Ring finger protein 213 assembles into a sensor for ISGylated proteins with antimicrobial activity

Abstract

ISG15 is an interferon-stimulated, ubiquitin-like protein that can conjugate to substrate proteins (ISGylation) to counteract microbial infection, but the underlying mechanisms remain elusive. Here, we use a virus-like particle trapping technology to identify ISG15-binding proteins and discover Ring Finger Protein 213 (RNF213) as an ISG15 interactor and cellular sensor of ISGylated proteins. RNF213 is a poorly characterized, interferon-induced megaprotein that is frequently mutated in Moyamoya disease, a rare cerebrovascular disorder. We report that interferon induces ISGylation and oligomerization of RNF213 on lipid droplets, where it acts as a sensor for ISGylated proteins. We show that RNF213 has broad antimicrobial activity in vitro and in vivo, counteracting infection with Listeria monocytogenes, herpes simplex virus 1, human respiratory syncytial virus and coxsackievirus B3, and we observe a striking co-localization of RNF213 with intracellular bacteria. Together, our findings provide molecular insights into the ISGylation pathway and reveal RNF213 as a key antimicrobial effector.

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“…RNF213 is expressed at baseline levels in many tissues (Liu et al, 2011), but is upregulated by LPS, TNFa and interferon (IFN), supporting the idea that inflammatory and innate immune signals might act as potential environmental triggers for MMD (Kobayashi et al, 2015;Ohkubo et al, 2015;Bosch et al, 2020;Sarkar and Thirumurugan, 2020). RNF213 is present in the cytosol (Liu et al, 2011), but overexpression, oleic acid treatment (Sugihara et al, 2019), interferon or LPS stimulation (Bosch et al, 2020;Thery et al, 2021) target the protein to the surface of lipid droplets (LDs), organelles that regulate lipid storage but also act as intracellular innate immune hubs (Bosch et al, 2020). Overexpression of RNF213 stabilizes LDs, removing adipose triglyceride lipase (ATGL) from their surface and functioning as a positive regulator of fat storage in the cells (Sugihara et al, 2019).…”

“…Most interestingly, we demonstrated that RNF213, through its binding to ISG15, has a broad antimicrobial activity in vitro and in vivo, counteracting infection against Listeria monocytogenes, human respiratory syncytial virus (RSV), coxsackievirus (CV) B3 and herpes simplex virus 1 (HSV-1). Along with a striking co-localization of RNF213 with intracellular bacteria, these findings uncovered a new role of RNF213 as a key antimicrobial effector (Thery et al, 2021). Similarly, it was recently shown that RNF213 counteracts Salmonella through the ubiquitylation of LPS, independent of its naming RING domain, but instead relying on an RZ finger in the E3 shell (Otten et al, 2021).…”

“…Interestingly, the same variants were previously shown to be required for oligomerization of RNF213 (Morito et al, 2014), suggesting that LD translocation of RNF213 is associated with its oligomerization. We recently found that this is indeed the case and showed that RNF213 oligomerizes on the surface of lipid droplets upon IFN induction (Thery et al, 2021).…”

“…To elucidate the molecular mechanisms underlying the antimicrobial activity of RNF213 we set out to identify proteins regulated by RNF213 by untargeted mass spectrometry-based proteome analysis. To this end, we knocked down RNF213 by a pool of siRNAs (siRNF213) in HeLa cells, conditions that were previously shown to promote infection with Listeria monocytogenes, HSV-1 and CVB3 (Thery et al, 2021). Since RNF213 is upregulated by type-I interferon we also included interferon-b (IFN-b) treatment in our screen, leading to four experimental conditions each analysed in quadruplicate (Figure 1A).…”

“…RNF213 is unique in combining a dynein-like ATPase core with a ubiquitin E3 ligase module (Kamada et al, 2011;Ahel et al, 2020) The ATPase core contains six AAA (ATPase associated with a variety of cellular activities) units, but different from other AAA ATPases the RNF213 ATPase core does not generate movement. Instead, it seems to behave as an elaborated molecular switch that is involved in the oligomerization of RNF213 on the surface of lipid droplets and likely also bacteria (Sugihara et al, 2019;Ahel et al, 2020;Otten et al, 2021;Thery et al, 2021). Additionally, the recent cryo-EM structure revealed that RNF213 does not depend on its naming RING domain to function as an E3 ubiquitin ligase (Ahel et al, 2020).…”