Browsing by Subject "Adaptor Proteins, Signal Transducing"
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Item ADAP1 Promotes Latent HIV-1 Reactivation by Tuning the KRAS-ERK-AP-1 Signaling-Transcriptional Axis(December 2021) Ramirez, Nora-Guadalupe Piña; Schoggins, John W.; D'Orso, Iván; Pfeiffer, Julie K.; Alto, NealImmune stimulation fuels cell signaling-transcriptional programs that induce biological responses to eliminate virus-infected cells. Yet, retroviruses that integrate into host cell chromatin, such as HIV-1, co-opt these programs to switch between latent and reactivated states. However, many regulatory mechanisms are still unfolding. As such, here I take advantage of the unique intrinsic reliance HIV-1 has on host cell signaling-transcriptional programs to discover undescribed cell signaling regulators. Specifically, I implemented a functional screening platform, given HIV-1 gene expression relies on CD4+ T cell activation state, to identify host factors modulating CD4+ T cell signaling-transcriptional axes and consequently HIV-1 fate. Among the hits, I focus on ADAP1 (ArfGAP with Dual PH Domains 1), a previously thought neuro-restricted factor, and discover it is an amplifier of select human CD4+ T cell signaling programs. Using physiological models, I characterize ADAP1 expression is low in naïve and memory CD4+ T cells, but largely induced upon immune stimulation where it interacts with the immune signalosome. Using complementary biochemical and cellular assays, I demonstrate ADAP1 directly stimulates the GTPase activity of KRAS to amplify CD4+ T cell signaling through targeted activation of ERK-AP-1 axis. In primary CD4+ T cells which I have genetically ablated ADAP1, I show loss of ADAP1 function blunts gene expression programs in response to stimulation thereby reducing CD4+ T cell expansion and dampening latent HIV-1 reactivation. Supporting the impact of these findings, I propose the reduced CD4+ T cell programs and proliferation upon ADAP1 loss validates Genome-wide Association Studies linking ADAP1 single nucleotide polymorphisms in non-coding enhancers to an altered T lymphocyte count trait, potentially attributed to ADAP1 haploinsufficiency. Through these combined experimental approaches, I was able to define ADAP1 as an unexpected tuner of CD4+ T cell activation programs and co-opted by HIV-1 to escape latency.Item B-Cell Adapter for Phosphoinositide 3-Kinase Is a Signaling Adapter in the Toll-Like Receptor/Interleukin-1 Receptor Superfamily(2014-02-17) Troutman, Ty Dale; van Oers, Nicolai S. C.; Pasare, Chandrashekhar; Hooper, Lora V.; Chen, Zhijian J.; Krämer, HelmutToll-like receptor (TLR)/Interleukin-1 receptor (IL1R) superfamily members share signaling components and (with the exception of TLR3) depend on the adapter myeloid differentiation primary response gene 88 (MyD88) for engagement of downstream pathways. Signals from the receptor to the adapter are transmitted through homotypic interaction of TIR (Toll-Interleukin-1 receptor) homology domains found in all TLR/IL1R family members and their adapters. The present work defines a novel TLR/IL1R signaling adapter, termed BCAP (B-cell adapter for PI3K), which was identified based on the presence of a cryptic N-terminal TIR domain. I show here that BCAP (B-cell adapter for PI3K) contains a functional TIR domain enabling its participation in the TLR signaling pathway. Through its TIR domain, BCAP associates with the TLR/IL1R signaling adapter MyD88, as well as the TLR signaling adapter toll-interleukin 1 receptor domain containing adapter protein (TIRAP). Importantly, BCAP plays an obligate role in linking TLRs to activation of phosphoinositide 3-kinase (PI3K) through recruitment of PI3K to the signaling complex and relief of inhibitory influences on PI3K activity. Importantly, BCAP selectively mediates TLR signaling towards the PI3K branch without affecting signaling to NFκB nor MAP kinases. In this capacity, BCAP inhibits secretion of inflammatory cytokines and regulates susceptibility to inflammatory colitis. Because the TLR/IL1R family shares signaling components, BCAP may also function in IL1R family signaling. To test this hypothesis, T cells were chosen as a model cell type responding to IL1R family signals. T helper cells utilize IL18 and IL1 (which engage the IL18R or the IL1R respectively, both IL1R family members) cytokines provided by myeloid cells to achieve optimal Th1 and Th17 effector capacities. I show here that BCAP intrinsically regulates differentiation of naïve T cells towards Th1 and Th17 effector lineages by participation in the IL1R family signaling pathways. Further, BCAP intrinsically regulates both T cell proliferation and survival during priming. The significance of this work lies in the revelation of a TLR signaling adapter serving as a node connecting TLRs to PI3K. Further, the findings here will increase the understanding of key signaling pathways involved in disease and inflammation.Item Biochemical Dissection of Innate Immune Signaling Mechanisms Mediated by MAVS and Inflammasomes(2014-10-23) Chen, Jueqi; Tu, Benjamin; Chen, Zhijian J.; Olson, Eric N.; Buszczak, MichaelProject 1: MAVS Recruits Multiple Ubiquitin E3 Ligases to Activate Antiviral Signaling Cascades The RIG-I antiviral pathway plays a pivotal role in innate immune response against RNA viruses. Upon virus infection, viral RNA in the cytoplasm is detected by the RIG-I family of receptors, which activates an adaptor protein called MAVS (mitochondrial antiviral signaling). MAVS in turn leads to the activation of two transcription factors, NF-κB and IRF3, which coordinately induce type-I interferons and proinflammatory cytokines that are critical in eliminating viral infection. However, the exact mechanism of NF-κB and IRF3 activation by MAVS is still largely unknown. In this study we show that activated MAVS recruits three ubiquitin E3 ligases TRAF6, TRAF2 and TRAF5 to the mitochondria through distinct TRAF-binding motifs upon virus infection. Mutations that disrupt these motifs in MAVS abrogated the recruitment of these E3 ligases, and abolished the ability of MAVS to activate the downstream signaling. Interestingly, virus-induced prion-like aggregation of MAVS is essential for its interaction with these TRAF proteins as well as the downstream activation. Genetic evidence has shown that these E3 ligases function redundantly to activate NF-κB and IRF3, so that they not only amplify the antiviral signal, but also serve as backup systems against specific viruses that can degrade components in the antiviral pathway. Identifying these key players in MAVS pathway will help us explore novel therapeutic targets for infectious diseases caused by RNA viruses. Project 2: Dissecting the Mechanism of NLRP3 Inflammasome Activation Inflammasome is a multi-protein oligomer that serves as a platform for caspase-1-depedent activation of proinflammatory cytokines and the induction of a specific form of cell death termed pyroptosis. A plethora of pathogen-associated-molecular patterns (PAMPs) and damage-associated-molecular-patterns (DAMPs) have been found to activate inflammasome through NLRP3, a Nod-like receptor protein. As a result, the dysfunction of NLRP3 is closely associated with various health problems including autoimmune diseases, neurodegenerative diseases, susceptibility to pathogen infection, and metabolic disorders. Given the chemical and structural diversity of the stimuli, and the lack of evidence that NLRP3 directly interacts with these stimuli, it has been hypothesized that NLRP3 activation is triggered by a common cellular signal, the identity of which is still a mystery. We have reconstituted NLRP3 inflammasome pathway in HEK293T cell and used this system to establish a cell-free assay, in which NLRP3 from stimulated cells was added to cells containing Asc and caspase-1 to activate the downstream signaling. With this assay, we were able to obtain highly purified active NLRP3 for further characterization, and have found that dephosphorylation plays an important role in NLRP3 inflammasome activation. Moreover, both biochemistry and imaging data has suggested that NLRP3 is translocated to specific subcellular structures after stimulation, of which the significance is still under investigation. Our final goal is to dissect the detailed mechanism of NLRP3 activation, which can provide insight into the prevention and treatment of various related human diseases.Item Key Roles of Ubiquitination and Phosphorylation in RIG-I/MAVS Viral Sensing Pathway(2013-04-17) Liu, Siqi 1983-; Olson, Eric N.; van Oers, Nicolai S. C.; De Martino, GeorgeRNA virus infections are detected by the RIG-I family of receptors, which induce the production of type-I interferons (IFNs) and other antiviral molecules through the mitochondrial membrane protein MAVS. We have recently shown that MAVS forms large prion-like aggregates in response to virus infection and that these aggregates are highly potent in activating the cytosolic kinases IKK and TBK1, which in turn activate NF-κB and IRF3, respectively, to induce IFNs. However, the mechanisms remain unknown. Here I showed that MAVS aggregates recruited several TRAF proteins, including TRAF2, TRAF3, TRAF5 and TRAF6, through two distinct TRAF binding motifs. Mutations of both motifs in MAVS that disrupted its binding to the TRAF proteins were necessary to abrogate its ability to activate IRF3 and induce IFNβ. These antiviral responses were also abolished in cells lacking TRAF2, 5, and 6, but not in those lacking individual TRAF protein. These TRAF proteins catalyze ubiquitination reactions that recruit NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. MAVS phosphorylation by the recruited kinases then brings IRF3 to the complex, where IRF3 is phosphorylated by TBK1. These results reveal that MAVS, through the recruitment of multiple E3 ligases, not only activate downstream kinases but also specifies IRF3 phosphorylation by TBK1.Item LRBA Restricts Murine Colitis by Regulating Interferon Responses and Autophagy(2018-11-27) Wang, Kuan-Wen; Pascual, Juan M.; Malter, James; Chen, Zhijian J.; Beutler, BruceLrba encodes lipopolysaccharide-responsive and beige like anchor (LRBA) protein, which was originally identified as an LPS-inducible gene in immune cells. LRBA putatively regulates the recycling and degradation of proteins critical for immune quiescence in human lymphocytes. However, recent studies showed that Lrba-deficient rodents had normal adaptive immune responses. Mutations in Lrba are associated with the immune deficiency, autoimmunity, and inflammatory bowel diseases (IBDs) in human; however it is unclear how LRBA regulates intestinal homeostasis and the cellular mechanisms it involves in. Here, I showed that LRBA is critical for both adaptive and innate immunity by regulating T cell, dendritic cells and intestinal epithelial cells to control inflammation. Lrba-deficient (Lrba-/-) mice showed a murine model colitis-DSS-induced colitis, and LRBA expression was essential in the hematopoietic (in both adaptive and non-adaptive) and non-hematopoietic compartment to prevent colitis susceptibility. LRBA regulates T cell homeostasis and activation, and Lrba-/- T cells skew to more effector subsets, especially Th1 and Th17 condition, but not to naïve and regulatory subsets (naïve T and regulatory T cells) in splenocytes and colon lamina propria lymphocytes. Furthermore, Lrba-/- naïve T cells showed a greater potential to become activated, lead to intestinal inflammation. Moreover, the possible mechanism that LRBA regulates T cell function is through PI3K/AKT/mTOR signaling. Bone marrow-derived dendritic cells (BMDCs) from Lrba-/- mice contribute to intestinal inflammation in an adaptive immune cells-independent manner. Lrba-/- BMDCs displayed an accumulated endosomal Toll-like receptor (TLR) ligands in the lysosomes, and led to excessive interferons (IFNs) signaling in response to endosomal TLR stimulation through IRF3/7 pathway that was PI3K/AKT/mTOR pathway-dependent. Furthermore, blocking the endosomal TLRs translocation to endolysosome/lysosome by genetically disturbs UNC93B1 to reduce enhanced IFN signaling that partially ameliorated the experimentally-induced colitis severity. Accumulated autophagosomes was observed in both BMDCs and intestinal epithelial cells in LRBA deficient condition. The possibly impaired autophagosome-lysosome fusion led to the defect of autophagy might be the reason for accumulated autophagosomes, further exaggerated a potential intracellular cell stress from dysfunctional organelles to dampen the inflammation from the dysregulated endosomal TLR signaling. Taking together, the possibility of LRBA functions in the process of vesicle fusion (endosome-autophagosome-lysosome) that suggests LRBA could be as a new potential linker between innate endosomal TLR signaling, adaptive immune functions and autophagy. Therefore, deficiency of LRBA can lead to excessive inflammatory responses and induction of colitis.Item Molecular Mechanisms and Functions of Estrogen Receptor Enhancers in Hormone-Dependent Gene Expression(2017-11-21) Murakami, Shino; Kim, Tae-Kyung; Kraus, W. Lee; Kliewer, Steven A.; Zhang, Chun-LiTranscription is a fundamental regulatory mechanism of biological processes in a range of physiological and pathological conditions. Transcription enhancers are DNA regulatory elements that regulate the expression of the target genes by accommodating transcription factor (TF) binding through sequence specificity. Estrogen receptor alpha (ERα) belongs to ligand-dependent nuclear receptor superfamily. Upon activation by estrogenic ligands, ERα binds to specific sites on chromatin, and assembles and activates enhancer complexes, which in turn lead to the transcription of target genes. Various molecular events have been associated with enhancer function, including coregulator recruitment, induction of enhancer-enriched histone modifications, nucleosome remodeling, enhancer-promoter chromatin interactions, and transcription activation at the enhancer, as well as the target gene promoter. However, we lack a clear understanding of the order of events, the specific roles of each coregulator and enhancer-enriched chromatin features, and the functional relationships among them. Using ERα in estrogen (E2)-regulated gene transcription as a model in combination with molecular and cellular biology, as well as genomic and computational approaches, my dissertation herein describes a series of studies elucidating the molecular mechanisms and functions of these evens that lead to ERα enhancer activation. Collectively, it demonstrates that (1) ERα enhancer assembly and activation is a dynamic process, (2) the temporally-defined recruitment and activation of key coregulators are required for successful activation of ERα enhancers, and (3) enhancer transcripts (eRNA) mark active enhancers. Lastly, I delineate the development of a new technology, single-cell Global Run-on Sequencing (scGRO-seq), to uncover the link between enhancer activity and target gene transcription at the single-cell level. Single-cell imaging and sequencing technologies have demonstrated the heterogeneous nature of gene expression and enhancer activity in a wide range of biological systems, including clonally-expanded populations of cultured cells. However, our understanding on the molecular basis of heterogeneous gene expression is limited because of a lack of technologies that allow us to simultaneously examine enhancer activity and target gene transcription at the single-cell level. scGRO-seq will overcome this problem by capturing active transcription at the enhancers, which is an indicative of enhancer activity, and at the target gene in the same cells.Item Nur77 Prevents Excessive Osteoclastogenesis by Inducing Ubiquitin Ligase Cbl-b to Mediate NFATc1 Self-Limitation(2020-07-21) Li, Xiaoxiao; Mendelson, Carole R.; Zinn, Andrew R.; Kliewer, Steven A.; Wan, YihongOsteoclasts are bone-resorbing cells essential for skeletal remodeling. However, over-active osteoclasts can cause bone degenerative disorders. Therefore, the level of NFATc1, the master transcription factor of osteoclast, must be tightly controlled. Although the activation and amplification of NFATc1 have been extensively studied, how NFATc1 signaling is eventually resolved is unclear. Here, we uncover a novel and critical role of the orphan nuclear receptor Nur77 in mediating an NFATc1 self-limiting regulatory loop to prevent excessive osteoclastogenesis. Nur77 deletion leads to low bone mass owing to augmented osteoclast differentiation and bone resorption. Mechanistically, NFATc1 induces Nur77 expression at late stage of osteoclast differentiation; in turn, Nur77 transcriptionally up-regulates E3 ubiquitin ligase Cbl-b, which triggers NFATc1 protein degradation. These findings not only identify Nur77 as a key player in osteoprotection and a new therapeutic target for bone diseases, but also elucidate a previously unrecognized NFATc1→Nur77→Cblb→NFATc1 feedback mechanism that confers NFATc1 signaling autoresolution.Item Phase Transitions of Multivalent Adaptor Proteins(2015-05-27) Banjade, Sudeep; Rice, Luke M.; Rosen, Michael K.; Liou, Jen; Ross, Elliott M.Eukaryotic cells efficiently organize their activities to achieve their functional capabilities. This organization of biochemical reactions is a direct result of the cells' ability to compartmentalize their molecules. For example, within a eukaryotic cell, compartments like the nucleus, the endoplasmic reticulum and the vacuoles exist, which are relatively well known for their specific functions. These aforementioned compartments are surrounded by membranes. However, for the past hundred years, we have also known about assemblies of biomolecules that are not bound by membranes. After the initial discovery of nuages, other structures such as Cajal bodies, the nucleolus, promyelocytic leukemia (PML) bodies, paraspeckles, etc., were also described as membraneless organelles. Furthermore, membranes themselves are self-assembled entities of lipids, proteins and carbohydrates. Additionally, within and on surface of membranes, molecules cluster into signaling compartments in many different biological pathways. Interactions between individual biomolecules have been studied comprehensively in biology. One of our goals as biophysicists is to attempt to propose physical properties that allow these interactions at the subnanometer scale to give rise to formation of cellular structures, the compartments that are listed above. This thesis proposes a hypothesis based on polymerization of multivalent proteins that causes these complexes to phase separate in solution. The behavior of multivalent proteins and their ligands to phase separate may be a general property that allows cells to regulate their activities in certain localized compartments. To study this larger goal, I used a specific example of proteins involved in creating the slit-diaphragm, which is the filtration barrier of our kidneys. Nephrin, an integral membrane protein at the slit-diaphragm, interacts with its partners Nck and N-WASP in a multivalent fashion. I show here that these interactions create large assemblies that phase separate into liquid droplets, both in solution and on membranes. I also find that the creation of these assemblies affects the downstream biochemical activity of N-WASP toward the Arp2/3 complex and actin. The widespread existence of multivalent molecules suggests that these findings may have broad corollaries in different biological systems.Item Regulation of Reparative Macrophage Transition by the B-cell Adapter for PI3K (BCAP)(2021-05-01T05:00:00.000Z) Irizarry-Caro, Ricardo A.; Satterthwaite, Anne B.; Pasare, Chandrashekhar; Street, Nancy E.; Tagliabracci, Vincent S.; Zaki, HasanMacrophages respond to microbial ligands and various noxious cues by initiating an inflammatory response aimed at eliminating the original pathogenic insult. Transition of macrophages from a pro-inflammatory state to a reparative state, however, is vital for resolution of inflammation and return to homeostasis. The molecular players governing this transition remain poorly defined. Here, we find that the reparative macrophage transition is dictated by B-cell adapter for PI3K (BCAP). Mice harboring a macrophage specific deletion of BCAP fail to recover from and succumb to DSS-induced colitis due to prolonged intestinal inflammation and impaired tissue repair. Following microbial stimulation, gene expression in WT macrophages switches from an early inflammatory signature to a late reparative signature, a process that is hampered in BCAP deficient macrophages. We find that absence of BCAP hinders inactivation of FOXO1 and GSK3b that contributes to their enhanced inflammatory state. BCAP deficiency also results in defective aerobic glycolysis and reduced lactate production. This translates into reduced histone lactylation and decreased expression of reparative macrophage genes. Thus, our results reveal BCAP to be critical cell intrinsic switch that regulates transition of inflammatory macrophages to reparative macrophages by imprinting epigenetic changes.Item Regulatory Mechanisms of Semaphorin/Plexin/Mical-Mediated F-actin Disassembly and Cellular Remodeling(2017-04-14) Rich, Shannon Kay Good; Johnson, Jane E.; Terman, Jonathan R.; Krämer, Helmut; Alto, NealDynamic changes to the actin cytoskeleton modify the shape of cells and their membranous extensions, and underlie diverse developmental and functional events in multiple tissues including migration, navigation, and connectivity. Semaphorins, together with their Plexin receptors, are a large family of extracellular cues that trigger complex cytoskeletal rearrangements to direct these cellular phenomena, but the mechanisms regulating their effects are poorly understood. Emerging evidence identifies Mical, a conserved oxidoreductase (Redox) enzyme, as a critical component in Semaphorin/Plexin signaling through its post-translational oxidation of F-actin, which promotes actin instability and disassembly. How this Mical-mediated redox regulation of actin dynamics is locally positioned and coordinated with the activity of other actin regulatory proteins to achieve specific, targeted effects on the cytoskeleton remains unknown. Therefore, as a part of my dissertation research, I used a genetic assay to begin to address these questions and search for proteins that could alter Semaphorin/Plexin/Mical signaling effects on the cytoskeleton. In this dissertation, I present my discovery of a functional interplay between Mical and two critical new interactors - cofilin, a well-known ubiquitous F-actin regulatory protein, and Sisyphus, an unconventional class XV myosin. With regards to cofilin, my in vivo genetic/functional assays reveal that cofilin activity is required for and enhances Semaphorin/Plexin/Mical-dependent cytoskeletal rearrangements and morphological changes. Additionally, in vitro biochemical assays demonstrate that cofilin preferentially binds Mical-oxidized actin and accelerates its disassembly. Together, these findings indicate that cofilin and Mical act as a functional pair in both neuronal and non-neuronal cells to rapidly and efficiently disassemble actin filaments. Similarly, my results reveal that Sisyphus is necessary and sufficient for triggering Semaphorin/Plexin/Mical-dependent F-actin disassembly/cellular remodeling. Moreover, using in vivo functional assays, I find that Sisyphus uses its myosin motor activity and the first MyTH4 domain of its C-terminal tail region to modify the subcellular localization of Mical. In this way, Sisyphus spatially controls Mical-dependent F-actin disassembly/cellular remodeling. Therefore, both cofilin and Sisyphus function to promote Mical-mediated F-actin disassembly; thereby, they act as critical regulators of Semaphorin/Plexin/Mical-mediated effects on cytoskeletal and morphological dynamics. Thus, my findings unveil novel molecular and biochemical mechanisms that orchestrate cellular, developmental, and neural biology.Item Roles of MAVs and MYD88 in Innate and Adaptive Immune Responses to Respiratory Syncytial Virus(2010-05-14) Bhoj, Vijay Garud; Chen, Zhijian J.Detection of invading viruses by pathogen recognition receptors (PRRs) first illicits an innate immune response which includes the production of anti-viral interferons. TLRs 3, 4, and 7/8 and the RLR, RIG-I, are the primary PRRs involved in the recognition of RNA viruses and they signal through the adaptors TRIF (TLR3, 4), MyD88 (TLR4, 7/8, 9), and MAVS (RIG-I). The innate response serves to limit viral spread and to activate adaptive immune responses which eventually clear the infection. We dissected the contribution of TLR- and RLR- mediated recognition in the host response to Respiratory Syncytial Virus, a common human pathogen. Deletion of Mavs abolished the induction of type I interferon (IFN-I) and other pro-inflammatory cytokines by RSV. Genome-wide expression profiling in the lung showed that the vast majority of RSV-induced genes depended on MAVS. Although Myd88 deficiency did not affect most RSVinduced genes, mice lacking both adaptors harbored higher and more prolonged viral load and exhibited more severe pulmonary disease than those lacking either adaptor alone. Surprisingly, Myd88-/-Mavs-/- mice were able to activate a subset of pulmonary DCs which traffic to the draining lymph node in response to RSV. These mice subsequently mounted a normal cytotoxic T lymphocyte (CTL) response and demonstrated delayed but effective viral clearance. These results provide an example of a normal and effective adaptive immune response in the absence of innate immunity mediated by MAVS and MyD88.Item Spindle Checkpoint Silencing by TRIP13(2017-10-30) Brulotte, Melissa Lynn; DeBose-Boyd, Russell A.; Yu, Hongtao; Luo, Xuelian; Burma, Sandeep; Roth, Michael G.The spindle checkpoint is important for maintaining genomic stability and preventing aneuploidy, a hallmark of cancer. The checkpoint ensures that chromosome segregation does not occur until all sister chromatids are correctly attached to the mitotic spindle during metaphase. When this requirement is met, the checkpoint must be silenced for the cell to proceed to anaphase. Thyroid hormone receptor interacting protein 13 (TRIP13) is a hexameric AAA+ ATPase involved in spindle checkpoint silencing. TRIP13 functions by initiating a conformational change in mitotic arrest deficient 2 (Mad2), a key component of the mitotic checkpoint complex (MCC). This TRIP13-mediated conformational change of Mad2 causes MCC disassembly and relieves inhibition of the anaphase promoting complex/cyclosome (APC/C). The interaction between TRIP13 and Mad2 is dependent on the p31comet adaptor protein. In my first project, I show that TRIP13-p31comet disrupts the MCC by local unfolding of Mad2. I identify a binding surface on human TRIP13 for p31comet-Mad2 and key TRIP13 residues involved in its conformational dynamics. I propose that the flexibility of the hinge region of TRIP13 is important for coupling its ATPase activity to substrate unfolding. The hinge region is conserved in other eukaryotic AAA+ ATPases, and may also be important for energetic coupling in those systems. I have also reconstituted the process of spindle checkpoint silencing in vitro. Importantly, I show that TRIP13 can disrupt the free MCC complex, but not MCC bound to APC/C, providing an explanation for the coordination of the multiple mechanisms that work together to achieve spindle checkpoint silencing. In my second project, to provide a tool for future mechanistic studies and to examine the oncogenic activity of TRIP13, I attempted to identify chemical inhibitors for TRIP13 through high-throughput screening. I identified a series of lead compounds that indirectly inhibited TRIP13 as pan-assay interference compounds. These compounds are redox cyclers that generate hydrogen peroxide, which covalently modifies protein residues such as cysteines and tryptophans. No other potent lead compounds were discovered. This study revealed that TRIP13 may be a difficult protein to target, and that large compound libraries should be prescreened for redox cyclers before they are used in high-throughput inhibitor screening.Item Structural Basis for the Activation of RIG-I/MAVS Antiviral Immune Signaling(2015-04-09) Xu, Hui; Rice, Luke M.; Chen, Zhijian J.; Jiang, Qiu-Xing; Rosen, Michael K.; Liu, QinghuaRetinoic acid inducible gene-I (RIG-I) is a key cytosolic pathogen RNA sensor that activates mitochondrial antiviral signaling protein (MAVS) to trigger rapid innate immune responses. Using RNAs of different lengths as model ligands, we showed that RIG-I oligomerized on dsRNA in an ATP hydrolysis-dependent and dsRNA length-dependent manner, which correlated with the strength of type-I interferon (IFN-I) activation. The obtained negative stain EM structure of full-length RIG-I in complex with a 5'ppp stem-loop RNA and the crystal structure of RIG-I/Ub complex elucidated a two-step oligomerization and conformational change of RIG-I for activation. RIG-I oligomers nucleate MAVS through homotypic interaction of the N-terminal caspase activation and recruitment domains (CARDs) and induce the formation of prion-like aggregates. The obtained cryoEM structure of left-handed helical filaments of MAVS CARD revealed specific interfaces between individual CARD subunits that are dictated by a combination of electrostatic and hydrophobic interactions and hydrogen bonding. Point mutations at multiple locations of these interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidated the structural mechanism of RIG-I activation by RNA and K63-linked ubiquitin chains as well as the activation of MAVS through polymerization, revealing a highly efficient signaling cascade for viral RNA sensing.Item T Cell Intrinsic BCAP Links IL1R to the PI3K-mTOR Pathway and Regulates Pathogenic Th17 Differentiation(2018-07-24) Deason, Krystin Leigh; Hooper, Lora V.; Pasare, Chandrashekhar; van Oers, Nicolai S. C.; Satterthwaite, Anne B.Toll-IL-1R homology (TIR) domains are found within adaptor proteins involved in the signaling of Toll like receptors (TLRs) and Interleukin 1 receptor (IL1R) families. Previous work by our lab identified a TIR domain in the protein B cell adaptor for phosphoinositide 3-kinase (BCAP) and determined a role for BCAP in the TLR signaling pathway in myeloid cells. Due to the shared use of TIR domains by TLR and IL1R signaling pathways, I hypothesized that BCAP would also be involved in signaling downstream of the IL1 family of receptors. The IL1 cytokine family has been shown to play a major role in T cell activation, survival, and differentiation; IL1b, in particular, plays a critical role in the differentiation of Th17 lineage cells. Here, I discovered that BCAP functions downstream of IL1R in CD4 T cells and thereby regulates Th17 lineage differentiation and function. IL1b-induced PI3K-Akt-mTOR signaling is compromised in BCAP deficient T cells which leads to decreased mTOR activation, decreased glycolysis, and defective Th17 lineage commitment. Transcriptional analysis of BCAP deficient CD4 T cells revealed that BCAP is critical for the expression of genes associated with pathogenic Th17 lineage cells. Mice specifically lacking BCAP in T cells have normal development of steady state Th17 cells in vivo yet have decreased development of pathogenic Th17 diseases, such as experimental autoimmune encephalomyelitis (EAE) and T cell transfer colitis. Further, the use of a potent inhibitor of mTOR, which is downstream of BCAP activation, mimics BCAP deficiency by preventing IL1b induced differentiation of pathogenic Th17 cells. This study establishes BCAP as a critical link between IL1R and the metabolic status of activated Th17 cells and further demonstrates that BCAP is critical for the generation of pathogenic Th17 cells in vitro and in vivo.