Proteome Profiling of RNF213 Depleted Cells Reveals Nitric Oxide Regulator DDAH1 Antilisterial Activity

Abstract

RNF213 is a large, poorly characterized interferon-induced protein. Mutations in RNF213 are associated with predisposition for Moyamoya disease (MMD), a rare cerebrovascular disorder. Recently, RNF213 was found to have broad antimicrobial activity in vitro and in vivo, yet the molecular mechanisms behind this function remain unclear. Using mass spectrometry-based proteomics and validation by real-time PCR we report here that knockdown of RNF213 leads to transcriptional upregulation of MVP and downregulation of CYR61, in line with reported pro- and anti-bacterial activities of these proteins. Knockdown of RNF213 also results in downregulation of DDAH1, which we discover to exert antimicrobial activity against Listeria monocytogenes infection. DDAH1 regulates production of nitric oxide (NO), a molecule with both vascular and antimicrobial effects. We show that NO production is reduced in macrophages from RNF213 KO mice, suggesting that RNF213 controls Listeria infection through regulation of DDAH1 transcription and production of NO. Our findings propose a potential mechanism for the antilisterial activity of RNF213 and highlight NO as a potential link between RNF213-mediated immune responses and the development of MMD.

Interferon-Inducible E3 Ligase RNF213 Facilitates Host-Protective Linear and K63-Linked Ubiquitylation of Toxoplasma gondii Parasitophorous Vacuoles

“…Recent work characterized the ubiquitin E3 ligase RNF213 as a novel antiviral and antibacterial protein ( 53 – 56 ). In line with its role as a host defense protein, we observed that RNF213 protein expression, already detectable in naive cells, was further induced by IFN-γ priming in A549 and a human foreskin fibroblast cell line (HFF-1) ( Fig.…”

Biallelic variants in NOS3 and GUCY1A3, the two major genes of the nitric oxide pathway, cause moyamoya cerebral angiopathy

“…Some years before, Erdmann et al reported a large family in which rare heterozygous mutations in GUCY1A3 and CCT7, the latter encoding a scaffold protein which stabilizes sGC, were associated with premature coronary heart disease [ 23 ]. Interestingly, recent experimental data suggest that the NO pathway is functionally connected at the cellular level to RNF213 , a giant ubiquitin ligase encoded by a major susceptibility gene in MMA [ 24 , 25 ]. Altogether, these data strongly suggest that NO pathway disruption leads to the development of steno-occlusive lesions in various vascular beds, as observed in MMA (homozygous LOF mutations) or coronary heart disease (heterozygous variants).…”

Multiomics and blood-based biomarkers of moyamoya disease: protocol of Moyamoya Omics Atlas (MOYAOMICS)

“…Moreover, serum proteomics has revealed dysregulated lipid metabolism in both ischemic and hemorrhagic MMD patients, emphasizing its significance in vascular changes [ 22 ].In addition, the characterization of proteins in Moyamoya angiopathy (MMA) between the brain surface and dura mater has identified Filamin A as a potential key player, offering initial insights into MMA’s unique intracranial vasculopathy [ 23 ]. Investigation into RNF213’s role in MMD and antimicrobial activity has highlighted changes in proteins like MVP, CYR61, and DDAH1 upon RNF213 knockdown, suggesting potential connections between RNF213, immune responses, and MMD development, possibly involving nitric oxide production in combating Listeria infection [ 24 ].…”