Quantitative Proteomics Reveals That ADAM15 Can Have Proteolytic-Independent Functions in the Steady State

Abstract

A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner.

High-Resolution Secretome Analysis of Chemical Hypoxia Treated Cells Identifies Putative Biomarkers of Chondrosarcoma

“…Thus, we treated SW 1353 chondrosarcoma cells with DMOG, a compound that stabilizes HIF-1α, thereby mimicking hypoxia [ 14 , 50 , 51 ], and performed a quantitative secretome analysis to identify proteins that were differentially released in response to hypoxia. SW 1353 cells represent a model that we have extensively investigated and the secretome of which we have fully characterized [ 52 , 53 ]. We found that approximately 50% of the total proteins were altered in response to hypoxia.…”

Identification of membrane proteins regulated by ADAM15 by SUSPECS proteomics

“…For instance, ADAM15 catalytic activity was required for claudin-1-dependent regulation of growth and mobility of breast cancer cells ( Mattern et al, 2019 ), but was not required for its role in promoting pathological neovascularization in a mouse model of oxygen-induced retinopathy ( Maretzky et al, 2014 ). Similarly, we found that ADAM15 regulates levels of TIMP-3 in a catalytic-independent manner ( Yang et al, 2022 ). For this reason, we here used a cutting-edge proteomic method that is specifically designed to enrich cell membrane proteins, and therefore could allow identification both of canonical ADAM15 substrates and proteins regulated by the proteinase through other catalytic-independent mechanisms.…”

“…Thus, we further investigated ADAM15-dependent shedding by using data-independent acquisition mass spectrometry (DIA-MS). DIA-MS is a recently-developed global MS-based strategy that allows a broader protein coverage than data-dependent acquisition (DDA) MS, which was previously used to investigate changes in the secretome of ADAM15-silenced cells ( Yang et al, 2020 ; Yang et al, 2022 ).…”

“…Although it has been proven to be a proteolytically active enzyme in vitro , the reported substrate repertoire of ADAM15 is still restricted to a few proteins, including FGFR2iiib ( Maretzky et al, 2009 ), MICB ( Duan et al, 2013 ), CD23 ( Fourie et al, 2003 ), E-cadherin ( Najy et al, 2008 ) and HB-EGF ( Schafer et al, 2004 ), and no proteins have been validated as ADAM15 substrates in vivo . We previously used a number of innovative proteomic methods in an attempt to identify transmembrane proteins released by ADAM15, either in the steady-state or upon overexpressing the proteinase, but failed to identify any putative substrates (( Yang et al, 2020 ; Yang et al, 2022 )). Similar methods to the ones we used have been effective for identifying substrates of sheddases, such as ADAM10 and BACE1 that release over 50 different proteins, and also for identifying substrates of signal peptide peptidase (SPP) sheddases that have a very limited substrate spectrum.…”

“…Despite its clear role in regulating cartilage homeostasis, little is known about how ADAM15 exerts its biological functions. A systemic secretome analysis using state-of-the-art mass spectrometry-based methods failed to identify ADAM15 substrates in chondrocytes and chondrocyte-like cells, arguing against the assumption that ADAM15 exerts its biological functions by releasing transmembrane proteins ( Yang et al, 2020 ; Yang et al, 2022 ). Additionally, in agreement with these studies, ADAM15 has been shown to regulate pathological neovascularization in the retina independently of its catalytic activity, indicating that ADAM15 has at least some functions that do not require its proteolytic potential ( Maretzky et al, 2014 ).…”