Interferon restores replication fork stability and cell viability in BRCA-defective cells via ISG15
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Lorenza Penengo
LAB HEAD: Lorenza Penengo
PROVIDER: PXD045154 | Pride | 2023-10-09
REPOSITORIES: Pride
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Project description:Germline BRCA1 or BRCA2 mutations (mtBRCA1 and mtBRCA2) dramatically increase risk for high-grade serous ovarian cancer (HGSOC), the most commonly diagnosed histotype. Other risk factors for this cancer, which originates primarily in the distal fallopian tube epithelium (FTE), implicate ovulation. To test whether mtBRCA1 or mtBRCA2 FTE cells respond differently to peri-ovulatory follicular fluid (FF) exposure than control patient FTE, gene expression profiles from primary FTE cultures were compared at baseline, 24h after FF exposure, and 24h after FF replacement with culture medium. Hierarchical clustering revealed both FF exposure and BRCA mutation status affect gene expression, with BRCA1 mutation having the greatest impact. Analysis revealed increased NFκB and EGFR signaling at baseline, with increased interferon signaling after recovery from FF exposure in mtBRCA1 samples. Inhibition of EGFR signaling and ISGylation by increased BRCA1 expression was verified in an immortalized FTE cell line, OE-E6/E7, stably transfected with BRCA1. Suppression of ISG15 and ISGylated protein levels by BRCA1 expression was found to be mediated by decreased NFκB signaling and was transiently suppressed by FF exposure. This study demonstrates increased NFκB signaling associated with decreased BRCA1 expression resulting in increased ISG15 and ISGylation following FF exposure, which could represent potential targets for chemoprevention.
2018-05-31 | GSE98699 | GEO
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
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Project description:Here, we applied an ISG15-GlyGly peptidomics approach on HeLa cells producing high levels of ISGylated proteins upon stimulation with IFN-α. After obtaining cellular lysates from wild-type and Isg15-/-cells, the samples were incubated with recombinant wild-type or mutant PLpro to catalyze deISGylation of host cellular proteins. After incubation with PLpro, we continued with trypsin digestion to generate diglycine-modified peptides that were subsequently enriched by immunoprecipitation and identified and quantified by LC-MS/MS. By comparing sites across all four conditions, we uncovered 118 ISGylation sites on 95 proteins as targets of the SARS-CoV-2 protease PLpro.
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Project description:The deubiquitylating enzyme USP18 is a major negative regulator of the interferon (IFN) signalling cascade. IFN pathways contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy and are often upregulated in certain tumours, including breast, lung, and colon tumours. USP18 is the predominant human protease that cleaves interferon-stimulated gene ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. In this study, using advanced proteomic techniques, we have expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line (HAP1) treated with type I IFN. Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. Our results suggest USP18 as a pharmacological target in cancer immunotherapy and radiotherapy.
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Project description:The pathways involved in suppressing DNA replication stress and the associated DNA damage are critical to maintaining genome integrity. The Mre11 complex is unique among double strand break (DSB) repair proteins for its association with the DNA replication fork. Here we show that Mre11 complex inactivation causes DNA replication stress and changes in the abundance of proteins associated with nascent DNA. One of the most highly enriched proteins at the DNA replication fork upon Mre11 complex inactivation was the ubiquitin like protein ISG15. Mre11 complex deficiency and drug induced replication stress both led to the accumulation of cytoplasmic DNA and the subsequent activation of innate immune signaling via cGAS-STING-Tbk1. This led to ISG15 induction and protein ISGylation, including constituents of the replication fork. ISG15 plays a direct role in preventing replication stress. Deletion of ISG15 was associated with replication fork stalling, tonic ATR activation, genomic aberrations, and sensitivity to aphidicolin. These data reveal a previously unrecognized role for ISG15 in mitigating DNA replication stress and promoting DNA replication fidelity.
2022-10-12 | PXD031770 | Pride
Project description:The ubiquitin-like modifier ISG15 can modulate host and viral proteins to restrict viral and microbial infections, and act as a cytokine. Its expression and conjugation are strongly up-regulated by type I interferons. Here we identify the deubiquitinating enzyme USP16 as an ISG15 cross-reactive protease. Ubiquitin-specific protease 16 (USP16) was found to react with an ISG15 activity-based probe in pull-down experiments using chronic myeloid leukaemia-derived human cells (HAP1). Supporting this finding, recombinant USP16 cleaved pro-ISG15 and ISG15 iso-peptide linked model substrates in vitro, as well as ISGylated substrates present in cell lysates. Moreover, the interferon-induced stimulation of ISGylation in human HAP1 cells was increased by knockdown or knockout of USP16. Depletion of USP16 did not affect interferon signaling, and interferon treatment did not affect USP16 expression or enzymatic activity either. A USP16-dependent ISG15 interactome was established by anti-ISG15 immunoprecipitation mass spectrometry (IP-MS), which indicated that the deISGylating function of USP16 may regulate metabolic pathways involving GOT1, ALDOA, SOD1 and MDH1, all of which were further confirmed to be deISGylated by USP16 in HEK293T cells. Together, our results indicate that USP16 may contribute to regulating the ISGylation status of a subset of proteins related to metabolism during type I interferon responses.
2023-11-17 | PXD043553 | Pride
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: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: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
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