SPATIOTEMPORAL REGULATION OF CAMP/PKA SIGNALING IN CELLULAR PROCESSES

Cells respond to extracellular stimuli by modulating intricately intertwined signaling pathways. To achieve specific responses, these pathways are tightly controlled in space and time by regulating second messenger concentrations and kinase activity dynamics. The 3'-5'-cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase (PKA) pathway is spatiotemporally compartmentalized at various levels in order to elicit specific cellular activities. At the second messenger level, cAMP concentration gradients are maintained by actively controlling its accumulation and degradation. PKA activity is also modulated in space and time by the spatiotemporal compartmentalization of upstream components, including cAMP gradients, and is itself spatially localized to specific subcellular compartments, contributing to highly specific substrate recognition. Moreover, interactions with signaling molecules from other signaling pathways serve as another source of regulation of the cAMP/PKA pathway. To study cAMP and PKA in their native environment, wherein the entire signaling network remains intact, we used fluorescence resonance energy transfer (FRET)-based biosensors to capture their endogenous signaling dynamics. This dissertation is composed of two parts. The first part consists of two studies, which investigate the role of spatiotemporally compartmentalized PKA signaling in two separately important biological processes. In the first study, we used FRET-based PKA reporter (AKAR4-Kras) to study how cells tune PKA activity to transition between different modes of migration in response to different degree of physical confinement. We demonstrate the importance of crosstalk between different signaling pathways in regulating PKA activity to enable cells to adapt to their microenvironment and migrate efficiently. In the second study we also employed FRET-based reporters to profile compartment-specific Akt activity dynamics in PC12 cells. We observed spatial differences of Akt as well as interesting temporal dynamics in response to EGF and NGF. While dissecting the molecular mechanisms governing compartment-specific Akt activity, we demonstrated a complex interaction between PKA and Akt that could regulate the differentiation of PC12 cells. The second portion of the dissertation involves developing enhanced BRET- and FRET-based reporters to sensitively monitor kinase activity (Bim-BRET-KARs) and cAMP levels (ICUE4), respectively. We demonstrated the ability of Bim-BRET-KARs in detecting various kinase activities with high spatial resolution, and the applicability of these reporters to be used in combination with fluorescent molecules and optogenetic tools. We also generated a cAMP reporter that can detect low cAMP levels that were previously undetectable by current FRET-based cAMP reporters. Taken together, the studies presented herein highlight various modes and roles of spatiotemporally compartmentalized cAMP/PKA signaling in modulating specific cellular processes. Moreover, utilizing the newly developed tools, we have elucidated dynamics of the cAMP/PKA signaling that we previously could not visualize. These studies enhance our understanding of the cAMP/PKA pathway and unveil unknown roles it plays in biological processes

EphB and ephrinB in pain signaling

The Ephrin type B receptors (EphB) and their membrane bound ephrinB ligands are involved in diverse facets of cell physiology and pathophysiology. EphB-ephrinB signaling mediates synapse formation and plasticity by regulating the insertion, localization and function of glutamate receptors in synaptic membranes. Whereas, EphB-ephrinB signaling at the excitatory glutamatergic synapses in the dorsal horns of the spinal cord, has been implicated in the pathophysiology of pain. Here, the key evidence that support the participation of EphB-ephrinB signaling in pain processes are highlighted. Then, a possible role for the pseudokinase EphB6 in the EphB-ephrinB pain signaling complex is considered. These pathways are currently being intensely studied to exploit selective therapeutic targets for pain relief

The role of cell death in the pathogenesis of autoimmune disease: HMGB1 and microparticles as intercellular mediators of inflammation

Cell death is critical to normal homeostasis, although this process, when increased aberrantly, can lead to the production of pro-inflammatory mediators promoting autoimmunity. Two novel intercellular mediators of inflammation generated during cell death are high mobility group box 1 (HMGB1) protein and microparticles (MPs). HMGB1 is a nuclear protein that functions in transcription when inside the nucleus but takes on pro-inflammatory properties when released during cell death. Microparticles are small, membrane-bound structures that extrude from cells when they die and contain cell surface proteins and nuclear material from their parent cells. MPs circulate widely throughout the vasculature and mediate long-distance communication between cells. Both MPs and HMGB1 have been implicated in the pathogenesis of a broad spectrum of inflammatory diseases, including the prototypic autoimmune conditions systemic lupus erythematosus and rheumatoid arthritis. Given their range of activity and association with active disease, both structures may prove to be targets for effective therapy in these and other disorders

Reactive oxygen species and age-related genes p66Shc, sirtuin, FoxO3 and klotho in senescence

Reactive oxygen species (ROS) superoxide and hydrogen peroxide perform important signaling functions in many physiological and pathophysiological processes. Cell senescence and organismal age are not exemptions. Aging-regulating genes p66shc, Sirtuin, FOXO3a and Klotho are new important factors which are stimulated by ROS signaling. It has been shown that ROS participate in initiation and prolongation of gene-dependent aging development. ROS also participate in the activation of protein kinases Akt/PKB and extracellular signal-regulated kinase ERK, which by themselves or through gene activation stimulates or retards cell senescence. Different retarding/stimulating effects of ROS might depend on the nature of signaling species—superoxide or hydrogen peroxide. Importance of radical anion superoxide as a signaling molecule with “super-nucleophilic” properties points to the possibility of the use of superoxide scavengers (SOD mimetics, ubiquinones and flavonoids) for retarding the development of aging

Insulin Substrate Receptor (IRS) proteins in normal and malignant hematopoiesis

The insulin receptor substrate (IRS) proteins are a family of cytoplasmic proteins that integrate and coordinate the transmission of signals from the extracellular to the intracellular environment via transmembrane receptors, thus regulating cell growth, metabolism, survival and proliferation. The PI3K/AKT/mTOR and MAPK signaling pathways are the best-characterized downstream signaling pathways activated by IRS signaling (canonical pathways). However, novel signaling axes involving IRS proteins (noncanonical pathways) have recently been identified in solid tumor and hematologic neoplasm models. Insulin receptor substrate-1 (IRS1) and insulin receptor substrate-2 (IRS2) are the best-characterized IRS proteins in hematologic-related processes. IRS2 binds to important cellular receptors involved in normal hematopoiesis (EPOR, MPL and IGF1R). Moreover, the identification of IRS1/ABL1 and IRS2/JAK2V617F interactions and their functional consequences has opened a new frontier for investigating the roles of the IRS protein family in malignant hematopoiesis. Insulin receptor substrate-4 (IRS4) is absent in normal hematopoietic tissues but may be expressed under abnormal conditions. Moreover, insulin receptor substrate-5 (DOK4) and insulin receptor substrate-6 (DOK5) are linked to lymphocyte regulation. An improved understanding of the signaling pathways mediated by IRS proteins in hematopoiesis-related processes, along with the increased development of agonists and antagonists of these signaling axes, may generate new therapeutic approaches for hematological diseases. The scope of this review is to recapitulate and review the evidence for the functions of IRS proteins in normal and malignant hematopoiesis

Exploring complex cellular membranes containing lipids, cholesterols, proteins, and gangliosides using molecular simulations

Domains of different thermodynamic phases manifest in the cell membrane as a consequence of the complex interactions between lipids and proteins. One of the outstanding challenges in membrane biophysics is to understand the role played by the structural and dynamical heterogeneity of membranes in supporting cellular function. In particular, there remain fundamental open questions related to the role of proteins in lipid domain formation, the effect of protein co-localization with lipid domains, and the nanoscopic structure of lipid domains. My dissertation research systematically investigated cellular membrane environments using all-atom and coarse-grained molecular simulations. Systems of incremental complexity, binary and ternary lipid model membranes, laterally heterogeneous membranes with proteins, and membranes with gangliosides were studied. With the aid of statistical mechanics and molecular simulation algorithms, critical insights were gained into cholesterol aggregation in model membranes. Cholesterol was found to populate a dimer ensemble with distinct sub-states in contrast to the idealistic view of face-flush cholesterol dimers. Further investigations characterized the inter- and intra-leaflet interactions of cholesterols, providing insights into possible trimer and tetramer formation. To probe the dynamic interplay of lipids and proteins in lipid raft-mimicking environments, we accurately modeled laterally heterogeneous membranes and explored the colocalization of transmembrane proteins. The proteins were observed to preferably co-localize at the domain boundaries, reducing the excess free energy of forming an interface. This observation has implications in transmembrane proteins known to be involved in the biogenesis of amyloid beta protein and believed to have activity dependent on localization in raft domains. Venturing beyond ternary lipid mixtures, membranes formed from quaternary lipid mixtures that approximate the surface of an artificial virus nanoparticle were examined. The effects of cations in mediating the interactions of negatively charged lipids were established through collaborative of experimental and simulation studies. Finally, development of force field parameters for sulfated poly-amido-saccharides and also validating existing cholesterol parameters across all available force fields were also undertaken as major methodological pursuits. Taken together these studies demonstrate the power of computer simulation, well-validated by experiment, to elucidate the structural and functional nature of complex biomolecular systems

Investigating the control of Listeria monocytogenes on uncured, no-nitrate-or-nitrite-added ready-to-eat meat products using natural antimicrobial ingredients and post-lethality interventions

Restrictions on the use of conventional antimicrobials, combined with the restricted use of nitrite and nitrate, have generated concerns over the perceived risk for foodborne illness associated with natural and organic RTE meat and poultry products. Thus, the use of natural antimicrobial interventions alone and in combination with post-lethality interventions as a means to inhibit the recovery and growth of Listeria monocytogenes in naturally cured RTE processed meats was the focus of the work reported in this dissertation. Natural antimicrobials evaluated were cranberry powder, vinegar, and vinegar and lemon juice concentrate. Post-lethality interventions studied were high hydrostatic pressure, lauric arginate, octanoic acid, and post-packaging thermal treatment. Parameters evaluated through 98 days of storage at 4C included viable L. monocytogenes on modified Oxford (MOX) and thin agar layer (TAL) media. The vinegar and vinegar and lemon juice concentrate ingredients exhibited strong bacteriostatic properties against L. monocytogenes whereas cranberry powder did not. However, none of these natural antimicrobial ingredients exhibited bactericidal properties under the conditions or these studies. Additionally, the high hydrostatic pressure, octanoic acid, and lauric arginate post-lethality interventions demonstrated significant bactericidal effects on initial numbers of L. monocytogenes whereas the post-packaging thermal treatment investigated did not. Nonetheless, although beneficial from the standpoint of initial lethality, none of these post-lethality interventions offered protection against the growth of surviving L. monocytogenes upon storage of the products and under the conditions of these studies. Furthermore, upon studying the combination of natural antimicrobial ingredients with the use of post-lethality interventions, results showed that implementing the high hydrostatic pressure, octanoic acid, or lauric arginate post-lethality interventions in combination with vinegar or vinegar and lemon juice concentrate, under the conditions of these studies, represent promising multiple-hurdle approaches for not only addressing the potential presence of L. monocytogenes in naturally cured RTE processed meat products, but also at inhibiting the potential recovery and growth of those cells that remain viable over the refrigerated storage of the products. The combinations of these hurdles represent effective options that could be instituted by manufacturers of organic and natural processed meat products in their L. monocytogenes control plans