StressRhomboid · Trapping intramembrane protease substrates in living cells: focus on RHBDL4 role in ERAD
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2020-07-29 → 2023-05-28
- EU contribution
- €224,934
- Participants
- 1
- Scheme
- MSCA-IF
Lines connect the coordinator with its partners.
Results in brief
Trapping intramembrane protease substrates in living cells: focus on RHBDL4 role in ERAD
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.
Data: CORDIS, © European Union
Project objective
Intramembrane proteolysis is increasingly understood to control many cellular processes but we still know little about the role of most intramembrane proteases. The major roadblock is the lack of a robust method of protease substrate discovery. In this action, I focus on RHBDL4, a poorly understood but highly conserved member of the rhomboid intramembrane serine protease family, which participates in ER associated degradation (ERAD), apoptosis and exocytosis, and which has been frequently implicated in cancer growth and metastasis.My three objectives are to establish a systematic approach to trapping and identifying rhomboid substrates; to use this to discover the substrates of human RHBDL4; and to explore the mechanism of how RHBDL4 participates in ER quality control.Matthew Freeman's group discovered rhomboids and is a leader in the field. I will also collaborate with Jason Chin in Cambridge, who has pioneered the use of genetically encoded unnatural amino acids (UAAs) to engineer proteins. I will adapt a technique recently published by the Chin lab for use in living cells. Using a cross-linking UAA analogue of the catalytic serine in RHBDL4, I will achieve unprecedented specificity and efficiency of substrate capture. I will thus covalently capture RHBDL4 substrates, which will then be identified by mass spectrometry. Hits will be functionally validated and, using a range of experimental conditions, I will distinguish substrates involved in ERAD from substrates cleaved in other RHBDL4-dependent processes.By establishing the first systematic assay for rhomboid substrates and investigating the role of RHBDL4 in ER quality control, I will pioneer a general approach to intramembrane protease substrate discovery; gain broader understanding of how RHBDL4 contributes to cellular quality control; and finally reveal pathophysiological roles of this human protein, thereby guiding possible future therapeutic targeting.
Original text from CORDIS.
Participants
- THE CHANCELLOR, MASTERS AND SCHOLARS OF THE UNIVERSITY OF OXFORD · OxfordCoordinatorUnited Kingdom
Links
Data: CORDIS, © European Union