High-Resolution Analysis and Functional Mapping of Cleavage Sites and Substrate Proteins of Furin in the Human Proteome

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Abstract

Background: There is a growing appreciation of the role of proteolytic processes in human health and disease, but tools for analysis of such processes on a proteome-wide scale are limited. Furin is a ubiquitous proprotein convertase that cleaves after basic residues and transforms secretory proproteins into biologically active proteins. Despite this important role, many furin substrates remain unknown in the human proteome. Methodology/Principal Findings: We devised an approach for proteinase target identification that combines an in silico discovery pipeline with highly multiplexed proteinase activity assays. We performed in silico analysis of the human proteome and identified over 1,050 secretory proteins as potential furin substrates. We then used a multiplexed protease assay to validate these tentative targets. The assay was carried out on over 3,260 overlapping peptides designed to represent P7-P1' and P4-P4' positions of furin cleavage sites in the candidate proteins. The obtained results greatly increased our knowledge of the unique cleavage preferences of furin, revealed the importance of both short-range (P4-P1) and long-range (P7-P6) interactions in defining furin cleavage specificity, demonstrated that the R-X-R/K/X-R↓ motif alone is insufficient for predicting furin proteolysis of the substrate, and identified ~490 potential protein substrates of furin in the human proteome. Conclusions/Significance: The assignment of these substrates to cellular pathways suggests an important role of furin in development, including axonal guidance, cardiogenesis, and maintenance of stem cell pluripotency. The novel approach proposed in this study can be readily applied to other proteinases. © 2013 Shiryaev et al.

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Shiryaev, S. A., Chernov, A. V., Golubkov, V. S., Thomsen, E. R., Chudin, E., Chee, M. S., … Cieplak, P. (2013). High-Resolution Analysis and Functional Mapping of Cleavage Sites and Substrate Proteins of Furin in the Human Proteome. PLoS ONE, 8(1). https://doi.org/10.1371/journal.pone.0054290

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