Interaction mapping of endoplasmic reticulum ubiquitin ligases identifies modulators of innate immune signalling

Abstract

Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly 5 defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 different stably expressed, full-length E3s. Bioinformatic analysis linked ER-E3s and their interactors to multiple homeostatic, regulatory, and metabolic pathways. Among these were four membrane-embedded interactors of RNF26, a polytopic E3 whose abundance is auto-regulated by ubiquitin-proteasome dependent degradation. RNF26 co-assembles with 10 TMEM43, ENDOD1, TMEM33 and TMED1 to form a complex capable of modulating innate immune signalling through the cGAS-STING pathway. This RNF26 complex represents a new modulatory axis of STING and innate immune signalling at the ER membrane. Collectively, these data reveal the broad scope of regulation and differential functionalities mediated by ER-E3s for both membrane-tethered and cytoplasmic processes. 15

Comparative multiplexed interactomics of SARS-CoV-2 and homologous coronavirus non-structural proteins identifies unique and shared host-cell dependencies

“…In particular, we identify the E3 ubiquitin ligase RNF5 interacting with all nsp4 homologs. RNF5 targets STING for degradation, which stabilizes retinoic acid-inducible gene-I (RIG-I) and mitochondrial antiviral-signaling protein (MAVS) interactions at MAMs, thereby inducing inteferon-1 and −3 production via IRF3 and NF-kB signaling 33 , 34 . RIG-I is one of the main viral RNA genome sensors in host cells; therefore, it is possible that nsp4 increases targeting of RNF5 to MAMs to inhibit downstream signaling of RIG-1.…”

“…These include components of UPR signaling (TMEM33) and ER-phagy (CCPG1). We also identify RNF5, an ER-localized E3 ubiquitin ligase known to modulate anti-viral innate immune signaling 33,34 , and VKORC1, which reduces Vitamin K, a key cofactor for several coagulation factor proteins 35 . Not surprisingly, given that nsp4 is a glycosylated protein, we also identify several members of the Nlinked glycosylation machinery (STT3B, MAGT1, CANX, DDOST) ( Figure 4D) in all three strains.…”

“…mitochondrial antiviral-signaling protein (MAVS) interactions at MAMs, thereby inducing inteferon-1 and -3 production via IRF3 and NF-kB signaling 33,34 . RIG-I is one of the main viral RNA genome sensors in host cells; therefore, it is possible that nsp4 increases targeting of RNF5 to MAMs to inhibit downstream signaling of RIG-1.…”

EVI/WLS function is regulated by ubiquitination and linked to ER-associated degradation by ERLIN2

“…It is known that EVI/WLS is ubiquitinated by UBE2J2 and CGRRF1 (Fenech et al, 2020; Glaeser et al, 2018) and that the UBE2J2 ortholog in yeast (Ubc6) is indispensable for priming a broad range of substrates with monoubiquitin or K11-linked ubiquitin dimers (Weber et al, 2016; Xu et al, 2009). However, so far the kind of ubiquitin linkage types present on EVI/WLS and the sites of its modification have not been identified, since the TUBEs used to detect ubiquitination in Figure 5D were non-selective and unable to distinguish between different ubiquitin linkage types.…”

“…Indeed, only about 20 to 30 endogenous substrates for mammalian regulatory ERAD have been reported and for most it is unclear how they are recognised, ubiquitinated, and linked to the ERAD machinery (Bhattacharya & Qi, 2019; Printsev et al, 2017). Furthermore, various studies focus on ubiquitination by HRD1 while other ER membrane resident E3 ligases remain poorly defined (Fenech et al, 2020).…”