A Method for Rapid Protease Substrate Evaluation and Optimization

Journal of Biological Chemistry

et al. 2008

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130

137

We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R2 and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R2 motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.

Section: Resultsmentioning

confidence: 99%

Substrate Cleavage Analysis of Furin and Related Proprotein Convertases

Remacle

Journal of Biological Chemistry

et al. 2008

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130

137

“…Previous studies have also verified the efficiency of the synthesizer and the synthetic scheme we used and the high quality of the synthesized peptides [31][32][33]39,40]. As a proof-of-principle, the same approach was used for the synthesis of the peptide sequences, which were used as the cleavage targets of trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease [41]. These additional data verified that the synthetic method and the follow-on peptide cleavage screening were applicable for the analyses of many endoprotease types…”

Section: Peptide Cleavage Screening Assaymentioning

confidence: 87%

Cleavage preference distinguishes the two-component NS2B–NS3 serine proteinases of Dengue and West Nile viruses

Kozlov

Ratnikov

et al. 2007

Biochemical Journal

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Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

“…The design of a centrifugation-based, parallel peptide synthesizer, the techniques for purification and characterization of the peptides, and the control and cleavage reaction parameters were all described in detail previously (14,(16)(17)(18)29). Briefly, peptide synthesis was performed in wells of a 96-well flat bottom polypropylene microtiter plate (Evergreen Scientific, Los Angeles, CA).…”

Section: Methodsmentioning

confidence: 99%

Switching the Substrate Specificity of the Two-Component NS2B-NS3 Flavivirus Proteinase by Structure-Based Mutagenesis

Ratnikov

Aleshin

et al. 2007

J Virol

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The flavivirus NS2B-NS3(pro)teinase is an essential element in the proteolytic processing of the viral precursor polyprotein and therefore a potential drug target. Recently, crystal structures and substrate preferences of NS2B-NS3pro from Dengue and West Nile viruses (DV and WNV) were determined. We established that the presence of Gly-Gly at the P1-P2 positions is optimal for cleavage by WNV NS3pro, whereas DV NS3pro tolerates well the presence of bulky residues at either P1 or P2. Structure-based modeling suggests that Arg 76 and Pro 131 -Thr 132 limit the P1-P2 subsites and restrict the cleavage preferences of the WNV enzyme. In turn, Leu 76 and Lys 131 -Pro 132 widen the specificity of DV NS3pro. Guided by these structural models, we expressed and purified mutant WNV NS2B-NS3pro and evaluated cleavage preferences by using positional scanning of the substrate peptides in which the P4-P1 and the P3-P4 positions were fixed and the P1 and P2 positions were each randomized. We established that WNV R76L and P131K-T132P mutants acquired DV-like cleavage preferences, whereas T52V had no significant effect. Our work is the first instance of engineering a viral proteinase with switched cleavage preferences and should provide valuable data for the design of optimized substrates and substrate-based selective inhibitors of flaviviral proteinases.Dengue and West Nile viruses (DV and WNV) are members of the flavivirus genus within the Flaviviridae family. Four distinct, but structurally similar, serotypes represent DV (13). Flaviviruses are transmitted to animals, including humans, by either mosquito or tick bites. Flaviviruses are encoded by a single-strand, positive-sense, 11-kb RNA genome, which serves as an mRNA for protein synthesis and as a template for RNA replication in the host cell. There are three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) encoded by the viral genome (21). Proteinases of the host (furin and secretase) and of the virus [NS3 serine (pro)teinase (NS3pro)] are required for the complete processing of the polyprotein precursor and its transformation into mature viral proteins (4,27,31,34). Full-length NS3 is a bifunctional protein: its N-terminal and C-terminal portions encode the NS3pro domain and the helicase (NS3hel) domain, respectively (33, 35). NS3pro is responsible for cleavage of the capsid protein C, as well as the boundaries between NS2A/NS2B, NS2B/NS3, NS3/NS4A, NS4A/ NS4B, and NS4B/NS5 (6, 24).NS2B is an essential cofactor of NS3pro, and it is located immediately upstream in the polyprotein precursor (5, 9, 25, 26). The NS2B sequence includes three to four transmembrane helices that anchor the NS2B-NS3 heterodimer to the endoplasmic reticulum membrane. In vitro, the cofactor activity of the 35-to 48-residue central portion of NS2B is approximately equivalent to that of the entire NS2B sequence (11,12,19). Mutations in the NS3pro cleavage motifs in the polyprotein precursor abolish viral infectivity (2). These characteris...