Abstract
Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P1′, glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates.
Highlights
Proteases represent one of the largest and most important protein families known, and their importance in processes that govern the life and death of a cell cannot be overestimated
We have created a convenient, label-free, and function-based protease assay, suitable for flow cytometry analysis and cell sorting, by utilizing sensitive short-lived fluorescent substrates expressed in Escherichia coli (E. coli)
The above-described strategy was used to examine the processing efficiency of the sequence specific tobacco etch virus protease (TEVp) on three closely related substrate reporters that only differed in the P19 position of the TEVp substrate peptide, ENLYFQX
Summary
Proteases represent one of the largest and most important protein families known, and their importance in processes that govern the life and death of a cell cannot be overestimated. Proteases are widely used as virulence factors by many infectious microorganisms, viruses and parasites [3]. Proteases and their substrates are of great interest as potential drug targets. In humans, proteases represent 5–10% of all drug targets [4,5]. The function of proteases is regulated either by controlling the spatial and temporal activity or through their ability to discriminate among potential substrates, of which the last is probably the most important mechanism. Efficient methods for characterization of proteases and their associated substrates could enhance our understanding of biological systems, which may result in new therapeutic strategies
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