Emerging technologies for protease engineering: New tools to clear out disease

Abstract

Proteases regulate many biological processes through their ability to activate or inactive their target substrates. Because proteases catalytically turnover proteins and peptides, they present unique opportunities for use in biotechnological and therapeutic applications. However, many proteases are capable of cleaving multiple physiological substrates. Therefore their activity, expression, and localization are tightly controlled to prevent unwanted proteolysis. Currently, the use of protease therapeutics has been limited to a handful of proteases with narrow substrate specificities, which naturally limits their toxicity. Wider application of proteases is contingent upon the development of methods for engineering protease selectivity, activity, and stability. Recent advances in the development of high-throughput, bacterial and yeast-based methods for protease redesign have yielded protease variants with novel specificities, reduced toxicity, and increased resistance to inhibitors. Here, we highlight new tools for protease engineering, including methods suitable for the redesign of human secreted proteases, and future opportunities to exploit the catalytic activity of proteases for therapeutic benefit. Biotechnol. Bioeng. 2017;114: 33-38. © 2016 Wiley Periodicals, Inc.

Multiple domains of bacterial and human Lon proteases define substrate selectivity

“…This high success rate (47.6%) demonstrates the reliability of our approach in identification of Lon substrates. Because Lon degrades only small number of proteins encoded by the Francisella proteome, this result also shows that Lon strictly selects its substrates as compared with other serine proteases with promiscuous substrate selectivity, such as trypsin and subtilisin 1 , 49 .…”

Aminopeptidase secreted by Chromobacterium sp. Panama inhibits dengue virus infection by degrading the E protein

“…While their antiviral potential has only been explored in topical applications against cold-causing rhinoviruses and, more recently, herpes simplex virus [ 31 ], the great amount of research on alternative-to-parenteral delivery methods for biologics brings promise on the ability to deliver therapeutic proteins through non-invasive routes such as orally or intranasally in the near future [ 32 – 34 ]. Other potential hindrances relate to their propensity for immunogenicity in the form of anti-drug antibodies and the lack of specificity of their proteolytic activity as measured by the number of putative substrates, but these obstacles have also been extensively addressed and strategies developed to overcome them [ 35 – 37 ]. For example, the botulinum toxins are also zinc metalloproteases initially characterized from bacteria: significant advances since their introduction have allowed for new-generation drugs with reduced immunogenicity and increased stability that can be absorbed transdermally [ 38 , 39 ].…”

Engineering Proteases for Mass Spectrometry‐Based Post Translational Modification Analyses

“…However, most proteases of non‐viral origin, especially those used in MS‐based proteomics, have complicated folding patterns and more relaxed specificity, which can lead to even more toxicity. Therefore, methods developed for TEV protease would need to be optimized to become applicable to non‐viral proteases . The use of a eukaryotic expression system in YESS technology might be a way to accommodate this complex folding requisite.…”

“…However, activity against peptide substrates does not necessarily translate to activity against fully folded proteins. Although the same primary sequence as selection peptide maybe present on the protein substrate, the level of protease activity against this protein might remain largely determined by specific substrate conformations . Therefore, high‐throughput screening/selection strategies developed in the near future should focus on fully folded protein targets, not peptides …”

“…The studies by Varadarajan et al and Yi et al have shown the evident benefits of incorporating a counter‐selection substrate within a screening strategy. Directed evolution platforms that use multiple non‐target and fully folded protein substrates along with selection substrate will further increase the chance of identifying variants with narrow specificities . One can even imagine developing a directed evolution platform where the ratio of PTM versus non‐PTM specific activity of a protease can be fine‐tuned to achieve the most optimal cleavage of respective residues.…”