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.

• Klíčová slova
• ADAM17, EGFR ligand release iRhom–ADAM17 complex structure, Ectodomain shedding, TNF signalling, iRhom, iRhom homology domain,
• MeSH
• buněčná membrána metabolismus   MeSH
• cytokiny metabolismus   MeSH
• membránové proteiny * metabolismus   MeSH
• modely strukturální MeSH
• protein ADAM17 metabolismus   MeSH
• transportní proteiny * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokiny MeSH
• membránové proteiny * MeSH
• protein ADAM17 MeSH
• transportní proteiny * MeSH

Membrane-tethered signalling proteins such as TNFα and many EGF receptor ligands undergo shedding by the metalloproteinase ADAM17 to get released. The pseudoproteases iRhom1 and iRhom2 are important for the transport, maturation and activity of ADAM17. Yet, the structural and functional requirements to promote the transport of the iRhom-ADAM17 complex have not yet been thoroughly investigated. Utilising in silico and in vitro methods, we here map the conserved iRhom homology domain (IRHD) and provide first insights into its structure and function. By focusing on iRhom2, we identified different structural and functional factors within the IRHD. We found that the structural integrity of the IRHD is a key factor for ADAM17 binding. In addition, we identified a highly conserved motif within an unstructured region of the IRHD, that, when mutated, restricts the transport of the iRhom-ADAM17 complex through the secretory pathway in in vitro, ex vivo and in vivo systems and also increases the half-life of iRhom2 and ADAM17. Furthermore, the disruption of this IRHD motif was also reflected by changes in the yet undescribed interaction profile of iRhom2 with proteins involved in intracellular vesicle transport. Overall, we provide the first insights into the forward trafficking of iRhoms which is critical for TNFα and EGF receptor signalling.

• Klíčová slova
• ADAM17, Ectodomain shedding, Growth factors, TNF, iRhom, iRhom homology domain,
• MeSH
• aminokyselinové motivy MeSH
• buněčné linie MeSH
• epidermální růstové faktory metabolismus   MeSH
• lidé MeSH
• malá interferující RNA metabolismus   MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mutageneze MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• poločas MeSH
• protein ADAM17 chemie   metabolismus   MeSH
• proteinové domény MeSH
• RNA interference MeSH
• signální transdukce MeSH
• TNF-alfa metabolismus   MeSH
• transport proteinů MeSH
• transportní proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• epidermální růstové faktory MeSH
• iRhom1 protein, mouse MeSH Prohlížeč
• iRhom2 protein, mouse MeSH Prohlížeč
• malá interferující RNA MeSH
• membránové proteiny MeSH
• protein ADAM17 MeSH
• TNF-alfa MeSH
• transportní proteiny MeSH