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. One clue was that DP apoptosis is blocked by proteasome inhibitors, implicating regulated protein degradation as a sensitizing event. 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. In subsequent studies we made the following findings: - Signaling via Tumor Necrosis Factor (TNF) receptor 2 (TNF-R2), but not TNF-R1, results in ubiquitination and degradation of the signaling intermediate TRAF2 (TRAF2 is required for coupling the TNF-R to JNK activation and, with TRAF5, to NF-kappaB). - TNF-R2 -mediated ubiquitination of TRAF2 is mediated by c-IAP1. - Expression of an """"""""E3-dead"""""""" c-IAP1 RING point mutant (a dominant negative) prevented TNF-alpha-induced TRAF2 degradation and inhibited apoptosis, demonstrating that c-IAP1 can actually be pro-apoptotic, probably by causing the degradation of TRAF2 and, perhaps, other anti-apoptotic molecules. - Stimulation via TNF-R2 results in the translocation of a c-IAP1/TRAF2 complex to the perinuclear ER, where it encounters a ubiquitin conjugating enzyme (E2), which cooperates with c-IAP1 to cause TRAF2 ubiquitination. - We were the first to characterize mice deficient in c-IAP1, and found that it has an obligate role in the ubiquitination and degradation of another member of the IAP family, c-IAP2. We have also found that ASK1, an important upstream enzyme in the MAP kinase signaling cascade, is a target for c-IAP1 in B cells stimulated with TNF. As a result, MAP kinase signaling is terminated in a timely fashion, moderating B cell responses. Another area in which we have studied ubiquitination is in signaling for activation of the important transcription factor NF-kappaB. NF-kappaB is sequestered in the cytoplasm in a complex with IkappaB. In the """"""""canonical"""""""" pathway of activation, signals converge on IkappaB kinase (IKK), which phosphorylates IkappaB resulting in IkappaB K48-linked polyubiquitination, IkappaB degradation by proteasomes, and migration of NF-kappaB to the nucleus. IKK has two enzymatically-active subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma or NEMO. NEMO is essential for NF-kappaB activation, and NEMO mutations or deficiency have been identified as the cause of incontinentia pigmenti (IP) and hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). The mechanism by which proximal cytokine receptor signals result in its NEMO-dependent activation remains largely unknown. Among the best-studied of such signaling pathways is that for TNF-alpha. TNF receptor 1 (TNF-R1) occupancy results in receptor trimerization and the serial recruitment of TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), receptor-interacting protein (RIP), TRAF2, and c-IAP1 and c-IAP2. RIP in particular is an essential intermediate for downstream activation of NF-kappaB. Upon stimulation with TNF-alpha, RIP binds to NEMO, which brings with it the other IKK components. The RIP that associates with TNF-R1 undergoes polyubiquitination, initially K63-linked, in lipid rafts;the K63-linked polyUb is subsequently removed by the de-ubiquitinating domain of A20 and K48-linked polyUb chains are added by the zinc finger region of A20, resulting in RIP degradation. NF-kappaB can also be activated by an independent, non-canonical pathway, in which the kinase NIK is stabilized, resulting in phosphorylation of p100, its processing to p52, and migration of p52 and RelB dimers to the nucleus where they drive gene transcription. In the past year we found that a fusion protein between c-IAP2 and MALT1, which is the major single cause of human MALT lymphoma, activates NF-kappaB by two mechanisms. It activates the canonical pathway by virtue of its paracaspase enzymatic activity (contributed by the MALT1 portion). It also activates non-canonical NF-kappaB, via a paracaspase-independent means, by stabilizing NIK levels. Importantly, E3-inactive c-IAP2 mutants not fused to any other protein do the same thing. 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. - c-IAP1 E3-defective c-IAP1 mice have been generated as well. They do not have an overt phenotype, nor have we elicited one as of yet. We have found that c-IAP1 levels are decreased in the knockin mice, and are exploring the possibility that c-IAP1 is stabilized by its ability to ubiquitinate itself or other proteins. We are in the process of generating double-deficient mice that lack c-IAP1 and c-IAP2 E3 activity. 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. In fact, we have found the optineurin binds to K63-linked polyUb much better than NEMO, and that it competes with NEMO for ubiquitinated RIP in TNF-stimulated cells. Acquisition of optineurin inhibits NF-kappaB activation, and forced knock-down of optineurin greatly enhances NF-kappaB activation. Given that NF-kappaB greatly enhances excitotoxic neuronal cell death, we have proposed that loss-of-function mutations in optineurin may in fact cause glaucoma due to enhanced retinal neuron cell death. 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 129 amino acids and cannot bind TBK1. Preliminary results suggest that have defective autophagosome formation in response to a veriety of stimuli, which is currently under investigation. We are also creating animals that lack the C-terminal ubiquitin-binding domain, and expect to be able to collect phenotypic data soon.

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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-?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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