[Show abstract][Hide abstract] ABSTRACT: The HECT-type E3 ligase Itch is increasingly being shown to have a vital role in immune regulation. Itch deficiency leads to deleterious inflammatory disorders both in mice and humans. By adding ubiquitin to the key signaling intermediates, Itch functions as a critical regulator of lymphocyte-cell activation, differentiation and immune tolerance. Also, Itch cooperates with deubiquitinating enzymes such as A20 and Cyld to terminate NF-κB signaling and prevent chronic inflammation. This review summarizes recent advances that highlight Itch's role in lymphocyte function and explores recent insights regarding its role as a regulator of inflammatory signaling. Immunology and Cell Biology advance online publication, The intracellular signaling events induced by external stimuli in immune cells are tightly regulated by post-translation modifications such as phosphorylation and ubiquitination. 1 These modifications allow the immune cells to exert dynamic responses against external threats without self-tissue damage associated with autoimmunity. 2 Attachment of ubiquitin serves as either a non-proteasomal modifica-tion or a termination signal for proteasomal degradation based on the number of attached ubiquitin chains and the topology of their linkage. Ubiquitin contains seven lysine residues, but only one of them is linked to the C-terminal glycine of another ubiquitin to form ubiquitin chains. Typically, the linkage occurs via either Lys63 (K63) or Lys48 (K48). K63-linked polyubiquitination leads to either non-proteasomal modifications, such as subcellular localization or protein– protein interactions; however, K48-linked polyubiquitination targets proteins for proteasomal degradation. 3 The process of ubiquitination is a complex biochemical process that involves a series of enzymes: E1, E2 and E3. First, ubiquitin is activated by E1, the ubiquitin-activating enzyme, to form a high-energy thioester linkage between its C-terminal glycine residue and an active cysteine on E1. Next, the thiol-linked ubiquitin is transiently transferred to E2, the ubiquitin-conjugating enzyme. An E3 ligase transfers the activated ubiquitin molecule from the E2 to a lysine residue on the substrate. 3,4 The human genome is estimated to encode 2 E1s, ~ 50 E2s and over 600 E3s. 5 Each E3 recognizes a set of substrates that share one or more ubiquitination signals, and an individual E3 cooperates with one or a few E2s. The E3 ligases are divided into two different groups based on their functional domains: the homology to the E6-associated protein carboxyl terminus (HECT) type E3s and the really interesting new gene (RING) type E3s. 2,3 Of the more than 600 human genes that encode ubiquitin E3 ligases, most belong to the RING family; however, only 28 belong to the HECT family. 6 The RING-type E3 ligases can be further divided into single-protein E3 ligases such as Cbl-b (Casitas B-lineage lymphoma), in which three RING-finger domains and a substrate-recruiting domain are found in one polypeptide chain, and multi-subunit E3 ligases such as SCF complex (S-phase kinase-associated protein 1(SKP)-cullin-1 (CUL1)-F-box-protein complex). 2 The substrate-recruiting compo-nent of the F-box protein associates with scaffold proteins SKP1 and CUL1, whereas the RING-finger domain only associates with protein RBX1. RBX1 binds ubiquitin-loaded E2. The F-box protein recruits a specific protein substrate and helps transfer ubiquitin from E2 to the substrate. 2 The HECT-type E3s contain a highly conserved region of roughly 350 amino acids called the HECT domain. This region allow for the transfer of ubiquitin to the final substrate. These ligases are characterized by an N-terminal Ca 2+ phospholipid-binding C2 domain, WW domains for protein–protein interaction and the C-terminal HECT domain. The HECT domain contains an evolutio-narily conserved cysteine, which allow it to form ubiquitin complexes and eventually attach ubiquitin to target substrates. 3 Itchy E3 ubiquitin protein ligase (Itch, encoded by Itch) belongs to the family of HECT-type E3 ligases, which predominantly target substrates for K48-linked ubiquitination. However, Itch has also been shown to target its substrates for K27-, K29-, K33-and K63-linked ubiquitination. 7–11 Recent molecular investigation has provided us with a better under-standing of how Itch can regulate a variety of signaling pathways that are linked to the immune system's inflammatory response by adding ubiquitin chains to target proteins. Figure 1 summarizes some of the Itch-specific substrates and associated proteins that have been identified in mouse. This review will focus on the role of Itch in the
Immunology and Cell Biology 02/2015; · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Understanding the developmental mechanisms of follicular helper T cells (TFH cells) in humans is relevant to the clinic. However, the factors that drive the differentiation of human CD4(+) helper T cells into TFH cells remain largely undefined. Here we found that transforming growth factor-β (TGF-β) provided critical additional signals for the transcription factors STAT3 and STAT4 to promote initial TFH differentiation in humans. This mechanism did not appear to be shared by mouse helper T cells. Developing human TFH cells that expressed the transcriptional repressor Bcl-6 also expressed RORγt, a transcription factor typically expressed by the TH17 subset of helper T cells. Our study documents a mechanism by which TFH cells and TH17 cells emerge together in inflammatory environments in humans, as is often observed in many human autoimmune diseases.
[Show abstract][Hide abstract] ABSTRACT: TNF-related apoptosis-inducing ligand receptor 2 [TRAIL-R2 or death receptor 5 (DR5)] is expressed at elevated levels in a broad range of solid tumors to mediate apoptotic signals from TRAIL or agonist antibodies. We tested the hypothesis that DR5 DNA vaccination will induce proapoptotic antibody to trigger apoptosis of tumor cells. BALB/c mice were electrovaccinated with DNA-encoding wild-type human DR5 (phDR5) or its derivatives. Resulting immune serum or purified immune IgG induced apoptosis in triple-negative breast cancer (TNBC) cells, which were also TRAIL sensitive. The proapoptotic activity of immune serum at dilutions of 0.5-2% was comparable to that of 1-2 μg/ml of TRAIL. Apoptotic activity of immune serum was enhanced by antibody crosslinking. Apoptotic cell death induced by anti-DR5 antibody was shown by the cleavage of PARP and caspase-3. In contrast, immune serum had no effect on the proliferation of activated human T cells, which expressed low levels of DR5. In vivo, hDR5 reactive immune serum prevented growth of SUM159 TNBC cells in severe combined immune-deficient mice. DR5-specific IFN-γ-secreting T cells were also induced by DNA vaccination. Furthermore, the feasibility to overcome immune tolerance to self DR5 was shown by the induction of mouse DR5-binding antibody after electrovaccination of BALB/c mice with pmDR5ectm-Td1 encoding a fusion protein of mouse DR5 and an immunogenic fragment of tetanus toxin. These findings support DR5 as a promising vaccine target for controlling TNBC and other DR5-positive cancers.
International Journal of Cancer 03/2012; 131(11):2562-72. · 6.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic inflammation has been strongly associated with tumor progression, but the underlying mechanisms remain elusive. Here we demonstrate that E3 ligase Itch and deubiquitinase Cyld formed a complex via interaction through 'WW-PPXY' motifs. The Itch-Cyld complex sequentially cleaved Lys63-linked ubiquitin chains and catalyzed Lys48-linked ubiquitination on the kinase Tak1 to terminate inflammatory signaling via tumor necrosis factor. Reconstitution of wild-type Cyld but not the mutant Cyld(Y485A), which cannot associate with Itch, blocked sustained Tak1 activation and proinflammatory cytokine production by Cyld(-/-) bone marrow-derived macrophages. Deficiency in Itch or Cyld led to chronic production of tumor-promoting cytokines by tumor-associated macrophages and aggressive growth of lung carcinoma. Thus, we have identified an Itch-Cyld-mediated regulatory mechanism in innate inflammatory cells.
[Show abstract][Hide abstract] ABSTRACT: Earlier, we demonstrated the essential role of Kruppel-like transcription factor, TIEG1, in TGF-β-induced regulatory T cell (Treg) development. In this article, we demonstrate that IL-6, which promotes Th17 development, abrogated TIEG1 nuclear translocation and inhibited TGF-β-induced Treg development. Tyrosine kinase Tyk2-mediated phosphorylation of TIEG1 at Tyr179 promoted noncanonical K-27-linked polyubiquitination, which inhibited TIEG1 nuclear translocation. To test the role of TIEG1-regulated Treg/Th17 development in antitumor immunity, we analyzed TRAMP-C2 tumor growth in TIEG1(-/-) mice. The defective Treg development and elevated Th17 response resulted in enhanced immune reactivity in the tumor and inhibition of TRAMP-C2 tumor growth in TIEG1(-/-) mice. Thus, our results uncovered a novel regulatory mechanism that modulates Tregs and may regulate tumor progression.
The Journal of Immunology 04/2011; 186(10):5638-47. · 5.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Allergic diseases and asthma are caused by dysregulated Th2-type immune responses, which drive disease development in susceptible individuals. Immune tolerance to allergens prevents inflammatory symptoms in the respiratory mucosa and provides protection against inflammation in the airways. Increasing evidence indicates that Foxp3+ regulatory T cells (Tregs) play a critical role in immune tolerance and control Th2-biased responses. Tregs develop in the thymus from CD4(+) T cells (natural Tregs) and also in the periphery by the conversion of naïve CD4(+) T cells (induced Tregs). Increased susceptibility to allergy and airway inflammation is hypothesized to result from impaired development and function of Tregs. Thus, strategies to induce allergen-specific Tregs hold great promise for treatment and prevention of asthma.
American Journal Of Pathology 08/2010; 177(2):525-31. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: E3 ligases Cbl-b and Itch have emerged as dominant "tolerogenic" regulators of T cells because their deficiency results in severe autoimmune diseases. Cbl-b and Itch ligase activity regulate T-cell anergy and development of Foxp3+ regulatory T cells (Treg) in the periphery by modulating key components of T-cell receptor (TCR) and transforming growth factor-beta (TGF-beta) signaling. Manipulation of Cbl-b and Itch activities may provide unique opportunities to develop future therapies for immune disorders such as autoimmunity and cancer.
Cancer Research 04/2010; 70(8):3009-12. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor-beta (TGF-beta) signaling in naive T cells induces expression of the transcription factor Foxp3, a 'master' regulator of regulatory T cells (T(reg) cells). However, the molecular mechanisms leading to Foxp3 induction remain unclear. Here we show that Itch-/- T cells were resistant to TGF-beta treatment and had less Foxp3 expression. The E3 ubiquitin ligase Itch associated with and promoted conjugation of ubiquitin to the transcription factor TIEG1. Itch cooperated with TIEG1 to induce Foxp3 expression, which was reversed by TIEG1 deficiency. Functionally, 'TGF-beta-converted' T(reg) cells generated from TIEG1-deficient mice were unable to suppress airway inflammation in vivo. These results suggest TIEG and Itch contribute to a ubiquitin-dependent nonproteolytic pathway that regulates inducible Foxp3 expression and the control of allergic responses.
[Show abstract][Hide abstract] ABSTRACT: The immune system is capable of mounting robust responses against invading pathogens but refrains from attacking self. Many studies have focused on tolerance induction of Th1 cells, whose failure results in development of autoimmune diseases. However, the molecular mechanisms governing tolerance induction in Th2 cells and its relation to allergic responses remain unclear. Here we used both in vivo and in vitro protocols to demonstrate that Th2 cells either containing a mitogen and extracellular kinase kinase 1 (MEKK1) mutant or lacking JNK1 or the E3 ubiquitin ligase Itch cannot be tolerized. In a mouse allergic model, injection of high-dose tolerizing antigen failed to block the development of airway inflammation in Itch-/- mice. This study suggests that MEKK1-JNK signaling regulates Itch E3 ligase-mediated tolerogenic process in Th2 cells. These findings have therapeutic implications for allergic diseases.
Journal of Clinical Investigation 05/2006; 116(4):1117-26. · 13.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The proinflammatory cytokine tumor necrosis factor (TNF) alpha signals both cell survival and death. The biological outcome of TNFalpha treatment is determined by the balance between NF-kappaB and Jun kinase (JNK) signaling; NF-kappaB promotes survival, whereas JNK enhances cell death. Critically, identity of a JNK substrate that promotes TNFalpha-induced apoptosis has been outstanding. Here we show that TNFalpha-mediated JNK activation accelerates turnover of the NF-kappaB-induced antiapoptotic protein c-FLIP, an inhibitor of caspase-8. This is not due to direct c-FLIP phosphorylation but depends on JNK-mediated phosphorylation and activation of the E3 ubiquitin ligase Itch, which specifically ubiquitinates c-FLIP and induces its proteasomal degradation. JNK1 or Itch deficiency or treatment with a JNK inhibitor renders mice resistant in three distinct models of TNFalpha-induced acute liver failure, and cells from these mice do not display inducible c-FLIP(L) ubiquitination and degradation. Thus, JNK antagonizes NF-kappaB during TNFalpha signaling by promoting the proteasomal elimination of c-FLIP(L).
[Show abstract][Hide abstract] ABSTRACT: It becomes increasingly evident that protein ubiquitination is closely linked to many aspects of the immune system and the E3 ubiquitin ligases play an important role in those processes via specific targeting of protein substrates. Gene database searching has indicated that there are several hundred E3 ubiquitin ligases, with the majority being RING finger-containing E3 ligases. It is conceivable that there will be very complicated interplay among the E3 ligases. Several E3 ligases may target the same substrate, or otherwise, one E3 ligase may target different substrates under diverse physiological conditions. In addition, the list of de-ubiquiting enzymes involved in immune regulation will increase in the near future. It has to be mentioned that biochemical and molecular approaches are still the main tools for identification of a substrate for a particular E3 ubiquitin ligase. The physiological relevance of such findings has to be correlated with genetic investigations. In any case, the field of research on protein ubiquitination in immune modulation will continue to be a hot topic in the years to come.
Advances in Experimental Medicine and Biology 02/2006; 584:207-17. · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In lymphocytes, integration of Ca2+ and other signalling pathways results in productive activation, while unopposed Ca2+ signalling leads to decreased responsiveness to subsequent stimulation (anergy). The Ca(2+)-regulated transcription factor NFAT has an integral role in both aspects of lymphocyte function. NFAT cooperates with the transcription factor AP-1 (Fos/Jun) to up-regulate genes involved in productive activation of lymphocytes. However, in the absence of AP-1, NFAT imposes an opposing genetic programme that leads to lymphocyte anergy. Anergy is implemented at least partly through proteolytic degradation of the key signalling proteins PKCtheta and PLCgamma1. Sustained Ca(2+)-calcineurin signalling increases mRNA and protein levels of the E3 ubiquitin ligases Itch, CblB and Grail and induces expression of Tsg1O1, the ubiquitin-binding component of the ESCRT1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promotes membrane translocation of Itch and the related protein Nedd4, resulting in PKCtheta and PLCgamma1 degradation. T cells from Itch- and CblB-deficient mice are resistant to anergy induction. Anergic T cells show impaired calcium mobilization after TCR triggering and are unable to maintain a mature immunological synapse. Thus Ca(2+)-calcineurin-NFAT signalling links gene transcription to a multi-step programme that leads to impaired signal transduction in anergic T cells.
Novartis Foundation symposium 02/2005; 267:165-74; discussion 174-9.
[Show abstract][Hide abstract] ABSTRACT: Antigen-specific immunotolerance limits the expansion of self-reactive T cells involved in autoimmune diseases. Here, we show that the E3 ubiquitin ligase Cbl-b is upregulated in T cells after tolerizing signals. Loss of Cbl-b in mice results in impaired induction of T cell tolerance both in vitro and in vivo. Importantly, rechallenge of Cbl-b mutant mice with the tolerizing antigen results in massive lethality. Moreover, ablation of Cbl-b resulted in exacerbated autoimmunity. Mechanistically, loss of Cbl-b rescues reduced calcium mobilization of anergic T cells, which was attributed to Cbl-b-mediated regulation of PLCgamma-1 phosphorylation. Our results show a critical role for Cbl-b in the regulation of peripheral tolerance and anergy of T cells.
[Show abstract][Hide abstract] ABSTRACT: Sustained calcium signaling induces a state of anergy or antigen unresponsiveness in T cells, mediated through calcineurin and the transcription factor NFAT. We show here that Ca(2+)-induced anergy is a multistep program that is implemented at least partly through proteolytic degradation of specific signaling proteins. Calcineurin increased mRNA and protein of the E3 ubiquitin ligases Itch, Cbl-b and GRAIL and induced expression of Tsg101, the ubiquitin-binding component of the ESCRT-1 endosomal sorting complex. Subsequent stimulation or homotypic cell adhesion promoted membrane translocation of Itch and the related protein Nedd4, resulting in degradation of two key signaling proteins, PKC-theta and PLC-gamma1. T cells from Itch- and Cbl-b-deficient mice were resistant to anergy induction. Anergic T cells showed impaired calcium mobilization after TCR triggering and were unable to maintain a mature immunological synapse, instead showing late disorganization of the outer ring containing lymphocyte function-associated antigen 1. Our results define a complex molecular program that links gene transcription induced by calcium and calcineurin to a paradoxical impairment of signal transduction in anergic T cells.