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Exploring Caspase Mutations and Post-Translational Modification by Molecular Modeling Approaches

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Published on: October 13, 2022

Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for

Maureen E Hill¹, Derek J MacPherson¹, Peng Wu¹

¹Department of Chemistry, 104 LGRT, 710 N. Pleasant St., University of Massachusetts , Amherst, Massachusetts 01003, United States.

ACS Chemical Biology

|April 1, 2016

Summary

Researchers engineered protease specificity using a novel reporter system. This method successfully reprogrammed caspase-7 to match caspase-6 specificity, offering new tools for biological research and therapeutics.

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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

Engineering protease specificity is crucial for understanding protease biology and developing new therapeutics.
Current methods for engineering protease specificity are limited, especially for intracellular proteases.

Purpose of the Study:

To develop a novel reporter system for selecting intracellular protease specificity.
To reprogram the specificity of caspase-7 to match that of caspase-6.

Main Methods:

Utilized a caged green fluorescent protein (CA-GFP) reporter for flow cytometry-based selection.
Employed saturation mutagenesis and directed evolution to alter caspase-7 specificity.
Determined the structures of evolved-specificity caspase-7 (esCasp-7).
Performed N-terminomics profiling of esCasp-7 against the human proteome.

Main Results:

Developed a system for selecting intracellular protease specificity.
Successfully converted caspase-7 specificity to match caspase-6 through directed evolution.
Identified nonobvious mutations and structural changes in esCasp-7, including active site loop reorganization.
esCasp-7 demonstrated global specificity similar to caspase-6 on natural protein substrates.
Identified a caspase-6 substrate, lamin C, potentially recognized via an exosite.

Conclusions:

The developed reporter system enables efficient engineering of protease specificity.
Reprogrammed caspases can distinguish between exosite-dependent and independent substrates.
This specificity reprogramming approach is potentially generalizable to a wide range of proteases.