Microfluidic Deep Mutational Scanning of the Human Executioner Caspases Reveals Differences in Structure and Regulation

Overview

Journal Cell Death Discov

Date 2022 Jan 11

PMID 35013287

Authors

Hridindu Roychowdhury

Philip A Romero

Affiliations

Soon will be listed here.

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 caspase-specific therapeutics.

Citing Articles

Droplet microfluidic screening to engineer angiotensin-converting enzyme 2 (ACE2) catalytic activity.

Okal E, Romero P, Heinzelman P J Biol Eng. 2025; 19(1):12.

PMID: 39901286 PMC: 11792573. DOI: 10.1186/s13036-025-00482-3.

A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins.

Stadelmann T, Heid D, Jendrusch M, Mathony J, Aschenbrenner S, Rosset S Nucleic Acids Res. 2024; 52(22):e103.

PMID: 39558174 PMC: 11662660. DOI: 10.1093/nar/gkae1052.

Understanding activity-stability tradeoffs in biocatalysts by enzyme proximity sequencing.

Vanella R, Kung C, Schoepfer A, Doffini V, Ren J, Nash M Nat Commun. 2024; 15(1):1807.

PMID: 38418512 PMC: 10902396. DOI: 10.1038/s41467-024-45630-3.

A probabilistic graphical model for estimating selection coefficient of missense variants from human population sequence data.

Zhao Y, Zhong G, Hagen J, Pan H, Chung W, Shen Y medRxiv. 2024; .

PMID: 38168397 PMC: 10760286. DOI: 10.1101/2023.12.11.23299809.

ProteinGym: Large-Scale Benchmarks for Protein Design and Fitness Prediction.

Notin P, Kollasch A, Ritter D, van Niekerk L, Paul S, Spinner H bioRxiv. 2023; .

PMID: 38106144 PMC: 10723403. DOI: 10.1101/2023.12.07.570727.

References

Firnberg E, Labonte J, Gray J, Ostermeier M . A comprehensive, high-resolution map of a gene's fitness landscape. Mol Biol Evol. 2014; 31(6):1581-92. PMC: 4032126. DOI: 10.1093/molbev/msu081. View

Haddox H, Dingens A, Bloom J . Experimental Estimation of the Effects of All Amino-Acid Mutations to HIV's Envelope Protein on Viral Replication in Cell Culture. PLoS Pathog. 2016; 12(12):e1006114. PMC: 5189966. DOI: 10.1371/journal.ppat.1006114. View

Agniswamy J, Fang B, Weber I . Conformational similarity in the activation of caspase-3 and -7 revealed by the unliganded and inhibited structures of caspase-7. Apoptosis. 2009; 14(10):1135-44. DOI: 10.1007/s10495-009-0388-9. View

Hacker H, Tekeste Sisay M, Gutschow M . Allosteric modulation of caspases. Pharmacol Ther. 2011; 132(2):180-95. DOI: 10.1016/j.pharmthera.2011.07.003. View

Pande J, Szewczyk M, Grover A . Phage display: concept, innovations, applications and future. Biotechnol Adv. 2010; 28(6):849-58. DOI: 10.1016/j.biotechadv.2010.07.004. View

Fowler D, Fields S . Deep mutational scanning: a new style of protein science. Nat Methods. 2014; 11(8):801-7. PMC: 4410700. DOI: 10.1038/nmeth.3027. View

Collins D, Neild A, DeMello A, Liu A, Ai Y . The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation. Lab Chip. 2015; 15(17):3439-59. DOI: 10.1039/c5lc00614g. View

Denault J, Salvesen G . Human caspase-7 activity and regulation by its N-terminal peptide. J Biol Chem. 2003; 278(36):34042-50. DOI: 10.1074/jbc.M305110200. View

Brentnall M, Rodriguez-Menocal L, De Guevara R, Cepero E, Boise L . Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis. BMC Cell Biol. 2013; 14:32. PMC: 3710246. DOI: 10.1186/1471-2121-14-32. View

10.

Hardy J, Lam J, Nguyen J, OBrien T, Wells J . Discovery of an allosteric site in the caspases. Proc Natl Acad Sci U S A. 2004; 101(34):12461-6. PMC: 514654. DOI: 10.1073/pnas.0404781101. View

11.

OBrien T, Lee D . Prospects for caspase inhibitors. Mini Rev Med Chem. 2004; 4(2):153-65. DOI: 10.2174/1389557043487448. View

12.

Starr T, Thornton J . Epistasis in protein evolution. Protein Sci. 2016; 25(7):1204-18. PMC: 4918427. DOI: 10.1002/pro.2897. View

13.

Nussinov R, Tsai C . Allostery in disease and in drug discovery. Cell. 2013; 153(2):293-305. DOI: 10.1016/j.cell.2013.03.034. View

14.

Bloom J, Lu Z, Chen D, Raval A, Venturelli O, Arnold F . Evolution favors protein mutational robustness in sufficiently large populations. BMC Biol. 2007; 5:29. PMC: 1995189. DOI: 10.1186/1741-7007-5-29. View

15.

Nicholls S, Chu J, Abbruzzese G, Tremblay K, Hardy J . Mechanism of a genetically encoded dark-to-bright reporter for caspase activity. J Biol Chem. 2011; 286(28):24977-86. PMC: 3137071. DOI: 10.1074/jbc.M111.221648. View

16.

Suzuki Y, Nakabayashi Y, Nakata K, Reed J, Takahashi R . X-linked inhibitor of apoptosis protein (XIAP) inhibits caspase-3 and -7 in distinct modes. J Biol Chem. 2001; 276(29):27058-63. DOI: 10.1074/jbc.M102415200. View

17.

Slee E, Adrain C, Martin S . Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem. 2000; 276(10):7320-6. DOI: 10.1074/jbc.M008363200. View

18.

Reynolds K, McLaughlin R, Ranganathan R . Hot spots for allosteric regulation on protein surfaces. Cell. 2011; 147(7):1564-75. PMC: 3414429. DOI: 10.1016/j.cell.2011.10.049. View

19.

Lamkanfi M, Festjens N, Declercq W, Vanden Berghe T, Vandenabeele P . Caspases in cell survival, proliferation and differentiation. Cell Death Differ. 2006; 14(1):44-55. DOI: 10.1038/sj.cdd.4402047. View

20.

McIlwain D, Berger T, Mak T . Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol. 2013; 5(4):a008656. PMC: 3683896. DOI: 10.1101/cshperspect.a008656. View