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

Hill ME, MacPherson DJ, Wu P, Julien O, Wells JA, Hardy JA
ACS Chem Biol 11 1603-12 (2016)
Related entries: 4zvo, 4zvp, 4zvr, 4zvs, 4zvt, 4zvu

Cited: 24 times
EuropePMC logo PMID: 27032039

Abstract

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here, 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, 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. This approach to specificity reprogramming should also be generalizable across a wide range of proteases.

Reviews citing this publication (5)

  1. Caspases and their substrates. Julien O, Wells JA. Cell Death Differ 24 1380-1389 (2017)
  2. Caspases rule the intracellular trafficking cartel. Duclos C, Lavoie C, Denault JB. FEBS J 284 1394-1420 (2017)
  3. Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity. Grinshpon RD, Shrestha S, Titus-McQuillan J, Hamilton PT, Swartz PD, Clark AC. Biochem J 476 3475-3492 (2019)
  4. Making the cut with protease engineering. Dyer RP, Weiss GA. Cell Chem Biol 29 177-190 (2022)
  5. N-Terminomics Strategies for Protease Substrates Profiling. Mintoo M, Chakravarty A, Tilvawala R. Molecules 26 4699 (2021)

Articles citing this publication (19)

  1. Multiple Mechanisms of Zinc-Mediated Inhibition for the Apoptotic Caspases-3, -6, -7, and -8. Eron SJ, MacPherson DJ, Dagbay KB, Hardy JA. ACS Chem Biol 13 1279-1290 (2018)
  2. Deorphanizing Caspase-3 and Caspase-9 Substrates In and Out of Apoptosis with Deep Substrate Profiling. Araya LE, Soni IV, Hardy JA, Julien O. ACS Chem Biol 16 2280-2296 (2021)
  3. Dual Site Phosphorylation of Caspase-7 by PAK2 Blocks Apoptotic Activity by Two Distinct Mechanisms. Eron SJ, Raghupathi K, Hardy JA. Structure 25 27-39 (2017)
  4. Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity. Blum TR, Liu H, Packer MS, Xiong X, Lee PG, Zhang S, Richter M, Minasov G, Satchell KJF, Dong M, Liu DR. Science 371 803-810 (2021)
  5. Caspase-7 uses RNA to enhance proteolysis of poly(ADP-ribose) polymerase 1 and other RNA-binding proteins. Desroches A, Denault JB. Proc Natl Acad Sci U S A 116 21521-21528 (2019)
  6. The CaspBase: a curated database for evolutionary biochemical studies of caspase functional divergence and ancestral sequence inference. Grinshpon RD, Williford A, Titus-McQuillan J, Clay Clark A. Protein Sci 27 1857-1870 (2018)
  7. Tri-arginine exosite patch of caspase-6 recruits substrates for hydrolysis. MacPherson DJ, Mills CL, Ondrechen MJ, Hardy JA. J Biol Chem 294 71-88 (2019)
  8. Caspase-6 Undergoes a Distinct Helix-Strand Interconversion upon Substrate Binding. Dagbay KB, Bolik-Coulon N, Savinov SN, Hardy JA. J Biol Chem 292 4885-4897 (2017)
  9. Sequential Glycosylation of Proteins with Substrate-Specific N-Glycosyltransferases. Lin L, Kightlinger W, Prabhu SK, Hockenberry AJ, Li C, Wang LX, Jewett MC, Mrksich M. ACS Cent Sci 6 144-154 (2020)
  10. Rare human Caspase-6-R65W and Caspase-6-G66R variants identify a novel regulatory region of Caspase-6 activity. Tubeleviciute-Aydin A, Zhou L, Sharma G, Soni IV, Savinov SN, Hardy JA, LeBlanc AC. Sci Rep 8 4428 (2018)
  11. Exogenous Introduction of Initiator and Executioner Caspases Results in Different Apoptotic Outcomes. Anson F, Thayumanavan S, Hardy JA. JACS Au 1 1240-1256 (2021)
  12. Evolution of the folding landscape of effector caspases. Shrestha S, Clark AC. J Biol Chem 297 101249 (2021)
  13. Caspase-9 Activation of Procaspase-3 but Not Procaspase-6 Is Based on the Local Context of Cleavage Site Motifs and on Sequence. Soni IV, Hardy JA. Biochemistry 60 2824-2835 (2021)
  14. A Nanopore Approach for Analysis of Caspase-7 Activity in Cell Lysates. Pham B, Eron SJ, Hill ME, Li X, Fahie MA, Hardy JA, Chen M. Biophys J 117 844-855 (2019)
  15. Rare CASP6N73T variant associated with hippocampal volume exhibits decreased proteolytic activity, synaptic transmission defect, and neurodegeneration. Zhou L, Nho K, Haddad MG, Cherepacha N, Tubeleviciute-Aydin A, Tsai AP, Saykin AJ, Jesper Sjöström P, LeBlanc AC. Sci Rep 11 12695 (2021)
  16. Caspase-Activated Oligonucleotide Probe. Yang L, Eberwine JH, Dmochowski IJ. Bioconjug Chem 31 2172-2178 (2020)
  17. Non-Apoptotic Caspase Activity Preferentially Targets a Novel Consensus Sequence Associated With Cytoskeletal Proteins in the Developing Auditory Brainstem. Weghorst F, Mirzakhanyan Y, Hernandez KL, Gershon PD, Cramer KS. Front Cell Dev Biol 10 844844 (2022)
  18. General Theory of Specific Binding: Insights from a Genetic-Mechano-Chemical Protein Model. McBride JM, Eckmann JP, Tlusty T. Mol Biol Evol 39 msac217 (2022)
  19. Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker. Alves J, Garay-Malpartida M, Occhiucci JM, Belizário JE. Biochem Cell Biol 95 634-643 (2017)


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