A structural model of the iRhom–ADAM17 sheddase complex reveals functional insights into its trafficking and activity

Abstract

Several membrane-anchored signal mediators such as cytokines (e.g. TNFα) and growth factors are proteolytically shed from the cell surface by the metalloproteinase ADAM17, which, thus, has an essential role in inflammatory and developmental processes. The membrane proteins iRhom1 and iRhom2 are instrumental for the transport of ADAM17 to the cell surface and its regulation. However, the structure–function determinants of the iRhom–ADAM17 complex are poorly understood. We used AI-based modelling to gain insights into the structure–function relationship of this complex. We identified different regions in the iRhom homology domain (IRHD) that are differentially responsible for iRhom functions. We have supported the validity of the predicted structure–function determinants with several in vitro, ex vivo and in vivo approaches and demonstrated the regulatory role of the IRHD for iRhom–ADAM17 complex cohesion and forward trafficking. Overall, we provide mechanistic insights into the iRhom–ADAM17-mediated shedding event, which is at the centre of several important cytokine and growth factor pathways.

Pathological mutations reveal the key role of the cytosolic iRhom2 N-terminus for phosphorylation-independent 14-3-3 interaction and ADAM17 binding, stability, and activity

“…As iRhom levels are a rate limiting factor for forward transport and maturation of ADAM17, increased expression of either human or murine wt-iRhom2 results in more mature ADAM17 (Fig. 3A-C), as shown before (Düsterhöft et al, 2021, Kahveci-Türköz et al, 2023. Consequently, the amount of mature ADAM17 on the cell surface is also elevated (Fig.…”

“…7). During maturation, the proteolytic removal of the ADAM17 prodomain may further decrease binding affinity, as our recent study identified the prodomain as an additional binding determinant between the ADAM17 ectodomain and the IRHD (Kahveci-Türköz et al, 2023). The reduced cohesion of the iRhom2-matureADAM17 complex on the cell surface appears to induce instability and accelerated turnover, culminating in lysosomal degradation (Fig.…”

“…To identify conserved sequence regions, the tool CONYAR ( Co nserved Y ou Ar e) was employed to perform high-throughput comparison of as many sequences of a target protein as possible (Kahveci-Türköz et al, 2023).…”

“…Precipitation experiments were done as described before (Düsterhöft et al, 2021, Kahveci-Türköz et al, 2023): Cells were lysed in 1 ml lysis buffer (50 mM Tris, 2 mM CaCl 2, 137 mM NaCl, 2 mM EDTA, 10 mM 1,10-Phenanthroline, pH 7.5) supplemented with cOmplete™ protease inhibitor (Sigma; 11697498001). The lysates were cleared by centrifugation at 16,000 x g for 20 minutes at 4 °C.…”

“…ADAM17-mediated shedding activity was measured by an alkaline phosphatase (AP)–based assay as described before (Düsterhöft et al, 2021, Kahveci-Türköz et al, 2023). In brief, we used cells transfected with ADAM17 substrates fused to an alkaline phosphatase to analyse shedding activity: The following ADAM17 substrates cloned in pCndA3.1 were utilised: AP-IL1R2, AP-TNFα, AP-AREG.…”