A disintegrin and metalloprotease (ADAM) 17 cleaves and releases membrane-tethered pro-forms of several signalling molecules from the plasma membrane, including the inflammatory cytokine tumour necrosis factor alpha (TNFα) and ligands of the epidermal growth factor receptor (EGFR). Due to the important functions of its substrates, ADAM17 activity has to be tightly controlled, and its misregulation has implications for inflammation and cancer. The multi-pass membrane proteins iRhom1 and iRhom2 are members of the conserved rhomboid-like superfamily and control ADAM17 activity by several mechanisms throughout the secretory pathway. First, iRhoms facilitate trafficking of the catalytically inactive proenzyme form of ADAM17 from the endoplasmic reticulum (ER) to the Golgi apparatus, where the inhibitory pro-domain of ADAM17 is removed. Subsequently, iRhoms exert a different form of control of ADAM17 at the plasma membrane, this time on stimulus-induced ADAM17 activity, its substrate specificity, and its stability. iRhoms ultimately regulate the release of ADAM17 substrates, and are consequently key players in TNFα and EGFR signalling. However, it remains unclear how iRhom function itself is regulated posttranslationally, and whether iRhoms require co-factors to exert their roles as ADAM17 regulators. The goal of my project was to shed light into these questions by identifying new iRhom interaction partners. I developed a mass spectrometry-based screen to identify new binding partners of human iRhoms using co-immunoprecipitation. The top hit of the screen was the poorly characterised FERM domain-containing protein 8 (FRMD8), which binds to both iRhom1 and iRhom2. FRMD8 was found to play a crucial role in the iRhom/ADAM17 pathway because FRMD8 knockdown and knockout in HEK293T cells significantly reduced the levels of mature ADAM17 and the release of ADAM17 substrates. The closely related metalloprotease ADAM10 was not affected by the loss of FRMD8, implying that FRMD8 is not a general regulator of ADAM metalloproteases. Interaction studies revealed that FRMD8 binds to the cytosolic N-terminus of iRhom2 throughout the entire secretory pathway. FRMD8 loss does not affect the ER-to-Golgi trafficking of iRhom2 but plays a role in stabilising iRhom2 at the plasma membrane by preventing the lysosomal degradation of both iRhom2 and mature ADAM17. Using human induced pluripotent stem cell (hiPSC)-derived macrophages, I showed that FRMD8 regulates mature ADAM17 levels and the ADAM17-dependent release of TNFα in human macrophages. Studies in FRMD8 knockout (KO) mice confirmed the reduced mature ADAM17 levels in all mouse tissues tested, further supporting the conclusion that FRMD8 is a novel regulator of the iRhom/ADAM17 pathway with physiological relevance in mammals. Finally, I showed that the interaction of FRMD8 and iRhom, which are both conserved from Drosophila to human, is also conserved. Furthermore, loss of the FRMD8 orthologue in flies, Bili, leads to motility defects and shows similarity to the loss of iRhom in flies. These results suggest that FRMD8 is a novel regulator of iRhom function in mammals and Drosophila.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:736117 |
| Date | January 2017 |
| Creators | Künzel, Ulrike |
| Contributors | Freeman, Matthew |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://ora.ox.ac.uk/objects/uuid:cf562c05-7039-46cf-a897-d6de87970bc9 |
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