Mapping ISG15 sites on GAPDH and PGK1 by AP-MS
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Hek-293t Cell, Epithelial Cell
SUBMITTER: 
Denzel Eggermont
LAB HEAD: Francis Impens
PROVIDER: PXD071724 | Pride | 2026-03-02
REPOSITORIES: Pride
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Project description:We employed a GlyGly-peptidomics approach to comprehensively map ISG15 modification sites in HeLa cells under three conditions: mock-transfected controls, IFN-induced cells, and cells transfected with the ISGylation machinery. Lysates from wild-type and Isg15 knockout (KO) cells were processed using S-trap, followed by trypsin digestion to generate diglycine-modified peptides. These peptides were enriched via immunoprecipitation and analyzed by LC-MS/MS for identification and quantification. Comparative analysis across conditions and genotypes revealed 671 ISG15 modification sites on 456 distinct proteins, expanding the HeLa ISGylome.
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Project description:To identify the site(s) of O-GlcNAcylation on PGK1, we transiently co-expressed Flag-tagged PGK1 and OGT in HEK293T cells. After immunoprecipitation using anti-Flag M2 beads and in-gel trypsin digestion, resulted peptides were subjected to mass spectrometry analysis
2019-11-06 | PXD015659 | Pride
Project description:ISG15 is an interferon-stimulated, ubiquitin-like protein, with anti-viral activity against several clinically relevant viruses. However, its precise mechanism of action during viral infection remains elusive. Upon infection, ISG15 conjugates to protein substrates in covalent manner. We previously found that ISG15 restricts Coxsackie virus (CV) infection both in vitro and in vivo. Here, we endeavored to map the in vivo ISGylome following CV 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 addition, we also quantified protein level changes in the host proteome following infection.
2024-05-23 | PXD032078 | Pride
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.
2021-10-12 | PXD026748 | 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: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: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 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:Interferon (IFN)-stimulated gene 15 (ISG15), a ubiquitin-like protein, is covalently conjugated to host immune proteins such as MDA5 and IRF3 in a process called ISGylation, thereby promoting type I interferon (IFN) induction to limit the replication of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, whether SARS-CoV-2 proteins can be directly targeted for ISGylation remains elusive. In this study, we identified the nucleocapsid (N) protein of SARS-CoV-2 as a major substrate of ISGylation catalyzed by the host E3 ligase HERC5; however, N ISGylation is readily removed through de-ISGylation by the papain-like protease (PLpro) activity of NSP3. Mass spectrometry analysis identified that the N protein undergoes ISGylation at four lysine residues (K266, K355, K387 and K388), and mutational analysis of these sites in the context of a SARS-CoV-2 replicon (N-4KR) abolished N ISGylation and alleviated ISGylation-mediated inhibition of viral RNA synthesis. Furthermore, our results indicated that HERC5 targets preferentially phosphorylated N protein for ISGylation to regulate its oligomeric assembly. These findings reveal a novel mechanism by which the host ISGylation machinery directly targets SARS-CoV-2 proteins to restrict viral replication and illuminate how an intricate interplay of host (HERC5) and viral (PLpro) enzymes coordinates viral protein ISGylation and thereby regulates virus replication.
2024-08-14 | PXD053712 | Pride
Project description:<p>Ubiquitin-like protein ISG15 (interferon-stimulated gene 15) has been implicated in the regulation of central carbon metabolism, but conflicting findings across experimental systems have limited mechanistic insight. Here, we applied a multi-omics approach in cells ectopically expressing the ISGylation machinery independent of immune stimuli, to generate a systematic view of ISGylation in metabolic control. We found that ISGylation predominantly modifies metabolic proteins, including key glycolytic enzymes, and is associated with suppression of the energy-producing phase of glycolysis. Tracer metabolomics indicated a bottleneck at glyceraldehyde-3-phosphate dehydrogenase (GAPDH), marked by accumulation of its substrate and depletion of downstream metabolites. Functional assays demonstrated that ISGylation directly impairs GAPDH activity, and structural analysis revealed that ISG15 modifications cluster near the active site and dimerization interfaces, critical for enzymatic function. These findings identify GAPDH as a central metabolic checkpoint regulated by ISGylation and uncover a direct post-translational mechanism by which ISG15 controls energy metabolism.</p>
2026-03-02 | MTBLS12619 | MetaboLights