Uncovering the proteolytic potential of orphan rhomboids

Intramembrane proteases regulate essential signalling and quality control functions by performing proteolysis of transmembrane proteins within the lipid bilayer. Rhomboids are intramembrane proteases that belong to a larger rhomboid-like superfamily comprising inactive pseudoproteases that have roles in wound healing, cell migration and development. However, many of their biological functions in mammals remain to be elucidated. This is perhaps best exemplified by RHBDL3, for which no substrates and therefore no cellular roles have been reported. The protease clearly performs an important function, as RHBDL3 knockout in mice is embryonically lethal. Additionally, RHBDL3 is highly expressed in the brain, and its expression levels increase with age and neurological disease. Collectively, these data hint that RHBDL3 regulates neuronal membrane proteins, but its molecular and cellular role is currently unknown. Importantly, without a validated substrate, it is unclear whether it is catalytically active. We have employed unbiased screening approaches in primary neurons to identify novel substrates and regulators of RHBDL3. With validation of these ongoing, we have turned to computational modelling approaches to gain fundamental insights into the molecular mechanisms underlying RHBDL3 function. These include AlphaFold predictions and molecular dynamics to simulate the protein in a lipid bilayer, supported by free energy calculations. We identify that RHBDL3 has distinct conformational features to other well-characterised rhomboids. These insights reveal that either RHBDL3 is inactive like the rhomboid pseudoproteases, or that potential regulatory mechanisms unlock RHBDL3 activity.