A consensus sequence for substrate hydrolysis by rhino virus 3C proteinase

Long, Audrey C.; Orr, David C.; Js, Cameron; Dunn, Ben M.; Kay, John

doi:10.1016/0014-5793(89)81619-9

Cited by 48 publications

(41 citation statements)

References 6 publications

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“…Substitution of P1Ј-Leu and P2Ј-Leu by Ala had smaller but still significant detrimental effects on cleavage. The P1 and P4 positions have previously been shown to be important in peptide substrates for HRV and HAV 3C proteases (38,39) and are the most highly conserved positions in the FMDV polyprotein cleavage sites (40). Whereas the deleterious effect of Ala substitutions for P2-Lys and P4Ј-Phe indicates that these residues contribute substantially to substrate recognition, it should be noted that they are not conserved in the polyprotein (Table II).…”

Section: Structure and Specificity Of Fmdv 3cmentioning

confidence: 88%

Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease

Birtley¹,

Knox

Jaulent

et al. 2005

Journal of Biological Chemistry

133

144

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Foot-and-mouth disease virus (FMDV) causes a widespread and economically devastating disease of domestic livestock. Although FMDV vaccines are available, political and technical problems associated with their use are driving a renewed search for alternative methods of disease control. The viral RNA genome is translated as a single polypeptide precursor that must be cleaved into functional proteins by virally encoded proteases. 10 of the 13 cleavages are performed by the highly conserved 3C protease (3C pro ), making the enzyme an attractive target for antiviral drugs. We have developed a soluble, recombinant form of FMDV 3C pro , determined the crystal structure to 1.9-Å resolution, and analyzed the cleavage specificity of the enzyme. The structure indicates that FMDV 3C pro adopts a chymotrypsin-like fold and possesses a Cys-His-Asp catalytic triad in a similar conformation to the Ser-His-Asp triad conserved in almost all serine proteases. This observation suggests that the dyad-based mechanisms proposed for this class of cysteine proteases need to be reassessed. Peptide cleavage assays revealed that the recognition sequence spans at least four residues either side of the scissile bond (P4 -P4) and that FMDV 3C pro discriminates only weakly in favor of P1-Gln over P1-Glu, in contrast to other 3C pro enzymes that strongly favor P1-Gln. The relaxed specificity may be due to the unexpected absence in FMDV 3C pro of an extended ␤-ribbon that folds over the substrate binding cleft in other picornavirus 3C pro structures. Collectively, these results establish a valuable framework for the development of FMDV 3C pro inhibitors.

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Section: Structure and Specificity Of Fmdv 3cmentioning

confidence: 88%

Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease

Birtley¹,

Knox

Jaulent

et al. 2005

Journal of Biological Chemistry

133

144

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“…hOSM was produced as a glutathione S-transferase fusion protein that has a recognition site for human rhinovirus protease 3C (HRV3C) (48,49) inserted in place of the thrombin site.…”

Section: Methodsmentioning

confidence: 99%

An Antagonist for the Leukemia Inhibitory Factor Receptor Inhibits Leukemia Inhibitory Factor, Cardiotrophin-1, Ciliary Neurotrophic Factor, and Oncostatin M

Vernallis

Hudson

Heath

1997

Journal of Biological Chemistry

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The leukemia inhibitory factor receptor (LIF-R) is activated not only by LIF, but also by cardiotrophin-1, ciliary neurotrophic factor with its receptor, and oncostatin M (OSM). Each of these cytokines induces the hetero-oligomerization of LIF-R with gp130, a signaltransducing subunit shared with interleukin-6 and interleukin-11. The introduction of mutations into human LIF that reduced the affinity for gp130 while retaining affinity for LIF-R has generated antagonists for LIF. In the current study, a LIF antagonist that was free of detectable agonistic activity was tested for antagonism against the family of LIF-R ligands. On cells that express LIF-R and gp130, all LIF-R ligands were antagonized. On cells that also express OSM receptor, OSM was not antagonized, demonstrating that the antagonist is specific for LIF-R. Ligand-triggered tyrosine phosphorylation of both LIF-R and gp130 was blocked by the antagonist. The antagonist is therefore likely to work by preventing receptor oligomerization.Functional overlap among cytokines often derives from shared receptor components. Interleukin-6 (IL-6), 1 IL-11, leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), ciliary neurotrophic factor (CNTF), and OSM (oncostatin M) have biological activities in common, and all require gp130 as a signal-transducing subunit (for review see Ref. 1). LIF (2-6), CT-1 (7), CNTF (8,9), and OSM (6, 10, 11) activate gp130 in complexes with LIF receptor (LIF-R). LIF-R is a transmembrane signaling subunit that is structurally related to gp130 and belongs to the same hematopoietin receptor family (4). Ligand binding drives the heterodimerization of LIF-R and gp130, which results in inter alia activation and tyrosine phosphorylation of the Jak-Tyk cytoplasmic tyrosine kinases (12,13). The Jaks in turn phosphorylate tyrosine residues in the cytoplasmic domains of both LIF-R and gp130 (8), allowing the recruitment of substrates with Src homology 2 domains including STAT3 and protein tyrosine phosphatase PTP1D (14 -17) (for review see Refs. 18 and 19). CNTF weakly activates LIF-R-gp130, becoming a potent agonist once first bound to a nonsignal-transducing subunit, CNTFR␣. In addition to LIF-Rgp130, OSM also activates a receptor complex unique to OSM made up of . OSM-R has recently been identified as a transmembrane signaling subunit related to . Thus, LIF-R participates in a subset of gp130-mediated responses that includes all known LIF, CT-1, and CNTF responses, as well as some responses to OSM.LIF-R is moderately expressed in testis, eye, skeletal muscle, ovary, uterus, thymus, brain, and fat and is highly expressed in liver and placenta (21). Activation of LIF-R-gp130 complexes regulates the differentiation and proliferation of a variety of cell lines as well as influencing the behavior of cultured neurons, hepatocytes, and adipocytes (for review see Ref. 22). Gene knockout experiments have demonstrated roles for LIF-R in development. LIF-RϪ/Ϫ and CNTFR␣Ϫ/Ϫ mice show a substantial loss of motor neurons (23,24). LIF-RϪ/Ϫ mice have also be...

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“…A wide range of techniques have previously been used for measurement of HRV-14 3C activity in vitro. These include (i) an in vitro translation assayusing viral polyprotein fragments as substrate (Heinz et al, 1996); (ii) HPLC methods with small synthetic peptides as substrates Long et al, 1989;Cordingley et al, 1990); and (iii) a magnetic bead assay using radiolabelled peptide (Wu & Abeles, 1995). The availability of these methods has greatly facilitated our understanding of viral 3C protease, especiallywith respect to its substrate specificity and amino acid preferences.These assaying methods, however, are not adequate for high throughput screening owing to their laborious and time consuming procedures.…”

Section: Introductionmentioning

confidence: 99%

Development of a Continuous Fluorescence Assay for Rhinovirus 14 3C Protease Using Synthetic Peptides

Wang

Cohen

et al. 1997

Antivir Chem Chemother

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SummaryRhinovirus 3C protease is an attractive target For therapeutic intervention owing to its important role in virion maturation and inFectivity. In order to Facilitate the identification of potential 3C protease inhibitors, we have developed a continuous Fluorescence assay using 5-[(2-aminoethyl}amino]naphthalene-l-sulphonic acid (Edans) as a Fluorescent donor and 4-(4-dimethylaminophenylazo}benzoic acid (Dabcyl) as a quenching acceptor. Several F1uorogenic peptide substrates For 3C protease were synthesized by both solution chemistry and solid phase peptide synthesis. One of the synthetic Edans/ Dabcyl substrates, with an amino acid sequence derived From the 2C/3A site of the virus polyprotein, yielded a 24-Fold increase in Fluorescence intensity after 3C cleavage. Data regarding substrate cleavage kinetics, assay sensitivity and optimization are presented. The application ofthisassay to the evaluation of 3C protease inhibitors is also shown.

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A consensus sequence for substrate hydrolysis by rhino virus 3C proteinase

Cited by 48 publications

References 6 publications

Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease

Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease

An Antagonist for the Leukemia Inhibitory Factor Receptor Inhibits Leukemia Inhibitory Factor, Cardiotrophin-1, Ciliary Neurotrophic Factor, and Oncostatin M

Development of a Continuous Fluorescence Assay for Rhinovirus 14 3C Protease Using Synthetic Peptides

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