ZIA BC 010779 (ZIA) | |||
---|---|---|---|
Title | Regulation and Consequences of Ubiquitination | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Ashwell, Jonathan | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $230,786 | Project Dates | 01/01/2006 - 00/00/0000 |
Fiscal Year | 2015 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Autoimmune Diseases (20.0%) Cancer (100.0%) |
N/A | ||
Research Type | |||
Normal Functioning Cancer Initiation: Oncogenes & Tumor Suppressor Genes |
|||
Abstract | |||
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. Studies on autopha |