Abstract
Disintegrin and metalloproteinases (ADAMs) 10 and 17 can release the extracellular part of a variety of membrane-bound proteins via ectodomain shedding important for many biological functions. So far, substrate identification focused exclusively on membrane-anchored ADAM10 and ADAM17. However, besides known shedding of ADAM10, we identified ADAM8 as a protease capable of releasing the ADAM17 ectodomain. Therefore, we investigated whether the soluble ectodomains of ADAM10/17 (sADAM10/17) exhibit an altered substrate spectrum compared to their membrane-bound counterparts. A mass spectrometry-based N-terminomics approach identified 134 protein cleavage events in total and 45 common substrates for sADAM10/17 within the secretome of murine cardiomyocytes. Analysis of these cleavage sites confirmed previously identified amino acid preferences. Further in vitro studies verified fibronectin, cystatin C, sN-cadherin, PCPE-1 as well as sAPP as direct substrates of sADAM10 and/or sADAM17. Overall, we present the first degradome study for sADAM10/17, thereby introducing a new mode of proteolytic activity within the protease web.
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MS data
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [95] partner repository with the data set identifier PXD013718.
Abbreviations
- ACN:
-
Acetonitrile
- ADAM:
-
A disintegrin and metalloprotease
- APP:
-
Amyloid precursor protein
- CTF:
-
C-terminal fragment
- DAPT:
-
N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester
- DCM:
-
Dilated cardiomyopathy
- dnp:
-
2,4-Dinitrophenyl
- ECM:
-
Extracellular matrix
- FCS:
-
Fetal calf serum
- HEK:
-
Human embryonic kidney
- LC–MS/MS:
-
Liquid chromatography–tandem mass spectrometry
- Mca:
-
7-Methyloxycoumarin-4-yl-acetyl
- MMP:
-
Matrix metalloproteinase
- PCPE-1:
-
Procollagen C-proteinase enhancer-1
- PSM:
-
Peptide scoring matches
- RFU:
-
Relative fluorescence unit
- SD:
-
Standard deviation
- TAILS:
-
Terminal amine isotopic labeling of substrates
- TCA:
-
Trichloracetic acid
- TFA:
-
Trifluoroacetic acid
- TIMP:
-
Tissue inhibitor of metalloproteinases
- TMT:
-
Tandem mass tag
- TR:
-
Technical replicate
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Acknowledgements
We thank William C. Claycomb (Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana) for providing HL-1 cells. We thank Björn Rabe from the Biochemical Institute of the University of Kiel for ADAM10−/−; 17−/− HEK293T cells.
Funding
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) SFB 877 (Proteolysis as a Regulatory Event in Pathophysiology, Projects A1, A9, A15 and Z2), BE 4086/2-2 (C.B.-P.), University of Lyon (C.M.), FOR2290 (S.F.L.), PI379/5-2 (C.U.P.), and BA1606/3-1 (J.W.B).
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Conceptualization, FS and CB-P; methodology, investigation, and data analysis, FS, AH, MS, JB, US, FP, RW, MB, DS-A, SR-J, CM, SFL, CUP, JWB, AT, and CB-P; writing, FS and CB-P.
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Scharfenberg, F., Helbig, A., Sammel, M. et al. Degradome of soluble ADAM10 and ADAM17 metalloproteases. Cell. Mol. Life Sci. 77, 331–350 (2020). https://doi.org/10.1007/s00018-019-03184-4
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DOI: https://doi.org/10.1007/s00018-019-03184-4
Keywords
- TAILS
- ADAM8
- ADAM10
- ADAM17
- Ectodomain shedding
- Proteolysis