Engineering of protease variants exhibiting altered substrate specificity

Saravanan, S.; Shin, Bae Hyun; Lee, Eui Seung; Rho, Seong-Hwan; Hwang, Wang-Taek; Lee, Yong Jae; Han, Hui; Kim, Jae Il; Park, Woo Jin

doi:10.1016/j.bbrc.2008.04.026

Cited by 21 publications

(22 citation statements)

References 13 publications

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“…While this might not be an issue for a well studied protease like TEVp whose substrate profile is well know, this can be an issue for less-characterized proteases. One way to determine the scissile bond is through mass spectrometry (MS) analysis of cleavage products from synthetic peptide substrates [32], [33]. However, we believe that our system also is amendable to MS-based identification of the exact cleavage site.…”

Section: Resultsmentioning

confidence: 99%

Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

2011

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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.

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Section: Resultsmentioning

confidence: 99%

Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

2011

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“…Studies from our laboratory and others have highlighted the importance of incorporating simultaneous counterselection substrates during the directed evolution of proteases to avoid isolating variants with relaxed specificity (14,20,36,37). Any number of counterselection substrate sequences can be added to the YESS substrate fusion construct to facilitate the engineering of narrow selectivity enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…Engineered proteases displaying some degree of novel specificity have been developed in a few instances either via structure-guided mutagenesis or through directed evolution (12)(13)(14)(15)(16)(17). In general, though, it has proved difficult to use rational design to generate highly active proteases with a desired new substrate selectivity, as mutations in one subsite typically disrupt the structure of neighboring subsites or of residues important for catalysis.…”

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confidence: 99%

Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries

Li¹,

Gebhard

Li³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

121

173

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Myriad new applications of proteases would be enabled by an ability to fine-tune substrate specificity and activity. Herein we present a general strategy for engineering protease selectivity and activity by capitalizing on sequestration of the protease to be engineered within the yeast endoplasmic reticulum (ER). A substrate fusion protein composed of yeast adhesion receptor subunit Aga2, selection and counterselection substrate sequences, multiple intervening epitope tag sequences, and a C-terminal ER retention sequence is coexpressed with a protease library. Cleavage of the substrate fusion protein by the protease eliminates the ER retention sequence, facilitating transport to the yeast surface. Yeast cells that display Aga2 fusions in which only the selection substrate is cleaved are isolated by multicolor FACS with fluorescently labeled antiepitope tag antibodies. Using this system, the Tobacco Etch Virus protease (TEV-P), which strongly prefers Gln at P1 of its canonical ENLYFQ↓S substrate, was engineered to recognize selectively Glu or His at P1. Kinetic analysis indicated an overall 5,000-fold and 1,100-fold change in selectivity, respectively, for the Glu-and His-specific TEV variants, both of which retained high catalytic turnover. Human granzyme K and the hepatitis C virus protease were also shown to be amenable to this unique approach. Further, by adjusting the signaling strategy to identify phosphorylated as opposed to cleaved sequences, this unique system was shown to be compatible with the human Abelson tyrosine kinase. directed evolution | method | protein engineering

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“…In a prior study, we had engineered the S2 pocket of 3CP and generated a number of variants [16]. From this group, 12 variants were selected that contained mutations at amino acids 145, 146, 147, and 155.…”

Section: Resultsmentioning

confidence: 99%

“…These proteases show a unique substrate specificity preference for a glutamine residue at the P1 site. We have previously utilized a yeast-based screening method, referred to as the Genetic Assay for Site-specific Proteolysis (GASP), to produce an engineered variant of HAV 3CP that can cleave a peptide substrate containing glutamine at the P1′ site more efficiently than its original substrate [16]. Motivated by this earlier success, we further extended the use of GASP to generate a polyQ-specific protease (PQP) using directed evolution approach.…”

Section: Introductionmentioning

confidence: 99%

An Engineered Viral Protease Exhibiting Substrate Specificity for a Polyglutamine Stretch Prevents Polyglutamine-Induced Neuronal Cell Death

et al. 2011

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BackgroundPolyglutamine (polyQ)-induced protein aggregation is the hallmark of a group of neurodegenerative diseases, including Huntington's disease. We hypothesized that a protease that could cleave polyQ stretches would intervene in the initial events leading to pathogenesis in these diseases. To prove this concept, we aimed to generate a protease possessing substrate specificity for polyQ stretches.Methodology/Principal FindingsHepatitis A virus (HAV) 3C protease (3CP) was subjected to engineering using a yeast-based method known as the Genetic Assay for Site-specific Proteolysis (GASP). Analysis of the substrate specificity revealed that 3CP can cleave substrates containing glutamine at positions P5, P4, P3, P1, P2′, or P3′, but not substrates containing glutamine at the P2 or P1′ positions. To accommodate glutamine at P2 and P1′, key residues comprising the active sites of the S2 or S1′ pockets were separately randomized and screened. The resulting sets of variants were combined by shuffling and further subjected to two rounds of randomization and screening using a substrate containing glutamines from positions P5 through P3′. One of the selected variants (Var26) reduced the expression level and aggregation of a huntingtin exon1-GFP fusion protein containing a pathogenic polyQ stretch (HttEx1(97Q)-GFP) in the neuroblastoma cell line SH-SY5Y. Var26 also prevented cell death and caspase 3 activation induced by HttEx1(97Q)-GFP. These protective effects of Var26 were proteolytic activity-dependent.Conclusions/SignificanceThese data provide a proof-of-concept that proteolytic cleavage of polyQ stretches could be an effective modality for the treatment of polyQ diseases.

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Engineering of protease variants exhibiting altered substrate specificity

Cited by 21 publications

References 13 publications

Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries

An Engineered Viral Protease Exhibiting Substrate Specificity for a Polyglutamine Stretch Prevents Polyglutamine-Induced Neuronal Cell Death

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