activities of HCV NS3 using 2'-O-methyl RNA substrates.

Abstract: The hepatitis C virus (HCV) NS3 protein contains an amino terminal protease (NS3 aa. 1-180) and a carboxyl terminal RNA helicase (NS3 aa. 181-631). NS3 functions as a heterodimer of NS3 and NS4A (NS3/4A). NS3 helicase, a nucleic acid stimulated ATPase, can unwind RNA, DNA, and RNA:DNA duplexes, provided that at least one strand of the duplex contains a single-stranded 3' overhang (this strand of the duplex is referred to as the 3' strand). We have used 2'-O-methyl RNA (MeRNA) substrates to study the mechanism of NS3 helicase activity and to probe the relationship between its helicase and RNA-stimulated ATPase activities. NS3/4A did not unwind double-stranded (ds) MeRNA. NS3/4A unwinds hybrid RNA:MeRNA duplex containing MeRNA as the 5' strand but not hybrid duplex containing MeRNA as the 3' strand. The helicase activity of NS3/4A was 50% inhibited by 40 nM single-stranded (ss) RNA but only 35% inhibited by 320 nM ss MeRNA. Double-stranded RNA was 17 times as effective as double-stranded MeRNA in inhibiting NS3/4A helicase activity, while the apparent affinity of NS3/4A for ds MeRNA differed from ds RNA by only 2.4-fold. However ss MeRNA stimulated NS3/4A ATPase activity similar to ss RNA. These results indicate that the helicase mechanism involves 3' to 5' procession of the NS3 helicase along the 3' strand and only weak association of the enzyme with the displaced 5' strand. Further, our findings show that maximum stimulation of NS3 ATPase activity by ss nucleic acid is not directly related to procession of the helicase along the 3' strand.

Flaviviridae :T he Viruses and Their Replication

Abstract: FLAVIVIRUSES 1103 Background and Classification 1103 Structure and Physical Properties of the Virion 1104 Binding and Entry 1105 Genome Structure 1106 Translation and Proteolytic Processing 1107 Features of the Structural Proteins 1108 Features of the Nonstructural Proteins 1109 RNA Replication 1112 Membrane Reorganization and the Compartmentalization of Flavivirus Replication 1112 Assembly and Release of Particles from Flavivirus-infected Cells 1112 Host Resistance to Flavivirus Infection 1113

Replication of hepatitis C virus

Abstract: Hepatitis C virus (HCV) afflicts more than 170 million people worldwide causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The recent development of complete cell-culture systems for HCV has accelerated the pace of hepatitis research. Specifically, these techniques have provided new insights into the virus lifecycle that are reviewed here. This should pave the way for developing bespoke and effective antiviral therapies and vaccines. Exciting progress has recently been made in understanding the replication of hepatitis C virus, a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. The development of complete cell-culture systems should now enable the systematic dissection of the entire viral lifecycle, providing insights into the hitherto difficult-to-study early and late steps. These efforts have already translated into the identification of novel antiviral targets and the development of new therapeutic strategies, some of which are currently undergoing clinical evaluation.

Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF

Abstract: Proteins interact with genomic DNA to bring the genome to life; and these interactions also define many functional features of the genome. SBF and MBF are sequence-specific transcription factors that activate gene expression during the G1/S transition of the cell cycle in yeast. SBF is a heterodimer of Swi4 and Swi6, and MBF is a heterodimer of Mbpl and Swi6 (refs 1, 3). The related Swi4 and Mbp1 proteins are the DNA-binding components of the respective factors, and Swi6 mayhave a regulatory function. A small number of SBF and MBF target genes have been identified. Here we define the genomic binding sites of the SBF and MBF transcription factors in vivo, by using DNA microarrays. In addition to the previously characterized targets, we have identified about 200 new putative targets. Our results support the hypothesis that SBF activated genes are predominantly involved in budding, and in membrane and cell-wall biosynthesis, whereas DNA replication and repair are the dominant functions among MBF activated genes. The functional specialization of these factors may provide a mechanism for independent regulation of distinct molecular processes that normally occur in synchrony during the mitotic cell cycle.

Replication of the hepatitis C virus

Abstract: Infection with the hepatitis C virus (HCV) is a major cause of chronic liver disease. HCV is an enveloped plus-strand RNA virus closely related to flavi- and pestiviruses. The first cloning of the HCV genome, about 10 years ago, initiated research efforts leading to the elucidation of the genomic organization and the definition of the functions of most viral proteins. Despite this progress the lack of convenient animal models and appropriate in vitro propagation systems have hampered a full understanding of the way the virus multiplies. This review summarizes our current knowledge about HCV replication and describes attempts pursued in the last few years to establish efficient and reliable cell culture systems.

In vitro folding of inclusion body proteins.

Abstract: Insoluble, inactive inclusion bodies are frequently formed upon recombinant protein production in transformed microorganisms. These inclusion bodies, which contain the recombinant protein in an highly enriched form, can be isolated by solid/liquid separation. After solubilization, native proteins can be generated from the inactive material by using in vitro folding techniques. New folding procedures have been developed for efficient in vitro reconstitution of complex hydrophobic, multidomain, oligomeric, or highly disulfide-bonded proteins. These protocols take into account process parameters such as protein concentration, catalysis of disulfide bond formation, temperature, pH, and ionic strength, as well as specific solvent ingredients that reduce unproductive side reactions. Modification of the protein sequence has been exploited to improve in vitro folding.