Transcriptome Regulation by PARP13 in Basal and Antiviral States in Human Cells (eCLIP)
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE215250 | GEO | 2023/09/11
REPOSITORIES: GEO
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Project description:Filamentous hemagglutinin (FHA), Bordetella pertussis' key adhesin, is both present at the cell surface and released in the extracellular milieu. To comprehend the role free FHA plays in the pathogenic process, we analyzed the global transcriptional changes in human peripheral blood mononuclear cells (PBMC) after exposure to that purified virulence factor. This analysis was undertaken with four different FHA preparations (FHA-1, FHA-2, FHA-3, and FHA-4), purified from four different B. pertussis strains. Cultures comprising 3x106 PBMC were stimulated for 0.5, 2, 4, and 6 hours with 5 ug/ml of each purified B. pertussis FHA. Control PBMC cultures were simultaneously treated with similar volumes of elution buffers (Mock-1 and Mock-2), and sampled at the same time points. The array experiment was composed of 27 samples. Most of the transcripts known to be part of the "common host-transcriptional response" that mediates inflammation were among the 1,235 array elements (representing 683 known unique genes) showing a greater than 3-fold change in transcript abundance between FHA-treated and untreated cells. We also identified an FHA-specific signature not observed in cells stimulated with B. pertussis LPS or heat-killed B. pertussis. This response, mounted by 13 genes, largely (69%, 9/13 genes) interferon (IFN)-regulated genes. Further analysis revealed that 18.3% (125 out of 683) of the FHA-responsive genes were in fact regulated by IFN. These included several major players of the antiviral IFN type I response, as well as three key components of the ISGylation pathway. Induction of the ubiquitin-like protein ISG15 and its specific protease USP18 was confirmed at the mRNA level by relative real-time polymerase chain reaction (RT-PCR). Western-blot analysis demonstrated the presence of both free ISG15 and several ISGylated conjugates in FHA-stimulated PBMC cell lysate. Intracellular FACS analysis provided further evidence that monocytes and a natural-killer (NK)-enriched cell population are the primary producers of ISG15 after FHA stimulation. Our results revealed new activities of free B. pertussis FHA: activation of the host IFN and ISGylation pathways. The consequences of such activation are yet to be resolved, but we hypothesize that FHA utilizes the ISGylation pathway or USP18 to regulate the IFN response, which in turn sensitizes the host to the infection and induces cell death. Groups of assays that are related as part of a time series. Infection: treatment with purified B. pertussis filamentous hemagglutinin (FHA) at 0.005 mg/mL Time: Samples were collected after various treatment time (hours) Keywords: time_series_design Computed
2008-10-29 | E-GEOD-8802 | biostudies-arrayexpress
Project description:The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it remains unclear how it regulates diverse processes. Here we characterized a mutation, R54P, critical for TRIM25 catalytic activity, which we successfully utilized to “trap” substrates. We demonstrated that TRIM25 targets proteins implicated in stress granule formation (G3BP1/2), nonsense-mediated mRNA decay (UPF1), and nucleoside synthesis (NME1). R54P abolishes TRIM25 inhibition of alphaviruses independently of the host interferon response, suggesting that this antiviral effect is a direct consequence of ubiquitination. Consistent with that, we observed diminished antiviral activity upon knockdown of several TRIM25-R54P specific interactors including NME1. Our findings highlight that multiple substrates mediate the cellular and antiviral activities of TRIM25, illustrating the multi-faceted role of this ubiquitination network in diverse biological processes.
2022-08-02 | PXD034024 | Pride
Project description:During infection, interferon production leads to upregulation of the interferon-stimulated gene 15 (ISG15) which encodes the ubiquitin-like protein ISG15. ISG15 has a profound role in restricting intracellular infection by viral and bacterial pathogens. It can exert its function either as a free intracellular or extracellular molecule, or as an ubiquitin-like conjugate to substrate proteins in a process called ISGylation. Here, we used a viral-like particle trapping (Virotrap) technology and GST pull down, both coupled to mass spectrometry, to screen for intracellular ISG15-binding proteins. As strongest hit, we identified RNF213, a very large interferon-induced protein targeted to lipid droplets and a genetic risk factor for Moyamoya disease, a rare cerebrovascular disorder. We verified binding of ISG15 and RNF213 with biochemical methods and imaging and found that interferon signaling induces ISGylation and oligomerization of RNF213 on the surface of lipid droplets, acting as a binding platform for ISGylated proteins. Moreover, we showed that knockdown of RNF213 promotes in vitro infection by Listeria monocytogenes, respiratory syncytial virus (RSV) and Coxsackie virus (CV) B3, suggesting a broad antimicrobial activity of RNF213. In contrast, overexpression of RNF213 restricted Listeria infection and was associated with a striking co-localization of RNF213 with intracellular bacteria. Infection experiments in RNF213 deficient mice corroborated these results in vivo, revealing a so far unknown function of RNF213 in the host defense against infection. Together, we report that RNF213 assembles into a binding platform for ISGylated proteins on the surface of lipid droplets, acting as a first step of an antimicrobial cellular pathway that processes ISGylated proteins. Our findings provide novel molecular insights into the ISGylation pathway and point towards an important role of the immune response to infection in the etiology of Moyamoya disease.
2021-08-16 | PXD018345 | Pride
Project description:During infection, interferon production leads to upregulation of the interferon-stimulated gene 15 (ISG15) which encodes the ubiquitin-like protein ISG15. ISG15 has a profound role in restricting intracellular infection by viral and bacterial pathogens. It can exert its function either as a free intracellular or extracellular molecule, or as an ubiquitin-like conjugate to substrate proteins in a process called ISGylation. Here, we used a viral-like particle trapping (Virotrap) technology and GST pull down, both coupled to mass spectrometry, to screen for intracellular ISG15-binding proteins. As strongest hit, we identified RNF213, a very large interferon-induced protein targeted to lipid droplets and a genetic risk factor for Moyamoya disease, a rare cerebrovascular disorder. We verified binding of ISG15 and RNF213 with biochemical methods and imaging and found that interferon signaling induces ISGylation and oligomerization of RNF213 on the surface of lipid droplets, acting as a binding platform for ISGylated proteins. Moreover, we showed that knockdown of RNF213 promotes in vitro infection by Listeria monocytogenes, respiratory syncytial virus (RSV) and Coxsackie virus (CV) B3, suggesting a broad antimicrobial activity of RNF213. In contrast, overexpression of RNF213 restricted Listeria infection and was associated with a striking co-localization of RNF213 with intracellular bacteria. Infection experiments in RNF213 deficient mice corroborated these results in vivo, revealing a so far unknown function of RNF213 in the host defense against infection. Together, we report that RNF213 assembles into a binding platform for ISGylated proteins on the surface of lipid droplets, acting as a first step of an antimicrobial cellular pathway that processes ISGylated proteins. Our findings provide novel molecular insights into the ISGylation pathway and point towards an important role of the immune response to infection in the etiology of Moyamoya disease.
2021-08-16 | PXD018346 | Pride
Project description:TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. Almost nothing is known about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Here, we reveal that RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25’s endogenous RNA targets and protein binding partners. Finally, we show that the RNAbinding activity of TRIM25 is important for its ubiquitin ligase function. These results reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor for their biological functions.
2017-10-19 | PXD007960 | Pride
Project description:Analysis of ISG15 conjugation (Isgylation)role as driver of poor prognosis in non HER2+ breast cancer tumours. The project includes an analysis of total proteome and ISGylome characterization of MDA-MB-231 cells, wild-type or knock-out for the ISGyltaion associated genes, ISG15, UBE2L6 and USP18. Additionally interactome analysis of the ISGylation target GDI2 was perfored in the same lines.
2021-11-03 | PXD025753 | Pride
Project description:Filamentous hemagglutinin (FHA), Bordetella pertussis' key adhesin, is both present at the cell surface and released in the extracellular milieu. To comprehend the role free FHA plays in the pathogenic process, we analyzed the global transcriptional changes in human peripheral blood mononuclear cells (PBMC) after exposure to that purified virulence factor. This analysis was undertaken with four different FHA preparations (FHA-1, FHA-2, FHA-3, and FHA-4), purified from four different B. pertussis strains. Cultures comprising 3x106 PBMC were stimulated for 0.5, 2, 4, and 6 hours with 5 ug/ml of each purified B. pertussis FHA. Control PBMC cultures were simultaneously treated with similar volumes of elution buffers (Mock-1 and Mock-2), and sampled at the same time points. The array experiment was composed of 27 samples. Most of the transcripts known to be part of the "common host-transcriptional response" that mediates inflammation were among the 1,235 array elements (representing 683 known unique genes) showing a greater than 3-fold change in transcript abundance between FHA-treated and untreated cells. We also identified an FHA-specific signature not observed in cells stimulated with B. pertussis LPS or heat-killed B. pertussis. This response, mounted by 13 genes, largely (69%, 9/13 genes) interferon (IFN)-regulated genes. Further analysis revealed that 18.3% (125 out of 683) of the FHA-responsive genes were in fact regulated by IFN. These included several major players of the antiviral IFN type I response, as well as three key components of the ISGylation pathway. Induction of the ubiquitin-like protein ISG15 and its specific protease USP18 was confirmed at the mRNA level by relative real-time polymerase chain reaction (RT-PCR). Western-blot analysis demonstrated the presence of both free ISG15 and several ISGylated conjugates in FHA-stimulated PBMC cell lysate. Intracellular FACS analysis provided further evidence that monocytes and a natural-killer (NK)-enriched cell population are the primary producers of ISG15 after FHA stimulation. Our results revealed new activities of free B. pertussis FHA: activation of the host IFN and ISGylation pathways. The consequences of such activation are yet to be resolved, but we hypothesize that FHA utilizes the ISGylation pathway or USP18 to regulate the IFN response, which in turn sensitizes the host to the infection and induces cell death. Groups of assays that are related as part of a time series. Infection: treatment with purified B. pertussis filamentous hemagglutinin (FHA) at 0.005 mg/mL Time: Samples were collected after various treatment time (hours) Keywords: time_series_design
2008-10-30 | GSE8802 | GEO
Project description:TRIM25 E3 ubiquitin ligase is a new RNA-binding protein. We present the first high-throughput analysis of the molecular interactomes of TRIM25. We show that TRIM25 is a bona fide RNA-binding protein that interacts with numerous coding and non-coding transcripts. This suggests that TRIM25 could play a role in the regulation of RNA metabolism.
2018-07-27 | GSE104949 | GEO
Project description:ISG15 is an interferon-stimulated, ubiquitin-like protein, with anti-viral and anti-bacterial activity, however its precise mechanism of action during viral or bacterial infection remains elusive. We previously found that ISG15 restricts Listeria infection both in vitro and in vivo and identified ISGylated proteins that could be responsible for the protective effect. Here, we endeavored to map the in vivo ISGylome following Listeria infection to mechanistically elucidate the function of ISG15 in host defense. To do so we combined a genetic approach employing a murine model of deregulated ISGylation with quantitative proteomics of immune-enriched endogenous ISG15 modification sites. In this way, we mapped 930 ISG15 sites in 438 proteins. In addition, we also quantified protein level changes in the host proteome following infection. Gene ontology analysis revealed that ISGylated proteins were mostly enriched in proteins which alter cellular metabolic processes, including four upstream modulators of the catabolic and antibacterial pathway of autophagy. Structural analysis further revealed that a number of ISG15 modifications can occur at catalytic sites or dimerization interfaces of enzymes. Finally, we showed that animals and cells with deregulated ISGylation have increased infection-induced autophagy through the modification of mTOR, WIPI2, and AMBRA1. Taken together, these findings ascribe a putative role of ISGylation to temporarily reprogram organismal metabolism following infection through direct modification of a variety of enzymes in the liver.
2019-11-28 | PXD011513 | Pride
Project description:ISG15 is primarily documented as an interferon-stimulated, ubiquitin-like protein (ubl), which has anti-viral activity. Although ISG15 was the founding member of the ubl protein family, very little is known about its function. We have found that ISG15 expression in non-phagocytic cells is dramatically induced upon Listeria infection and that surprisingly this induction can be Type I Interferon independent. Listeria-mediated ISG15 induction depends on the cytosolic surveillance pathway, which senses bacterial DNA and signals through STING, TBK1, IRF3 and IRF7. Most importantly, we observed that ISG15 expression restricts Listeria infection both in vitro and in vivo. We then made use of Stable Isotope Labeling in tissue culture (SILAC) to identify the ISGylated proteins that could be responsible for the ISG15-mediated protective effect. Our SILAC analysis revealed that overexpression of ISG15 leads to a striking ISGylation of integral membrane proteins of the endoplasmic reticulum and Golgi apparatus, which correlates with increased canonical secretion of cytokines. Taken together, our data reveal a previously uncharacterized signaling pathway that restricts Listeria infection and acts via ISGylation, reinforcing the view that ISG15 is a key component of the innate immune arsenal of the mammalian host.
2015-07-24 | PXD001805 | Pride