Kinetics, inhibition and oligomerization of Epstein‐Barr virus protease

Buisson, Marlyse; Rivail, Lucie; Hernandez, Jean‐François; Jamin, Marc; Martinez, Jean; Ruigrok, Rob W.H.; Burmeister, W.P.

doi:10.1016/j.febslet.2006.11.006

Cited by 5 publications

(4 citation statements)

References 36 publications

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“…It has been established in other proteases that catalytic function is dependent on dimerization. For example, the catalytic ability of the Epstein-Barr protease has previously been shown to be dependent on the extent of dimerization (11), and HIV-1 protease oligomerization inhibitors are currently in development (12).…”

mentioning

confidence: 99%

Single Mutation on the Surface of Staphylococcus aureus Sortase A Can Disrupt Its Dimerization

Zhu

Standland

et al. 2008

Biochemistry

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Staphylococcus aureus Sortase A (SrtA) is an important Gram-positive membrane enzyme which catalyzes the anchoring of many cell surface proteins conserved with the LPXTG sequence. Recently SrtA has been demonstrated to be a dimer with a Kd of 55 microM in vitro. Herein, we show that a single point mutation of amino acid residue on the surface of SrtA can completely disrupt the dimerization. Native polyacrylamide gel electrophoresis and analytical gel filtration chromatography were used to detect the dimer-monomer equilibrium of SrtA mutants. Circular dichroism spectrum experiments were performed to study the conformational change of each SrtA mutant. An enzyme activity assay confirmed that all the SrtA mutants were active in vitro. Our results not only are important for understanding the SrtA protein self-associating mechanism but also provided the necessary starting materials for the study of sortase A pathway in vivo, which may have significant implications for discovering microbial physiology and give a potential target for novel Gram-positive antibiotics.

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confidence: 99%

Single Mutation on the Surface of Staphylococcus aureus Sortase A Can Disrupt Its Dimerization

Zhu

Standland

et al. 2008

Biochemistry

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“…HPLC-grade solvents were used for HPLC and LC/MS analyses. The Fmoc-protected norstatine analogue of alanine [(3S)-3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-2-hydroxybutanoic acid], which was used as the precursor of the -ketoacyl residue, was synthesized from Fmoc-Ala-OH as described previously (Buisson et al, 2006).…”

Section: Synthesis Of Mam-117 An A-ketoamide Inhibitor Of Pvs1mentioning

confidence: 99%

3D structures of the Plasmodium vivax subtilisin-like drug target SUB1 reveal conformational changes to accommodate a substrate-derived α-ketoamide inhibitor

Martinez,

Batista,

Maurel

et al. 2023

Acta Crystallogr D Struct Biol

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The constant selection and propagation of multi-resistant Plasmodium sp. parasites require the identification of new antimalarial candidates involved in as-yet untargeted metabolic pathways. Subtilisin-like protease 1 (SUB1) belongs to a new generation of drug targets because it plays a crucial role during egress of the parasite from infected host cells at different stages of its life cycle. SUB1 is characterized by an unusual pro-region that tightly interacts with its cognate catalytic domain, thus precluding 3D structural analysis of enzyme–inhibitor complexes. In the present study, to overcome this limitation, stringent ionic conditions and controlled proteolysis of recombinant full-length P. vivax SUB1 were used to obtain crystals of an active and stable catalytic domain (PvS1Cat) without a pro-region. High-resolution 3D structures of PvS1Cat, alone and in complex with an α-ketoamide substrate-derived inhibitor (MAM-117), showed that, as expected, the catalytic serine of SUB1 formed a covalent bond with the α-keto group of the inhibitor. A network of hydrogen bonds and hydrophobic interactions stabilized the complex, including at the P1′ and P2′ positions of the inhibitor, although P′ residues are usually less important in defining the substrate specificity of subtilisins. Moreover, when associated with a substrate-derived peptidomimetic inhibitor, the catalytic groove of SUB1 underwent significant structural changes, particularly in its S4 pocket. These findings pave the way for future strategies for the design of optimized SUB1-specific inhibitors that may define a novel class of antimalarial candidates.

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“…Biochemical and x-ray crystallography studies revealed that EBV PR is active as a homodimer. Dimerization is crucial for the stabilization of the active site in each of the monomers [25,[27][28][29]. The EBV PR, present in mature virions, could elicit a specific T-cell-mediated immune response in vivo [21,30].…”

Section: Original Articlementioning

confidence: 99%

Inhibition of Epstein–Barr virus replication by small interfering RNA targeting the Epstein–Barr virus protease gene

Larrat

Morand²,

Bas³

et al. 2008

Antiviral Therapy

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Background The Epstein–Barr virus (EBV) protease (PR), coded by the BVRF2 gene, is essential for the maturation of the viral capsid and viral DNA packaging during the late stage of the EBV lytic cycle. Like the other herpesvirus serine PRs, EBV PR could be a target for the inhibition of EBV replication. To date, no data have been reported on the inhibition of EBV PR messenger RNA (mRNA) by small interfering RNA (siRNA). Methods In this study, siRNAs targeting EBV PR were delivered to the epithelial 293 cell line stably transfected with the complete B95-8 EBV episome. EBV DNA and PR mRNA were quantified by real-time PCR in cells and supernatant, protein expression was assessed by immunoblotting, and production of EBV infectious particles in the culture medium was measured by Raji cell superinfection. Results The EBV PR mRNA within the cells was reduced by 73%, the PR protein by 35% and the amount of virus in the cell supernatant was drastically decreased by 86% or 95%, depending on the method. Conclusions The strong effect of the siRNA targeting EBV PR on EBV replication attests to the crucial role played by EBV PR in the production of infectious particles and suggests that targeting this enzyme can be a new strategy against EBV-associated diseases where virus replication occurs.

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Kinetics, inhibition and oligomerization of Epstein‐Barr virus protease

Cited by 5 publications

References 36 publications

Single Mutation on the Surface of Staphylococcus aureus Sortase A Can Disrupt Its Dimerization

Single Mutation on the Surface of Staphylococcus aureus Sortase A Can Disrupt Its Dimerization

3D structures of the Plasmodium vivax subtilisin-like drug target SUB1 reveal conformational changes to accommodate a substrate-derived α-ketoamide inhibitor

Inhibition of Epstein–Barr virus replication by small interfering RNA targeting the Epstein–Barr virus protease gene

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