Intracellular FRET-based Screen for Redesigning the Specificity of Secreted Proteases

Abstract

Proteases are attractive as therapeutics given their ability to catalytically activate or inactivate their targets. However, therapeutic use of proteases is limited by insufficient substrate specificity, since off-target activity can induce undesired side-effects. In addition, few methods exist to enhance the activity and specificity of human proteases, analogous to methods for antibody engineering. Given this need, a general methodology termed protease evolution via cleavage of an intracellular substrate (PrECISE) was developed to enable engineering of human protease activity and specificity toward an arbitrary peptide target. PrECISE relies on coexpression of a protease and a peptide substrate exhibiting Förster resonance energy transfer (FRET) within the endoplasmic reticulum of yeast. Use of the FRET reporter substrate enabled screening large protease libraries using fluorescence activated cell sorting for the activity of interest. To evolve a human protease that selectively cleaves within the central hydrophobic core (KLVF↓F↓AED) of the amyloid beta (Aβ) peptide, PrECISE was applied to human kallikrein 7, a protease with Aβ cleavage activity but broad selectivity, with a strong preference for tyrosine (Y) at P1. This method yielded a protease variant which displayed up to 30-fold improvements in Aβ selectivity mediated by a reduction in activity toward substrates containing tyrosine. Additionally, the increased selectivity of the variant led to reduced toxicity toward PC12 neuronal-like cells and 16-1000-fold improved resistance to wild-type inhibitors. PrECISE thus provides a powerful high-throughput capability to redesign human proteases for therapeutic use.

Mass spectrometry-based determination of Kallikrein-related peptidase 7 (KLK7) cleavage preferences and subsite dependency

“…Although mutant KLK7 (mKLK7) carrying the single mutation (S195A) in the catalytic triad has been widely used in in vitro cell-based protease research 14 , the activity has not been measured using more highly sensitive in-depth proteomics approaches. We observed that the single mKLK7 has residual chymotryptic-like activity by cleaving C-terminal to Tyrosine (Y), Leucine (L) and Phenylalanine (F) at the P1 position in line with active KLK7 (k cat /K M  = 7.93 × 10 2  s −1 M −1 ).…”

Evolution of a mass spectrometry-grade protease with PTM-directed specificity

“…Examples include chemoselective pSer side chain modification to establish pSer-dependent tryptic cleavage sites (7,8), and directed evolution of novel proteolytic cleavage activity, yielding a pTyr-dependent subtilisin mutant (9) and a suite of OmpT mutants that cleaved the Ala-Arg (10), sTyr-Arg (11), or nTyr-Arg (12) P1-P1′ peptide bonds; P1-P1′ cleavage junction requirements precluded further implementation. Proteolytic specificity has also been successfully evolved in other enzymes (13,14), but the MS workhorse enzyme, trypsin, has remained unexplored. Although the trypsin family is the textbook example of divergent evolution (the highly homologous family members possess diverse cleavage specificities), its network of specificity-determining residues-a constellation of approximately four point mutations (15)(16)(17) and two 4-residue surface-exposed loops (18)-is daunting.…”

FRET Microscopy in Yeast

“…When mEos3.1 was partially photoconverted from its green to red form by blue light, the two mEos3.1 forms became the FRET donor and acceptor, showing FRET during protein aggregation. In addition, yeast was also used as a screening platform for the evolution of proteases specifically targeting amyloid β peptide, here fused to CyPet-YPet FPs for FRET readout of its cleavage [59] (see scheme in Figure 3A).…”

“…However, when monitoring of FRET biosensors is established, the ease of genetic and experimental manipulations of yeast opens many possibilities for a potential interference and subsequent analysis of studied events, either in single cells or cell population. Abscisic acid ABACUS1 sensors Cerulean-Citrine [57] Prion proteins nucleation AmFRET mEos3.1 (FACS) [58] Amyloid β cleavage by evolved protease PrECISE CyPet-Ypet (FACS) [59] PolII promoter activity IMAGEtags (RNA aptamers) Cy3-Cy5 (sensitized emission) [60] MAPK signaling pathway yEKAREV CFP-YPet [61] cAMP/PKA signaling pathway Epac2-camps (Epac2) AKAR3 CFP-YFP CFP-cpVenus [62] Force for chromosome segregation Ndc80 tension sensor CFP-YPet [63] Force for endocytic vesicle formation molecular tension sensors in Sla2 protein mTq2-mNG [64,66] 1 If a method other than ratiometric FRET was used, it is specified in parentheses.…”