Serpin 2a Is Induced in Activated Macrophages and Conjugates to a Ubiquitin Homolog

Department of Immunology, University of Washington, Seattle, WA 98185, USA.
The Journal of Immunology (Impact Factor: 4.92). 04/2002; 168(5):2415-23. DOI: 10.4049/jimmunol.168.5.2415
Source: PubMed


After i.p. infection of mice with the intracellular bacterium Mycobacterium bovis bacillus Calmette-Guérin, macrophages recovered from the peritoneal cavity display classical signs of immune activation. We have identified a member of the serine protease inhibitor (serpin) family which is highly induced in macrophages during bacillus Calmette-Guérin infection. Serpin 2a (spi2a) expression is also induced in macrophages in vivo during infection with Salmonella typhimurium and Listeria monocytogenes, and in vitro by a variety of bacteria and bacterial products. The cytokine IFN-gamma also induces spi2a expression in macrophages, and this induction is synergistic with bacterial products. We also demonstrate here that a ubiquitin homolog, IFN-stimulated gene of 15-kDa (ISG15), is strongly induced during in vitro and in vivo activation of macrophages and that it conjugates to spi2a in activated macrophages. The ISG15-spi2a conjugates were identified by tandem mass spectrometry and contained spi2a conjugated to either one or two molecules of ISG15. Whereas spi2a was induced by either bacterial products or IFN-gamma, ISG15 was induced only by bacterial products. Although many protein targets have been described for ubiquitin conjugation, spi2a is the first ISG15-modified protein to be reported. Macrophage activation is accompanied by the activation of a variety of proteases. It is of interest that a member of the serine protease inhibitor family is concomitantly induced and modified by a ubiquitin-like protein.

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    • "The role of endosomal cathepsins has been described in several extensive reviews [66,67]. Only some of the endogenous inhibitors (cystatin F and Spia3g) are also up regulated, while cystatin C is down regulated [65,68]. Serpina3g (Spia3g) is highly induced in macrophages during bacillus Calmette-Guérin infection as well as in infection with Salmonella typhimurium and Listeria monocytogenes [68]. "
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    ABSTRACT: Proteinases and their inhibitors play essential functional roles in basic biological processes in both hosts and pathogens. Endo/lysosomal cathepsins participate in immune response in pathogen recognition and elimination. They are essential for both antigen processing and presentation (host adaptive immune response) and activation of endosomal toll like receptors (innate immune response). Pathogens can produce proteases and also natural inhibitors to subvert the host immune response. Several pathogens are sensed through the intracellular pathogen recognition receptors, but only some of them use the host proteolytic system to escape into the cytosol. In this review, I provide an update on the most recent developments regarding the role of proteinases and their inhibitors in the initiation and regulation of immune responses.
    Current Protein and Peptide Science 12/2012; 13(8). DOI:10.2174/138920312804871102 · 3.15 Impact Factor
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    • "Protein modification by ubiquitin and ubiquitin-like proteins, including ISG15, SUMO and Nedd8, Fat 10, has been shown to be important for many cellular processes, such as cell cycle, stress response and immune response [4]. ISG15 has been shown to be upregulated upon type I interferon stimulation, microbial infections and tumor growth [4,5,22,23]. Expression of ISG15 is reported to be controlled by transcriptional factors Stat, NFkb, p53 and JNK [4]. "
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    ABSTRACT: Dengue virus (DENV) and West Nile virus (WNV), close siblings of the Flaviviridae family, are the causative agents of Dengue hemorraghic shock or West Nile meningoencephalitis respectively. Vaccines against these two flaviviruses are currently unavailable. Interferon- Stimulated Gene 15 (ISG15), encoding an ubiquitin-like protein, is significantly induced by type I interferons or viral infections. Its roles in viral infections, however, vary with viruses, being either anti- or pro-viral. The exact roles of ISG15 in DENV and WNV infections remain unknown. In the current study, we evaluated the relevancies of ISG15 to DENV and WNV infection of a mouse macrophage cell line RAW264.7. Quantitative PCR showed that mouse Isg15 was dramatically induced in DENV or WNV- infected RAW264.7 cells compared with non-infected cells. Isg15 and two other Jak-Stat related genes, Socs1 and Socs3, were silenced using siRNA mediated RNA interference. The intracellular DENV and WNV loads, as determined by quantitative PCR, were significantly higher in Isg15 silenced cells than control cells. The expression levels of interferon beta 1 (Ifnb1) were increased significantly in Isg15, Socs1 or Socs3 siRNA treated cells. Further investigation indicated that protein modification by ISG15, so called ISGylation, was significantly enhanced in DENV-infected cells compared to that in non-infected cells. These findings suggest that ISG15 plays an anti-DENV/WNV function via protein ISGylation.
    Virology Journal 10/2011; 8(1):468. DOI:10.1186/1743-422X-8-468 · 2.18 Impact Factor
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    • "One study in support of this hypothesis demonstrated that overexpression of UBE1L stimulates PML/RARα degradation in NB4 APL cells treated with retinoic acid [22]. However, more recent work has demonstrated that direct ISG15 conjugation to Serpin 2a, JAK, or STAT1 does not increase their respective rates of degradation [23], [24], suggesting that the role of ISGylation in PML-RARα degradation may be indirect. On the other hand, it is becoming increasingly clear that ISG15 exerts its biological effect by interfering with the ubiquitin pathway. "
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    ABSTRACT: Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients.
    PLoS ONE 01/2011; 6(1):e16422. DOI:10.1371/journal.pone.0016422 · 3.23 Impact Factor
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