Expression, Reinigung, strukturelle und biochemische Charakterisierung der Hämagglutinin-spaltenden Proproteinkonvertase PC5
Biologische und Biomimetische Chemie
Virologie
Zusammenfassung der Projektergebnisse
The family of proprotein convertases (PCs) comprises nine members, whose catalytic sites are highly conserved. PCs are involved in the activation of viral proteins and bacterial toxins during infections. The short- or mid-term treatment with PC inhibitors in acute viral or bacterial infectious diseases could be a promising therapeutic strategy. However, the selectivity profile of most known PC inhibitors is limited as they inhibit several PCs in a similar range, e.g. in the case of furin and PC5. The recognition motif of other kexin-like PCs than furin has been less investigated and remains not fully understood. In case of PC5 only limited data are available on its substrate specificity and properties. The recognition motif of PC5 was investigated in enzyme kinetic measurements using FRET- and AMC-substrates. Furin and PC5 are described to activate the hemagglutinin (HA) of highly pathogenic avian influenza virus H5 and H7 strains at a mulitbasic cleavage site. Therfore, a series of FRET-substrates derived from the natural occurring H1-H18 of influenza A and B hemagglutinin sequences were synthesized to examine the cleavage rates by PC5. Interestingly PC5 and furin show almost the same cleavage behavior favoring the same substrates. Based on these data the synthesis of substrate-analog inhibitors seems to be challenging. More differences in substrate processing where found for the AMC-substrates. The best PC5 substrates of this series are compounds Ac-Arg-Arg-Tle-Lys-Arg-AMC and Ac-Arg-Arg-Arg-Tle-Lys-Arg-AMC possessing Vmax/KM values over 8 000 RFU/(M^-1s-1), whereas furin favors H-Arg-Arg-Tle-Lys-Arg-AMC. Regarding PC5´s role in HA activation, first the expression of both proprotein convertases was examined by qPCR. Furin and PC5 are both expressed in equal amounts on mRNA level in lung tissue. From the examined cell lines only HEK293 and Calu-3 resemble this expression pattern. Despite a similar cleavage pattern found for the hemagglutinin derived substrates, PC5 seems to play only a minor role in the activation of hemagglutinin of a novel occurred highly pathogenic H7N9 virus and the controls H7N1 and H5N1. Infection studies in Calu-3 cells with reassortant viruses harboring the HA of the highly pathogenic H7N9 mutant indicate that the main activating protease seems to be furin. However, in general PC5 is able to cleave the hemagglutinin in overexpression experiments as shown in a fusion assay.
Projektbezogene Publikationen (Auswahl)
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“Activating proteases of a novel emerging H7N9 influenza A virus”, 6th International Influenza Meeting, 02. – 04. September 2018, Münster, Deutschland
Kornelia Hardes
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“Activating proteases of a novel emerging H7N9 influenza A virus”, Gordon Research Conference und Gordon Research Seminar “Proteolytic Enzymes and Their Inhibitors”, 02. – 08. Juni 2018, Barga, Italien
Kornelia Hardes
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“Cleavability of hemagglutinin-derived fluorogenic substrates by proteases”, Frontiers in Medicinal Chemistry, 11. – 14. März 2018, Jena, Deutschland
Kornelia Hardes
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“Protease specificity of influenza A virus H7N9 hemagglutinin with multibasic cleavage site”, 28th Annual Meeting of the Society for Virology, 14. – 17. März 2018, Würzburg, Deutschland
Kornelia Hardes
