The primary problem addressed by this project is the lack of understanding regarding the role of Rhomboid protease 4 (RHBDL4) in human cells. RHBDL4 belongs to a family of proteases known as rhomboids. These enzymes play critical roles in various cellular processes, including protein regulation, cell signalling, and membrane protein trafficking. In addition to RHBDL4, other notable members of the rhomboid protease family in humans include RHBDL1, RHBDL2, RHBDL3 and PARL. Rhomboid proteases are essential for numerous physiological and pathological processes, such as embryonic development, mitochondrial dynamics, and host-pathogen interactions. Understanding the substrates of rhomboid proteases is crucial to unravel their functional roles. Traditional methods for substrate discovery often fall short in the case of rhomboid proteases due to their unique mechanisms of action, where the proteolysis happens within the plane of the membrane. RHBDL4 is especially difficult case because of its localization in the endoplasmic reticulum (ER) which renders many substrate discovery methods impotent. Therefore, development of new substrate discovery method that would suit this scenario is of utmost importance.
As a ubiquitously expressed protein, RHBDL4 is likely crucial for fundamental cellular processes. Unveiling its role can unlock valuable insights into basic cellular functions and potentially uncover therapeutic targets for combating various diseases.
The first objective was to develop a protease substrate discovery assay that utilized a newly discovered substrate trapping mechanism. This innovative approach involved genetic replacement of the active site serine with an unnatural amino acid. The second objective aimed to investigate the role of RHBDL4 in the ER stress. ER stress is a cellular state associated with numerous diseases, including neurodegenerative disorders and diabetes. The third objective was to find components of ERAD associated RHBDL4 complex.
In collaboration with LMB Cambridge, the project established a protease trapping assay. This breakthrough technique enabled the discovery of novel and surprising substrates, the majority of which were found to be soluble.
The project's findings, published in the prestigious journal Nature, led to a shift in the direction of inquiry. Instead of solely focusing on RHBDL4's role in ER stress, we redirected their attention to the identified substrates. This strategic shift holds promise for uncovering new avenues of research and deepening our understanding of cellular processes influenced by RHBDL4.
