期刊
MOLECULAR & CELLULAR PROTEOMICS
卷 4, 期 5, 页码 626-636出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/mcp.M500004-MCP200
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资金
- NHLBI NIH HHS [HL56621] Funding Source: Medline
- NIGMS NIH HHS [GM54051] Funding Source: Medline
Proteases regulate numerous biological processes with a degree of specificity often dictated by the amino acid sequence of the substrate cleavage site. To map protease/substrate interactions, a 722-member library of fluorogenic protease substrates of the general format Ac-Ala-X-X-(Arg/Lys)-coumarin was synthesized (X = all natural amino acids except cysteine) and microarrayed with fluorescent calibration standards in glycerol nano-droplets on glass slides. Specificities of 13 serine proteases ( activated protein C, plasma kallikrein, factor VIIa, factor IXa beta, factor XIa and factor alpha XIIa, activated complement C1s, C1r, and D, tryptase, trypsin, subtilisin Carlsberg, and cathepsin G) and 11 papain-like cysteine proteases ( cathepsin B, H, K, L, S, and V, rhodesain, papain, chymopapain, ficin, and stem bromelain) were obtained from 103,968 separate microarray fluorogenic reactions (722 substrates x 24 different proteases x 6 replicates). This is the first comprehensive study to report the substrate specificity of rhodesain, a papain-like cysteine protease expressed by Trypanasoma brucei rhodesiense, a parasitic protozoa responsible for causing sleeping sickness. Rhodesain displayed a strong P-2 preference for Leu, Val, Phe, and Tyr in both the P-1 = Lys and Arg libraries. Solution-phase microarrays facilitate protease/substrate specificity profiling in a rapid manner with minimal peptide library or enzyme usage.
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