The metalloproteinase ADAM10 requires its activity to sustain surface expression

Anke Seifert,Andreas Ludwig,Daniela Yildiz,Armando Rossello,Michael G Tomlinson,Chek Z Koo,Stefan Düsterhöft,Elisa Nuti,Justyna Wozniak,Doretta Cuffaro

doi:10.1007/s00018-020-03507-w

Abstract

The metalloproteinase ADAM10 critically contributes to development, inflammation, and cancer and can be controlled by endogenous or synthetic inhibitors. Here, we demonstrate for the first time that loss of proteolytic activity of ADAM10 by either inhibition or loss of function mutations induces removal of the protease from the cell surface and the whole cell. This process is temperature dependent, restricted to mature ADAM10, and associated with an increased internalization, lysosomal degradation, and release of mature ADAM10 in extracellular vesicles. Recovery from this depletion requires de novo synthesis. Functionally, this is reflected by loss and recovery of ADAM10 substrate shedding. Finally, ADAM10 inhibition in mice reduces systemic ADAM10 levels in different tissues. Thus, ADAM10 activity is critically required for its surface expression in vitro and in vivo. These findings are crucial for development of therapeutic ADAM10 inhibition strategies and may showcase a novel, physiologically relevant mechanism of protease removal due to activity loss.

Highlights

Surface-expressed proteases of the disintegrin and metalloproteinase (ADAM) family function as critical regulators of physiological and pathophysiological processes during development, inflammation, and cancer [1, 2]
USA), ikarugamycin was from biomol (Hamburg, Germany), recombinant human tissue inhibitor of metalloproteinases (TIMP)-1 (970-TM-010) was from R&D Systems (Minneapolis, USA), E. coli pHrodo (P35366), EZ-LinkTM Sulfo-NHS-LC-Biotin, and Aldehyde/Sulfate latex beads were from Thermo Fisher Scientific (Waltham, USA), Streptavidin-Sepharose High Performance was from GE Healthcare (Chicago, USA) and the ADAM10 substrate peptide (PRYEAYKMG) was from peptides & elephants (Hennigsdorf, Germany)
We verified that this effect was time- and concentration-dependent reaching about 15% residual ADAM10 expression after 24 h treatment with 10 μM GI (Fig. 1b, c)

Summary

Introduction

Surface-expressed proteases of the disintegrin and metalloproteinase (ADAM) family function as critical regulators of physiological and pathophysiological processes during development, inflammation, and cancer [1, 2]. Many more cleavage events have been found to depend on ADAM10 [1] This includes adhesion molecules of the cadherin family, members of the epidermal growth factor family (EGF and betacellulin), low affinity IgE receptor CD23, ephrins, and inflammatory mediators such as transmembrane chemokines (CXCL16 and CX3CL1) [7]. By regulating these substrates, ADAM10 is involved in endothelial permeability regulation, leukocyte migration [7, 8], transactivation of cancer cells [9], fibrogenesis, immune responses, and infectious diseases [10, 11]

Methods

Results

Conclusion