The iRhom homology domain is indispensable for ADAM17-mediated TNF\u03b1 and EGF receptor ligand release

Stefan Düsterhöft,Christian Preisinger,Henrike Ohm,Jana Kopkanova,Juliane Lokau,Selcan Kahveci-Türköz,Andreas Ludwig,Radislav Sedlacek,Petr Kasparek,Matthew Freeman,Christoph Garbers,Shixin Liu,Anke Seifert,Justyna Wozniak

doi:10.1007/s00018-021-03845-3

Abstract

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.

Highlights

Maintaining balanced regulation of cell-to-cell communication is the basis of multicellularity
The pseudoprotease iRhom is indispensable for ADAM17 maturation and thereby plays critical regulatory functions in inflammatory and proliferative diseases that depend on ADAM17-mediated shedding of cytokines, growth factors and their receptors
Using in silico and in vitro analysis of human and murine iRhom2 constructs we analysed the luminal iRhom homology domain (IRHD) and could show that this domain and its structural integrity is crucial for ADAM17 binding

Summary

Introduction

Maintaining balanced regulation of cell-to-cell communication is the basis of multicellularity. A disintegrin and metalloproteinases (ADAM) plays a crucial role in the regulation. Of many signalling pathways such as in developmental and regenerative processes by releasing ligands of the epidermal growth factor receptor (EGFR) [2, 3]. Systemic absence of ADAM17 activity in mice causes developmental defects and lethality [3]. ADAM17 is instrumental in the inflammatory response. This includes the release of tumour necrosis factor alpha (TNFα) [4, 5] and its receptors as well as the interleukin 6 (IL6) receptor [6,7,8]. Dysregulation of ADAM17 activity is implicated in pathologies such as chronic inflammation and cancer progression [7,8,9,10]

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Conclusion