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

Abstract

<jats:p>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 defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 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 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.</jats:p>

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

“…We did not yet identify the E3 ubiquitin ligase which is required for UBE2N-mediated ubiquitination of EVI/WLS, but our data and recently published high-throughput studies indicate that it is presumably not an ER-membrane associated protein, but rather cytosolic (Fenech et al, 2020;Leto et al, 2019;Glaeser et al, 2018). It should also be considered that earlier in vitro and structural studies showed that UBE2N~ubiquitin together with UBE2V2 can adopt its active conformation even in the absence of an E3 ligase, suggesting E3 independent ubiquitin chain elongation (McKenna et al, 2001;Pruneda et al, 2011).…”

“…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).…”

Synthetic lethality between TP53 and ENDOD1

“…Previous studies have demonstrated that ENDOD1 is a cytoplasmic nuclease ( Figure 1a) involved in innate immunity as part of the RNF26-mediated cGAS-STING pathway 6 . The unexpected identification of ENDOD1 as a PARPi-responsive protein lead us to examine its potential role in DNA repair.…”

The journey of Ca2+ through the cell – pulsing through the network of ER membrane contact sites

“…In addition to cholesterol-associated formation of ER-endosomes MCSs, ubiquitin-mediated events, such as those regulated by the ER-embedded ubiquitin ligase ring finger-containing protein 26 (RNF26), ubiquitin-conjugating enzymes E2 J1 (UBE2J1) and sequestosome 1 (SQSTM1; also known as p62) complex (Cremer et al, 2020 preprint;Jongsma et al, 2016) can also support reversible connections between these organelles. Interestingly, recent proteomic findings suggest that RNF26 resides in a complex with various members of the ER Ca 2+ efflux machinery, including IP3R1 and SigmaR1 (Fenech et al, 2020). The observed dynamics of lysosomes at IP3R clusters parallel those of endolysosomes that reversibly associate with the ER via In the course of their maturation, endosomes travel into the cell interior (towards the minus-end of microtubules); this requires active transport that is mediated by the cholesterol-sensitive RILP-dynein complex, or the Ca 2+ -responsive ALG2-dynein complex.…”