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• W4253697777 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract ISG15 is an interferon-stimulated, linear di-ubiquitin-like protein, with anti-viral activity. The role of ISG15 during bacterial infection remains elusive. We show that ISG15 expression in nonphagocytic cells is dramatically induced upon Listeria infection. Surprisingly this induction can be type I interferon independent and 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 in vitro and in vivo. We made use of stable isotope labeling in tissue culture (SILAC) to identify ISGylated proteins that could be responsible for the protective effect. Strikingly, infection or overexpression of ISG15 leads to ISGylation of ER and Golgi proteins, which correlates with increased secretion of cytokines known to counteract infection. Together, our data reveal a previously uncharacterized ISG15-dependent restriction of Listeria infection, reinforcing the view that ISG15 is a key component of the innate immune response. https://doi.org/10.7554/eLife.06848.001 eLife digest Listeria monocytogenes is a bacterium that can cause serious food poisoning in humans. Infections with this bacterium can be particularly dangerous to young children, pregnant women, the elderly, and individuals with weakened immune systems because they are more susceptible to developing serious complications that can sometimes lead to death. The bacteria infect cells in the lining of the human gut. Cells that detect the bacteria respond by producing proteins called interferons and other signaling proteins that activate the body's immune system to fight the infection. One of the genes that the interferons activate encodes a protein called ISG15, which helps to defend the body against viruses. However, it is not clear what role ISG15 plays in fighting bacterial infections. Here, Radoshevich et al. studied the role of ISG15 in human cells exposed to L. monocytogenes. The experiments show that ISG15 levels increase in the cells, but that the initial increase does not depend on Interferon proteins. Instead, ISG15 production is triggered by an alternative pathway called the cytosolic surveillance pathway, which is activated by the presence of bacterial DNA inside the cell. Further experiments found that ISG15 can counteract the infections of L. monocytogenes both in cells grown in cultures and in living mice. ISG15 modifies other proteins in the cell to promote the release of proteins called cytokines that help the body to eliminate the bacteria. Radoshevich et al.'s findings reveal a new role for ISG15 in fighting bacterial infections. A future challenge will be to understand the molecular details of how ISG15 triggers the release of cytokines. https://doi.org/10.7554/eLife.06848.002 Introduction Listeria monocytogenes is a food-borne pathogen that can cause enteric infections. In addition, in immunocompromised individuals it can cross the blood–brain barrier and in pregnant women the feto-placental barrier potentially leading to cases of meningitis and septicemia. To be fully virulent, Listeria must evade macrophage killing, enter and replicate in epithelial cells and spread from cell to cell. Towards these aims Listeria subverts a number of normal host cell functions in order to promote its own replication and dissemination through a plethora of well-characterized virulence factors (Cossart and Lebreton, 2014). Conversely, Listeria induces a rapid and sterilizing CD8+ T cell-mediated adaptive immune response that has been extensively characterized (Lara-Tejero and Pamer, 2004; Pamer, 2004). A more recent area of investigation has been the innate immune response to the pathogen (Stavru et al., 2011). Since Listeria is able to survive and replicate in the cytosol, several groups have sought to elucidate how bacteria are sensed within macrophages and more recently within nonphagocytic cells. Once Listeria has escaped from the phagosome, its multidrug efflux pumps secrete small molecules leading to activation of an IRF3-dependent cytosolic surveillance pathway (CSP), resulting in type I interferon activation (Crimmins et al., 2008). One of these small molecules, cyclic-di-AMP, is sufficient to activate interferon β production in macrophages (Woodward et al., 2010). In nonphagocytic cells, type I interferon induction seems to emanate from sensing of triphosphorylated RNA molecules via a RIG-I and MAVS-dependent pathway (Abdullah et al., 2012; Hagmann et al., 2013). Type I interferon production subsequently leads to autocrine or paracrine activation of interferon-stimulated genes (ISGs). We have recently shown that Listeria also activates the type III interferon pathway (Lebreton et al., 2011; Bierne et al., 2012), a pathway which was discovered much later than type I interferon (Kotenko et al., 2003; Sheppard et al., 2003). The type III interferon receptor has a more limited tissue expression pattern than the receptor for type I interferon but activates a signaling pathway similar to that of the type I interferon receptor. Several laboratories including ours have recently contributed to the understanding of the type III interferon-dependent response to intracellular viral and bacterial infections. Strikingly, the type III response occurs via peroxisomal MAVS (Dixit et al., 2010; Odendall et al., 2014). The role of one particular ISG, ISG15, during bacterial infection remains elusive. ISG15 is a linear di-ubiquitin-like molecule (ubl) that is conserved from zebrafish to human; however, it is much less well characterized than other ubls (Bogunovic et al., 2013). It can conjugate to over 300 cellular proteins and can also function as a cytokine to induce interferon-γ production in peripheral blood mononuclear cells (D'Cunha et al., 1996; Giannakopoulos et al., 2005; Zhao et al., 2005). Since Listeria, as other pathogenic bacteria, often targets post-translational modifications during infection (Bonazzi et al., 2008; Ribet and Cossart, 2010; Ribet et al., 2010), we were interested in investigating the interplay between the interferon-stimulated ubl ISG15 and Listeria. ISG15 plays an important role in the innate immune response to viruses. Isg15 expression becomes rapidly upregulated, and the protein is subsequently conjugated to cellular and/or viral targets following type I interferon induction (Zhang and Zhang, 2011). Mice deficient in ISG15 are susceptible to infection with Influenza, Sindbis, and Herpes viruses (D'Cunha et al., 1996; Lenschow et al., 2005, 2007). Furthermore, many viruses encode proteins that specifically impair ISGylation (Frias-Staheli et al., 2007). ISG15 seems to be unique among ubls, as it can both modify specific target proteins and non-specifically modify proteins cotranslationally (Frias-Staheli et al., 2007; Durfee et al., 2010; Zhao et al., 2010). Since ISG15 is strongly induced by type I interferon, which is produced during bacterial infection, we aimed to decipher whether ISG15 is induced during Listeria infection and if so whether ISGylation acts as a means of host defense against invading bacteria. Here, we show that in nonphagocytic cells ISG15 is dramatically induced upon Listeria infection and that, surprisingly, early induction can be type I interferon independent. Listeria-mediated ISG15 induction depends on the CSP, which senses bacterial DNA and signals through STING, TBK1, IRF3, and IRF7. Most importantly, we demonstrate that ISG15 counteracts Listeria infection both in vitro and in vivo. We identified protein targets of ISGylation following overexpression of ISG15 using stable isotope labeling in tissue culture (SILAC) analysis and uncovered a prominent enrichment in integral membrane proteins of the endoplasmic reticulum and Golgi apparatus. This enrichment correlated with an increase in canonical secretion of cytokines known to control infection, highlighting a new mechanism of regulation of the host response to an intracytosolic pathogen. Results ISG15 is induced by L. monocytogenes infection both in vitro and in vivo To test whether ISG15 and ISGylation are induced upon bacterial infection, we infected HeLa cells with Listeria. Upon L. monocytogenes infection, ISG15 was massively induced, whereas incubation with the related non-pathogenic bacterium, Listeria innocua, did not lead to an increase in ISG15 production (Figure 1A). We subsequently monitored ISG15 expression in cells infected with Listeria over time. ISG15 protein levels increased relatively rapidly; the unconjugated protein was already present at 6 hr post infection and accumulated steadily throughout the infection (Figure 1B). We next investigated whether ISG15 induction following Listeria infection also occurs in vivo. After 72 hr of systemic sub-lethal Listeria infection in mice, there was a robust induction of ISG15 and ISGylated conjugates in infected liver tissue, revealing that Listeria infection leads to ISG15 induction both in vitro and in vivo (Figure 1C). Figure 1 with 1 supplement see all Download asset Open asset ISG15 is induced by Listeria monocytogenes infection both in vitro and in vivo and ISG15 induction can be type I interferon independent. (A) HeLa cells were lysed and immunoblotted with α-ISG15, α-EF-Tu (EF-Tu is a prokaryotic translation elongation factor that we use as an indicator of infection level), and α-ACTIN following 12 or 24 hr of interferon β treatment at 1000 units/ml, infection with L. monocytogenes for 18 hr at multiplicity of infection (MOI) of 10 or 25 bacteria per human cell, and incubation with Listeria innocua at MOI of 10 or 25 bacteria per human cell. (B) HeLa cells were lysed and immunoblotted with α-ISG15, α-EF-Tu, and α-ACTIN following a time course of L. monocytogenes infection from 3 to 24 hr, interferon β treatment for 24 hr was used as a positive control for ISGylation. (C) Liver tissue from mice injected with saline or infected with Listeria for 72 hr was lysed and immunoblotted with α-ISG15, each lane corresponds to a distinct animal (* indicates background band). (D) Relative fold change by qRT-PCR of ISG15 transcript following time course of infection with Listeria. (E) Relative fold change of ISG15 by qRT-PCR compared with the interferon β transcript over time course of infection with Listeria; data represented in a logarithmic scale. (F) Cells were lysed and immunoblotted with α-ISG15 and α-ACTIN following pre-treatment with the viral protein B18R followed by interferon α2 treatment (1000 μ/ml) or Listeria infection. (G) 2fTGH and U5A (IFNAR2−/−) cells were lysed and immunoblotted with α-ISG15 and α-ACTIN following Listeria infection for 18 hr. https://doi.org/10.7554/eLife.06848.003 ISG15 induction can be type I interferon independent Since ISG15 protein levels increased relatively rapidly and ISG15 is known to be transcriptionally induced in response to interferon, we monitored transcript levels of both ISG15 and IFNB1 by quantitative real time PCR (qRT-PCR). Interestingly, we found that the two transcripts are concomitantly induced after 3 hr of infection with Listeria (Figure 1D,E). This concomitant induction led us to hypothesize that ISG15 could be induced in an interferon-independent manner during Listeria infection. In order to test this hypothesis, we used the viral protein B18R to block signaling from the interferon receptor (Chairatvit et al., 2012). The protein acts similarly to a blocking antibody. When cells are pretreated with B18R, the viral protein inhibits binding of interferon to its receptor, which is thus prevented from signaling. Following pretreatment with B18R, HeLa cells were either stimulated with interferon or infected with Listeria to assess whether the bacterial-ISG15 induction was dependent on secreted interferon signaling in an autocrine or paracrine manner. We observed that bacteria-induced ISG15 production was not diminished by B18R pretreatment in stark contrast to the interferon-induced ISG15 signal, which was almost entirely abrogated by B18R pretreatment (Figure 1F). To confirm the B18R results, we took advantage of a human fibrosarcoma cell line, 2fTGH, from which interferon-unresponsive mutants have been isolated (Pellegrini et al., 1989). The U5A clone lacks a functional IFNAR2 receptor (IFNAR2−/−) and thus is impaired in type I interferon receptor signaling (Lutfalla et al., 1995). 2fTGH and U5A cells were both highly permissive to Listeria infection (Figure 1—figure supplement 1). Strikingly in the U5A cells (defective for type I interferon binding and signaling), as in 2fTGH cells, there is still a robust ISG15 response to Listeria infection (Figure 1G). Since B18R treatment does not inhibit type III interferon signaling, ISG15 induction could arise via type III interferon receptor activation (Bandi et al., 2010). However, 2fTGH cells are unresponsive to type III interferon (Zhou et al., 2007). Therefore, the ISG15 protein induction that we observed is independent of both type I and type III interferon signaling. Taken together, our results show that ISG15 can be induced by Listeria in an interferon-independent manner in human nonphagocytic cells. We thus sought to determine how ISG15 was induced and what consequences ISG15 expression had on the cell and on infection. Cytosolic Listeria induces ISG15 To determine which signaling pathway was responsible for the Listeria-induced ISG15 transcript and to help identify the cellular compartment in which bacteria are sensed, we made use of Listeria strains that are impaired at different stages of infection. Incubation of cells with L. innocua, a non-pathogenic Listeria species which cannot invade cells, did not induce ISG15 induction, demonstrating that external pathogen recognition receptors were not involved (Figure 2A). We then used a strain of L. innocua that expresses Internalin B (InlB), a L. monocytogenes virulence factor that mediates entry into nonphagocytic cells (Dramsi et al., 1995). These bacteria enter the cell and are entrapped in a membrane-bound phagosome, but lack the required virulence factors to escape from it. This strain was also unable to induce an ISG15 signal, suggesting that the pathogen recognition receptors that survey the phagosome are not sufficient for ISG15 induction (Figure 2A). Listeria's hemolysin, listeriolysin O (LLO) is an extremely potent virulence factor, which triggers vacuolar escape of the bacterium as well as a plethora of changes in the host cell (Hamon et al., 2012). Strikingly, we found that the Δhly mutant was able to potently induce ISG15 (Figure 2A). Thus, LLO is not necessary for ISG15 induction. However, in several human epithelial cell lines the mutant that lacks LLO (Δhly) can still escape into the cytosol (Portnoy et al., 1988; Marquis et al., 1995). Listeria expresses two phospholipases that can compensate for the lack of LLO in the Δhly mutant in order to free the bacterium from the phagosome in human epithelial cells (Marquis et al., 1995; Smith et al., 1995). To assess whether Listeria trapped in the phagosome could induce ISG15, we constructed a triple mutant (lacking LLO, PLCA, and PLCB) of Listeria. This mutant is unable to escape the phagosome of human epithelial cells (Figure 2—figure supplement 1). Single mutants (either in PLCA or LLO), which escape into the cytosol, induce a strong ISG15 signal relative to non-infected cells (Figure 2B). In contrast, the triple mutant that is confined to the phagosome does not induce ISG15 (Figure 2B). We thus conclude that only cytoplasmic bacteria induce ISG15. In fact, the only other mutant to induce less ISG15 production was the ΔactA mutant (Figure 2A). This mutant is unable to spread from cell to cell and cannot escape autophagic recognition, degradation, and lysis (Gouin et al., 2005; Yoshikawa et al., 2009). As a result bacterial load is much lower compared to wild-type bacteria, providing an explanation for the reduced ISG15 signal (Figure 2A). Taken together, our results reveal that ISG15 induction stems from cytosolic bacteria. Figure 2 with 2 supplements see all Download asset Open asset Listeria induces ISG15 via the cytosolic surveillance pathway (CSP) which senses bacterial DNA. (A) Cells were lysed and immunoblotted with the indicated antibodies following infection with various mutants of Listeria: Listeria strain EGD (MOI 10), Δhly (MOI 10), ΔactA (MOI of 50), and L. innocua + InlB (MOI of 100). Different MOIs were used at the outset in an attempt to equalize Colony forming units (CFUs) at the end of the experiment. CFUs per ml of intracellular bacteria following infection were determined by serial dilution after 18 hr of infection. (B) As before cells were lysed and immunoblotted with the indicated antibodies following infection with Listeria strain EGD-e PrfA* (MOI 10), ΔplcA (MOI 10), Δhly (MOI of 10), and a triple mutant of ΔhlyΔplcAΔplcB (MOI of 100). CFUs per ml of intracellular bacteria following infection were determined by serial dilution after 24 hr of infection. (C) HeLa cells were treated with siRNA pools targeting the indicated mRNA or siControl for 72 hr. Cells were then infected for 18 hr (MOI 10), lysed, and immunoblotted with α-ISG15 and α-ACTIN antibodies. Values were generated using ImageJ to quantify relative levels of induction of ISG15 compared to ACTIN. Values from three independent experiments are displayed (average ± SEM). siRNA knockdown was confirmed with qRT-PCR from a well of a technical replicate. Statistical significance calculated using ANOVA followed by Bonferonni's multiple comparison test against siControl. (D) Cells were lysed and immunoblotted with α-ISG15 and α-ACTIN following permeablization without Cyclic-diAMP, with 1 μM Cyclic-diAMP or with 10 μM Cyclic-diAMP. Average ± SEM; fold change of ISG15 or interferon β normalized to GAPDH levels following permeablization without Cyclic-diAMP, with 1 μM Cyclic-diAMP or with 10 μM Cyclic-diAMP. (E) Cells were lysed and immunoblotted with α-ISG15 and α-ACTIN following transfection with 800 ng of Listeria genomic DNA, Listeria genomic DNA treated with DNAase or total Listeria RNA for 24 hr; Average ± SEM; values were generated using ImageJ to quantify relative levels of induction of ISG15 compared to ACTIN in three independent experiments. Statistical significance calculated using ANOVA followed by Bonferonni's multiple comparison test against transfection control. Statistical significance is indicated as follows: NS, nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001. https://doi.org/10.7554/eLife.06848.005 ISG15 is induced via the CSP following sensing of cytosolic DNA In order to determine which pathway was essential for ISG15 induction, we performed an siRNA screen of innate immune molecules that are known to be involved in bacterial sensing (Figure 2C). As for the experiments described above, we used HeLa cells for the siRNA screen. Although HeLa cells are reported to lack STING (Burdette and Vance, 2013), the ATCC line we worked with expressed STING mRNA, as evidenced by qRT-PCR. We were able to specifically extinguish this signal with siRNA (Figure 2—figure supplement 2). Our data showed that the ISG15 signal was clearly dependent on IRF3, IRF7, STING, and TBK1, implicating the CSP. In further support of an interferon-independent signal, depleting STAT1, which is a critical mediator of type I and III interferon signaling did not abrogate the ISG15 signal (Figure 2C). In non-immune cells interferon induction has been linked to sensing of triphosphorylated RNA by RIG-I (Abdullah et al., 2012; Hagmann et al., 2013); however, in our experimental conditions, RIG-I did not seem to be required for the ISG15 signal. Instead, it seems that direct ISG15 induction occurs through a pathway similar to the CSP in macrophages. We next sought which PAMP was necessary and sufficient for ISG15 induction. We transfected cells with either Listeria genomic DNA, Listeria genomic DNA pre-treated with DNAse, Listeria total RNA, or we permeabilized cells in the presence of cyclic-di-AMP. To control whether the cyclic di-AMP was biologically active and reached the cytosol of the cells, we assessed IFNB1 levels by qRT-PCR (Figure 2D). As expected Listeria cyclic di-AMP led to an increase in IFNB1 transcript levels. Although IFNB1 induction with cyclic di-AMP was lower than that reported for murine macrophages (Woodward et al., 2010), it was similar to that reported for human phagocytes and monocytes (Hansen et al., 2014). Listeria genomic DNA was the only PAMP sufficient for an increase in ISG15 levels (Figure 2E), whereas ISG15 levels following cytosolic exposure to cyclic-di-AMP did not increase (Figure 2D). Collectively, these data implicate that the CSP can directly induce ISG15 after sensing of bacterial DNA in the cytosol of cells in a pathway that requires STING, TBK1, IRF3, and IRF7. ISG15 protects against Listeria infection in vitro and in vivo We next assessed whether ISG15 has a functional effect on infection. We created a retroviral construct that expresses an epitope-tagged version of ISG15 (3XFlag-6His-ISG15). We then infected cells that stably express 3XFlag-6His-ISG15 with Listeria. As a control, cells were retrovirally transduced with pBabe puro empty vector. For the same multiplicity of infection (MOI) after 3 hr of infection, stable overexpression of ISG15 resulted in 50% fewer cytosolic bacteria as compared to control cells (Figure 3A). We then assessed bacterial uptake by differentiating between the bacteria that are inside the cell or those that remain on the surface (inside-out staining) and did not observe a difference between control and ISG15-overexpressing cells for invasion (Figure 3—figure supplement 1A). During a time course of infection in these cells at 7 and 12 hr, there were still 50% fewer bacteria, and by 24 hr, the levels of bacteria had equalized between the two cell lines (Figure 3—figure supplement 1B). This time course suggests that ISG15 does not impact bacterial replication as an active bacterial clearance mechanism would (Figure 3—figure supplement 1B). We then knocked down ISG15 during infection. This increased bacterial load by nearly twofold after 15 hr (Figure 3B, Figure 3—figure supplement 2). These data strongly suggest that ISG15 plays a role in protection against Listeria infection following uptake. To further explore this phenotype in primary cells, we isolated mouse embryonic fibroblasts (MEFs) from wild-type and Isg15−/− embryos. We infected these cells with Listeria and we observed a fivefold increase in bacterial load in Isg15−/− MEFs compared to wild-type MEFs for the same MOI (Figure 3C,D). Interestingly, Isg15−/− MEFs are not susceptible to other intracellular pathogens such as Shigella flexneri and Salmonella typhimurium, and in HeLa cells only Staphylococcus aureus is able to induce as much ISG15 as Listeria (Figure 3—figure supplement 1C,D). Indeed, S. flexneri and S. typhimurium induce very little ISG15. We next examined whether ISG15 could also play a role during Listeria infection in vivo by assessing the susceptibility of Isg15−/− animals to the pathogen during systemic infection (Osiak et al., 2005). ISG15-deficient mice exhibited a significant increase in bacterial load compared to wild-type animals in both the spleen and liver after 72 hr of systemic sub-lethal Listeria infection (Figure 3E,F). Taken together, these data demonstrate that ISG15 restricts Listeria infection both in vitro and in vivo. Figure 3 with 2 supplements see all Download asset Open asset ISG15 protects against Listeria infection in vitro and in vivo. (A) Percentage of bacteria inside HeLa cells, infected at an MOI of 25, which were transduced with empty vector (Control) or stably express ISG15 after 3 hr of infection; CFUs of bacteria within control cells were normalized to 100%, data shown is AVG ± SEM. Statistical significance were determined using two tailed t-test. (B) Percentage of bacteria inside HeLa cells which have been transfected with siControl or siISG15 (15 hr of infection, MOI 25). siControl cells were normalized to 100%, data is shown as AVG ± SEM. Statistical significance determined using two tailed t-test. (C) Primary mouse embryonic fibroblasts (MEFs, Isg15+/+ or Isg15−/−) were infected with Listeria for 4 hr at an MOI of 10. CFUs of bacteria within Isg15+/+ cells were normalized to 100%, and error bars represent ± SEM. Statistical significance determined using two tailed t-test. (D) Data shown is average CFUs per ml ± SEM in Isg15+/+ MEFs vs Isg15−/− MEFs following 4 hr of infection, MOI 10. (E) and (F) Isg15+/+ or Isg15−/− mice were infected intravenously with 5 × 105 of Listeria strain EGD. The liver and spleen were isolated following 72 hr of infection, and CFUs per organ were calculated by serial dilution and replating; circles or squares depict individual animals. The line denotes AVG ± SEM. Significance for in vivo data determined using Mann–Whitney test. (G) RNASeq data of significantly upregulated ISG15-related genes compared with non-infected controls in LoVo cells following Listeria infection for 24 hr. (H) Cells were lysed and immunoblotted with the indicated antibodies following infection with Listeria for 3 hr. (I) Data shown is average CFUs per ml ±SEM in Isg15+/+ MEFs vs Isg15−/− MEFs and in Ube1L+/+ and Ube1L−/− MEFs following 4 hr of infection, using an MOI of 10. Statistical significance is indicated as follows: NS, nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001. https://doi.org/10.7554/eLife.06848.008 ISGylation machinery is induced by Listeria and ISGylation protects against infection Since ISG15 can mediate its protective effect through conjugation-dependent or conjugation-independent mechanisms, we wanted to determine if conjugation contributed to ISG15's role in defense against Listeria. Therefore, we initially assessed whether Listeria could induce the enzymes required for ISGylation. To this end, we performed RNASeq of Lovo cells infected with Listeria for 24 hr compared to uninfected cells. The RNASeq data has been uploaded to Array Express with the accession E-MTAB-3649 (Radoshevich et al., 2015b). We found that UBE1L (E1), UBE2L6 (E2), HERC5 and TRIM25 (E3s) mRNA are all significantly upregulated following Listeria infection, as is the deconjugating enzyme USP18 (Figure 3G). In order to assess whether UBE2L6 and TRIM25 are induced as rapidly as ISG15 at the protein level, we monitored their expression by immunoblot at 3 hr post infection (Figure 3H). Interestingly, TRIM25 already displayed increased expression relative to loading control at 3 hr post infection. UBE2L6 was present in these cells at this time point as well. We then sought to determine whether conjugation-incompetent MEFs would have a higher bacterial load following Listeria infection. Primary MEFs that lack UBE1L, and thus can not form ISG15 conjugates, have a much higher bacterial burden following Listeria infection than wild-type MEFs at 4 hr post infection. This phenotype mirrors the bacterial burden of Isg15−/− MEFs, clearly indicating that ISG15's role in host defense against Listeria in vitro requires ISGylation (Figure 3I). ISGylation modifies ER and Golgi proteins and increases canonical secretion Since ISG15 is a ubiquitin-like modifier that is known to be covalently linked to hundreds of cellular and several viral substrates (Giannakopoulos et al., 2005; Zhao et al., 2005), our goal was to identify which substrates following overexpression of ISG15 could account for the protective effect detected in the context of Listeria infection and used a proteomic approach. We made use of SILAC coupled with LC-MS/MS and compared cells that express empty vector, cells that express ISG15 and cells that express ISG15 that were treated with interferon, the primary inducer of ISGylation (Figure 4A,B). We identified thirty ISGylated proteins modified following overexpression of ISG15 (Figure 4C–E, Figure 4—source data 1, depicted in blue). The proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014) via the PRIDE partner repository with the data set identifier PXD001805 (Radoshevich et al., 2015a). Interestingly, these proteins have not yet been reported to be targets of ISGylation. Following interferon treatment, ISG15 modified twelve additional proteins distinct from those that were ISGylated after overexpression without treatment (Figure 4C–E, Figure 4—source data 1, depicted in red). Three of these proteins are known targets of ISGylation following interferon treatment in the aforementioned screens. The other nine are novel substrates of ISGylation. To gain more insight into the role of ISGylation of the modified substrates following overexpression, we performed a gene ontology (GO) analysis of the thirty ISGylated proteins. To our surprise over 80% of ISG15-target proteins are integral membrane proteins (Figure 5A). Even more intriguingly they are known to be primarily localized to the endoplasmic reticulum and Golgi apparatus and/or are critical for glycosylation, ER morphology and ER to Golgi trafficking (e.g., the oligosaccharyl-transferase (OST) complex, RTN4, ATL3, SEC22B, ERGIC1, and ERGIC3; Figure 4C). One of the proteins enriched following ISG15 overexpression is Magnesium Transporter 1 (MAGT1). MAGT1 is critical for T cell activation and patients with a deletion in" @default.
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• W4253697777 title "Decision letter: ISG15 counteracts Listeria monocytogenes infection" @default.
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