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Oxidation matters: The ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation
ARTICLE IN PRESS
G Model MIMM-4033; No. of Pages 4
Molecular Immunology xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Review
Oxidation matters: The ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation夽 Annika Warnatsch 1 , Theresa Bergann, Elke Krüger ∗ Charité – Universitätsmedizin Berlin, Charité CrossOver, Institut für Biochemie CCM, Virchowweg 6, 10117 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 30 September 2012 Accepted 8 October 2012 Available online xxx Keywords: Antigen presentation MHC class I Virus infection Oxidative stress Immunoproteasome
a b s t r a c t During innate immune responses the delicate balance of protein synthesis, quality control and degradation is severely challenged by production of radicals and/or the massive synthesis of pathogen proteins. The regulated degradation of ubiquitin-tagged proteins by the ubiquitin proteasome system (UPS) represents one major pathway for the maintenance of cellular proteostasis and regulatory processes under these conditions. In addition, MHC class I antigen presentation is strictly dependent on an appropriate peptide supply by the UPS to efficiently prime CD8+ T cells and to initiate an adaptive immune response. We here discuss recent efforts in defining the link between innate immune mechanisms like cytokine and ROS production, the induction of an efficient adaptive immune response and the specific involvement of the UPS therein. Cytokines and/or infections induce translation and the production of free radicals, which in turn confer oxidative damage to nascent as well as folded proteins. In parallel, the same signaling cascades are able to accelerate the protein turnover by the concomitantly induced ubiquitin conjugation and degradation of such damaged polypeptides by immunoproteasomes. The ability of immunoproteasomes to efficiently degrade such oxidant-damaged ubiquitylated proteins protects cells from accumulating toxic ubiquitin-rich aggregates. At the same time, this innate immune mechanism facilitates a sufficient peptide supply for MHC class I antigen presentation and connects it to initiation of adaptive immunity. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction 1.1. The ubiquitin proteasome system The ubiquitin proteasome system (UPS) plays a central role in maintaining the protein homeostasis of cells by the ubiquitinmediated degradation of short-lived, misfolded, oxidant- or otherwise damaged proteins by the proteasome. This is an essential mechanism for the regulation of numerous important cellular processes, such as gene transcription, DNA repair, apoptosis, cell cycle regulation, cell differentiation and signaling, as well as the generation of antigenic peptides presented by major histocompatibility (MHC) class I molecules (Goldberg, 2007). The ubiquitylation of protein substrates takes place in a three step enzymatic cascade. The E1 enzyme activates ubiquitin in an ATP-dependent manner and transfers it via a thioester bond on an E2 ubiquitin-conjugating enzyme, which shifts ubiquitin to a substrate-specific E3-ligase. E3-ligases facilitate the covalent
夽 This article belongs to Special Issue on Antigen Processing and Presentation. ∗ Corresponding author. Tel.: +49 30 450528317. E-mail address: [email protected] (E. Krüger). 1 Present address: MRC National Institute for Medical Research, The Ridgeway, Mill Hill, NW7 1AA London, UK.
modification of substrates with several ubiquitin moieties with a chain link via lysine 48 in the ubiquitin sequence labeling proteins for degradation by the 26S proteasome. The 26S proteasome consists of the 20S core complex and at least one regulatory 19S subunit attached to the core end. The 19S subunit recognizes and unfolds poly-ubiquitylated protein substrates prior to their degradation by the 20S core, which represents a barrel-shaped structure constituted of four stacked heptameric rings by the assembly of nonidentical ␣ and  subunits (␣1–7 1–7 1–7 ␣1–7 ). Three of -subunits, namely 1, 2 and 5, harbor the active sites of the proteasome conveying caspase-like, trypsin-like and chymotrypsin-like cleavage specificities, which enables the proteasome to cleave behind almost every residue within a protein (Goldberg, 2007). Apart from the standard proteasome (s-proteasome) different isoforms of the proteasome are described. The appearance of a certain isoform in immune cells and immune tissue led to the designation immunoproteasome (i-proteasome). I-proteasomes are constitutively expressed in immune relevant cells and formed upon exposure to interferons (IFNs) and other cytokines that induce the expression of i-subunits, 1i/Lmp2, 2i/Mecl-1 and 5i/Lmp7. Isubunits replace the catalytically active standard  subunits within the 20S core. Both, s-proteasomes as well as i-proteasomes, are able to produce MHC class I ligands as part of the adaptive immune response. In most instances i-proteasomes generate antigenic
0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.10.007
Please cite this article in press as: Warnatsch, A., et al., Oxidation matters: The ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation. Mol. Immunol. (2012), http://dx.doi.org/10.1016/j.molimm.2012.10.007
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peptides more efficiently and at a higher rate. In vitro experiments as well as in vivo mice studies underline a major role for i-proteasomes during an immune response to bacterial as well as viral infections. The increased capacity of i-proteasomes compared to s-proteasomes to generate antigenic peptides results either from an increased chymotrypsin-like activity following the incorporation of the 5i subunit or from conformational changes improving the substrate accessibility to the active sites (Ebstein et al., 2012). 1.2. Oxidative stress during innate immunity During an innate immune response phagocytes produce large amounts of reactive oxygen species (ROS) as well as reactive nitrogen species (RNS) through activation of the NADPH oxidase 2 (Nox2) and the inducible nitric oxide synthetase (iNOS). Moreover, also non-immune cells produce ROS and RNS upon exposure to cytokines by activation of the NADPH oxidases Nox1 or Nox4. Upon IFN␥ signaling fibroblasts and epithelial cells induce Nox1 and almost all cell types express Nox4 (Katsuyama, 2010). In phagoytes ROS production by Nox2 serves an antimicrobial function for elimination of the pathogens by phagosomes. Low levels of ROS produced by the phagocyctic Nox2 in dendritic cells have been shown to play a role for the adaptive immune response by supporting cross-presentation. Thereby, Nox2 is involved in the adaptive immune response (Kotsias et al., 2012). Besides foreign pathogens, ROS and RNS attack also host cellular molecules, because free radicals are not selective. Accordingly, proteins from invaders and host cells are equally damaged due to the high chemical reactivity of radicals. However, the acknowledged functions of ROS were not only restricted to the direct antimicrobial activity against pathogens, but are also involved in the regulation of signaling pathways (Katsuyama, 2010). Protein modifications through oxidative stress include amino acid carbonylation, cross-linking of disulfide bonds and cleavage of peptide backbones. Consequently, oxidant-damaged proteins tend to aggregate in large and dynamic ubiquitin-rich cellular inclusions called aggresome-like induced structures (ALIS) (Kriegenburg et al., 2011). Irreversibly oxidant-damaged proteins are potentially toxic and require elimination, which is mediated by autophagy or the UPS in order to preserve cell viability upon oxidative stress. Besides the induction of a variety of antioxidant enzymes, the removal of oxidant-damaged proteins by the UPS is meanwhile established as an important cytoprotective mechanism in the oxidative stress response (Kriegenburg et al., 2011). 1.3. Defective ribosomal products A limitation for the generation of MHC class I ligands, which can be presented to CD8+ T cells in order to induce adaptive immunity, represents the availability of substrates for proteasomal degradation due to their stability and/or localization. Newly synthesized defective ribosomal products (DRiPs) serve as the major substrate for the proteasome to generate MHC class I ligands (Yewdell, 2011). DRiPs occur due to errors in transcription, translation, folding and due to oxidative damage. During IFN-signaling and viral infections the protein synthesis rate as well as oxidative stress is enhanced resulting in elevated levels of DRiPs, thus adapting the levels of DRiPs to the increased requirements for MHC class I peptide supply. The DRiP hypothesis therefore delivers an explanation on how long-lived, stable and also compartmentalized viral proteins can be fed into the MHC class I antigen presentation pathway, so that the cellular immune systems gains early access to viral proteins (Yewdell, 2011).
2. Current status 2.1. I-proteasomes and cytokine regulation There is increasing evidence for novel concepts of i-proteasome function beyond the generation of peptide ligands for MHC class I antigen presentation. Several studies exist describing the impact of i-proteasome function in the regulation of pro-inflammatory cytokine secretion. Thus, reduced levels of pro-inflammatory cytokines were detected in response to influenza A infection in dendritic cells of 1i/Lmp2 knockout mice (Ebstein et al., 2012). Similarly, after CVB3 infection an impaired activation of NF-B signaling was shown in 5i/Lmp7-deficient mice (Opitz et al., 2011). As a greater proteolytic capacity was attributed to the iproteasome compared to the s-proteasome. These observations were in line with the finding of a higher turnover of the NFB inhibitory molecule I-B in i-proteasome-overexpressing cells (Seifert et al., 2010). Besides NF-B activation, an influence of i-proteasome function on various other inflammatory signaling pathways was demonstrated. Thus, TLR-4-mediated NO production by macrophages in response to bacterial LPS was hampered in i-proteasome knockout mice. Furthermore, even IFN signal transduction was affected due to a mutation in the PSMB8 gene as shown by the upregulation of IFN␥-inducible factors in CANDLE syndrome. In contrast, impaired i-proteasome function by chemical proteasome inhibition or deletion of the immunosubunit Lmp7 has been shown to attenuate experimental colitis (Ebstein et al., 2012). Another central function of the UPS is represented by the modulation of cell cycle control. UPS-dependent degradation of cyclin-dependent kinase inhibitors like p21 or of the tumor suppressor protein p53 can trigger prolonged cell cycle progression. In this context, especially i-proteasomes have been connected with the maintenance of proliferation, as T cell expansion has been found impaired in i-proteasome knockout mice (Ebstein et al., 2012). 2.2. UPS in inflammation and infection Beyond their crucial role for the generation of MHC class I ligands in infected cells other cellular functions have been implicated for i-proteasomes due to the observation of their induction in noninfected cells in response to various stimuli (Yewdell, 2011). This notion was further strengthened by the discovery of the constitutive expression of i-proteasomes subunits in a wide variety of non-immune cells, for example in large and small intestinal cells and in human kidney (Ebstein et al., 2012). Recently, i-proteasomes have been shown to contribute to the elimination of oxidant-damaged proteins and therefore to protect cells against inflammation-induced oxidative stress (Kruger and Kloetzel, 2012). IFNs trigger a transient accumulation of ubiquitin conjugates by the combined action of IFN-induced radical production, the up-regulation of translation, the up-regulation of the ubiquitin-conjugation capacity by UBE2L6 and a transient decrease in proteolytic activity. As soon as a certain level of iproteasomes has been formed the higher degradation capacity of i-proteasomes facilitates the degradation of these ubiquitinconjugates, which are mainly DRiPs (Seifert et al., 2010). The above-mentioned involvement of i-proteasomes in the proper cellular response to stress conditions was corroborated by findings made in animal models and cell lines deficient in i-proteasomes. I-proteasome-deficient retina cells were shown to be more susceptible to oxidation-induced cell death. Accordingly, in response to IFN-induced oxidative stress 5i/Lmp7-deficient cells displayed a significantly increased accumulation of poly-ubiquitin conjugates compared to wild type cells. Moreover, an augmented formation of ubiquitin-rich inclusions was detected in inflammation-challenged livers or brains of i-proteasome-deficient mice (Seifert et al., 2010).
Please cite this article in press as: Warnatsch, A., et al., Oxidation matters: The ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation. Mol. Immunol. (2012), http://dx.doi.org/10.1016/j.molimm.2012.10.007
G Model MIMM-4033; No. of Pages 4
ARTICLE IN PRESS A. Warnatsch et al. / Molecular Immunology xxx (2012) xxx–xxx
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In line with these experimental findings, i-proteasome induction in the course of particular diseases associated with intracellular accumulations of abnormal proteins has been explained with the enduring requirement to maintain cellular protein homeostasis in the disease-affected tissues (Ebstein et al., 2012). Aberrant function of i-proteasomes by genetic mutations in the human 5i/Lmp7 gene PSMB8 has been implicated in a number of auto-inflammatory diseases. Such syndromes are characterized by impaired formation of i-proteasomes paralleled by reduced chymotrypsin-like proteolytic activity, enhanced levels of ubiquitylated and oxidized proteins and as a consequence an increase in pro-inflammatory cytokines in patients’ sera (Goldbach-Mansky, 2012). During viral infection i-proteasomes are essential for the protection against oxidative protein damage. The investigation of coxsackievirus-induced myocarditis revealed exacerbated tissue damage in 5i/Lmp7 knockout mice due to elevated levels of oxidized proteins. Therefore, infection-induced formation of i-proteasomes prevents inflammation- and oxidation-mediated protein damage (Opitz et al., 2011). These newly identified functions of the UPS place it in a central position to both control the innate immune reaction and to protect against overshooting inflammatory responses.
3. Future perspectives As discussed above, protein homeostasis is severely challenged by production of radicals and/or the massive synthesis of pathogen proteins during the immune response. Therein the UPS and in particular i-proteasomes are important to preserve the vitality of cells and tissues during innate immune responses and ensures the appropriate peptide supply for MHC class I antigen presentation. We have evidence that this holds true for the efficient presentation of immundominant viral epitopes derived from structural proteins of the virion of human cytomegalo- and influenza virus within 2 h post infection and independent of translation. Radical production by Nox4 resulted in oxidant-damage of virion proteins and their concomitant UBE2L6-dependent ubiquitin conjugation, their subsequent i-proteasome-mediated degradation and the generation of virus-specific epitopes (see Fig. 1). Thus, radical production in target cells gives the immune system access to structural proteins from the virus particle independent of their de novo synthesis and links this innate mechanism to MHC class I antigen presentation (our unpublished results). In this context it is interesting to note that UBE2L6 has been identified as an important player in cross presentation of viral epitopes in dendritic cells (Ebstein et al., 2012). However, our understanding of the interplay of radical producing enzymes, the UPS and antigen processing or preservation of proteostasis is far from complete. Why i-proteasomes are not always expressed to counteract accumulation of DRiPs and other substrates is unclear. Possibly, constitutive i-proteasome expression is deleterious in contexts other than the immune system. Nevertheless, many cell types express low levels of 5i/LMP7 resulting in proteasome subtypes containing standardand immunosubunits. IFN-signaling not only induces oxidative stress by NOX enzymes, DRiPs and i-proteasomes, but also triggers the adaptation of the UPS to these immune challenges by induction of a specific set of E2s and E3s for marking pathogen- and damaged-proteins with ubiquitin. In addition, the alternative proteasome activator PA28 is induced and forms PA28-20S-PA28 proteasome complexes or hybrid proteasomes (PA28-20S-19S). Like i-proteasomes, PA28 has been shown to improve the peptide supply for MHC class I antigen presentation. Moreover PA28 has been implicated in clearance
Fig. 1. Oxidative stress increases epitope processing from DRiPs and non-DRiPs. Upon an immunological challenge interferons as well as virus infection itself induce protein synthesis and oxidative stress. Radicals produced by Nox and iNOS cause oxidative damage of newly synthesized proteins, resulting in DRiPs, as well as of long-lived viral and cellular proteins (non-DRiPs). Due to a concomitant induction of the ubiquitylation machinery these oxidant-damaged proteins are labeled with ubiquitin for degradation by the proteasome. The reduced degradation capacity of s-proteasomes compared to i-proteasomes results in the accumulation of an overwhelming amount of damaged and misfolded proteins and the formation of ubiquitin-rich aggregates/ALIS. In contrast, the induction of i-proteasomes leads to the efficient degradation of oxidant-damaged proteins, thereby protecting cells from accumulating toxic proteins, and moreover in case of viral infection results in the generation of virus-derived antigenic peptides, triggering an efficient adaptive immune response.
of protein aggregates in response to oxidative stress. Thus, proteasome compositions in cells and tissues are heterogeneous and do not follow the simple differentiation into s- and i-proteasomes. It is likely that cells and tissues can adapt their UPS to changing proteolytic requirements during the immune response. To address all of these issues will be a future challenge. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB740, KR1915/4-1). References Ebstein, F., Kloetzel, P.M., Kruger, E., Seifert, U., 2012. Emerging roles of immunoproteasomes beyond MHC class I antigen processing. Cellular and Molecular Life Sciences: CMLS 69, 2543–2558. Goldbach-Mansky, R., 2012. Immunology in clinic review series; focus on autoinflammatory diseases: update on monogenic autoinflammatory diseases: the role of interleukin (IL)-1 and an emerging role for cytokines beyond IL-1. Clinical and Experimental Immunology 167, 391–404. Goldberg, A.L., 2007. Functions of the proteasome: from protein degradation and immune surveillance to cancer therapy. Biochemical Society Transactions 35, 12–17.
Please cite this article in press as: Warnatsch, A., et al., Oxidation matters: The ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation. Mol. Immunol. (2012), http://dx.doi.org/10.1016/j.molimm.2012.10.007
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Katsuyama, M., 2010. NOX/NADPH oxidase, the superoxide-generating enzyme: its transcriptional regulation and physiological roles. Journal of Pharmacological Sciences 114, 134–146. Kotsias, F., Hoffmann, E., Amigorena, S., Savina, A., 2012. Reactive oxygen species production in the phagosome: impact on antigen presentation in dendritic cells. Antioxidants & Redox Signaling, Sep 11. (Epub ahead of print). Kriegenburg, F., Poulsen, E.G., Koch, A., Kruger, E., Hartmann-Petersen, R., 2011. Redox control of the ubiquitin-proteasome system: from molecular mechanisms to functional significance. Antioxidants & Redox Signaling 15, 2265–2299. Kruger, E., Kloetzel, P.M., 2012. Immunoproteasomes at the interface of innate and adaptive immune responses: two faces of one enzyme. Current Opinion in Immunology 24, 77–83.
Opitz, E., Koch, A., Klingel, K., Schmidt, F., Prokop, S., Rahnefeld, A., Sauter, M., Heppner, F.L., Volker, U., Kandolf, R., Kuckelkorn, U., Stangl, K., Kruger, E., Kloetzel, P.M., Voigt, A., 2011. Impairment of immunoproteasome function by beta5i/LMP7 subunit deficiency results in severe enterovirus myocarditis. PLoS Pathogens 7, e1002233. Seifert, U., Bialy, L.P., Ebstein, F., Bech-Otschir, D., Voigt, A., Schroter, F., Prozorovski, T., Lange, N., Steffen, J., Rieger, M., Kuckelkorn, U., Aktas, O., Kloetzel, P.M., Kruger, E., 2010. Immunoproteasomes preserve protein homeostasis upon interferoninduced oxidative stress. Cell 142, 613–624. Yewdell, J.W., 2011. DRiPs solidify: progress in understanding endogenous MHC class I antigen processing. Trends in Immunology 32, 548–558.
Please cite this article in press as: Warnatsch, A., et al., Oxidation matters: The ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation. Mol. Immunol. (2012), http://dx.doi.org/10.1016/j.molimm.2012.10.007
G Model MIMM-4033; No. of Pages 4
Molecular Immunology xxx (2012) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Review
Oxidation matters: The ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation夽 Annika Warnatsch 1 , Theresa Bergann, Elke Krüger ∗ Charité – Universitätsmedizin Berlin, Charité CrossOver, Institut für Biochemie CCM, Virchowweg 6, 10117 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 30 September 2012 Accepted 8 October 2012 Available online xxx Keywords: Antigen presentation MHC class I Virus infection Oxidative stress Immunoproteasome
a b s t r a c t During innate immune responses the delicate balance of protein synthesis, quality control and degradation is severely challenged by production of radicals and/or the massive synthesis of pathogen proteins. The regulated degradation of ubiquitin-tagged proteins by the ubiquitin proteasome system (UPS) represents one major pathway for the maintenance of cellular proteostasis and regulatory processes under these conditions. In addition, MHC class I antigen presentation is strictly dependent on an appropriate peptide supply by the UPS to efficiently prime CD8+ T cells and to initiate an adaptive immune response. We here discuss recent efforts in defining the link between innate immune mechanisms like cytokine and ROS production, the induction of an efficient adaptive immune response and the specific involvement of the UPS therein. Cytokines and/or infections induce translation and the production of free radicals, which in turn confer oxidative damage to nascent as well as folded proteins. In parallel, the same signaling cascades are able to accelerate the protein turnover by the concomitantly induced ubiquitin conjugation and degradation of such damaged polypeptides by immunoproteasomes. The ability of immunoproteasomes to efficiently degrade such oxidant-damaged ubiquitylated proteins protects cells from accumulating toxic ubiquitin-rich aggregates. At the same time, this innate immune mechanism facilitates a sufficient peptide supply for MHC class I antigen presentation and connects it to initiation of adaptive immunity. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction 1.1. The ubiquitin proteasome system The ubiquitin proteasome system (UPS) plays a central role in maintaining the protein homeostasis of cells by the ubiquitinmediated degradation of short-lived, misfolded, oxidant- or otherwise damaged proteins by the proteasome. This is an essential mechanism for the regulation of numerous important cellular processes, such as gene transcription, DNA repair, apoptosis, cell cycle regulation, cell differentiation and signaling, as well as the generation of antigenic peptides presented by major histocompatibility (MHC) class I molecules (Goldberg, 2007). The ubiquitylation of protein substrates takes place in a three step enzymatic cascade. The E1 enzyme activates ubiquitin in an ATP-dependent manner and transfers it via a thioester bond on an E2 ubiquitin-conjugating enzyme, which shifts ubiquitin to a substrate-specific E3-ligase. E3-ligases facilitate the covalent
夽 This article belongs to Special Issue on Antigen Processing and Presentation. ∗ Corresponding author. Tel.: +49 30 450528317. E-mail address: [email protected] (E. Krüger). 1 Present address: MRC National Institute for Medical Research, The Ridgeway, Mill Hill, NW7 1AA London, UK.
modification of substrates with several ubiquitin moieties with a chain link via lysine 48 in the ubiquitin sequence labeling proteins for degradation by the 26S proteasome. The 26S proteasome consists of the 20S core complex and at least one regulatory 19S subunit attached to the core end. The 19S subunit recognizes and unfolds poly-ubiquitylated protein substrates prior to their degradation by the 20S core, which represents a barrel-shaped structure constituted of four stacked heptameric rings by the assembly of nonidentical ␣ and  subunits (␣1–7 1–7 1–7 ␣1–7 ). Three of -subunits, namely 1, 2 and 5, harbor the active sites of the proteasome conveying caspase-like, trypsin-like and chymotrypsin-like cleavage specificities, which enables the proteasome to cleave behind almost every residue within a protein (Goldberg, 2007). Apart from the standard proteasome (s-proteasome) different isoforms of the proteasome are described. The appearance of a certain isoform in immune cells and immune tissue led to the designation immunoproteasome (i-proteasome). I-proteasomes are constitutively expressed in immune relevant cells and formed upon exposure to interferons (IFNs) and other cytokines that induce the expression of i-subunits, 1i/Lmp2, 2i/Mecl-1 and 5i/Lmp7. Isubunits replace the catalytically active standard  subunits within the 20S core. Both, s-proteasomes as well as i-proteasomes, are able to produce MHC class I ligands as part of the adaptive immune response. In most instances i-proteasomes generate antigenic
0161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2012.10.007
Please cite this article in press as: Warnatsch, A., et al., Oxidation matters: The ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation. Mol. Immunol. (2012), http://dx.doi.org/10.1016/j.molimm.2012.10.007
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peptides more efficiently and at a higher rate. In vitro experiments as well as in vivo mice studies underline a major role for i-proteasomes during an immune response to bacterial as well as viral infections. The increased capacity of i-proteasomes compared to s-proteasomes to generate antigenic peptides results either from an increased chymotrypsin-like activity following the incorporation of the 5i subunit or from conformational changes improving the substrate accessibility to the active sites (Ebstein et al., 2012). 1.2. Oxidative stress during innate immunity During an innate immune response phagocytes produce large amounts of reactive oxygen species (ROS) as well as reactive nitrogen species (RNS) through activation of the NADPH oxidase 2 (Nox2) and the inducible nitric oxide synthetase (iNOS). Moreover, also non-immune cells produce ROS and RNS upon exposure to cytokines by activation of the NADPH oxidases Nox1 or Nox4. Upon IFN␥ signaling fibroblasts and epithelial cells induce Nox1 and almost all cell types express Nox4 (Katsuyama, 2010). In phagoytes ROS production by Nox2 serves an antimicrobial function for elimination of the pathogens by phagosomes. Low levels of ROS produced by the phagocyctic Nox2 in dendritic cells have been shown to play a role for the adaptive immune response by supporting cross-presentation. Thereby, Nox2 is involved in the adaptive immune response (Kotsias et al., 2012). Besides foreign pathogens, ROS and RNS attack also host cellular molecules, because free radicals are not selective. Accordingly, proteins from invaders and host cells are equally damaged due to the high chemical reactivity of radicals. However, the acknowledged functions of ROS were not only restricted to the direct antimicrobial activity against pathogens, but are also involved in the regulation of signaling pathways (Katsuyama, 2010). Protein modifications through oxidative stress include amino acid carbonylation, cross-linking of disulfide bonds and cleavage of peptide backbones. Consequently, oxidant-damaged proteins tend to aggregate in large and dynamic ubiquitin-rich cellular inclusions called aggresome-like induced structures (ALIS) (Kriegenburg et al., 2011). Irreversibly oxidant-damaged proteins are potentially toxic and require elimination, which is mediated by autophagy or the UPS in order to preserve cell viability upon oxidative stress. Besides the induction of a variety of antioxidant enzymes, the removal of oxidant-damaged proteins by the UPS is meanwhile established as an important cytoprotective mechanism in the oxidative stress response (Kriegenburg et al., 2011). 1.3. Defective ribosomal products A limitation for the generation of MHC class I ligands, which can be presented to CD8+ T cells in order to induce adaptive immunity, represents the availability of substrates for proteasomal degradation due to their stability and/or localization. Newly synthesized defective ribosomal products (DRiPs) serve as the major substrate for the proteasome to generate MHC class I ligands (Yewdell, 2011). DRiPs occur due to errors in transcription, translation, folding and due to oxidative damage. During IFN-signaling and viral infections the protein synthesis rate as well as oxidative stress is enhanced resulting in elevated levels of DRiPs, thus adapting the levels of DRiPs to the increased requirements for MHC class I peptide supply. The DRiP hypothesis therefore delivers an explanation on how long-lived, stable and also compartmentalized viral proteins can be fed into the MHC class I antigen presentation pathway, so that the cellular immune systems gains early access to viral proteins (Yewdell, 2011).
2. Current status 2.1. I-proteasomes and cytokine regulation There is increasing evidence for novel concepts of i-proteasome function beyond the generation of peptide ligands for MHC class I antigen presentation. Several studies exist describing the impact of i-proteasome function in the regulation of pro-inflammatory cytokine secretion. Thus, reduced levels of pro-inflammatory cytokines were detected in response to influenza A infection in dendritic cells of 1i/Lmp2 knockout mice (Ebstein et al., 2012). Similarly, after CVB3 infection an impaired activation of NF-B signaling was shown in 5i/Lmp7-deficient mice (Opitz et al., 2011). As a greater proteolytic capacity was attributed to the iproteasome compared to the s-proteasome. These observations were in line with the finding of a higher turnover of the NFB inhibitory molecule I-B in i-proteasome-overexpressing cells (Seifert et al., 2010). Besides NF-B activation, an influence of i-proteasome function on various other inflammatory signaling pathways was demonstrated. Thus, TLR-4-mediated NO production by macrophages in response to bacterial LPS was hampered in i-proteasome knockout mice. Furthermore, even IFN signal transduction was affected due to a mutation in the PSMB8 gene as shown by the upregulation of IFN␥-inducible factors in CANDLE syndrome. In contrast, impaired i-proteasome function by chemical proteasome inhibition or deletion of the immunosubunit Lmp7 has been shown to attenuate experimental colitis (Ebstein et al., 2012). Another central function of the UPS is represented by the modulation of cell cycle control. UPS-dependent degradation of cyclin-dependent kinase inhibitors like p21 or of the tumor suppressor protein p53 can trigger prolonged cell cycle progression. In this context, especially i-proteasomes have been connected with the maintenance of proliferation, as T cell expansion has been found impaired in i-proteasome knockout mice (Ebstein et al., 2012). 2.2. UPS in inflammation and infection Beyond their crucial role for the generation of MHC class I ligands in infected cells other cellular functions have been implicated for i-proteasomes due to the observation of their induction in noninfected cells in response to various stimuli (Yewdell, 2011). This notion was further strengthened by the discovery of the constitutive expression of i-proteasomes subunits in a wide variety of non-immune cells, for example in large and small intestinal cells and in human kidney (Ebstein et al., 2012). Recently, i-proteasomes have been shown to contribute to the elimination of oxidant-damaged proteins and therefore to protect cells against inflammation-induced oxidative stress (Kruger and Kloetzel, 2012). IFNs trigger a transient accumulation of ubiquitin conjugates by the combined action of IFN-induced radical production, the up-regulation of translation, the up-regulation of the ubiquitin-conjugation capacity by UBE2L6 and a transient decrease in proteolytic activity. As soon as a certain level of iproteasomes has been formed the higher degradation capacity of i-proteasomes facilitates the degradation of these ubiquitinconjugates, which are mainly DRiPs (Seifert et al., 2010). The above-mentioned involvement of i-proteasomes in the proper cellular response to stress conditions was corroborated by findings made in animal models and cell lines deficient in i-proteasomes. I-proteasome-deficient retina cells were shown to be more susceptible to oxidation-induced cell death. Accordingly, in response to IFN-induced oxidative stress 5i/Lmp7-deficient cells displayed a significantly increased accumulation of poly-ubiquitin conjugates compared to wild type cells. Moreover, an augmented formation of ubiquitin-rich inclusions was detected in inflammation-challenged livers or brains of i-proteasome-deficient mice (Seifert et al., 2010).
Please cite this article in press as: Warnatsch, A., et al., Oxidation matters: The ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation. Mol. Immunol. (2012), http://dx.doi.org/10.1016/j.molimm.2012.10.007
G Model MIMM-4033; No. of Pages 4
ARTICLE IN PRESS A. Warnatsch et al. / Molecular Immunology xxx (2012) xxx–xxx
3
In line with these experimental findings, i-proteasome induction in the course of particular diseases associated with intracellular accumulations of abnormal proteins has been explained with the enduring requirement to maintain cellular protein homeostasis in the disease-affected tissues (Ebstein et al., 2012). Aberrant function of i-proteasomes by genetic mutations in the human 5i/Lmp7 gene PSMB8 has been implicated in a number of auto-inflammatory diseases. Such syndromes are characterized by impaired formation of i-proteasomes paralleled by reduced chymotrypsin-like proteolytic activity, enhanced levels of ubiquitylated and oxidized proteins and as a consequence an increase in pro-inflammatory cytokines in patients’ sera (Goldbach-Mansky, 2012). During viral infection i-proteasomes are essential for the protection against oxidative protein damage. The investigation of coxsackievirus-induced myocarditis revealed exacerbated tissue damage in 5i/Lmp7 knockout mice due to elevated levels of oxidized proteins. Therefore, infection-induced formation of i-proteasomes prevents inflammation- and oxidation-mediated protein damage (Opitz et al., 2011). These newly identified functions of the UPS place it in a central position to both control the innate immune reaction and to protect against overshooting inflammatory responses.
3. Future perspectives As discussed above, protein homeostasis is severely challenged by production of radicals and/or the massive synthesis of pathogen proteins during the immune response. Therein the UPS and in particular i-proteasomes are important to preserve the vitality of cells and tissues during innate immune responses and ensures the appropriate peptide supply for MHC class I antigen presentation. We have evidence that this holds true for the efficient presentation of immundominant viral epitopes derived from structural proteins of the virion of human cytomegalo- and influenza virus within 2 h post infection and independent of translation. Radical production by Nox4 resulted in oxidant-damage of virion proteins and their concomitant UBE2L6-dependent ubiquitin conjugation, their subsequent i-proteasome-mediated degradation and the generation of virus-specific epitopes (see Fig. 1). Thus, radical production in target cells gives the immune system access to structural proteins from the virus particle independent of their de novo synthesis and links this innate mechanism to MHC class I antigen presentation (our unpublished results). In this context it is interesting to note that UBE2L6 has been identified as an important player in cross presentation of viral epitopes in dendritic cells (Ebstein et al., 2012). However, our understanding of the interplay of radical producing enzymes, the UPS and antigen processing or preservation of proteostasis is far from complete. Why i-proteasomes are not always expressed to counteract accumulation of DRiPs and other substrates is unclear. Possibly, constitutive i-proteasome expression is deleterious in contexts other than the immune system. Nevertheless, many cell types express low levels of 5i/LMP7 resulting in proteasome subtypes containing standardand immunosubunits. IFN-signaling not only induces oxidative stress by NOX enzymes, DRiPs and i-proteasomes, but also triggers the adaptation of the UPS to these immune challenges by induction of a specific set of E2s and E3s for marking pathogen- and damaged-proteins with ubiquitin. In addition, the alternative proteasome activator PA28 is induced and forms PA28-20S-PA28 proteasome complexes or hybrid proteasomes (PA28-20S-19S). Like i-proteasomes, PA28 has been shown to improve the peptide supply for MHC class I antigen presentation. Moreover PA28 has been implicated in clearance
Fig. 1. Oxidative stress increases epitope processing from DRiPs and non-DRiPs. Upon an immunological challenge interferons as well as virus infection itself induce protein synthesis and oxidative stress. Radicals produced by Nox and iNOS cause oxidative damage of newly synthesized proteins, resulting in DRiPs, as well as of long-lived viral and cellular proteins (non-DRiPs). Due to a concomitant induction of the ubiquitylation machinery these oxidant-damaged proteins are labeled with ubiquitin for degradation by the proteasome. The reduced degradation capacity of s-proteasomes compared to i-proteasomes results in the accumulation of an overwhelming amount of damaged and misfolded proteins and the formation of ubiquitin-rich aggregates/ALIS. In contrast, the induction of i-proteasomes leads to the efficient degradation of oxidant-damaged proteins, thereby protecting cells from accumulating toxic proteins, and moreover in case of viral infection results in the generation of virus-derived antigenic peptides, triggering an efficient adaptive immune response.
of protein aggregates in response to oxidative stress. Thus, proteasome compositions in cells and tissues are heterogeneous and do not follow the simple differentiation into s- and i-proteasomes. It is likely that cells and tissues can adapt their UPS to changing proteolytic requirements during the immune response. To address all of these issues will be a future challenge. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft (SFB740, KR1915/4-1). References Ebstein, F., Kloetzel, P.M., Kruger, E., Seifert, U., 2012. Emerging roles of immunoproteasomes beyond MHC class I antigen processing. Cellular and Molecular Life Sciences: CMLS 69, 2543–2558. Goldbach-Mansky, R., 2012. Immunology in clinic review series; focus on autoinflammatory diseases: update on monogenic autoinflammatory diseases: the role of interleukin (IL)-1 and an emerging role for cytokines beyond IL-1. Clinical and Experimental Immunology 167, 391–404. Goldberg, A.L., 2007. Functions of the proteasome: from protein degradation and immune surveillance to cancer therapy. Biochemical Society Transactions 35, 12–17.
Please cite this article in press as: Warnatsch, A., et al., Oxidation matters: The ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation. Mol. Immunol. (2012), http://dx.doi.org/10.1016/j.molimm.2012.10.007
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Please cite this article in press as: Warnatsch, A., et al., Oxidation matters: The ubiquitin proteasome system connects innate immune mechanisms with MHC class I antigen presentation. Mol. Immunol. (2012), http://dx.doi.org/10.1016/j.molimm.2012.10.007