A study of human furin specificity using synthetic peptides derived from natural substrates, and effects of potassium ions.
We explored furin substrate requirements in addition to the motif R-X-K/R-R using synthetic fluorescent resonance energy transfer (FRET) decapeptides. These decapeptides were derived from furin cleavage sites in viral coat glycoproteins and human and bacterial protein precursors. The hydrolysis by furin of most substrate was activated by K(+) ion, whereas kosmotropic anions of the Hofmeister series were inhibitors. The analysis of furin hydrolytic activity showed that its efficiency is highly dependent on the particular combinations of amino acids at different substrate positions. There is a clear interdependence of furin subsites that must be taken in account in determining its specificity and also for the design of inhibitors. However, clear preferences were detected for substrates with S at P(1)', and V at P(2)', at P(3)' the amino acids D, S, L and A are almost equally frequent. In the non-prime subsites the best substrates presented S and H at P(6); basic amino acids at P(5); and no clear tendency at P(3). Interestingly, two amino acid substitutions on the prime side of the peptide derived from H5N1 influenza hemagglutinin furin processing site highly improved its hydrolysis. These modifications are possible by single point mutations, suggesting a potential yield of a more infectious virus.
Izidoro
Mario A
MA
Department of Biophysics, Escola Paulista de Medicina, UNIFESP, São Paulo 04044-020, Brazil.
Gouvea
Iuri E
IE
Santos
Jorge A N
JA
Assis
Diego M
DM
Oliveira
Vitor
V
Judice
Wagner A S
WA
Juliano
Maria A
MA
Lindberg
Iris
I
Juliano
Luiz
L
D000595:Amino Acid Sequence; D031541:Fluorescence Resonance Energy Transfer; D045683:Furin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology*; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects
D009842:Oligopeptides; D012965:Sodium Chloride; D011189:Potassium Chloride; D045683:Furin