Browsing by Subject "Signal Transduction"
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Item B Cell Signaling and Bioinformatics: Revealing Components of the MHC Class II Antigen Processing and Presentation Pathway(2005-05-11) Lee, Jamie Anne; Scheuermann, Richard H.Stimulation of mature B lymphocytes by extracellular ligands induces phenotypic changes through complex signal transduction pathways. Gene expression is altered as a result of these changes and re-programs the cell to undergo differentiation, activation, effecter function, anergy, and/or apoptosis. Gene expression microarrays are used to determine expression levels of a large number (tens of thousands) of genes simultaneously, resulting in a gene expression profile of the experimental sample. Microarray data must be appended with biological information in order to be interesting, and this field of microarray bioinformatics is rapidly expanding. These studies prompted the development of a bioinformatics tool termed CLASSIFI (Cluster Assignment for Biological Inference), which identifies statistically significant co-clustering of genes with similar Gene Ontology annotation within microarray gene clusters. CLASSIFI was used to analyze microarray results from two B cell projects from the Alliance for Cellular Signaling (AfCS): 1) the BAFF/CD40L project, which evaluates the effects of BAFF and CD40L on primary mouse B cells in long-term cultures, and 2) the B cell single ligand screen project, which evaluates the effects of 32 single ligands on primary mouse B cells in short-term cultures. CLASSIFI was able to identify significant overrepresentation of related genes within gene clusters for both of these data sets and facilitates hypothesis generation as to the biological process affected by a specific ligand. As CLASSIFI is strictly a statistical tool that aids in hypothesis generation, experimental validation of hypotheses was performed. The B cell single ligand screen microarray and CLASSIFI analysis followed by experimental validation revealed a biological process specific to B cell antigen receptor stimulation but not LPS or CD40L stimulation - antigen processing and presentation - and provides the groundwork for new discoveries in this field. As a result, several putative components were identified that are not currently known to play a role in antigen processing and presentation in B cells.Item Biochemical and Biophysical Characterization of the Allosteric Equilibrium of the Wiskott-Aldrich Syndrome Protein(2005-12-20) Leung, Daisy W.; Rosen, Michael K.Proteins provide the essential building blocks of signal transduction pathways governing many biological processes. They are dynamic entities endowed with properties that allow them to respond to changes in the cell while maintaining specificity and fidelity of signaling in a crowded intracellular environment. Many signaling proteins are regulated by a subset of allostery where intramolecular interactions modulate the conformational equilibrium between an uninhibited, inactive state and an active state. Relief of autoinhibition then requires that covalent modification or binding events shift the equilibrium to favor the active state. However, the structural, biochemical, and biophysical properties of many autoinhibited systems have not been characterized. Thus, an understanding of how binding events are coupled to effector activation remains incomplete. Previous work in our lab has described a framework based upon classical descriptions of allostery with which we can examine the regulation and activation of the Wiskott-Aldrich Syndrome protein (WASP) by the Rho-family GTPase Cdc42. Results from this work revealed that a simple two-state model can predict the hydrogen exchange behavior and binding affinity for Cdc42-GTP of WASP proteins over a range of folding stabilities. The goals of my thesis have been: 1) to further develop the quantitative twostate model of the allosteric regulation of WASP, 2) to understand how binding of Cdc42 is coupled to WASP activation toward Arp2/3 complex-mediated actin polymerization, and 3) to understand the implications of the GTPase nucleotide switch on effector activation. In order to expand upon the range of WASP protein folding stabilities and to, more importantly, examine the relationship between Cdc42 binding and WASP activity towards the Arp2/3 actin nucleation complex, I generated a different series of WASP proteins by introducing mutations in the autoinhibited core of a more physiologically relevant WASP construct, which I used to parameterize our two-state model. Model predictions of WASP affinity for Cdc42, activity toward Arp2/3 complex, and activation by Cdc42 are all borne out experimentally and are functions of the two-state allosteric equilibrium of WASP. Application of the model to Cdc42-GDP revealed that the ratio of binding affinities for the inactive and active states of WASP is significantly smaller for Cdc42-GTP than for Cdc42-GDP. Thus, the GTP-bound state of Cdc42 is more effective at distinguishing between the two states of WASP, converting Cdc42-GDP from a partial agonist to a full agonist of WASP. Therefore, the nucleotide switch in Cdc42 is not only based upon a change in affinity for the two states of WASP, but also the efficiency of coupling between the binding and allosteric equilibria of WASP. These properties have important implications for how specificity and fidelity of signaling can be maintained in a crowded intracellular environment, ensuring that only Cdc42-GTP can activate WASP and signal downstream.Item Biochemical Characterization of the Yersinia Effector Protein, Yopjbiochemical Characterization of the Yersinia(2007-05-22) Mukherjee, Sohini; Orth, KimYersinia species, the causal agent of plague and gastroenteritis, uses a variety of type III effector proteins to target eukaryotic signaling systems. The effector YopJ disrupts the mitogen-activated protein kinase and the nuclear factor κ B signaling pathways used in innate immune response by preventing activation of the family of mitogen-activated protein kinase kinases. The catalytic domain of YopJ is similar to Clan CE of cysteine proteases, and mutating the putative catalytic cysteine disrupts YopJ's inhibitory activity. YopJ binds mitogenactivated protein kinase kinases, including MKK1 through MKK6, and the related kinase, IκB kinase beta, however, the mechanism by which this binding leads to inactivation of these kinases is unknown. An in vitro cell-free signaling system was developed to recapitulate the inhibition of eukaryotic signaling by YopJ. Mass spectrometric studies were undertaken to determine the biochemical nature of modification of the mitogen-activated protein kinase kinases in the presence of YopJ. Based on the observations, a simple, molecular mechanism utilized by YopJ to block the signaling pathways was discovered. YopJ acted as an acetyltransferase, using acetyl coenzyme A, to modify the critical serine and threonine residues in the activation loop of mitogen-activated protein kinase kinases and thereby blocking phosphorylation. The acetylation on the kinase directly competed with phosphorylation, preventing activation of the modified protein. An essential characteristic feature of bacterial effector proteins is that they usurp or mimic a eukaryotic activity and refine this activity to produce an extremely efficient mechanism to combat eukaryotic signaling. Therefore, modification of amino acids, other than lysine, by acetylation could be a commonly used eukaryotic mechanism that has been undetected previously. The acetylation of these amino acids may compete with various other types of posttranslational modifications, such as ubiquitination, SUMOylation and glycosylation. Several questions that still need to be addressed are: Is this modification reversible? What are the eukaryotic proteins that add and remove this type of posttranslational modification? How do bacterial effectors use this activity? The characterization of a bacterial effector as a serine or threonine acetyltransferase presents a previously unknown paradigm to be considered for other biological signaling pathways.Item Biochemical Mechanism of Protein Kinase Activation by the Ubiquitination System(2008-05-13) Xia, Zong-Ping; Chen, ZhijianThe 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 Casein Kinase I Transduces WNT Signals(2005-08-11) Peters, John Michael; Graff, JonathanWnt signaling controls a diverse array of processes including cell growth, oncogenesis, and development. Components of the Wnt cascade are altered in several human cancers including colon cancers and melanomas. We set out to identify novel components of this signal transduction pathway via expression cloning in Xenopus embryos. This assay identified a protein kinase, casein kinase I (CKI), as a novel component of the Wnt signaling pathway. We first showed that expression of CKI produces Wnt phenotypes. First, CKI induced completely formed second axes, or Siamese twins, when injected into embryos. Second, embryos which had been ventralized by UV-irradiation were completely rescued by CKI, producing normal embryos. In order to extend these observations, we tested CKI in three biochemical assays of Wnt signaling. First, CKI expression stabilized b-catenin protein levels, a hallmark of Wnt signaling. Second, CKI induced the expression of Siamois and Xnr3, direct transcriptional targets of Wnt signaling. Finally, CKI expression dorsalized the ventral marginal zone in Xenopus, inducing markers of the Spemann organizer which is a product of Wnt signaling. We next investigated the ability of CKI inhibitors to block Wnt signaling in vivo. Two dominant negative forms of CKI and a specific pharmacological inhibitor of CKI all blocked the ability of Xwnt8 to induce dorsal axes and target genes in embryos. These data demonstrate that CKI function is required for transduction of Wnt signals. Additionally, we also demonstrated that CKI function is required for Wnt signaling in the nematode C. elegans, proving that CKI function in this pathway is conserved from invertebrates to vertebrates. We then investigated the biochemical mechanism of CKI function. Epistasis experiments indicated that CKI acts between Dishevelled and GSK-3. Yeast twohybrid assays showed that CKI strongly binds Dishevelled. Through in vivo phosphorylation experiments, we showed that CKI increases Disheveled phosphorylation and in vitro experiments showed that CKI can directly phosphorylate Dishevelled. To extend our studies we performed further experiments using additional CKI isoforms and found that many other isoforms can also reproduce markers of Wnt signaling. In summary, through a combination of gain-of-function and loss-of-function studies in invertebrates and vertebrates, we have shown that CKI is a novel and conserved component of the Wnt signaling pathway.Item CDC14 Coordinates Cyclin Destruction With the Onset of Cytokinesis(2004-08-19) Bembenek, Joshua Nathaniel; Yu, HongtaoThe Cdc14 family of protein phosphatases operate during the final stages of mitosis in various organisms. The Cdc14 phosphatases are downstream components of two homologous signaling pathways: the mitotic exit network (MEN) of S. cerevisiae and septation initiation network (SIN) of S. pombe. Studies of these pathways have revealed divergent roles of Cdc14. In the MEN pathway, Cdc14 is required for cyclin degradation by dephosphorylating Cdh1. The dephosphorylated form of Cdh1 binds to and activates a ubiquitin ligase known as the anaphase-promoting complex (APC/C), which then ubiquitinates mitotic cyclins, targeting them for degradation by the 26S proteosome. In contrast, Cdc14 of the SIN is dispensable for cyclin degradation, but plays an important role during cytokinesis. Two Cdc14 homologues are found in vertebrates, hCdc14A and hCdc14B. I have investigated the regulation of Cdc14 phosphatases to obtain insights into the mechanisms of mitotic exit in higher eukaryotes. Biochemical studies demonstrate that recombinant hCdc14A and hCdc14B can dephosphorylate human Cdh1 and stimulate APC/CCdh1 ligase activity in vitro. Since both the MEN and SIN pathways control Cdc14 localization, I have examined the regulation of the subcellular localization of hCdc14A, hCdc14B and the budding yeast Cdc14. In HeLa cells, hCdc14A localizes to the centrosome whereas hCdc14B is nucleolar during interphase. Both hCdc14 homologues localize to the centrosome and midbody during mitosis. In budding yeast, Cdc14p localizes to the nucleolus during most of the cell cycle and is released in late anaphase when it localizes to the centrosome and the bud neck. The subcellular localization the Cdc14 homologues in HeLa cells is regulated by a nuclear export signal. S. cerevisiae strains carrying only NES mutant CDC14 alleles are capable of degrading mitotic cyclins and escaping mitosis. However, they exhibit a temperature-sensitive phenotype at 37°C because they fail to complete cytokinesis and lack centrosome and bud neck localization of Cdc14. This demonstrates that the Cdc14 phosphatases are regulated by nucleocytoplasmic shuttling. Collectively, my work strongly suggests that the Cdc14 phosphatases play a conserved role in coordinating the destruction of mitotic cyclins with the execution of cytokinesis.Item Control of Skeletal Muscle Fiber Types by Calcium Signaling Pathways(2002-08-01) Hai, Wu; William, R. SandersDifferent patterns of motor nerve activity drive distinctive programs of gene expression in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myofiber subtypes. Previous studies have demonstrated that calcineurin activity is required to maintain slow myofiber identity. I am interested in determining the transcription factors downstream of calcineurin and other calcium-regulated signaling pathways in the control of myofiber specialization. By analyzing two fiber type-specific enhancers, I was able to demonstrate that there are functional NFAT (nuclear factor of activated T cells) and MEF2 (myocyte-specific enhancer factor 2) binding sites within the enhancer of troponin I slow, and both sites are required for slow fiber specific activity of this enhancer. Next, I identified MEF2 as a target of calcineurin in cultured myogenic cells. Calcineurin physically interacts with MEF2 and dephosphorylates MEF2. C-terminal transactivation domain, but not N-terminal DNA binding domain of MEF2, responds to calcineurin activation. The use of "MEF2 indicator" transgenic mice that harbor a MEF2-dependent lacZ transgene enabled us to monitor the endogenous activities of MEF2 transcription factors. MEF2 is selectively active in slow and oxidative myofibers. Calcineurin is both necessary and sufficient for MEF2 activation in skeletal muscles. I also found a dose-response relationship between calcineurin activity and expression level of slow, oxidative fiber-specific and MEF2 target genes. Furthermore, I observed that functional activity of MEF2 transcription factors was stimulated by sustained periods of endurance exercise or low-frequency motor nerve pacing in a calcineurin-dependent manner. In addition to calcineurin, CaMKs (calcium, calmodulin-dependent kinases) also transduce their signaling through MEF2. CaMKIV synergistically activates MEF2-dependent gene expression together with calcineurin. Transgenic mice expressing constitutively active CaMKIV in their skeletal muscles showed increased percentage of slow and oxidative myofibers, which was accompanied by increased mitochondrial biogenesis mediated through the upregulation of PGC-1 (PPARg co-activator). Taken together, these findings delineate a molecular pathway in which MEF2 and NFAT integrate signaling inputs from multiple calcium-regulated pathways in the control of skeletal muscle fiber types.Item Control of the Interferon Regulatory Factor - 3 Antiviral Pathway by The Hepatitis C Virus Ns3/4a Protease(2007-05-22) Foy, Eileen; Gale, Michael Jr.Hepatitis C Virus (HCV) is a major human pathogen that affects 200 million people worldwide. A majority of people exposed to HCV become chronically infected. In order to persist, the virus must encode mechanisms to subvert host immune defenses. We hypothesized that HCV disrupts critical host intracellular antiviral signaling pathways that culminate in activation of the antiviral response. Using the cell-based replicon system, HCV was found to inhibit the activation of critical intracellular signaling pathways. Our studies identified the NS3/4A protein as an interferon regulatory factor 3 (IRF-3) and NF-KB antagonist and further mapped this activity to the NS3/4A serine protease domain. HCV antagonism of IRF-3 and NF- KB prevents the expression of interferon (IFN) and IFN-stimulated genes required for viral clearance and the generation of a HCV-specific adaptive immune response. Upstream signaling components of IRF-3 were unknown, therefore, the NS3/4A protease was utilized as a tool to elucidate putative upstream signaling components. Characterization of Toll-like receptor 3 (TLR3) in HCV infection indicated that the NS3/4A protease cleaves TRIF, an essential adaptor protein required for TLR3 signaling. However the TLR3 pathway was not found to be essential for generating the intracellular antiviral response. Furthermore, these studies identified the retinoic acid inducible gene-I (RIG-I) as an intracellular viral double-stranded (ds)RNA sensor through molecular cloning and the characterization of a HCV replication permissive cell line defective in IRF-3 activation. The NS3/4A protease disrupted IRF-3 signaling through both the RIG-I pathway as well as the homologous MDA-5 pathway during viral infection. Lastly, these studies identified novel roles for TBK1, TANK and NEMO as signal transduction intermediates in the intracellular dsRNA response pathways. The HCV NS3/4A protease is critical for proper viral polyprotein processing, in addition to antagonism of intracellular antiviral signaling pathways. Therefore, the HCV NS3/4A protease is an ideal target for novel therapeutics. Indeed, the HCV replicon could be effectively eliminated from a persistently infected cell line through the use of an experimental NS3/4A protease inhibitor. These results offer great promise for the effective treatment of chronic HCV infection.Item Design of a Website and Signaling Map Template Acting as a Database Interface and Providing a Visual Explanation of Cellular Signaling Networks an Their Operative Components(2004-12-30) Wable, Lisa Jo; Calver, LewisThe intention of this thesis is to document and describe the design of a website and signaling map template wherein the maps are standardized, expandable in detail, and act as a database interface while providing a visual explanation of cellular signaling networks and their operative components. This template was designed to provide scientific researchers in the cell signaling community with a unique method of presenting cell signaling networks (maps) within the web-based environment that can be used by persons with little web design experience. It was further designed to offer the scientific community a new way to access cell signaling network (map) information which provides the viewer/researcher multiple levels of map detail, consistent map formats, and a map interface that taps into a large database with information on specific molecules and isoforms, narratives related to biochemical and dynamic processes, references, illustrations, animations, or other information which the webpage author may feel relevant to the specific network. For this thesis, development of the website and a set of sample pages with maps on chemotaxis were constructed for the Alliance for Cellular Signaling (AfCS). A manual was written and assembled for use by key AfCS members to use the template and thus expand the Signaling Maps pages of the AfCS website, and the sample pages from this thesis project were posted online for members to view and evaluate. This thesis explores the needs of the cell signaling community and the AfCS, discusses the unique environment of the internet as a medium for displaying and sharing cell signaling pathway information, and documents the research, creative process, and discovery that went into the creation of the template and sample web pages.Item FOXO is the Mediator Linking Temporal Differentiation and the Insulin Signaling Pathway(2005-12-20) Lah, Carol Joonhyun; Cameron, ScottThe timing of differentiation is crucial for the correct development of an organism, because specific pathways can be used reiteratively to differentiate cells. Until recently, the molecular mechanism behind the temporal control of differentiation has remained elusive. Bateman and McNeill (2004) revealed a novel role for the insulin/insulin-like growth factor receptor (InR) pathway in regulating the timing of differentiation in neuronal photoreceptor cells in the Drosophila compound eye. The link between the InR pathway and temporal differentiation is significant, because of the implication that external factors, e.g. nutrition, are tightly coupled to the timing of differentiation. This proposal tests the hypothesis that FOXO, a crucial component of the InR pathway, mediates the regulation of developmental timing. The aims are the following: 1. Observe if dFOXO mutants affect temporal differentiation in the Drosophila eye. 2. Perform epistasis experiments to determine if dFOXO is downstream of other insulin signaling components. 3. Analyze the downstream targets of dFOXO that may play a role in neuronal differentiation.Item From Flies to Mice: Drosophila as a Model System to Study Fat Biology(2006-12-20) Suh, Jaemyoung; Graff, Jonathan M.Adipose tissues are found in a wide range of animal species ranging from invertebrates to mammals and are essential for many aspects of the animal life cycle. Fat biology has taken on a new and urgent importance as it is intimately linked to one of the most prevelant and costly health problems of our time - obesity. Model organisms are a powerful resource for the discovery of genes critical to human health and disease. Thus far, the utility of Drosophila melanogaster as a model system to study fat biology has not been critically evaluated. My studies highlight the potential of this system to uncover new genes and pathways that may impact our current understanding of adipose biology. Hedgehog signals regulate invertebrate and vertebrate development, yet the role of the cascade in adipose development was undefined. I found that fat body specific transgenic activation of Hedgehog signaling inhibited fly fat formation. Conversely, fat body specific Hedgehog blockade stimulated fly fat formation. Strikingly, the anti-adipogenic effect of Hedgehog signaling was conserved in mammalian adipogenic models in both sufficiency and necessity tests. Hedgehog signals elicit this function early in adipogenesis, upstream of PPARgamma, potentially acting as a molecular switch diverting preadipocytes and mesenchymal prescursors away from adipogenesis and towards osteogenesis. In another study, I investigated the role of Adipose, an evolutionarily conserved gene isolated from naturally occurring obese flies. Through gain- and loss-of-function studies in flies, mammalian cell culture, and mice I showed that the Adipose pathway plays a conserved role in obesity and diabetes. Furthermore, I found that Adipose controls the activity of PPARgamma, a central regulator of adipogenesis and the target of the thiazolidinedione class of anti-diabetic drugs. Adipose inhibits PPARgamma function by directly binding Med23, a component of the Mediator Complex, which connects transcription factors with RNA polymerase II. Taken together, I have shown that both the Hh and Adp pathway have evolutionary conserved functions in fat formation. These results support the idea that Drosophila is a useful system to study adipose biology and discoveries in flies will lead to biological insights relevant to mammals and the treatment of obesity and diabetes.Item Functional Genomics Based Interrogation of Cell-Fate Determination Pathways(2011-08-26T17:34:56Z) Jacob, Leni Susan; Lum, LawrenceThe Hedgehog (Hh) and Wnt signal transduction pathways are master regulators of embryogenesis and tissue renewal and represent anticancer therapeutic targets. Using genome-wide RNA interference screening in murine cultured cells, I established previously unknown associations between these signaling pathways and genes linked to developmental malformations, diseases of premature tissue degeneration, and cancer. I identified functions in both pathways for the multitasking kinase Stk11 (also known as Lkb1), a tumor suppressor implicated in lung and cervical cancers. Stk11 loss resulted in disassembly of the primary cilium, a cellular organizing center for Hh pathway components, thus dampening Hh signaling. Loss of Stk11 also induced aberrant signaling through the Wnt pathway. Chemicals that targeted the Wnt acyltransferase Porcupine or that restored primary cilia length by inhibiting the tubulin deacetylase HDAC6 (histone deacetylase 6) countered deviant pathway activities driven by Stk11 loss. My study demonstrates that Stk11 is a critical mediator in both the Hh and the Wnt pathways, and that functional genomics based approaches to dissecting cell-fate determination pathways may support the development of targeted therapeutic strategies.Item Hepatitis C Virus NS3/4A Protease and the Intracellular Antiviral Response: Mapping Complex Virus-Host Interactions(2009-06-17) Johnson, Cynthia L.; Gale, Michael J.Virus infection triggers an innate immune response characterized by host cell production of interferon (IFN). Intermediates of viral replication, including dsRNA, initiate a signaling cascade that is amplified within the cell and alerts neighboring cells of viral invaders. Recognition of dsRNA intermediates occurs through retinoic acid inducible gene-I (RIG-I). RIG-I elicits an antiviral state by binding to the IFN-beta Promoter Stimulator-1 (IPS-1) adaptor protein, activating the latent downstream transcription factors IRF-3 and NF-kappaB. These transcription factors bind to the promoter region of effector genes including IFN-beta, producing an antiviral amplification loop within and around the infected cell. This response is critical for immunity to infection. Hepatitis C virus (HCV) is a serious global health problem with 170 million people chronically infected. HCV persistence is linked to viral regulation of innate host defenses by the nonstructural 3/4A protein complex (NS3/4A) cleavage of IPS-1. NS3 structural composition includes an amino-terminal serine protease and a carboxy-terminal RNA helicase. A structure-function analysis of NS3/4A truncation and deletion mutations was conducted. Mutants lacking the helicase domain retained the ability to control RIG-I signaling, but this regulation was abrogated by truncation of the protease domain. Furthermore, treatment of HCV-infected cells with a NS3/4A protease inhibitor prevented IPS-1 proteolysis, restored RIG-I signaling, and decreased viral protein levels. These results indicate that the NS3/4A protease domain alone can target IPS-1 on the mitochondrial membrane. Current dogma holds that NS3/4A is located on the endoplasmic reticulum, thus the mechanism of NS3/4A targeting IPS-1, a mitochondrial membrane protein, remains unexplained. We have shown that NS3/4A distributes on mitochondria independently of the previously identified NS4A membrane localization motif, in a manner dependent on the first twenty amino acids of the NS3 protease domain. The functional domains of IPS-1 that direct the immune response have not been elucidated. We conducted a structure-function study of IPS-1 that revealed distinct processes of IRF-3 and NF-kappaB activation. Mutational analyses further identified areas of IPS-1 critical for mitochondrial localization, dimerization, and uncoupling IRF-3 and NF-kappaB signaling. These findings improve our understanding of IPS-1 function in innate immunity to virus infection.Item Heterotrimeric G Protein Beta : Γ (gamma) Bound to a Biologically Active Peptide : Structural Definition of a Preferred Protein in Interaction Surface(2005-05-04) Davis, Tara Lynne; Sprang, Stephen R.Heterotrimeric G proteins transmit information within and throughout eukaryotic cells. These proteins serve crucial functions in diverse signaling pathways, including visual transduction and adenylyl cyclase regulation. Heterotrimers are composed of Α , Β, and Γ subunits. GΑ is a guanosine triphosphatase (GTPase); GΒ and GΓ have no intrinsic enzymatic activity and form an obligate heterodimer that participates in independent signaling interactions when released from GΑ . GΒΓ signaling partners include GΑ , phosphotidylinositol 3 kinase, phosducin, phospholipase C Β, adenylyl cyclase, G protein receptor kinase, G protein regulated inwardly rectifying channels, and N and P/Q type calcium channels. The 2.7?ystal structure of GΒ1Γ 2 bound to peptide (SIGKAFKILGYPDYD) obtained from a random peptide library has been solved. This peptide inhibits GΒΓ mediated activation of phospholipase C Β and phosphatidylinositol-3-kinase Γ and binds GΒ with sub-micromolar IC50. GΒ is a even-stranded Β-propeller protein containing WD-40 repeats. SIGK forms a helical structure that binds to the "top" face of the GΒ propeller. The peptide binds the same face of GΒ as GΑ , and mutations of GΒ in this region abrogate peptide binding. In addition, the SIGK peptide binds GΒ using residues sampled by other GΒΓ target proteins, both in crystal structures and in mutational analyses. No large conformational changes in GΒ are seen upon SIGK binding, and many of the biological effects seen for the SIGK peptide can be explained by simple competition for the top binding surface on GΒ. The SIGK peptide delineates a region on the "top" face of GΒ that is functionally a hot spot for GΒΓ target binding. However, this face of GΒ contains a mixture of polar and nonpolar contacts at the binding surface that allows it to interact with proteins of diverse sequence and secondary structure in a unique way compared to other hot spot proteins. The SIGK·GΒ1Γ 2 structure provides insight into the way in which GΒ is able to sustain a range of interactions with multiple binding partners.Item Integrating functional genomics, proteomics and computational analysis for the characterization of cellular networks(2008-09-18) Komurov, Kakajan; White, Michael A.As the study of biological systems progresses from a molecular level to a systems level, the development of new methodology for efficient data acquisition has been a key challenge of biological research in recent years. While development of novel high throughput experimental platforms is essential for an accurate large-scale data collection, novel theoretical methodology is indispensable for proper analysis and interpretation of these data. My projects aim at both, developing novel theoretic-analytical methodology for the analysis of functional patterns in biological networks, and also establishing a high throughput experimental platform for the study of signaling pathways. I have developed a generalized method for the analysis of functional organization in complex networks. This method makes use of several novel metrics used to characterize a node's status in the network. After the nodes are clustered according to their characteristics, statistically significant organizational patterns are revealed by random simulations of the network. Using this approach, I have found important characteristics of eukaryotic protein interaction networks that have direct implications in cellular phenomena like robustness and the efficiency of information processing. I have identified an entirely new class of functional modules with unique properties that contribute to the variability in cellular phenotypes. In addition, my analyses have uncovered a distinct pattern of organization in the protein network (called "rich club connectivity") that provides mechanistic explanations for some cell biological phenomena. This work not only reveals a highly organized functional dynamic layout of the protein interaction network, but also refines and/or corrects several notions proposed by previous studies. Functional genomic screens are a powerful tool for finding novel components of biological networks. However, in order to make these screens effective for assays that may require multiple readouts, it is necessary to channel the assay to another high throughput platform. Here, I used high throughput RNAi as a loss-of-function screen, and reverse-phase protein arrays as a high throughput readoutItem Leptin Action on Depamine Neurons: Biochemical and Behavioral Analysis(2009-01-14) Trinko III, Joseph Richard; DiLeone, Ralph J.It has been demonstrated that there are brain regions commonly activated by hedonic foods and drugs of abuse, and therefore, potentially common mechanisms underlying behaviors associated with them. Additionally, the metabolic state of an animal can affect drug seeking behavior. The discovery that leptin receptor (Lepr) is expressed on dopamine neurons in the ventral tegmental area (VTA) provides a link between metabolic state and rewarding behavior. By using a multitude of techniques, the functional roles for Lepr signaling on dopamine neurons have been assessed. By activating Lepr in the VTA via direct infusion of leptin, or conversely reducing Lepr signaling in the VTA with viral-mediated shRNAi, a role for VTA leptin signaling in feeding behavior was demonstrated. Lepr activation in the VTA leads to intracellular signaling pathways similar to that observed in the hypothalamus. These studies also identify differences in signaling between these two brain regions, specifically the phosphorylation of AMP-activated protein kinase (AMPK), which is opposite to what is observed in the hypothalamus. Interestingly, food restriction was found to differentially affect the signaling pathways in the VTA. Preliminary evidence also suggests a role for Lepr in saccharin and cocaine seeking behaviors. Rats with attenuated Lepr signaling in the VTA demonstrate persistent saccharin and cocaine seeking. Conversely, leptin infusions reduced lever pressing during drug withdrawal. These results are indicative of a potential role for leptin in drug and food seeking behavior. In sum, these results suggest a neuronal mechanism by which this key metabolic signal can modify both food and drug intake.Item Mesothelial and Mural Cell Contribution to Vascular Development through PDGF Signaling(2009-06-17) French, Wendy Joy; Tallquist, MichelleVascular development during embryogenesis and adulthood occurs through vasculogenesis and angiogenesis. Vasculogenesis is the de novo formation of blood vessels from mesoderm precursor cells. Angiogenesis is the formation of new vessels from existing vessels. Both processes involve hematopoietic, endothelial, and mural cells for the formation of mature, stable vasculature. While hematopoietic and endothelial cell contributions and function in vascular development have been extensively studied identifying the VEGF and TGF families as major contributors, the role of mural cells has not been clearly defined. The platelet derived growth factor beta (PDGFR beta) is essential for mural cell recruitment and expansion. Deletion of PDGFR beta leads to perinatal lethality resulting from vascular defects attributed to severe decreases in mural cells. PDGFR beta is a receptor tyrosine kinase with high homology in signal activation to PDGFR alpha. Downstream signaling pathway activation includes PI3 kinase, Src, RasGAP, Grb2, Shp-2, and PLC?gamma for the regulation of cellular functions.The focus of this research was to determine the temporal and functional requirements of PDGFR signaling in mural cells. To address the temporal requirements for PDGFR beta, genetic manipulation was used to delete the receptor in precursor and differentiated mural cells. In addition, mutant mice were generated with the additional deletion of PDGFR alpha to address the potential for compensatory or cooperative function between the two receptors. These studies identified a cooperative role for PDGFR?alpha and PDGFR beta in yolk sac mesothelial cells. Mutant mice were lethal around E10.5 with disrupted yolk sac vascular remodeling and extracellular matrix composition. The PDGFR regulate collagen matrix through regulation of matrix metalloproteinase activity and thus disrupt integrin activation. The functional role of PDGFR?beta in mural cells was addressed by signaling point mutants targeting and disrupting specific downstream pathways. These studies resulted in a progressive decrease in mural cells that correlated to the number of disrupted PDGFR-beta signaling pathways. Together these analyses demonstrate PDGFR and mural cells are essential for vascular development and maintenance.Item Protein Phosphatase Inhibitor-1 and Cdk5: of Molecules and Memory(2007-06-12) Nguyen, Baochan; Bibb, James A.Protein phosphatase inhibitor-1 and cyclin-dependent kinase 5 (Cdk5) have been independently implicated in synaptic plasticity, learning, and memory. We began our studies with the identification, confirmation, and characterization of a novel Cdk5-dependent phosphorylation site (Ser6) on inhibitor-1. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser6 were mediated by Cdk5 and protein phosphatases 2A (PP-2A) and 1 (PP-1), respectively. Additionally, protein phosphatase 2B (PP-2B) contributed to dephosphorylation under conditions of high Ca2+. Functionally, Cdk5-dependent phosphorylation of inhibitor-1 intramolecularly impaired dephosphorylation and deactivation of the protein, placing the activities of Cdk5 and protein kinase A (PKA) in synergism in the negative regulation of PP-1. These studies uncovered a potential new regulatory mechanism for Cdk5. Investigation revealed that depolarization differentially regulates the Cdk5-dependent sites of inhibitor-1 and its homologue dopamine- and cAMP-regulated phosphoprotein (DARPP-32) in a cofactor- and N-methyl-D-aspartate (NMDA) receptor-independent manner. Effects on DARPP-32 were Ca2+-mediated and PP-2A-dependent, while effects on inhibitor-1 were nonselectively cation-mediated and either partially PP-2B-dependent or independent of the major serine/threonine phosphatases, depending on the site. Given the uncertain role of inhibitor-1 in learning and memory, we next focused on identifying behaviors and substrates impacted by inhibitor-1 function. Mice constitutively lacking inhibitor-1 displayed enhanced neurogenesis and mildly impaired habituation, but normal contextual fear and novelty learning. Furthermore, levels of hippocampal inhibitor-1 were increased by voluntary wheel running, a stimulus for neurogenesis. Thus, inhibitor-1 may function in an anti-neurogenic mechanism and be more important in the direct or indirect modulation of dopamine-dependent behaviors than in the mnemonic functions of the hippocampus. Using a whole-cell patch clamp approach, we also attempted to identify electrical properties of dentate granule cells that might be affected by Cdk5-dependent phosphorylation of inhibitor-1. Most promising among the results was a reduction in the ability of granule cells lacking inhibitor-1 to faithfully respond to high-frequency trains of stimuli. Granule cell excitability was also increased by pharmacological inhibition of Cdk5 with roscovitine. Finally, in a related study, we helped firmly establish a role for Cdk5 in hippocampal synaptic plasticity by demonstrating that conditional loss of Cdk5 enhances NMDA receptor-mediated currents, particularly of the NR2B type.Item Regulation and Function of Apoer2 and Vidlr in the Central Nervous and Reproductive Systems(2009-06-15) Bowen, Irene Masiulis; Herz, JoachimThe LDL receptor gene family is an evolutionarily ancient family of membrane spanning proteins composed of a diverse collection of receptors that share common domains and mediate a variety of organismal functions. Members of this family, Apoer2 and Vldlr, are highly similar receptors and are both involved in the Reelin signaling pathway. This pathway controls neuronal migration and organization of the brain during development as well as synaptic function in adulthood. Although both receptors partially compensate for one another during Reelin signaling, the generation of knockin mutant animals has allowed us to begin to uncover the unique and independent functions of these receptors that have not yet been explored. For example, a single amino acid mutation in Vldlr determines binding of the Pafah1b complex to the cytoplasmic tail of the receptor. Mice expressing this L838R mutation were created using ES cell homologous recombination techniques and will be useful in exploring the effects of the mutation in brain development and synaptic function in vivo. Vldlr independent functions that are unique to Apoer2 have been explored in more depth. These include binding of scaffolding proteins PSD95 and JIPs to an alternatively spliced exon within the cytoplasmic tail of Apoer2, which is not found in any other LDLR family member. This exon has been shown to mediate increased LTP induction after Reelin stimulation. Apoer2 knockin animals, expressing splice variants lacking another alternatively spliced exon encoding the O-linked sugar domain, show receptor stabilization through the inhibition of Apoer2 proteolytic processing. This causes increased Apoer2 levels in both the brain and testis. While lack of exon 16 has no known consequences in the testis, it does cause disrupted synaptic transmission and defective LTP induction after reelin stimulation in the brain. Apoer2 was recently found to be involved in selenium uptake into the testis and brain. Through the use of various Apoer2 intracellular domain mutant mice, a differentiation was made between the Apoer2 functional domains involved in cell signaling and the function of the receptor in selenium endocytosis. Further use of the aforementioned knockin mouse lines will help uncover previously unexplored functions of Vldlr and Apoer2 and advance our understanding of their potential involvement in neurological diseases such as Alzheimer's disease.Item Revealing Regulation and Organization of Signaling Networks by Scaffolding Proteins(2007-05-23) Swanik, Jackie Thomas; White, MichaelUnderstanding the molecular mechanism that controls how cells respond to their environment is of major biological significance. The RAS/RAF signal transduction pathway is a good model with which to investigate this question as the major catalytic components of the pathway have been identified and it has been shown to elicit a wide variety of cellular responses such as proliferation, migration, differentiation, and apoptosis. The pathway is a three-tiered MAPK kinase cascade consisting of RAF, MEK, and ERK. Although the pathway has been extensively studied, pathway regulation is not completely understood. Genetic studies aimed at understanding pathway regulation have identified candidate scaffolding proteins. We used transient loss of function analysis to assess the contribution of the scaffolding proteins, Suppressor of RAS-8 (Sur-8) and Kinase Suppressor of RAS (KSR), to ligand mediated RAS/RAF signal transduction pathway activation. We show that Sur-8 and KSR are integral components of the RAS/RAF signal transduction pathway in mammalian cells. In addition, they display ligand specific coupling in that Sur-8 is required for EGF induced MEK activation while KSR is involved in LPA mediated MEK activation. to ligand mediated RAS/RAF signal transduction pathway activation. We show that Sur-8 and KSR are integral components of the RAS/RAF signal transduction pathway in mammalian cells. In addition, they display ligand specific coupling in that Sur-8 is required for EGF induced MEK activation while KSR is involved in LPA mediated MEK activation. Investigation of the molecular mechanism of action of Sur-8 and KSR found that Sur-8 is required for both EGF induced RAF-1 and B-RAF activation while KSR is involved in EGF induced RAF-1 activation. Additionally, Sur-8 contributes to RAF-1 localization as well as being associated with a RAF-1 activating kinase. Futhermore, we found that KSR does not impact LPA induced MEK activation through either RAF-1 or B-RAF activation and even though it impacts EGF induced RAF-1 activation it is not a limiting component to EGF induced MEK activation. In this study, we show that a function of scaffolding proteins in the RAS/RAF signal transduction pathway is to contribute to ligand specific coupling of MEK/ERK to distinct stimuli.