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. Importantly, we identified XIAP and c-IAP1 as ubiquitin protein ligases (E3s), enzyme 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 have have found - accumulation of B cells, especially of the marginal zone phenotype, and IgA hypergammaglobulinemia. - increased gut-associated lymphoid tissue (GALT) and lymphocyte inflitrates in the lung. - B cell hyperproliferation and relative insensitivity to growth factor-withdrawal apoptosis. - 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. In the past year we 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, where as 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. - the 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 are exploring the possibility that this is because of defective signaling via 4-1BB, an anti-apoptotic co-stimulatory molecule on activated/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 the first 152 amino acids and cannot bind TBK1 as well as optineurin knockin mice that lace the 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. Studies on autophagy in cells from these animals are ongoing.

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National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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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-κB 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-κB 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-κB 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
Wagner, S; Carpentier, I; Rogov, V et al. (2008) Ubiquitin binding mediates the NF-kappaB inhibitory potential of ABIN proteins. Oncogene 27:3739-45
Srinivasula, Srinivasa M; Ashwell, Jonathan D (2008) IAPs: what's in a name? Mol Cell 30:123-35
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

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