Our involvement in the regulation and consequences of ubiquitination began in the course of studies aimed at understanding why double positive (DP) thymocytes are so sensitive to pro-apoptotic stimuli. We found that induction of DP apoptosis, regardless of the molecular pathway, resulted in the degradation of XIAP and c-IAP1, proteins of the Inhibitor of APoptosis (IAP) family. Notably, we identified XIAP and c-IAP1 as ubiquitin protein ligases (E3s), enzymes involved in the addition of Ub to target proteins. This activity was dependent upon a motif called the RING domain. We are currently working on the following: We have generated mice in which we knocked-in an E3-defective c-IAP2 (it contains a point mutation in the RING domain). We found (1) accumulation of B cells, especially of the marginal zone phenotype, and IgA hypergammaglobulinemia, (2) increased gut-associated lymphoid tissue (GALT) and lymphocyte inflitrates in the lung, (3) B cell hyperproliferation and relative insensitivity to growth factor-withdrawal apoptosis (4) spontaneous B cell NF-kappaB activity via the non-canonical pathway (upregulation of NIK). The E3-defective c-IAP2 also prevents c-IAP1 from ubiqutinating/degrading NIK, because only one c-IAP molecule can bind TRAF2 (a component of the inhibitory complex that includes NIK) at a time. The phenotype of these B cells is similar to that of human MALT lymphomas. We propose that the loss of c-IAP2 E3 activity, which accompanies the generation of the c-IAP2/MALT1 fusion protein, is a major contributor to disease by activating non-canonical NF-kappaB. c-IAP2 E3-defective T cells, unlike wild type T cells, are hyperresponsive to TCR occupancy in the absence costimulation. As a result, infection of these mice with a normally avirulent strain of Toxoplasma gondii led to death via cytokine storm. These results strongly suggest that non-cannonical NF-kappaB acivation is a costimulation signaling pathway. We also found that T cells from p100 knockout mice, which cannot activate the non-canonical pathway, are also costimulation independent. This is because p100 binds p65 and is a negative regulator of the canonical pathway. Furthermore, p100 levels are decreased in the c-IAP2 knockin mice (due to constitutive processing to the stimulatory p52 form). Therefore, we have identified the balance between p100 and p52 as a key regulator of the ability of T cells to respond to TCR-mediated activation. c-IAP1 E3-defective c-IAP1 mice have been generated as well. They do not have an overt phenotype. Interestingly, whereas non-canonical NF-kB is normal in B cells and B cell proliferation is normal, T cells have modestly eleveated non-canonical NF-kB and increased proliferation (intermediate between wild type and c-IAP2 E3-defective mice). Therefore, there a tissue-specific differences in c-IAP uses. In the past year we have found that c-IAP2 knockin mice mount a normal immune response to virus (LCMV) but fail to maintain memory T cells. This is because the antigen-specific T cells die in vivo. We identified defective signaling via 4-1BB, an anti-apoptotic co-stimulatory molecule on activated/memory T cells, as the cause. Currently we are working on the contribution of metabolic abnormalities to the death of memory T cells. Optineurin is a protein whose mutation is responsible for a subset of adult-onset primary open angle glaucoma. Optineurin contains a motif highly homologous to the Ub-binding motif in NEMO. Optineurin also binds Tank-binding kinase 1 (TBK1), a kinase upstream of type 1 inferferon production, in an inducible fasion, and has recently been implicated as a key factor in autophagy to Salmonella. We have generated optineurin knockin mice that lack C-terminal exons and cannont bind polyubiquitin. The latter express very little optineurin protein. We have found that these mice have defective IFN Type 1 production in response to signaling via TLR3 and TLR4, as well as to infection with virus. Studieson autophagy in cells from these animals are ongoing.
| Giardino Torchia, Maria Letizia; Munitic, Ivana; Castro, Ehydel et al. (2015) c-IAP ubiquitin protein ligase activity is required for 4-1BB signaling and CD8(+) memory T-cell survival. Eur J Immunol 45:2672-82 |
| Giardino Torchia, Maria Letizia; Conze, Dietrich B; Jankovic, Dragana et al. (2013) Balance between NF-?B p100 and p52 regulates T cell costimulation dependence. J Immunol 190:549-55 |
| Munitic, Ivana; Giardino Torchia, Maria Letizia; Meena, Netra Pal et al. (2013) Optineurin insufficiency impairs IRF3 but not NF-?B activation in immune cells. J Immunol 191:6231-40 |
| Giardino Torchia, Maria Letizia; Conze, Dietrich B; Ashwell, Jonathan D (2013) c-IAP1 and c-IAP2 redundancy differs between T and B cells. PLoS One 8:e66161 |
| Srinivasula, Srinivasa M; Ashwell, Jonathan D (2011) A20: more than one way to skin a cat. Mol Cell 44:511-2 |
| Conze, Dietrich B; Zhao, Yongge; Ashwell, Jonathan D (2010) Non-canonical NF-?B activation and abnormal B cell accumulation in mice expressing ubiquitin protein ligase-inactive c-IAP2. PLoS Biol 8:e1000518 |
| Lo, Yu-Chih; Lin, Su-Chang; Rospigliosi, Carla C et al. (2009) Structural basis for recognition of diubiquitins by NEMO. Mol Cell 33:602-15 |
| Wu, Chuan-Jin; Ashwell, Jonathan D (2008) NEMO recognition of ubiquitinated Bcl10 is required for T cell receptor-mediated NF-kappaB activation. Proc Natl Acad Sci U S A 105:3023-8 |
| Ashwell, Jonathan D (2008) TWEAKing death. J Cell Biol 182:15-7 |
| Conze, Dietrich B; Wu, Chuan-Jin; Thomas, James A et al. (2008) Lys63-linked polyubiquitination of IRAK-1 is required for interleukin-1 receptor- and toll-like receptor-mediated NF-kappaB activation. Mol Cell Biol 28:3538-47 |
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