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Title: Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7 was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate,more » lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less
 [1] ;  [1] ;  [1] ;  [2] ;  [2] ;  [1]
  1. Univ. of Massachusetts, Amherst, MA (United States)
  2. Univ. of California, San Francisco, CA (United States)
Publication Date:
Grant/Contract Number:
AC02-98CH10886; AC02-06CH11357
Accepted Manuscript
Journal Name:
ACS Chemical Biology
Additional Journal Information:
Journal Volume: 11; Journal Issue: 6; Journal ID: ISSN 1554-8929
American Chemical Society (ACS)
Research Org:
Univ. of Massachusetts, Amherst, MA (United States)
Sponsoring Org:
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; apoptosis; conformational change; cysteine protease; caspase reporter; GFP; selection; flow cytometry; exosite; substrate binding; specificity; directed evolution; protease/protein engineering
OSTI Identifier: