Browsing by Subject "NF-kappa B"
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Item The Art of Viral Oncogenesis: Lessons from Human Papillomavirus and Polyomavirus Transformed Cancers(2020-05-01T05:00:00.000Z) Zhao, Jiawei; Xu, Jian; Banaszynski, Laura; Pfeiffer, Julie K.; Wang, RichardViruses account for about 15% of all human cancer. Understanding viral oncogenesis can substantially broaden our general knowledge on the molecular mechanisms of carcinogenesis. In this dissertation, I focused on two types of DNA oncoviruses, human papillomavirus (HPV) and polyomavirus (HPyV), and identified novel mechanisms by which these two types of viruses cause human cancers. In HPV transformed cancer cells, I identified a novel circular RNA species, circE7, that spans and encodes the HPV E7 oncoprotein. I later demonstrated that circE7 translated E7 protein accounts for a substantial proportion of the E7 protein in a HPV transformed cancer cell line, and whose absence significantly impacts cancer cell proliferation in vitro and in vivo. In Merkel Cell Polyomavirus (MCPyV) transformed MCC cancer cells, I identified the activation of non-canonical NF-κB pathway activation by the MCPyV small T (ST) oncoprotein. I further demonstrated that the ectopically activated non-canonical NF-κB pathway is required for cell growth in low serum. The inhibition of non-canonical NF-κB signaling by a small peptide inhibitor also resulted in impaired cell growth in vitro and in vivo due to ER stress mediated apoptosis, suggesting a novel therapeutic intervention strategy for viral positive MCC (VP-CC) patients.Item Attenuation of The Host Innate Cytokine Response By The Human Cytomegalovirus Immediate-Early 2 Protein Ie86(2006-12-20) Taylor, Roger Travis; Bresnahan, Wade A.Human cytomegalovirus infects a majority of the human population and is a significant cause of life-long morbidity and mortality in neonates and patients with an impaired immune system. Human cytomegalovirus infection has a profound effect on host cell, and expression of new viral proteins interferes with the ability of the host response to effectively limit virus persistence and the initation of a latent infection. This aim of this dissertation was to identify how human cytomegalovirus attenuates the host innate immune response early during infection. Specifically, I have employed genetic and biochemical approaches to identify the HCMV immediate-early 2 protein, IE86, as an interferon beta antagonist. IE86 expression also blocks the expression of a number of proinflammatory chemokines, including RANTES, MIG and IL-8 during human cytomegalovirus infection. I have further demonstrated that IE86 mediates the attenuation of cytokine and chemokine expression by targeting the nuclear factor kappa B pathway early during infection. Using gel shift analysis I have demonstrated that IE86 blocks nuclear factor kappa B DNA binding to target promoters, including the interferon beta promoter. Since IE86 is one of the first viral proteins to be expressed during human cytomegalovirus infection, it can rapidly block cytokine and chemokine expression thereby suppressing the antiviral response and limiting the recruitment of effecter cells. The attenuation of the innate immune response by IE86 likely enhances virus replication and contributes to persistence within the host. This work addresses a number of unanswered questions about human cytomegalovirus's interactions with the host, and has identified a previously unrecognized mechanism employed by human cytomegalovirus to evade the host immune response. A better understanding of IE86's ability to attenuate cytokine expression may be key to designing novel antiviral therapy or development of an effective vaccine to prevent human cytomegalovirus infection and disease.Item Biochemical and Genetic Studies on CC2D1A, a new NF-κB Activator and a Regulator of Synaptic Functions(2010-05-14) Zhao, Meng; Chen, Zhijian J.CC2D1A is an evolutionarily conserved gene from worm to human. It belongs to a new protein family with four DM14 domains at the NH2 terminus and a C2 domain at the COOH terminus. The function of this protein family remains largely unknown. CC2D1A has been identified as a new NF-κB activator through a large scale screen of human genes by Matsuda et al. Here I show that the conserved DM14 and C2 domains of CC2D1A are important for NF-κB activation. CC2D1A activates the IKK complex and NF-κB target genes through several key components in the canonical pathway including ubiquitin-conjugating enzyme UBC13, a RING domain ubiquitin ligase TRAF2, a protein kinase TAK1, and an essential regulator of IKK complex, NEMO. CYLD, a deubiquitination enzyme specific for Lysine-63 linked polyubiquitin chains, negatively regulates the activity of CC2D1A. These results suggest that CC2D1A activates NF-κB through the canonical IKK pathway. In an attempt to identify the physiological function of CC2D1A, I generated CC2D1A knockout mice. The KO animals die right after birth apparently due to their inability to breathe. Histological analysis identified no significant anatomical defects. In particular, brain, heart and muscle are normal with regard to morphology. In addition, neuromuscular junction at the diaphragm is formed in the absence of Cc2d1a. Human patients with mutations in the gene suffer from mental retardation, implying that Cc2d1a functions in the central nervous system (CNS). Here I show that Cc2d1a expression is enriched in the brain. Deletion of Cc2d1a impairs synapse maturation and function in cortical neurons. Our study may help understand the molecular basis of some human diseases such as mental retardation.Item Biochemical Mechanism of Protein Kinase Activation by the Ubiquitination System(2008-05-13) Xia, Zong-Ping; Chen, Zhijian J.The NF-κB signaling pathway is important for immune, inflammatory and stress responses of cells and can be activated by a variety of extracellular stimuli. In the IL1R/TLR signaling pathway, NF-κB is activated through activation of TAK1-IKK cascade by TRAF6 and Ubc13/Uev1A in a polyubiquitinationdependent manner. Mechanistically how TAK1 is activated by the TRAF6- Ubc13/Uev1A dependent polyubiquitination system is unknown. Whether TAK1 and IKK kinases can be activated by other than TRAF6-Ubc13/Uev1A is an open question. By inactivating ubiquitin activating enzyme E1 and ubiquitin conjugating enzyme E2 using NEM to further dissect the system, studies on how TAK1 is activated by the TRAF6-Ubc13/Uev1A system have revealed that 1) polyubiquitination step and kinase activation step can be un-coupled; 2) Polyubiquitination step generates unanchored K63-linked polyubiquitin chains as the kinase activators; 3) Coiled-coil domain of TRAF6 is required for synthesis of active polyubiquitin chains. Through biochemical fractionation, I purified UbcH5 as another E2 that works with TRAF6 to directly activate IKK. Mechanistic analysis on how TRAF6/UbcH5 activates IKK reveals that TRAF6-UbcH5 synthesize mixedlinkage- linked unanchored polyubiquitin chains and this polyubiquitin chains function as direct activators for IKK activation.Item Mechanisms Governing NF-κB Regulation by the Anti-Inflammatory Protein A20(2013-05-31) Skaug, Brian; Chen, Zhijian J.; Cobb, Melanie H.; Sternweis, Paul C.; Yarovinsky, FelixA20 is a potent anti-inflammatory protein that inhibits NF-ΚB, and A20 dysfunction is associated with autoimmunity and B-cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the IκB kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20’s 7th zinc finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a novel mechanism of IKK inhibition and a means by which polyubiquitin chains can specify a signaling outcome.Item Molecular and Genetic Analysis of Parkin in Microglia Activation and Inflammation-Related Neurodegeneration(2010-05-14) Tran, Thi Anh; Tansey, Malú G.Parkinson’s disease (PD) is a progressive, neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Genetic mutations account for only 5-10% of PD cases. Oxidative stress and inflammation have both been linked to sporadic PD. Inflammation-induced injury to dopaminergic neurons can be significantly attenuated by impairment of microglial activation. In addition, previous studies from our lab reported that parkin-/- mice are more susceptible to inflammation-induced degeneration of nigral DA neurons. Therefore, inflammatory responses are a critical determinant of DA neuronal survival. Microglia support neuronal survival by providing trophic factors and phagocytosing debris. However, with chronic inflammation glia release chemical mediators which are toxic to surrounding neurons. Our data provide evidence that Parkin is a negative regulator of microglial activation. parkin-/- mice display increased cytokine expression in the midbrain and increased cytokines in the serum suggesting parkin-/- mice are basally inflamed. Parkin loss-of-function mutations are linked to autosomal recessive PD. The parkin gene encodes an E3 ubiquitin ligase linked to mitochondrial dysfunction. Most studies on Parkin concentrate on its role in neurons, however, we hypothesize that Parkin function in microglial activation and inflammatory signaling also affect DA neuron survival. Our biochemical analyses of primary wild type microglia show Parkin expression is negatively regulated by inflammatory stimuli. Pharmacological or genetic inhibition of NF-κB, a transcription factor activated by inflammatory stimulation, blocks the inflammation-induced decrease in Parkin levels. Additionally, our data suggests that NF-κB may bind the parkin promoter, further implicating Parkin function in the inflammatory activation pathway. These novel findings suggest that in a normal cell experiencing inflammation, the decreased expression of Parkin, which has been shown to antagonize apoptotic signaling cascades, may render the cell more susceptible to death. Additionally, sources of inflammation including environmental triggers, infection, or traumatic injury could cause a normal individual to have the same susceptibility to PD as an individual with an inherited mutation, because inflammation leads to Parkin loss of function.Item NF-kB Mediates Cartilage Degradation induced by Trauma Injury and IL-1(2013-01-22) Carter, Kristen; Kashyap, Meghana; Sauer, Brent C.; Chen, Christopher T.BACKGROUND: IL-1 is one of the major pro-inflammatory cytokines responsible for cartilage degradation. Several studies have shown that IL-1 mediates the upregulation of tissue degradation through the NF-κB and Mitogen-activated protein kinase (MAPK, p38) pathways, but its role in cartilage degradation after blunt trauma injury is not clear. The objective of this study was to determine the roles of NF-κB and p38 in IL-1- induced cell death, proteoglycan (PG) degradation, nitric oxide (NO) production, and related gene upregulation in cartilage after blunt injury. METHODS: Full-thickness cartilage plugs were obtained from mature bovine knees (>18 mo) and pre-cultured in DMEM. The signaling pathways (p38 and NF-kB) were inhibited by pretreatment with 10μM SB202190 for p38 (p38i) and 50μM BAY117085 for NF-κB (IκBi) for 1 hour. Samples in Injury and Injury+IL-1 groups received impact injury with impact energy of 15J/cm2. IL-1 and Injury+IL-1 groups were treated with 1 ng/ml IL-1. Cell viability was assessed using fluorescein diacetate and propidium iodine. The mRNA from cartilage was isolated using Trizol and RNeasy Mini kit (Qiagen), reverse transcribed, and analyzed using qPCR to determine pro-inflammatory cytokine and tissue remodeling genes (IL- 6, MMP-3, TIMP-3). All gene expression was normalized to GAPDH. The medium was analyzed for proteoglycan (PG) release/loss and nitric oxide (NO) production using dimethylmethylene blue (DMMB) and Greiss assays, respectively. RESULTS: Increased cell death was found in the Injury and Injuy+IL-1 groups. Increase of PG loss was found in IL-1, Injury and Injury+IL-1 treated groups (37%, 104% and 126%, respectively). Significant decreases (69-73%) of PG loss were found in all IκBi treated groups, while little or no changes were found in the p38 groups. Results from the qPCR analysis supported the findings. IκBi treatment reduced MMP-3 upregulation induced by IL-1 and Injury, while there was minimal change with the p38 inhibitor. Similarly, NO production was also decreased in the IκBi treated groups. DISCUSSION and CONCLUSION: Our study suggests that the NF-κB signaling pathway plays a greater role than p38 in IL-1 mediated PG loss and NO production in cartilage after trauma injury. Future studies are needed to determine the time-course response and specific NF-κB mediators for downstream regulation, as well as the effects in long-term therapeutic treatment to ameliorate the progress of post traumatic osteoarthritis.Item Role of I Kappa B Kinase Alpha and I Kappa B Kinase Beta in the Development and Function of B and T Lymphocytes(2002-12-19) Ren, Hong; Gaynor, Richard B.Transcription factor NF-κB plays a key role in regulating the expression of genes involved in the control of the inflammatory and immune response. NF-κB binds to a group of inhibitory proteins, IκBs, in the cytoplasm of non-stimulated cells. Activation of NF-κB is regulated by the IκB kinase complex that phosphorylates IκB proteins, leading to their polyubiquitination and degradation. The released NF-κB molecules translocate into the nucleus and activate gene transcription. IκB kinase complex contains two catalytic subunits, IκB kinase α (IKKα) and IκB kinase β (IKKβ), and a regulatory subunit, IκB kinase γ (IKKγ) or NEMO. To evaluate the functions of IKKα and IKKβ in the development and function of the immune system, transgenic mice expressing dominant negative forms of IKKα and IKKβ specifically in their B cells or T cells were generated. Phenotypic analysis of transgenic mice expressing dominant negative IKKβ in the B cells revealed that the proliferation of B cells from these mutant mice in response to B cell mitogens was reduced due to impaired cell cycle progression. Accordingly, in vitro secretion of immunoglobulins by the mutant B cells in response to these mitogens was also decreased. In addition, these mice displayed selective defects in the production of specific immunoglobulin subclasses in response to type 2 but not type 1 T cell independent antigens. Moreover, the levels of certain immunoglobulin subclasses were reduced in mutant mice challenged with a T cell dependent antigen. These results indicate that IKKβ is critical for the proliferation of B cells and the control of some aspects of the humoral response. Transgenic mice expressing one or both of the dominant negative IKK specifically in T cells exhibited distinct phenotypes in thymocyte proliferation, cytokine production, and cell survival. Proliferation of thymic T cells from IKKβ mutant mice and IKKα/β mutant mice was markedly reduced due to impaired cell cycle progression. In addition, inhibition of both IKKα and IKKβ appeared to suppress the expression of multiple cytokines by thymocytes. Furthermore, apoptosis of the double positive thymocytes induced by the administration of anti-CD3 antibody was significantly reduced in transgenic mice expressing dominant negative IKKβ, but increased in mice expressing only dominant negative IKKα. These results indicate that IKKα and IKKβ play different roles in regulating the activation and survival of T cells.Item The Role of the cGAS-STING Pathway in Cancer and Autoimmunity(2021-05-01T05:00:00.000Z) Yum, Seoyun; Hooper, Lora V.; Cobb, Melanie H.; Fu, Yang-Xin; Chen, Zhijian J.The DNA sensor cyclic GMP-AMP synthase (cGAS) detects cytosolic DNA derived from infections, cancer, or aberrant self-DNA accumulation. DNA-bound cGAS synthesizes cGAMP, which activates the stimulator of interferon genes (STING) protein to induce type I interferons, inflammatory cytokines, and autophagy. The cGAS-STING pathway played a critical role in cancer immune surveillance by detecting tumor-derived DNA. Furthermore, stimulating the pathway using STING agonists conferred antitumor effects in preclinical tumor models. The induction of type I interferons through the transcription factor IRF3 is considered a major outcome of STING activation that drives immune responses against tumors, but the roles of IFN-independent functions of STING in cancer are not well understood. Here, I generated STING mouse strains with mutations that confer selective loss of STING functions. Using these STING-mutant mice, I show that TBK1 recruitment to STING promotes antitumor effects that are both type I interferon-dependent and -independent. In addition, using mice with selective deletion of the cGAS genes in specific immune cells, I show that dendritic cells, but not macrophages, are responsible for the cancer immune surveillance. By staining intracellular cGAMP, I show that tumor-infiltrating immune cells with greater pinocytosis ability take up cGAMP efficiently. When aberrantly activated, the cGAS-STING pathway can lead to autoimmune diseases. cGAS was responsible for the autoimmunity in mice deficient in intracellular DNase and was shown to be activated in some SLE patients. In order to develop a therapeutic strategy targeting cGAS-mediated autoimmune diseases, I tested if the AAV-CRISPR system targeting the cGAS gene can confer a therapeutic effect in TREX1-deficient mice. In addition, I tested if cGAS drives disease pathogenesis in a mouse SLE model, which closely mimics the human SLE disease. Furthermore, I designed an ImageStream assay to detect the activation of the cGAS-STING pathway in SLE patient PBMCs. In addition to the role in diseases, I observed cell-specific functions of the cGAS-STING pathway in promoting the endosome-to-cytosol protein delivery and mediating T cell death. Altogether, these results improve our understanding of the role of the cGAS-STING pathway in cancer and autoimmune diseases.Item Signal Specific Ubiquitination and Degradation of IkBa(2003-10-08) Hakala, Kevin William; Kodadek, Thomas J.The transcription factor Nuclear Factor kB (NF-kB) is retained in the cytoplasm by the action of its inhibitor IkB. Upon phosphorylation by the IKK complex, IkB is rapidly ubiquitinated and targeted for 26S proteasome mediated degradation, thus liberating NF-kB for transport to its nuclear destination. The current project was initiated to reconstitute this pathway in vitro by using the purified ubiquitination and degradation machinery to degrade IkBa, and activate NF-kB. While signal dependant IkBa ubiquitination was achieved early in the project, this substrate was not degraded by a number of different 26S protein preparations. Instead, an integral or associated isopeptidase activity was observed with each 26S preparation. The development of new 26S protein purification methods has enabled the isolation of highly purified 26S proteins that exhibits low degradative activity towards the ubiquitinated IkBa substrate without excess isopeptidase activity. In an effort to increase substrate degradation, the IkBa ubiquitination reaction was carefully scrutinized. The current literature reports that Ubch5 is the relevant E2 that works in conjunction with the IkBa SCFᔲCP E3 complex, however, Cdc34/Ubc3 can also ubiquitinate IkBa, and may also be a relevant E2. While both E2s carry out in vitro signal dependant ubiquitination of IkBa, the ubiquitin conjugates made by Ubc3 are specific for Lysine-48 linked isopeptide bonds, whereas Ubch5 is able to utilize a variety of ubiquitin surface Lysine residues in isopeptide bond formation. Because K-48 linked ubiquitin conjugates are believed to target substrates for 26S mediated degradation, it was not surprising to find that my 26S proteasome preparations exhibited higher levels of IkBa degradation when ubiquitin conjugation reactions were carried out with Ubc3 instead of Ubch5. Using small interfering RNA to knock down the protein levels of each E2 in vivo, we have found that Ubc3 has no effect on IkBa degradation, whereas the Ubc5/7 double knockdown exhibits partial inhibition of IkBa degradation which is comparable to knocking down the levels of the IkBa E3 specificity factor ᔲCP. The completion of this project has established an in vitro ubiquitination and degradation system that will be instrumental for future studies aimed at determining how the 26S proteasome unfolds and degrades its protein substrates.Item Ubiquitin Mediated Regulation of NF-kappa B Signaling(2008-05-13) Pineda, Gabriel; Chen, Zhijian J.NF-κB signaling is involved in many vital cellular functions such as immunity, cell proliferation, inflammation, and apoptosis. The activation of NF-κB signaling requires the process of ubiquitination. K63-and K48-linked ubiquitin chains have been shown to have distinct roles and biological function in NF-κB signaling. K63-linked ubiquitin chains are required for the activation of TAK1, which leads to the activation of IKK. Activation of IKK leads to K48-linked ubiquitination, and the subsequent proteasomal degradation of IκBalpha . Two important areas of research focusing on ubiquitin regulation of NF-κB signaling are addressed in this dissertation. The areas addressed include understanding how ubiquitinated substrates are targeted for proteasomal degradation and how CYLD negatively regulates NF-κB signaling. In these studies, I investigated the molecular mechanisms involved in the regulation of IκBalpha degradation. Using a siRNA approach, NPL4 was shown to be required for IκBalpha degradation. In vitro proteasomal degradation assays demonstrated that the NPL4 complex is required for IκBalpha degradation. Evidence from both in vitro and in vivo studies suggest NPL4 is required for IκBalpha degradation, but not for IKK activation. These results suggest NPL4 is working at a step after ubiquitination of IκBalpha , but before proteasomal degradation. I propose that ubiquitinated IκBalpha is targeted to the proteasome by an interaction between the NPL4 complex that is mediated through the zinc finger domain of NPL4. The cylindromatosis tumor suppressor gene (CYLD) encodes a 110 kDa deubiquitination enzyme that negatively regulates NF-κB signaling. Loss-of-function mutations in CYLD lead to the disease Familial Cylindromatosis, which is characterized by the formation of benign skin tumors that originate from the head and neck of individuals afflicted with the disease. Here I present in vitro evidence that CYLD inhibits both TAK1 and IKK activation by TRAF6 in a cell free system. I also demonstrate, using a highly purified in vitro system, that CYLD specifically cleaves K63 linked ubiquitin chains and harbors endoproteolytic activity. Furthermore, the third CAPGLY domain of CYLD was shown to be a novel ubiquitin binding domain. My results provide biochemical evidence that CYLD functions as a K63 deubiquitinase to attenuate NF-κB signaling.