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.

Keywords: ADAM15; ADAMs; metalloproteinases; proteomics; secretome.

Figure 1

Secretome analysis of ADAM15 knockdown HTB84 cells. (A) HTB94 cells were transfected with 20 nM siRNA targeting ADAM15 (siADAM15) or same concentration of non-targeting control RNA (NT). ADAM15 expression was measured by qPCR at 0, 24, 48, and 72 h after treatment with ADAM15 or NT siRNAs, and displayed as a percentage of ADAM15 expression in cells transfected with NT siRNAs at each time point. Protein levels of ADAM15 in HTB94 cells treated with NT siRNAs (control) or ADAM15 siRNAs were analysed by immunoblotting at 72 h (insert). (B) Number of proteins annotated as a membrane or secreted proteins identified by secretome analysis or hiSPECS analysis in the conditioned media of ADAM15 knockdown HTB94 cells (left). Topology of single-span membrane proteins detected by secretome or SPECS analysis in the conditioned media of ADAM15 knockdown cells (right). (C) Volcano plot showing the −log₁₀ of p-values versus the log₂ of protein ratio between ADAM15 KD and HTB94 control cells (ct) of proteins detected in the conditioned media by serum-free secretome analysis (n = 6). AXL, which is the only protein above the FDR curves (displayed as black hyperbolic curves) is displayed as a blue solid dot, while proteins below the FDR are displayed by grey open dots. (D) Protein levels of full-length AXL in the cell lysates (flAXL) or shed AXL conditioned media (sAXL) of HTB94 cells treated with non-targeting or ADAM15 siRNAs (siADAM15) cells analysed by immunoblotting. (E) Bands corresponding to sAXL in the conditioned media from 3 independent experiments were quantified and normalized to the mean of sAXL values in conditioned media of cells treated with non-targeting siRNAs. A two-sided Student’s t-test was used to statistically evaluate changes in sAXL (** p < 0.01). (F) AXL mRNA expression levels in HTB94 cells treated with non-targeting or ADAM15 siRNAs plotted as relative 2^−ΔΔCT (*** p < 0.005, Student’s t-test) (G). Volcano plot showing the −log₁₀ of p-values versus the log₂ of protein ratio between ADAM15 KD and HTB94 control cells (ct) of proteins detected in the conditioned media by hiSPECS analysis (n = 6).

Figure 2

Secretome analysis of ADAM15-overexpressing HTB84 cells. (A) HTB94 cells were transiently transfected with ADAM15 or GFP as a control, and expression was evaluated by immunoblotting. (B) Number of proteins annotated as a membrane or secreted proteins detected in the secretome of ADAM15-overexpressing cells. (C) Topology of single-span membrane proteins detected in the secretome of ADAM15-overexpressing cells. (D) Volcano plot showing the −log₁₀ of p-values versus the log₂ of protein ratio between ADAM15-overexpressing and GFP transfected HTB94 control cells of 1077 proteins (n = 6). The hyperbolic curves indicate the false discovery rate. Proteins above these lines are considered significantly regulated, while proteins below are considered not regulated. TIMP-3 was found in all ADAM15-overexpressing replicates, while not being detected in GFP-expressing samples (insert) (E) Log2 of LFQ intensities for TIMP3 detected in ADAM15-overexpressing HTB94 cells.

Figure 3

ADAM15 regulates levels of TIMP-3. (A) Levels of TIMP-3 and clusterin in the conditioned media and ADAM15 and actin in the cell lysates were evaluated by immunoblotting in ADAM15-overexpressing HTB94 cells and controls. (B) Densitometric quantifications of TIMP-3 and clusterin in the conditioned media of ADAM15 transfected and control cells are displayed as mean values ± standard deviation (* p < 0.05, ** p < 0.01, *** p < 0.005, Student’s t-test). (C) Similarly, levels of TIMP-3 and clusterin in the conditioned media, and ADAM15 and actin in the lysate of HTB94 cells transfected with ADAM15 E/A or controls were evaluated by immunoblotting and densitometric quantification (D).