Discovery and validation of novel substrates for the rhomboid protease RHBDL2

Thesis

Rhomboid proteases are a group of intramembrane proteases from the rhomboid-like superfamily. In mammals they include RHBDL1 -4, which are located along of the secretory pathway, and PARL, which is located in the mitochondria. Rhomboid proteases are highly conserved and, like other intramembrane proteases, they cleave their substrates within their transmembrane domains (TMD). However, not much is known about the biological role of rhomboid proteases, due to the very small number of validated substrates. To this end, the main project of my DPhil aimed at developing a comprehensive genetic screen that employed cell sorting to identify new potential substrates of RHBDL2. The assay was based on the use of Fluorescence-activated cell sorting (FACS) and used a decrease in the fluorescence as a readout for rhomboid cleavage. Firstly, I tested each step of the screen to ensure that this new methodology could work using control TMDs. After a long period of troubleshooting, I conducted the screen by using a newly generated lentiviral library that encodes for the TMDs of all human type I single-pass transmembrane proteins which are the most common substrates for RHBDL2. The screen was performed in the presence of either RHBDL2 or its inactive mutant, RHBDL2 SA, that acted as the negative control. The cells expressing the TMDs in both conditions were firstly labelled with a fluorescent antibody, then divided into 4 different bins according to their fluorescence. The cell populationsin the bins were sorted and used for Nanopore sequencing. Analysis of the sequencing data aimed at identifying TMDs whose fluorescence decreased upon co-expression of RHBDL2. Overall, 7 TMDs were identified that showed a significant decrease of their average fluorescence. In this work, I will present the preliminary validation of 4 of them.

In parallel to this main project, I have also investigated the relationship between RHBDL2 and a newly identified substrate, VE-Cadherin. I found that both the TMD and the full-length protein were cleaved by RHBDL2. Moreover, I have also attempted at mapping the rhomboid cleavage site. 

Overall, in this thesis I am going to discuss my work in the identification of novel rhomboid substrates and the validation of newly discovered ones

Authors: Zarcone , L

• DOI: 10.5287/ora-exxe4rqx0
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English