Figure - available from: Nature Communications
This content is subject to copyright. Terms and conditions apply.
RNF213 counteracts HSV-1 infection a HeLa cells were infected up to 72 h with eGFP-expressing recombinant herpes simplex virus 1 (HSV-1) at MOI 0.1. Forty eight hours prior to infection, cells were transfected with a pool of siRNAs targeting RNF213 (siRNF213) or a pool of scrambled siRNAs (siScramble) as control. The viral load was determined by monitoring the GFP signal in each condition every 24 h to generate a viral growth curve (right panel, representative viral growth curve from a single experiment, n = 4 technical replicates, curve connecting AVG, two-tailed unpaired t-test comparing siRNF213 to siScramble, a.u. arbitrary units). The area under the curve (AUC) was calculated for each growth curve and the average AUC of three independent experiments is shown relative to the siScramble control (left panel, AVG ± SEM, n = 3 independent experiments, two-tailed unpaired t-test). b HSV-1 infection experiment performed as in a, except that 16 h prior to infection cells were treated with interferon (IFN)-α (right panel, representative viral growth curve from a single experiment, n = 4 technical replicates, curve connecting AVG, two-tailed unpaired t-test comparing siRNF213 to siScramble; left panel, AVG ± SEM, n = 3 independent experiments, two-tailed unpaired t-test). Knockdown of RNF213 leads to significantly higher HSV-1 infection levels. c Immunoblots against RNF213, HSV-VP5, and MXA with tubulin as loading control confirmed knockdown of RNF213, HSV-1 infection and interferon-α treatment, respectively, in the experiments shown in a, b. d HSV-1 infection experiment performed as in a, except that 24 h prior to infection at MOI 0.05 cells were transfected with plasmids encoding 3xFLAG-RNF213 or MXB or with an empty vector (mock) as control (right panel, representative viral growth curve from a single experiment, AVG ± SEM, n = 4 technical replicates, curve connecting AVG, two-tailed unpaired t-test comparing RNF213 or MXB overexpression to mock). The average AUC of two independent experiments is shown relative to the mock control (left panel, AVG ± SEM, n = 2 independent experiments, two-tailed unpaired t-test). Overexpression of RNF213 leads to significantly lower HSV-1 infection levels. e Immunoblots against FLAG, HSV-VP5, and MXB with tubulin as loading control confirmed HSV-1 infection and expression of FLAG-RNF213 and MXB in the experiments shown in d. In a, b, e asterisks indicate p values with *p < 0.05, **p < 0.01, and ****p < 0.0001. Source data are provided as a Source Data file.
Source publication
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 interacto...
Citations
... Therefore, we can infer that in moyamoya disease, STK3 will promote the proliferation of VSMCs through the KRAS signaling pathway, HEDGEHOG signaling pathway and others in a similar way. In addition, although there was no obvious relationship between STK3 and RNF213, there was a significant negative correlation between STK3 and ISG15(interferon-stimulating gene 15) 35 . ISG15 has been reported to function as a crucial interacting agent of RNF213. ...
Moyamoya disease (MMD) is a cerebrovascular narrowing and occlusive condition characterized by progressive stenosis of the terminal portion of the internal carotid artery and the formation of an abnormal network of dilated, fragile perforators at the base of the brain. However, the role of PANoptosis, an apoptotic mechanism associated with vascular disease, has not been elucidated in MMD. In our study, a total of 40 patients’ genetic data were included, and a total of 815 MMD-related differential genes were screened, including 215 upregulated genes and 600 downregulated genes. Among them, DNAJA3, ESR1, H19, KRT18 and STK3 were five key genes. These five key genes were associated with a variety of immune cells and immune factors. Moreover, GSEA (gene set enrichment analysis) and GSVA (gene set variation analysis) showed that the different expression levels of the five key genes affected multiple signaling pathways associated with MMD. In addition, they were associated with the expression of MMD-related genes. Then, based on the five key genes, a transcription factor regulatory network was constructed. In addition, targeted therapeutic drugs against MMD-related genes were obtained by the Cmap drug prediction method: MST-312, bisacodyl, indirubin, and tropanyl-3,5-dimethylbenzoate. These results suggest that the PANoptosis-related genes may contribute to the pathogenesis of MMD through multiple mechanisms.
... Different from ubiquitination, ISGylation does not lead to degradation of substrates, implying ISGylation leads to consequence distinct from that of ubiquitination. Although no universal role has been proposed for protein ISGylation, both "gain of function" and "loss of function" effects had been characterized for ISGylated proteins [35][36][37][38][39]. Moreover, ISGylation of certain proteins could inhibit protein-protein interactions (PPIs) between ISG15-conjugated proteins and their interacting partners [35,40]. ...
Interferon-stimulated gene product 15 (ISG15) can be conjugated to substrates through ISGylation. Currently, the E3 ligase for porcine ISGylation remains unclear. Here, we identified porcine HERC5 and HERC6 (pHERC5/6) as ISGylation E3 ligases with pHERC6 acting as a major one by reconstitution of porcine ISGylation system in HEK-293 T cell via co-transfecting E1, E2 and porcine ISG15(pISG15) genes. Meanwhile, our data demonstrated that co-transfection of pISG15 and pHERC5/6 was sufficient to confer ISGylation, suggesting E1 and E2 of ISGylation are interchangeable between human and porcine. Using an immunoprecipitation based ISGylation analysis, our data revealed pHERC6 was a substrate for ISGylation and confirmed that K707 and K993 of pHERC6 were auto-ISGylation sites. Mutation of these sites reduced pHERC6 half-life and inhibited ISGylation, suggesting that auto-ISGylation of pHERC6 was required for effective ISGylation. Conversely, sustained ISGylation induced by overexpression of pISG15 and pHERC6 could be inhibited by a well-defined porcine ISGylation antagonist, the ovarian tumor (OTU) protease domain of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-nsp2 and PRRSV-nsp1β, further indicating such method could be used for identification of virus-encoded ISG15 antagonist. In conclusion, our study contributes new insights towards porcine ISGylation system and provides a novel tool for screening viral-encoded ISG15 antagonist.
... Considering the emerging knowledge on the antiviral functions of lipid droplets, recent findings highlight that the Ring finger protein 213 (RNF213), implicated in moyamoya arteriopathy (MA), exhibits antimicrobial activity against RSV, Listeria monocytogenes, herpes simplex virus 1 (HSV-1), and coxsackievirus B3 (CVB3). RNF213 is a substrate for ISGylation, forming oligomers on lipid droplets and acting as a sensor for ISGylated proteins [44]. Furthermore, it has been reported that ISGylation of the Stimulator of Interferon Genes (STING) protein facilitates its oligomerization, which is essential for the STING-mediated type I IFN induction in DNA sensing of viruses. ...
... Furthermore, it has been reported that ISGylation of the Stimulator of Interferon Genes (STING) protein facilitates its oligomerization, which is essential for the STING-mediated type I IFN induction in DNA sensing of viruses. In the context of human immunodeficiency virus type 1 (HIV-1) infection, a recent study shows that ISG15 deficiency exacerbates infection rates due to lack of ISGylation of the STING protein and suppression of the STING-dependent DNA sensing pathway [45]. is a substrate for ISGylation, forming oligomers on lipid droplets and acting as a sensor for ISGylated proteins [44]. Furthermore, it has been reported that ISGylation of the Stimulator of Interferon Genes (STING) protein facilitates its oligomerization, which is essential for the STING-mediated type I IFN induction in DNA sensing of viruses. ...
The Interferon Stimulated Gene 15 (ISG15), a unique Ubiquitin-like (Ubl) modifier exclusive to vertebrates, plays a crucial role in the immune system. Primarily induced by interferon (IFN) type I, ISG15 functions through diverse mechanisms: (i) covalent protein modification (ISGylation); (ii) non-covalent intracellular action; and (iii) exerting extracellular cytokine activity. These various roles highlight its versatility in influencing numerous cellular pathways, encompassing DNA damage response, autophagy, antiviral response, and cancer-related processes, among others. The well-established antiviral effects of ISGylation contrast with its intriguing dual role in cancer, exhibiting both suppressive and promoting effects depending on the tumour type. The multifaceted functions of ISG15 extend beyond intracellular processes to extracellular cytokine signalling, influencing immune response, chemotaxis, and anti-tumour effects. Moreover, ISG15 emerges as a promising adjuvant in vaccine development, enhancing immune responses against viral antigens and demonstrating efficacy in cancer models. As a therapeutic target in cancer treatment, ISG15 exhibits a double-edged nature, promoting or suppressing oncogenesis depending on the tumour context. This review aims to contribute to future studies exploring the role of ISG15 in immune modulation and cancer therapy, potentially paving the way for the development of novel therapeutic interventions, vaccine development, and precision medicine.
... This gene encodes a protein with AAA+ ATPase and E3 ubiquitin ligase domains, which are associated with functions like angiogenesis, autophagy, autoimmunity, and lipid metabolism [135]. RNF213 has been identified as an interferon-induced megaprotein with antimicrobial activity against various viruses [136]. In Madin Darby canine kidney cells, RNF213 genes were upregulated when exposed to acid in a metabolic acidosis model, suggesting a potential role as a ubiquitin ligase [137]. ...
Simple Summary
Benign prostatic hyperplasia (BPH) is a common condition in older intact dogs, characterized by nonmalignant growth of the prostate gland. However, diagnosis and treatment strategies vary widely. Clinical proteomics is a rapidly growing field in both human and veterinary medicine. It has the potential to help us discover new therapeutic targets and biomarkers for diagnosis, prognosis, and treatment efficacy. However, there has been a notable gap in our understanding regarding changes in the serum proteome of dogs with BPH. This study aimed to compare serum proteome profiles of dogs with BPH to healthy dogs and evaluate the impact of castration. This research represents the first investigation into serum proteome profiling in dogs with BPH before and after castration. The findings suggest that specific serum proteins may be associated with the development and progression of canine BPH. These proteins hold the potential to serve as non-invasive biomarkers for diagnosing the disease. Further study is needed to identify and validate diagnostic biomarkers for BPH in dogs, including proteins involved in related pathways, as numerous alterations in serum proteins were observed post-castration.
Abstract
BPH is the most prevalent prostatic condition in aging dogs. Nevertheless, clinical diagnosis and management remain inconsistent. This study employed in-solution digestion coupled with nano-liquid chromatography tandem mass spectrometry to assess serum proteome profiling of dogs with BPH and those dogs after castration. Male dogs were divided into two groups; control and BPH groups. In the BPH group, each dog was evaluated at two time points: Day 0 (BF subgroup) and Day 30 after castration (AT subgroup). In the BF subgroup, three proteins were significantly upregulated and associated with dihydrotestosterone: solute carrier family 5 member 5, tyrosine-protein kinase, and FRAT regulator of WNT signaling pathway 1. Additionally, the overexpression of polymeric immunoglobulin receptors in the BF subgroup hints at its potential as a novel protein linked to the BPH development process. Conversely, alpha-1-B glycoprotein (A1BG) displayed significant downregulation in the BF subgroup, suggesting A1BG’s potential as a predictive protein for canine BPH. Finasteride was associated with increased proteins in the AT subgroup, including apolipoprotein C-I, apolipoprotein E, apolipoprotein A-II, TAO kinase 1, DnaJ homolog subfamily C member 16, PH domain and leucine-rich repeat protein phosphatase 1, neuregulin 1, and pseudopodium enriched atypical kinase 1. In conclusion, this pilot study highlighted alterations in various serum proteins in canine BPH, reflecting different pathological changes occurring in this condition. These proteins could be a source of potential non-invasive biomarkers for diagnosing this disease.
... 7 The pathophysiology of RNF213 gene-related moyamoya angiopathy is detailed in Table 1. [21][22][23][24] ...
Moyamoya disease (MMD) is a rare cerebrovascular disorder characterized by the progressive narrowing and occlusion of the intracranial internal carotid arteries, leading to the formation of abnormal collateral vessels. MMD primarily affects the cerebrovascular system, and evidence suggests it is associated with various neuropsychiatric outcomes. This manuscript aims to provide an overview of the current understanding of MMD, including its epidemiology, pathophysiology, clinical manifestations, and diagnosis. Furthermore, it explores the emerging research on the neuropsychiatric sequelae of MMD, such as cognitive impairment, psychiatric disorders, and quality of life. The manuscript concludes with the challenges in managing MMD-related neuropsychiatric outcomes and potential avenues for future research.
... 21 Recent molecular studies have pointed out that RNF213 may play a role as an antimicrobial protein with important functions in the immune system. 22,23 Last, there are other genes that have been implicated in the development of MMS, such as mutations in the genes ACTA2 and GUCY1A3 in rare cases. [24][25][26] ...
... 32 Recent studies have indicated that RNF213 functions as an antimicrobial protein with important purposes in the immune system. 22,23 Together, these findings point out a potential association of immune-related responses as second hits to trigger moyamoya vasculopathy and open new avenues for future investigation. ...
Adult moyamoya disease and syndrome are rare disorders with significant morbidity and mortality. A writing group of experts was selected to conduct a literature search, summarize the current knowledge on the topic, and provide a road map for future investigation. The document presents an update in the definitions of moyamoya disease and syndrome, modern methods for diagnosis, and updated information on pathophysiology, epidemiology, and both medical and surgical treatment. Despite recent advancements, there are still many unresolved questions about moyamoya disease and syndrome, including lack of unified diagnostic criteria, reliable biomarkers, better understanding of the underlying pathophysiology, and stronger evidence for treatment guidelines. To advance progress in this area, it is crucial to acknowledge the limitations and weaknesses of current studies and explore new approaches, which are outlined in this scientific statement for future research strategies.
... However, it was not clear whether monomeric or oligomeric RNF213 associates with LDs until recently. Indeed, some researchers have found that RNF213 oligomerization is associated with LDs in cells treated with type-1 interferon (IFN) (Thery et al., 2021). These findings indicate a deep relationship between RNF213 and LDs, which might be important for the antimicrobial infection function of RNF213. ...
... The association of RNF213 with LDs was increased upon IFN treatment, accompanied by a notably enhanced appearance of a smear of ISGylated proteins. Mechanistically, they confirmed that IFN-induced RNF213 associated with ISGylated proteins on the surface of LDs in vitro (Thery et al., 2021). Furthermore, Thery and colleagues used herpes simplex virus (HSV-1), respiratory syncytial virus (RSV), and coxsackievirus (CVB3), all of which are ISG15-sensitive viruses. ...
... Knockdown of RNF213 in HeLa cells increased the genome replication of the virus and the expression level of viral proteins. However, it is unclear whether RNF213 binds ISGylated proteins on LDs in the three viruses resembling the vitro experiment and requires further verification (Thery et al., 2021). The work of Thery and colleagues suggests that the RNF213-ISG15 association in LDs plays an important role in the antiviral process ( Figure 2A). ...
Ring finger protein 213 (RNF213) is a large E3 ubiquitin ligase with a molecular weight of 591 kDa that is associated with moyamoya disease, a rare cerebrovascular disease. It is located in the cytosol and perinuclear space. Missense mutations in this gene have been found to be more prevalent in patients with moyamoya disease compared with that in healthy individuals. Understanding the molecular function of RNF213 could provide insights into moyamoya disease. RNF213 contains a C3HC4-type RING finger domain with an E3 ubiquitin ligase domain and six AAA+ adenosine triphosphatase (ATPase) domains. It is the only known protein with both AAA+ ATPase and ubiquitin ligase activities. Recent studies have highlighted the role of RNF213 in fighting against microbial infections, including viruses, parasites, bacteria, and chlamydiae. This review aims to summarize the recent research progress on the mechanisms of RNF213 in pathogenic infections, which will aid researchers in understanding the antimicrobial role of RNF213.
... Virotrap, on the contrary, traps the protein complexes in VLPs, protecting them during purification. Moreover, the GAG grid-like structure creates an avidity effect for the bait, and thus in general, Virotrap allows to detect weaker and transient interactions (35,55). As both methods detected known PPIs, one cannot question the quality of both datasets. ...
Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared with their canonical counterparts. Such proteoforms can have altered localizations, stabilities, and functions. Although proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. In addition, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues, as well as tissue-specific expression, highlighting their biological relevance. Protein–protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared with their canonical counterparts, thus further expanding the functional diversity of proteomes.
... Briefly, we immunoprecipitated ISG15 from mGCs expressing FLAG-ISG15 and then identified these immunoprecipitates through mass spectrometry (MS) (Fig. 5A). ISG15-binding candidates included ring finger protein 213 (RNF213), which is known as an ISG15 interactor and sensor of ISGylated substrates [20], C Forty-four hours after PMSG injection, the number of antral follicles was counted in Isg15 +/+ and Isg15 −/− ovaries. D Ten hours after PMSG/hCG injection, the number of pre-ovulatory follicles was counted in Isg15 +/+ and Isg15 −/− ovaries. ...
... To explore the role of ISG15-mediated ISGylation in ovulation, we performed mass spectrometric analysis and identified 57 potential proteins interacting with ISG15 in mGCs. In addition to the previously known RNF213 [20], we also identified novel proteins as potential ISGylation substrates, namely ribosomal protein S3 (RPS3) and protease serine 23 (PRSS23). RPS3, a DNA repair endonuclease and ribosomal protein, induces apoptosis through caspase-dependent JNK activation [35]. ...
Background
ISGylation is a post-translational protein modification that regulates many life activities, including immunomodulation, antiviral responses, and embryo implantation. The exact contribution of ISGylation to folliculogenesis remains largely undefined.
Results
Here, Isg15 knockout in mice causes hyperfertility along with sensitive ovarian responses to gonadotropin, such as increases in cumulus expansion and ovulation rate. Moreover, ISG15 represses the expression of ovulation-related genes in an ISGylation-dependent manner. Mechanistically, ISG15 binds to ADAMTS1 via the ISG15-conjugating system (UBA7, UBE2L6, and HERC6), ISGylating ADAMTS1 at the binding sites Lys309, Lys593, Lys597, and Lys602, resulting in ADAMTS1 degradation via a 20S proteasome-dependent pathway.
Conclusion
Taken together, the present study demonstrates that covalent ISG15 conjugation produces a novel regulatory axis of ISG15-ADAMTS1 that enhances the degradation of ADAMTS1, thereby compromising ovulation and female fertility.
... Interestingly, in a preliminary proteomic study of virion-enriched cell extracts (data not shown), we found the Ring Finger Protein 213 (RNF213) among the less abundant cellular proteins in Isg15 2/2 samples. This protein was described as a sensor of ISGylated proteins, which showed antimicrobial activity against bacteria and viruses in vitro and in vivo (49). This observation suggests that the interaction between ISG15 and RNF213 might have a relevant role in the antiviral response against VACV. ...
Viruses have developed many different strategies to counteract immune responses, and Vaccinia virus (VACV) is one of a kind in this aspect. To ensure an efficient infection, VACV undergoes a complex morphogenetic process resulting in the production of two types of infective virions: intracellular mature virus (MV) and extracellular enveloped virus (EV), whose spread depends on different dissemination mechanisms. MVs disseminate after cell lysis, whereas EVs are released or propelled in actin tails from living cells. Here, we show that ISG15 participates in the control of VACV dissemination. Infection of Isg15-/- mouse embryonic fibroblasts with VACV International Health Department-J (IHD-J) strain resulted in decreased EV production, concomitant with reduced induction of actin tails and the abolition of comet-shaped plaque formation, compared to Isg15+/+ cells. Transmission electron microscopy revealed the accumulation of intracellular virus particles and a decrease in extracellular virus particles in the absence of interferon-stimulated gene 15 (ISG15), a finding consistent with altered virus egress. Immunoblot and quantitative proteomic analysis of sucrose gradient-purified virions from both genotypes reported differences in protein levels and composition of viral proteins present on virions, suggesting an ISG15-mediated control of viral proteome. Lastly, the generation of a recombinant IHD-J expressing V5-tagged ISG15 (IHD-J-ISG15) allowed us to identify several viral proteins as potential ISG15 targets, highlighting the proteins A34 and A36, which are essential for EV formation. Altogether, our results indicate that ISG15 is an important host factor in the regulation of VACV dissemination. IMPORTANCE Viral infections are a constant battle between the virus and the host. While the host's only goal is victory, the main purpose of the virus is to spread and conquer new territories at the expense of the host's resources. Along millions of years of incessant encounters, poxviruses have developed a unique strategy consisting in the production two specialized "troops": intracellular mature virions (MVs) and extracellular virions (EVs). MVs mediate transmission between hosts, and EVs ensure advance on the battlefield mediating the long-range dissemination. The mechanism by which the virus "decides" to shed from the primary site of infection and its significant impact in viral transmission is not yet fully established. Here, we demonstrate that this process is finely regulated by ISG15/ISGylation, an interferon-induced ubiquitin-like protein with broad antiviral activity. Studying the mechanism that viruses use during infection could result in new ways of understanding our perpetual war against disease and how we might win the next great battle.
