Periodic Reporting for period 1 - StressRhomboid (Trapping intramembrane protease substrates in living cells: focus on RHBDL4 role in ERAD) Reporting period: 2020-07-29 to 2022-07-28 Summary of the context and overall objectives of the project 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. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far I started the project by testing expression of RHBDL4 with an unnatural aminoacid in the active site, which was successful. Next I spent a couple of weeks in LMB visiting Jason Chin's lab where we did a pilot experiment. We showed that RHBDL4 can be crosslinked to its model substrate using the DAP methodology. I then attempted to create a plasmid to stably introduce the components of the DAP incorporation machinery into the cell with the aminoacyltransferase expression inducible by tetracycline addition. This avenue was not successful as I never saw the expression of the reporter construct. At this time I also repeatedly attempted to create endogenously tagged RHBDL4, unsuccessfully. As our collaborator Shan Tang had a custom machine built to illuminate the cells with UV, it made sense to have her take lead on the optimization of the RHBDL4 crosslinking and purification for mass spectrometry. Once the preliminary mass spec data started coming in I was focusing on substrate validation. I created inducible FlpIn Hek cell lines that had either WT or inactive RHBDL4 under tetracycline control and used this system to look for cleavage of endogenous proteins. I did not find any substrates cleaved in this system. We then focused more on the secretion, looking for the cleavage products in the medium. The results of this work were published in 2022 in nature. To follow up on the substrates we identified, I looked whether the cleavage products are degraded by proteasome, which would suggest that these are cleaved in the context of ERAD, but that was not the case. Therefore, I shifted my focus away from the originally planned work connected to ERAD and started creating reagents to look at the context in which these novel substrates are cleaved. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In this project we developed a novel method for protease susbstrate discovery and used it to identify new substrates of RHBDL4. This is clearly a major leap not only in methodology, but also in our understanding of RHBDL4 - an odd intramembrane protease that seems to prefer soluble substrates. We identified some major chaperones as RHBDL4 substrates. This would be very unlikely without the new method, as these molecules are usually overlooked in purifications as contaminants. Our work is opening a possibility of major chaperone secretion regulation via RHBDL4 function. This project will have major impact on basic science inquiry avenues which in time could inform translational science and development of therapeutics. image-dap.png
