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Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation

Hridindu Roychowdury, View ORCID ProfilePhilip A. Romero
doi: https://doi.org/10.1101/2021.06.08.447609
Hridindu Roychowdury
1Department of Biochemistry, University of Wisconsin--Madison, Madison, WI, USA
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Philip A. Romero
1Department of Biochemistry, University of Wisconsin--Madison, Madison, WI, USA
2Department of Chemical & Biological Engineering, University of Wisconsin--Madison, Madison, WI, USA
3The University of Wisconsin Carbone Cancer Center, Madison, WI, USA
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  • ORCID record for Philip A. Romero
  • For correspondence: promero2@wisc.edu
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Abstract

The human caspase family comprises 12 cysteine proteases that are centrally involved in cell death and inflammation responses. The members of this family have conserved sequences and structures, highly similar enzymatic activities and substrate preferences, and overlapping physiological roles. In this paper, we present a deep mutational scan of the executioner caspases CASP3 and CASP7 to dissect differences in their structure, function, and regulation. Our approach leverages high-throughput microfluidic screening to analyze hundreds of thousands of caspase variants in tightly controlled in vitro reactions. The resulting data provides a large-scale and unbiased view of the impact of amino acid substitutions on the proteolytic activity of CASP3 and CASP7. We use this data to pinpoint key functional differences between CASP3 and CASP7, including a secondary internal cleavage site, CASP7 Q196 that is not present in CASP3. Our results will open avenues for inquiry in caspase function and regulation that could potentially inform the development of future caspasespecific therapeutics.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted June 08, 2021.
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Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation
Hridindu Roychowdury, Philip A. Romero
bioRxiv 2021.06.08.447609; doi: https://doi.org/10.1101/2021.06.08.447609
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Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation
Hridindu Roychowdury, Philip A. Romero
bioRxiv 2021.06.08.447609; doi: https://doi.org/10.1101/2021.06.08.447609

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