Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.

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 hopef...

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Autores principales:	George Kostallas, Per-Åke Löfdahl, Patrik Samuelson
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d641cb4316774a8ea295665d464fbb90
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spelling	oai:doaj.org-article:d641cb4316774a8ea295665d464fbb902021-11-18T07:00:19ZSubstrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.1932-620310.1371/journal.pone.0016136https://doaj.org/article/d641cb4316774a8ea295665d464fbb902011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/21267463/?tool=EBIhttps://doaj.org/toc/1932-6203Site-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.George KostallasPer-Åke LöfdahlPatrik SamuelsonPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 1, p e16136 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q George Kostallas Per-Åke Löfdahl Patrik Samuelson Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.
description	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.
format	article
author	George Kostallas Per-Åke Löfdahl Patrik Samuelson
author_facet	George Kostallas Per-Åke Löfdahl Patrik Samuelson
author_sort	George Kostallas
title	Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.
title_short	Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.
title_full	Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.
title_fullStr	Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.
title_full_unstemmed	Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.
title_sort	substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/d641cb4316774a8ea295665d464fbb90
work_keys_str_mv	AT georgekostallas substrateprofilingoftobaccoetchvirusproteaseusinganovelfluorescenceassistedwholecellassay AT perakelofdahl substrateprofilingoftobaccoetchvirusproteaseusinganovelfluorescenceassistedwholecellassay AT patriksamuelson substrateprofilingoftobaccoetchvirusproteaseusinganovelfluorescenceassistedwholecellassay
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Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.

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