Biomedical subjects

T Hesson

Publications and source records attributed to T Hesson.

6 recordsLinked to original sources

PubMed2001

Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies.

RNA helicases represent a family of enzymes that unwind double-stranded (ds) RNA in a nucleoside triphosphate (NTP)-dependent fashion and which are required in all aspects of cellular RNA metabolism and processing. The hepatitis C virus (HCV) non-structural 3 (NS3) protein possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion of the 631 amino acid residue bifunctional enzyme. The HCV NS3 RNA helicase is of key importance in the life cycle of HCV, which makes it a target for the development of therapeutics. However, neither the precise mechanism nor the substrate structure has been defined for this enzyme. For nuclear magnetic resonance (NMR)-based drug discovery methods and for mechanistic studies we engineered, prepared and characterized various truncated constructs of the 451-residue HCV NS3 RNA helicase. Our goal was to produce smaller fragments of the enzyme, which would be amenable to solution NMR techniques while retaining their native NTP and/or nucleic acid binding sites. Solution conditions were optimized to obtain high-quality heteronuclear NMR spectra of nitrogen-15 isotope-labeled constructs, which are typical of well-folded monomeric proteins. Moreover, NMR binding studies and functional data directly support the correct folding of these fragments.

Adenosine Triphosphatases↗

PubMed2000

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

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.

3' Untranslated Regions↗

PubMed1997

Structure of the hepatitis C virus RNA helicase domain.

Helicases are nucleotide triphosphate (NTP)-dependent enzymes responsible for unwinding duplex DNA and RNA during genomic replication. The 2.1 A resolution structure of the HCV helicase from the positive-stranded RNA hepatitis C virus reveals a molecule with distinct NTPase and RNA binding domains. The structure supports a mechanism of helicase activity involving initial recognition of the requisite 3' single-stranded region on the nucleic acid substrate by a conserved arginine-rich sequence on the RNA binding domain. Comparison of crystallographically independent molecules shows that rotation of the RNA binding domain involves conformational changes within a conserved TATPP sequence and untwisting of an extended antiparallel beta-sheet. Location of the TATPP sequence at the end of an NTPase domain beta-strand structurally homologous to the 'switch region' of many NTP-dependent enzymes offers the possibility that domain rotation is coupled to NTP hydrolysis in the helicase catalytic cycle.

Amino Acid Sequence↗

PubMed1992

Isolation and purification of a biologically active human platelet-derived growth factor BB expressed in Escherichia coli.

Human platelet-derived growth factor (PDGF) was expressed in Escherichia coli from a high-level cytoplasmic expression vector. A cDNA fragment encoding the mature form of the human PDGF B chain (hPDGF-B) was cloned into a plasmid under transcriptional control of the inducible E. coli Tac promoter. Expression of hPDGF-B from the final construct, pTacBIq, is regulated by the lactose repressor (LacIq). Upon induction, a polypeptide of approximately 14 kDa that had the same molecular mass and immunoreactivity as authentic hPDGF-B was produced. The production of recombinant hPDGF-B was significantly increased in an E. coli strain (CAG629) defective in expression of the lon protease. Expression of hPDGF-B in the CAG629 strain accounted for approximately 1% of total cell protein. In this system, hPDGF-B is expressed as an insoluble, intracellular protein and can readily be obtained in a partially purified form after differential centrifugation. Amino acid sequence determination of the purified protein has verified that the amino-terminal portion of the recombinant PDGF is correct. After renaturation into dimers, the purified recombinant hPDGF is fully functional in assays for receptor binding and mitogenesis.

Base Sequence↗

PubMed1985

Immunological evidence that inactive renin is prorenin.

Antibody raised to a synthetic dodecapeptide, corresponding to the C-terminal portion of the human renin pro-segment, was tested for its ability to recognize highly purified human inactive or active (mature) renins; immune complexes were detected by precipitation with protein A-Sepharose. Serial antibody dilutions caused identical binding of renal or plasma inactive renins but failed to bind active renin. In contrast, antibody to active renin recognized both active and inactive forms. Reversible acid activation of inactive renin enhanced its binding to the anti-prorenin antibody, whereas irreversible trypsin activation significantly reduced binding. Binding was abolished following prolonged exposure to trypsin or if inactive renin was acidified prior to trypsin treatment. These results indicate that inactive renin shares immunochemical determinants with prorenin; they suggest that acidification alters the conformation of the pro-segment and that trypsin can convert the molecule both to fully mature renin and to intermediate form(s).

Animals↗

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Biomedical Engineering↗