Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation.

BACKGROUND: Cell death and survival processes must be tightly regulated to ensure proper tissue homeostasis and prevent excessive inflammation and tissue damage. Death receptors, including TNF-R1, can induce either immunogenic (necroptosis) or non-immunogenic (apoptosis) cell death and relay proliferative / cell survival signaling by activating NFκB and MAPK cascades. In a recent report, we identified the metalloproteinase ADAM15 as a possible TNF-responding enzyme, leading to the hypothesis that it regulates either cell survival or death cascades. METHODS: CRISPR/Cas-9 was used to knock out the adam15 gene. Loss of gene expression was validated by Western blot and flow cytometry in U937 and Jurkat cells. NFκB, MAPK signaling, and cell death cascades were monitored by Western blot, flow cytometry, and enzyme assays. A bottom-up proteome analysis was performed to elucidate cellular processes affected by ADAM15 loss. The subcellular localization of ADAM15 was monitored by microscopy and immuno-magnetic fractionation. RESULTS: We identified ADAM15 as a regulator of necroptosis, leaving apoptosis and cell survival signaling unaffected. Loss of ADAM15 resulted in abrogated necroptosis, as evidenced by the application of death ligands TNF, TRAIL, FasL, and TL1a, as well as the BH3 mimetic Obatoclax. We observed enhanced basal Caspase-8 activity, which was not cytotoxic, and partial RIPK1 proteolysis. The loss of ADAM15 was verified in a proteome screen, which revealed alterations in various molecular pathways, including autophagy, organelle trafficking, and sorting. We observed ADAM15 in intracellular compartments, which in part have a lysosomal protein signature. We observed enhanced surface expression of TNF-R1, proposing it as a possible ADAM15 substrate. CONCLUSIONS: ADAM15 is a previously unknown regulator of necroptosis, likely due to its role in modulating intracellular organelle sorting processes. Its proteolytic activity and possible scaffolding capacity for recruiting adaptor molecules make it a veritable drug target. The activation or deactivation of ADAM15 may be exploited to modulate various disease conditions.