Protease Substrate-Independent Universal Assay for Monitoring Digestion of Native Unmodified Proteins.

Emmiliisa Vuorinen,Huda Habib,Harri Härmä,Morteza Malakoutikhah,Kari Kopra,Randa Mahran,Nazia Hassan,Salla Valtonen

doi:10.3390/ijms22126362

Emmiliisa Vuorinen, Huda Habib + Show 6 more

Open Access

https://doi.org/10.3390/ijms22126362

Copy DOI

Abstract

Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases.

Highlights

Proteases are vital enzymes that modify proteins by cutting them at specific digestion sites
The protease and substrate protein remain intact and require no label or additional tags enabling the fulfillment of criteria for a label-free assay technique
We present the method for monitoring protease activity using several proteases and a panel of substrate proteins

Summary

Introduction

Proteases are vital enzymes that modify proteins by cutting them at specific digestion sites. This is achieved by hydrolyzing a peptide bond either between two amino acids in the middle of the peptide chain (endopeptidase) or at the terminus of the peptide (exopeptidases). Even within a single protease group, the substrate specificity can vary significantly [1] This sequence preference leads to differences in enzyme specificity as the longer the target sequence is, the more specific the protease typically is [2]. This highlights the fact that proteases even within one group digest a variety of substrates, [1,3] and it is complex to develop an universal assay to monitor the activity of multiple proteases [4]

Methods

Results

Conclusion