Abstract
Mass spectrometry-based proteomics has been used successfully to identify substrates for proteases. Identification of protease substrates at the cell surface, however, can be challenging since cleavages are less abundant compared to other cellular events. Precise methods are required to delineate cleavage events that take place in these compartmentalized areas. This article by up-and-coming scientist Dr. Amy Weeks, an Assistant Professor at the University of Wisconsin-Madison, provides an overview of methods developed to identify protease substrates and their cleavage sites at the membrane. An overview is presented with the pros and cons for each method and in particular the N-terminomics subtiligase-TM method, developed by Dr. Weeks as a postdoctoral fellow in the lab of Dr. Jim Wells at University of California, San Francisco, is described in detail. Subtiligase-TM is a genetically engineered subtilisin protease variant that acts to biotinylate newly generated N termini, hence revealing new cleavage events in the presence of a specific enzyme, and furthermore can precisely identify the cleavage P1 site. Importantly, this proteomics method is compatible with living cells. This method will open the door to understanding protein shedding events at the biological membrane controlled by proteases to regulate biological processes.
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References
Abrahmsen L, Tom J, Burnier J, Butcher KA, Kossiakoff A, Wells JA (1991) Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution. Biochemistry 30:4151–4159. https://doi.org/10.1021/bi00231a007
Bausch-Fluck D, Hofmann A, Bock T, Frei AP, Cerciello F, Jacobs A, Moest H, Omasits U, Gundry RL, Yoon C, Schiess R, Schmidt A, Mirkowska P, Härtlová A, Eyk JEV, Bourquin J-P, Aebersold R, Boheler KR, Zandstra P, Wollscheid B (2015) A mass spectrometric-derived cell surface protein atlas. PLoS One 10:e0121314. https://doi.org/10.1371/journal.pone.0121314
Black RA, Rauch CT, Kozlosky CJ, Peschon JJ, Slack JL, Wolfson MF, Castner BJ, Stocking KL, Reddy P, Srinivasan S, Nelson N, Boiani N, Schooley KA, Gerhart M, Davis R, Fitzner JN, Johnson RS, Paxton RJ, March CJ, Cerretti DP (1997) A metalloproteinase disintegrin that releases tumour-necrosis factor-α from cells. Nature 385:729–733. https://doi.org/10.1038/385729a0
Blobel CP (2005) ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Bio 6:32–43. https://doi.org/10.1038/nrm1548
Damme PV, Martens L, Damme JV, Hugelier K, Staes A, Vandekerckhove J, Gevaert K (2005) Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis. Nat Methods 2:771–777. https://doi.org/10.1038/nmeth792
Dix MM, Simon GM, Cravatt BF (2008) Global mapping of the topography and magnitude of proteolytic events in apoptosis. Cell 134:679–691. https://doi.org/10.1016/j.cell.2008.06.038
Gevaert K, Goethals M, Martens L, Damme JV, Staes A, Thomas GR, Vandekerckhove J (2003) Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nat Biotechnol 21:566–569. https://doi.org/10.1038/nbt810
Griswold AR, Cifani P, Rao SD, Axelrod AJ, Miele MM, Hendrickson RC, Kentsis A, Bachovchin DA (2019) A chemical strategy for protease substrate profiling. Cell Chem Biol 26:901.e6-907.e6. https://doi.org/10.1016/j.chembiol.2019.03.007
Hemming ML, Elias JE, Gygi SP, Selkoe DJ (2009) Identification of β-secretase (BACE1) substrates using quantitative proteomics. PLoS One 4:e8477. https://doi.org/10.1371/journal.pone.0008477
Hughes CS, Moggridge S, Müller T, Sorensen PH, Morin GB, Krijgsveld J (2019) Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc 14:68–85. https://doi.org/10.1038/s41596-018-0082-x
Jackson HW, Defamie V, Waterhouse P, Khokha R (2017) TIMPs: versatile extracellular regulators in cancer. Nat Rev Cancer 17:38–53. https://doi.org/10.1038/nrc.2016.115
Jefferson T, Čaušević M, Keller auf dem U, Schilling O, Isbert S, Geyer R, Maier W, Tschickardt S, Jumpertz T, Weggen S, Bond JS, Overall CM, Pietrzik CU, Becker-Pauly C (2011) Metalloprotease meprin β generates nontoxic N-terminal amyloid precursor protein fragments in vivo. J Biol Chem 286:27741–27750. https://doi.org/10.1074/jbc.m111.252718
Khokha R, Murthy A, Weiss A (2013) Metalloproteinases and their natural inhibitors in inflammation and immunity. Nat Rev Immunol 13:649–665. https://doi.org/10.1038/nri3499
Kleifeld O, Doucet A, Keller auf dem U, Prudova A, Schilling O, Kainthan RK, Starr AE, Foster LJ, Kizhakkedathu JN, Overall CM (2010) Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products. Nat Biotechnol 28:281–288. https://doi.org/10.1038/nbt.1611
Kuhn P, Koroniak K, Hogl S, Colombo A, Zeitschel U, Willem M, Volbracht C, Schepers U, Imhof A, Hoffmeister A, Haass C, Roßner S, Bräse S, Lichtenthaler SF (2012) Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons. Embo J 31:3157–3168. https://doi.org/10.1038/emboj.2012.173
Kuhn P-H, Colombo AV, Schusser B, Dreymueller D, Wetzel S, Schepers U, Herber J, Ludwig A, Kremmer E, Montag D, Müller U, Schweizer M, Saftig P, Bräse S, Lichtenthaler SF (2016) Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function. Elife 5:e12748. https://doi.org/10.7554/elife.12748
Lichtenthaler SF, Lemberg MK, Fluhrer R (2018) Proteolytic ectodomain shedding of membrane proteins in mammals—hardware, concepts, and recent developments. Embo J. https://doi.org/10.15252/embj.201899456
Loureiro J, Lilley BN, Spooner E, Noriega V, Tortorella D, Ploegh HL (2006) Signal peptide peptidase is required for dislocation from the endoplasmic reticulum. Nature 441:894–897. https://doi.org/10.1038/nature04830
MacDonald JI, Munch HK, Moore T, Francis MB (2015) One-step site-specific modification of native proteins with 2-pyridinecarboxyaldehydes. Nat Chem Biol 11:326–331. https://doi.org/10.1038/nchembio.1792
Mahrus S, Trinidad JC, Barkan DT, Sali A, Burlingame AL, Wells JA (2008) Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini. Cell 134:866–876. https://doi.org/10.1016/j.cell.2008.08.012
Moss ML, Jin S-LC, Milla ME, Burkhart W, Carter HL, Chen W-J, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH, Leesnitzer MA, McCauley P, McGeehan G, Mitchell J, Moyer M, Pahel G, Rocque W, Overton LK, Schoenen F, Seaton T, Su J-L, Warner J, Willard D, Becherer JD (1997) Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-α. Nature 385:733–736. https://doi.org/10.1038/385733a0
Munro KM, Nash A, Pigoni M, Lichtenthaler SF, Gunnersen JM (2016) Functions of the Alzheimer’s disease protease BACE1 at the synapse in the central nervous system. J Mol Neurosci 60:305–315. https://doi.org/10.1007/s12031-016-0800-1
Puente XS, Sánchez LM, Overall CM, López-Otín C (2003) Human and mouse proteases: a comparative genomic approach. Nat Rev Genet 4:544–558. https://doi.org/10.1038/nrg1111
Rawlings ND, Barrett AJ, Thomas PD, Huang X, Bateman A, Finn RD (2017) The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res. https://doi.org/10.1093/nar/gkx1134
Saeed M, Kapell S, Hertz NT, Wu X, Bell K, Ashbrook AW, Mark MT, Zebroski HA, Neal ML, Flodström-Tullberg M, MacDonald MR, Aitchison JD, Molina H, Rice CM (2020) Defining the proteolytic landscape during enterovirus infection. PLOS Pathog 16:e1008927. https://doi.org/10.1371/journal.ppat.1008927
Shema G, Nguyen MTN, Solari FA, Loroch S, Venne AS, Kollipara L, Sickmann A, Verhelst SHL, Zahedi RP (2018) Simple, scalable, and ultrasensitive tip-based identification of protease substrates. Mol Cell Proteomics 17:826–834. https://doi.org/10.1074/mcp.tir117.000302
Simon GM, Dix MM, Cravatt BF (2009) Comparative assessment of large-scale proteomic studies of apoptotic proteolysis. ACS Chem Biol 4:401–408. https://doi.org/10.1021/cb900082q
Tam EM, Morrison CJ, Wu YI, Stack MS, Overall CM (2004) Membrane protease proteomics: isotope-coded affinity tag MS identification of undescribed MT1–matrix metalloproteinase substrates. Proc Natl Acad Sci USA 101:6917–6922. https://doi.org/10.1073/pnas.0305862101
Tian S, Huang Q, Fang Y, Wu J (2011) FurinDB: a database of 20-residue furin cleavage site motifs, substrates and their associated drugs. Int J Mol Sci 12:1060–1065. https://doi.org/10.3390/ijms12021060
Tüshaus J, Müller SA, Kataka ES, Zaucha J, Monasor LS, Su M, Güner G, Jocher G, Tahirovic S, Frishman D, Simons M, Lichtenthaler SF (2020) An optimized quantitative proteomics method establishes the cell type-resolved mouse brain secretome. EMBO J 39:105693. https://doi.org/10.15252/embj.2020105693
Venne AS, Vögtle F-N, Meisinger C, Sickmann A, Zahedi RP (2013) Novel highly sensitive, specific, and straightforward strategy for comprehensive N-terminal proteomics reveals unknown substrates of the mitochondrial peptidase Icp55. J Proteome Res 12:3823–3830. https://doi.org/10.1021/pr400435d
Weeks AM, Byrnes JR, Lui I, Wells JA (2021) Mapping proteolytic neo-N termini at the surface of living cells. Proc Natl Acad Sci USA 118:e2018809118. https://doi.org/10.1073/pnas.2018809118
Weeks AM, Wells JA (2019) Subtiligase-catalyzed peptide ligation. Chem Rev 120:3127–3160. https://doi.org/10.1021/acs.chemrev.9b00372
Weeks AM, Wells JA (2018) Engineering peptide ligase specificity by proteomic identification of ligation sites. Nat Chem Biol 14:50–57. https://doi.org/10.1038/nchembio.2521
Weng SSH, Demir F, Ergin EK, Dirnberger S, Uzozie A, Tuscher D, Nierves L, Tsui J, Huesgen PF, Lange PF (2019) Sensitive determination of proteolytic proteoforms in limited microscale proteome samples. Mol Cell Proteomics 18:2335–2347. https://doi.org/10.1074/mcp.tir119.001560
Wollscheid B, Bausch-Fluck D, Henderson C, O’Brien R, Bibel M, Schiess R, Aebersold R, Watts JD (2009) Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins. Nat Biotechnol 27:378–386. https://doi.org/10.1038/nbt.1532
Acknowledgements
I thank members of the Weeks lab, S. Coyle, and D. Sashital for helpful discussions. Research in the Weeks lab is supported by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund (1017065) and a Steenbock Career Award from the University of Wisconsin.
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Weeks, A.M. Spatially Resolved Tagging of Proteolytic Neo-N termini with Subtiligase-TM. J Membrane Biol 254, 119–125 (2021). https://doi.org/10.1007/s00232-021-00171-4
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DOI: https://doi.org/10.1007/s00232-021-00171-4