Structure-Function Analysis of iRhoms

Structure-function analysis of the iRhom Homology Domain (IRHD)

The IRHD is one of the most evolutionarily conserved structural subunits of iRhoms. However, until now, neither the structure nor the function of the IRHD was known. Our initial in silico structural analyses revealed that it appears to be a folding pattern that is not yet known (unknown fold). Our attempt to produce the IRHD of iRhom2 in recombinant soluble form in different expression systems for structure elucidation and in vitro characterisation was ultimately successful. However, the protein yield was not yet sufficient for experiments and therefore needs to be significantly optimised to be able to use this for experiments.

In parallel, we analysed the IRHD in silico. In doing so, we identified a highly conserved motif in an apparently unstructured (no secondary structure) region of the IRHD. Through cell biology, molecular biology and biochemistry experiments, we found that the integrity of this motif is critical for iRhom2 function in vitro, ex vivo and in vivo [1]. A single point mutation within this motif is sufficient to abolish ER-Golgi transport of iRhom2. Neither the overall structure of the IRHD was affected nor the binding to ADAM17 was affected by this point mutation. Using different approaches, we have shown that mutations within this conserved motif are not simply retained by the ER quality control system in the ER, nor are they degraded by ER-dependent degradation. Rather, mutations within this motif dramatically increase the half-life of iRhom2 and bound ADAM17. We showed that iRhom2 constructs with these mutations can even reach the ER-Golgi intermediate compartment (ERGIC), but are unable to enter the Golgi. Since this motif is obviously necessary for the entry of the iRhom-ADAM17 complex into the Golgi, we named it iCERES (iRhom conserved ER to Golgi export sequence). Since the intracellular transport of the iRhom2-ADAM17 complex is negatively affected by mutations in iCERES, neither ADAM17 maturation nor ADAM17-mediated shedding can occur. We could also show that this is not only the case in iRhom2 but also in iRhom1. In addition, we were also able to show that different drastic mutations at different positions in iCERES lead to different effects on the transport of iRhom2.

Although we have not yet been able to elucidate the structure of IRHD experimentally, we were able to use the recently published database of AlphaFold (https://www.alphafold.ebi.ac.uk/) to analyse the structural prediction of IRHD deposited there. In this possible structure, iCERES is positioned within a valley at the surface of the IRHD. This is consistent with our hypothesis that iCERES represents an interaction platform. Our goal now is to further understand the molecular mechanisms behind iCERES. An important step here is the search for possible interaction partners for which iCERES could represent a binding site.

In general, the study of the underlying mechanism of iCERES is not only interesting for the biological significance of iRhoms and ADAM17, but also for the general understanding of ER-to-Golgi transport. Many proteins leave the ER after folding without requiring further regulatory mechanisms. Instead, specific signals are required to keep ER-localised proteins in the ER. However, more and more proteins are being identified that require an additional signal sequence, such as the C-mannosylation motif, to leave the ER. In this respect, iCERES also emerges as a new signal motif that is important for transport into the Golgi.

References

[1] Düsterhöft, S., et al. (2021). "The iRhom homology domain is indispensable for ADAM17-mediated TNFalpha and EGF receptor ligand release." Cell Mol Life Sci 78(11):5015-5040. DOI: 10.1007/s00018-021-03845-3

Analysis of regulatory regions in the cytosolic N-terminus of iRhoms

Another important substructure of iRhom is the cytosolic N-terminus, which accounts for almost 50% of the total iRhom protein. The N-terminus is also the region where the largest sequence differences between iRhom1 and iRhom2 can be found. Since it has already been shown that iRhom1 and iRhom2 appear to regulate ADAM17 differently and are partly responsible for substrate specificity, this region is of great relevance for targeting ADAM17 activity. It is known that phosphorylation of the N-terminus leads to the binding of 14-3-3 proteins, which activates ADAM17-dependent shedding. In addition, the N-terminus has a binding site for the iRhome interactor FRMD8. This interaction stabilises the iRhome-ADAM17 complex at the cell surface. Although the phosphorylation sites and the FRMD8 binding site are known, the underlying mechanisms are not yet fully understood. Moreover, these sites represent only a small part of the N-terminus. For the rest of the long N-terminus, it is not yet known which (regulatory) functions are performed by these areas. For example, there is a short sequence motif in the N-terminus of iRhom2 that, when mutated in humans, leads to the rare Howel-Evans syndrome. This disease manifests in affected individuals as hyperkeratosis in combination with squamous cell carcinoma of the oesophagus (tylosis with oesophageal carcinoma: TOC). It is known that this mutation leads to a hyperactivation of ADAM17. However, the molecular mechanisms behind this are not yet understood.