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Mapping the Substrate Recognition Landscapes of Metalloproteases Using Comprehensive Mutagenesis

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Matrix Metalloproteases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1579))

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Abstract

Protease specificity is controlled by exosites, which capture and orient the substrate, and the active site, which binds substrate residues near the P1–P1′ scissile bond and catalyzes peptide hydrolysis. Techniques used to identify critical contact points between a protease and its substrate can be time consuming and labor-intensive. Screening tools such as phage display have been revitalized with the emergence of high-throughput sequencing technology, and can be used to interrogate protease substrate specificity. This article will outline a method for creating and screening a comprehensive mutagenesis substrate phage display library. High-throughput sequencing of uncleaved phage at various reaction time points enables k cat/K M determination for every possible single amino acid substitution at each position of the substrate, providing unprecedented resolution for the interaction between a protease and its substrate.

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References

  1. Chan CE, Lim AP, MacAry PA, Hanson BJ (2014) The role of phage display in therapeutic antibody discovery. Int Immunol 26:649–657

    Article  CAS  PubMed  Google Scholar 

  2. Chen S, Heinis C (2015) Phage selection of bicyclic peptides based on two disulfide bridges. Methods Mol Biol 1248:119–137

    Article  CAS  PubMed  Google Scholar 

  3. Whitney M, Crisp JL, Olson ES, Aguilera TA, Gross LA, Ellies LG, Tsien RY (2010) Parallel in vivo and in vitro selection using phage display identifies protease-dependent tumor-targeting peptides. J Biol Chem 285:22532–22541

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kridel SJ, Chen E, Kotra LP, Howard EW, Mobashery S, Smith JW (2001) Substrate hydrolysis by matrix metalloproteinase-9. J Biol Chem 276:20572–20578

    Article  CAS  PubMed  Google Scholar 

  5. Hills R, Mazzarella R, Fok K, Liu M, Nemirovskiy O, Leone J, Zack MD, Arner EC, Viswanathan M, Abujoub A, Muruganandam A, Sexton DJ, Bassill GJ, Sato AK, Malfait AM, Tortorella MD (2007) Identification of an ADAMTS-4 cleavage motif using phage display leads to the development of fluorogenic peptide substrates and reveals matrilin-3 as a novel substrate. J Biol Chem 282:11101–11109

    Article  CAS  PubMed  Google Scholar 

  6. Eckhard U, Huesgen PF, Schilling O, Bellac CL, Butler GS, Cox JH, Dufour A, Goebeler V, Kappelhoff R, Keller UA, Klein T, Lange PF, Marino G, Morrison CJ, Prudova A, Rodriguez D, Starr AE, Wang Y, Overall CM (2016) Active site specificity of the matrix metalloproteinase family: proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses. Matrix Biol 49:37–60

    Article  CAS  PubMed  Google Scholar 

  7. Chen EI, Kridel SJ, Howard EW, Li W, Godzik A, Smith JW (2002) A unique substrate recognition profile for matrix metalloproteinase-2. J Biol Chem 277:4485–4491

    Article  CAS  PubMed  Google Scholar 

  8. Ratnikov BI, Cieplak P, Gramatikoff K, Pierce J, Eroshkin A, Igarashi Y, Kazanov M, Sun Q, Godzik A, Osterman A, Stec B, Strongin A, Smith JW (2014) Basis for substrate recognition and distinction by matrix metalloproteinases. Proc Natl Acad Sci U S A 111(40):E4148–E4155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Huxley-Jones J, Clarke TK, Beck C, Toubaris G, Robertson DL, Boot-Handford RP (2007) The evolution of the vertebrate metzincins; insights from Ciona intestinalis and Danio rerio. BMC Evol Biol 7:63

    Article  PubMed  PubMed Central  Google Scholar 

  10. Shendure J, Lieberman Aiden E (2012) The expanding scope of DNA sequencing. Nat Biotechnol 30:1084–1094

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Tinberg CE, Khare SD, Dou J, Doyle L, Nelson JW, Schena A, Jankowski W, Kalodimos CG, Johnsson K, Stoddard BL, Baker D (2013) Computational design of ligand-binding proteins with high affinity and selectivity. Nature 501:212–216

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kitzman JO, Starita LM, Lo RS, Fields S, Shendure J (2015) Massively parallel single-amino-acid mutagenesis. Nat Methods 12:203–206

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Fowler DM, Araya CL, Fleishman SJ, Kellogg EH, Stephany JJ, Baker D, Fields S (2010) High-resolution mapping of protein sequence-function relationships. Nat Methods 7:741–746

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Starita LM, Young DL, Islam M, Kitzman JO, Gullingsrud J, Hause RJ, Fowler DM, Parvin JD, Shendure J, Fields S (2015) Massively parallel functional analysis of BRCA1 RING domain variants. Genetics 200:413–422

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kretz CA, Dai M, Soylemez O, Yee A, Desch KC, Siemieniak D, Tomberg K, Kondrashov FA, Meng F, Ginsburg D (2015) Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13. Proc Natl Acad Sci U S A 112:9328–9333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Fowler DM, Araya CL, Gerard W, Fields S (2011) Enrich: software for analysis of protein function by enrichment and depletion of variants. Bioinformatics 27:3430–3431

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Glenn TC (2011) Field guide to next-generation DNA sequencers. Mol Ecol Resour 11:759–769

    Article  CAS  PubMed  Google Scholar 

  18. Desch KC, Kretz C, Yee A, Gildersleeve R, Metzger K, Agrawal N, Cheng J, Ginsburg D (2015) Probing ADAMTS13 substrate specificity using phage display. PLoS One 10:e0122931

    Article  PubMed  PubMed Central  Google Scholar 

  19. Scott JK, Smith GP (1990) Searching for peptide ligands with an epitope library. Science 249:386–390

    Article  CAS  PubMed  Google Scholar 

  20. Love MI, Huber W, and Anders S (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15:550

    Google Scholar 

  21. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Araya CL, Fowler DM, Chen W, Muniez I, Kelly JW, Fields S (2012) A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function. Proc Natl Acad Sci U S A 109:16858–16863

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hochberg YBY (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B 57:289–300

    Google Scholar 

  24. Shendure J, Akey JM (2015) The origins, determinants, and consequences of human mutations. Science 349:1478–1483

    Article  CAS  PubMed  Google Scholar 

  25. Gnad F, Baucom A, Mukhyala K, Manning G, Zhang Z (2013) Assessment of computational methods for predicting the effects of missense mutations in human cancers. BMC Genomics 14(Suppl 3):S7

    PubMed  PubMed Central  Google Scholar 

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Acknowledgments

I would like to thank David Ginsburg (University of Michigan) for critical review of the manuscript. Colin A. Kretz holds a McMaster University Department of Medicine Internal Career Award. This work was also supported by the Judith Graham Pool Fellowship from the National Hemophilia Foundation, awarded to Colin A. Kretz.

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Authors and Affiliations

  1. Thrombosis and Atherosclerosis Research Institute and Department of Medicine, McMaster University, Room C5-117, 237 Barton St. East, Hamilton, ON, Canada, L8L 2X2

    Colin A. Kretz

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  1. Colin A. Kretz
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Correspondence to Colin A. Kretz .

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Editors and Affiliations

  1. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia

    Charles A. Galea

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Kretz, C.A. (2017). Mapping the Substrate Recognition Landscapes of Metalloproteases Using Comprehensive Mutagenesis. In: Galea, C. (eds) Matrix Metalloproteases. Methods in Molecular Biology, vol 1579. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6863-3_11

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  • DOI: https://doi.org/10.1007/978-1-4939-6863-3_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6861-9

  • Online ISBN: 978-1-4939-6863-3

  • eBook Packages: Springer Protocols

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