title: Characterisation and Modulation of CTLA4 Stability and Trafficking: Focus on Reversible Ubiquitylation
Creators: name: Tey, Pei Yee
Publisher: name: University of Liverpool Repository
Year: 2024

description: The immune checkpoint protein CTLA4 opposes CD28-dependent T-cell signalling by interacting with and removing their common ligands, CD80 and CD86, from the antigen-presenting cells (APCs) via trans-endocytosis. Despite the need for cell surface appearance to engage with its ligands, CTLA4 is mostly found in the endosomal compartments, whilst its endosomal sorting itinerary is poorly defined. Ubiquitylation provides a sorting signal by which receptors interact with the endosomal sorting complexes required for transport (ESCRT) machinery. Recent studies show that CTLA4 can be ubiquitylated upon CD80 binding or T-cell activation. However, whether ubiquitylation governs CTLA4 endosomal sorting is unknown, and this project aimed to explore how ubiquitylation controls the fate of CTLA4. 

Here, I use two cancer cell lines, the melanoma A2058 cells and lung NCI-H520 cells, that endogenously express CTLA4. I also create a panel of isogenic HeLa S3 Flp-In cell lines expressing wildtype and CTLA4 lysine mutants to allow for accurate comparison. I show that LRBA, a protein known to mediate CTLA4 retrieval from the lysosomal sorting pathway in T cells, differentially regulates CTLA4 levels in distinct cancer cell lines. Cycloheximide chase experiments show that CTLA4 has an unusually high turnover rate across cancer cell lines, with a half-life of less than 1 hour, making it one of the most short-lived membrane proteins known. I demonstrate that CTLA4 turnover in the lysosome relies on its ubiquitylation at lysine (Lys) residues 203 and 213 and is sensitive to the inhibition of the ubiquitin-activating E1 enzyme UBA1. Unexpectedly, the CTLA4 mutant lacking all the lysine residues (K-null) in its cytoplasmic domain can still be degraded, albeit at a much slower rate. Analysing K-null ubiquitylation under non-reducing conditions reveals some residual ubiquitylation, indicative of non-conventional ubiquitylation at a non-lysine residue. Inhibition of the v-ATPase only partially rescues CTLA4 levels during translational arrest but also uncovers a minor pool that is released from the cells via exosomes. Importantly, this exosomal CTLA4 displays its ligand binding domain to the extracellular environment. 

I show that endosomal deubiquitylating enzyme (DUB), USP8, is essential to reverse CTLA4 ubiquitylation. Depletion of USP8 significantly increases CTLA4 ubiquitylation in cancer cells, mouse CD4-positive T cells and cancer cell-derived exosomes but delays its degradation. I demonstrate that CTLA4 downregulation requires the USP8 endosomal adapter protein, HD-PTP, but not the ESCRT-0 components. Depletion of HD-PTP prevents USP8 recruitment to CTLA4 and recapitulates the main phenotypes of USP8 depletion, leading to an increase in CTLA4 ubiquitylation and a delay in its turnover, but differs with respect to CTLA4 incorporation into exosomes. I show that only the re-expression of wild-type USP8, but not a catalytically inactive or endosomal localisation-deficient ∆MIT domain mutant, can correct CTLA4 turnover or reverse its ubiquitylation. By performing a UbiCRest analysis, I demonstrate that CTLA4 has a complex ubiquitin architecture composed of the conventional Lys63- and more atypical Lys27- and Lys29-linked polyubiquitin chains, which may contribute to its rapid turnover in the lysosomes. Finally, I describe efforts to create and characterise a reporter HeLa S3 Flp-In cell line expressing Cas9 and SNAP-CTLA4-HA, which can be used for an unbiased CRISPR/Cas9 screen to search for E3 ligases targeting CTLA4 in the future.