The SH2D5 signaling molecule is a previously uncharacterized adaptor-like protein, containing an N-terminal phosphotyrosine binding (PTB) domain and a noncanonical Src Homology-2 (SH2) domain. With an antibody that I developed, I have shown that SH2D5 is highly enriched throughout adult brain regions. Furthermore, SH2D5 is localized to purkinjie cells in the cerebellum, the cornu ammonis (CA) of the hippocampus and pyramidal cells in the cortex. Despite harbouring two potential phosphotyrosine (pTyr) recognition domains, SH2D5 binds minimally to pTyr ligands. To discover the interaction partners of SH2D5 I conducted an immunoprecipitation/ mass spectrometry (IP/MS) screen from cultured Human Embryonic Kidney (HEK) 293T and Neuro2A cells along with murine brain lysates. These experiments revealed novel binding partners to SH2D5 including a prominent association with the RacGAP protein, Breakpoint Cluster Region protein (BCR), which is also highly expressed in brain. I have defined the interaction between SH2D5 and BCR and show that the PTB domain of SH2D5 engages an NxxF motif located within the N-terminal region of BCR. To address the biological significance of SH2D5, I utilized an siRNA approach to deplete the neuroblastoma cell-line, B35, of iii SH2D5. In these assays, B35 cells display a cell rounding phenotype and grow in a lattice formation. Furthermore, upon SH2D5 depletion these cells display low levels of activated Rac, associated with cell rounding. Taken together, these data reveal the first characterization of the SH2D5 signaling protein, its novel interaction with BCR and phenotype in neuronal-like cells. These data signify a biological function for SH2D5 in neurobiologic signaling perhaps applicable to learning and memory.

The SH2D5 signaling molecule is a previously uncharacterized adaptor-like protein, containing an N-terminal phosphotyrosine binding (PTB) domain and a noncanonical Src Homology-2 (SH2) domain. With an antibody that I developed, I have shown that SH2D5 is highly enriched throughout adult brain regions. Furthermore, SH2D5 is localized to purkinjie cells in the cerebellum, the cornu ammonis (CA) of the hippocampus and pyramidal cells in the cortex. Despite harbouring two potential phosphotyrosine (pTyr) recognition domains, SH2D5 binds minimally to pTyr ligands. To discover the interaction partners of SH2D5 I conducted an immunoprecipitation/ mass spectrometry (IP/MS) screen from cultured Human Embryonic Kidney (HEK) 293T and Neuro2A cells along with murine brain lysates. These experiments revealed novel binding partners to SH2D5 including a prominent association with the RacGAP protein, Breakpoint Cluster Region protein (BCR), which is also highly expressed in brain. I have defined the interaction between SH2D5 and BCR and show that the PTB domain of SH2D5 engages an NxxF motif located within the N-terminal region of BCR. To address the biological significance of SH2D5, I utilized an siRNA approach to deplete the neuroblastoma cell-line, B35, of iii SH2D5. In these assays, B35 cells display a cell rounding phenotype and grow in a lattice formation. Furthermore, upon SH2D5 depletion these cells display low levels of activated Rac, associated with cell rounding. Taken together, these data reveal the first characterization of the SH2D5 signaling protein, its novel interaction with BCR and phenotype in neuronal-like cells. These data signify a biological function for SH2D5 in neurobiologic signaling perhaps applicable to learning and memory.

Precise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions. Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension. Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.

Precise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions. Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension. Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.

<p><a> Worldwide, as many as 1 billion people suffer from neurological disorders. Fundamentally, neurological disorders are caused by dysregulation of biochemical signaling within neurons, leading to deficits in learning and memory formation. To identify better preventative and therapeutic strategies for patients of neurological disorders, we require a better understanding of how biochemical signaling is regulated within neurons.</a></p> <p> Biochemical signaling at the connections between neurons, called synapses, regulates dynamic shifts in a synapse’s size and connective strength. Called synaptic plasticity, these shifts are initiated by calcium ion (Ca²⁺) flux into message-receiving structures called dendritic spines. Within dendritic spines, Ca²⁺ binds sensor proteins such as calmodulin (CaM). Importantly, Ca²⁺/CaM may bind and activate a wide variety of proteins, which subsequently facilitate signaling pathways regulating the dendritic spine’s size and connective strength. </p> <p>In this thesis, I use computational models to characterize molecular mechanisms regulating Ca²⁺-dependent protein signaling within the dendritic spine. Specifically, I explore how Ca²⁺/CaM differentially activates binding partners and how these binding partners transduce signals downstream. For this, I present deterministic models of Ca²⁺, CaM, and CaM-dependent proteins, and in analyzing model output I demonstrate in-part that competition for CaM-binding alone may be sufficient to set the Ca²⁺ frequency-dependence of protein activation. Subsequently, I adapt my deterministic models into particle-based, spatial-stochastic frameworks to quantify how spatial effects influence model output, showing evidence that spatial gradients of Ca²⁺/CaM may set spatial gradients of activated proteins downstream. Additionally, I incorporate into my models the most detailed model to-date of Ca²⁺/CaM-dependent protein kinase II (CaMKII), a multi-subunit protein essential to synaptic plasticity. With this detailed model of CaMKII, my analysis suggests that the many subunits of CaMKII provide avidity effects that significantly increase the protein’s effective affinity for binding partners, particularly Ca²⁺/CaM. Altogether, this thesis provides a detailed analysis of Ca²⁺-dependent signaling within dendritic spines, characterizing molecular mechanisms that may be useful for the development of novel therapeutics for patients of neurological disorders. </p>

Double stranded RNA (dsRNA), the genomic material of some viruses and a replication intermediate in others, is recognized by multiple signaling receptors that initiate the anti-viral response1. Viruses have developed mechanisms to circumvent the anti-viral response by targeting components of the signaling pathway. An example of one such pathway is the TLR3 signaling pathway, which contains a kinase complex that activates interferon regulatory factor 3 (IRF3), leading to production of type I interferons. The kinase complex consists of a scaffold protein, NAK-associated protein 1 (NAP1), and two kinases, TANK-binding kinase 1 (TBK-1) and IκB kinase epsilon (IKKε). A fourty residue sequence in NAP1 was discovered that mediated its interaction with TBK1 and IKKε, termed the kinase binding domain (KBD)1. However, the function of NAP1 in mediating kinase activation is unknown and understanding this is the long-term goal of this project. The goal of this thesis was to test the dependency of NAP1’s dimeric structure on mediating interactions with the kinases. Biochemical characterization of recombinant targets was completed using size-exclusion chromatography (SEC) and NAP1 KBD WT eluted as a dimeric species. CFP/YFP/Alexa Fluor 546 fusion proteins of the NAP1 KBD and scaffold binding motif (SBM) of the kinases, TBK-1 and IKKε, were generated to assess interactions using fluorescence resonance energy transfer (FRET). NAP1 KBD directly interacts with TBK1 and IKKε, with low micromolar affinity in vitro. Mutagenesis was attempted to identify the residues necessary for NAP1 dimerization and any effect dimerization may have on kinase recognition. This thesis shows data to support that NAP1 KBD forms stable homo-oligomers and directly interacts with a small C-terminal portion of TBK1 and IKKε.

Regulator of G Protein Signaling 4 (RGS4) mediates motor defects in Parkinson's disease. Small molecule RGS4 inhibitors (e.g. CCG-50014) modify buried cysteine residues, but the structural and dynamic mechanisms underpinning specificity of inhibitors for RGS4 within the RGS family are poorly understood. We used NMR and other biophysical methods to examine ligand-induced structural changes and the dynamics of unliganded RGS4 and RGS8 that allow ligand binding. NMR and fluorescence spectroscopy data reveal details of the hidden, excited conformational state of RGS4 that exposes Cys148, one of the buried cysteines bound by inhibitors. We further show that specificity of RGS4 inhibitors is driven by differential accessibility of the target cysteine compared to its equivalent in RGS8. Cys148 is buried beneath the lid at the center the α4-α7 helix bundle, and this bundle is destabilized in RGS4 compared to RGS8. Notably, helix 6 is highly destabilized in RGS4 compared to RGS8 and is likely the key mediator of access to Cys148. Our findings provide key insight into the mechanism of allosteric RGS4 inhibition and show that dynamics drive inhibitory specificity among RGS proteins.

Background: RGS9-deficient mice show drug-induced dyskinesia but normal locomotor activity under unchallenged conditions. Results: Genes related to Ca2+ signaling and their functions were regulated in RGS9-deficient mice. Conclusion: Changes in Ca2+ signaling that compensate for RGS9 loss-of-function can explain the normal locomotor activity in RGS9-deficient mice under unchallenged conditions. Significance: Identified signaling components may represent novel targets in antidyskinetic therapy. The long splice variant of the regulator of G-protein signaling 9 (RGS9-2) is enriched in striatal medium spiny neurons and dampens dopamine D2 receptor signaling. Lack of RGS9-2 can promote while its overexpression prevents drug-induced dyskinesia. Other animal models of drug-induced dyskinesia rather pointed towards overactivity of dopamine receptor-mediated signaling. To evaluate changes in signaling pathways mRNA expression levels were determined and compared in wild-type and RGS9- deficient mice. Unexpectedly, expression levels of dopamine receptors were unchanged in RGS9-deficient mice, while several genes related to Ca2+ signaling and long-term depression were differentially expressed when compared to wild type animals. Detailed investigations at the protein level revealed hyperphosphorylation of DARPP32 at Thr34 and of ERK1/2 in striata of RGS9-deficient mice. Whole cell patch clamp recordings showed that spontaneous synaptic events are increased (frequency and size) in RGS9-deficient mice while long-term depression is reduced in acute brain slices. These changes are compatible with a Ca2+-induced potentiation of dopamine receptor signaling which may contribute to the drug-induced dyskinesia in RGS9-deficient mice.

In order to function, proteins must be folded into their native shape. While this can sometimes occur spontaneously, the process can be hindered by thermodynamic barriers, trapped intermediates, and aggregation prone hydrophobic interactions. Molecular chaperones are proteins that help client proteins or substrates overcome these barriers so that they can be folded properly. One such chaperone is the chaperonin CCT, a large MDa protein made up of 16 paralogous subunits that form a double ring structure. CCT encapsulates its substrates in a central cavity, where they are sequestered and folded, using ATP binding and hydrolysis to drive conformational changes in the CCT-substrate complex. CCT mediates the folding of many substrates involved in a variety of cellular process, including the cytoskeletal proteins actin and tubulin, and the G protein subunit Gabg, which signals downstream of GPCRs in a variety of cellular processes. We showed that CCT is responsible for folding the b-propeller containing proteins, mLST8 and Raptor, which are subunits of the mTOR complexes. The mTOR complexes (mTORC1 and mTORC2) are master regulators of cell growth and survival by controlling processes such as protein synthesis, energy metabolism, cell survival pathways and autophagy. CCT folds mLST8 and Raptor and help them assemble into the mTOR complexes. As a result, CCT is required for functional mTOR signaling. Furthermore, we solved a 4.0 Ǻ resolution structure of mLST8 bound to CCT. Surprisingly, mLST8 is found in the center of the folding cavity, in between the rings, despite previous evidence suggesting that substrates bind only in the apical domains. Given its role in folding and assembling the mTOR complexes, G proteins, and many other proteins involved in cell survival pathways, CCT has been implicated in cancer. CCT upregulation often correlates with a worse prognosis, likely because uncontrolled growth requires increased chaperone capacity. The peptide CT20P has been shown to have cytotoxic effects in cancer cells, likely through its binding to CCT. We characterized CT20P, showing that it binds to CCT and inhibits its substrate-folding functions in cells. We specifically showed that a GFP-CT20P fusion protein inhibited the assembly of two important signaling complexes Gbg and mTORC1. These results show that CT20P is a useful inhibitor for the study of CCT function.

Corals are cnidarian animals that build the founding structures of tropical reefs, which survival depends upon the obligate symbiotic association to photosynthetic dinoflagellate algae in the family Symbiodiniaceae. As corals are facing increasing environmental and anthropogenic stress, understanding the molecular principles governing this unique symbiotic association is crucial to predict their adaptive potential. Due to logistic, costly, and experimental difficulties of working with corals, we use the sea anemone Aiptasia (sensu Exaiptasia pallida) as a tractable model organism for the molecular study of cnidarian-algal symbiosis. A major advantage of Aiptasia is that it establishes a facultative symbiotic association with Symbiodiniaceae algae, that is, this anemone can be maintained in an aposymbiotic (symbiont-free) state, allowing for comparison of symbiotic and ‘control’ aposymbiotic processes. The main aim of this dissertation was to investigate the signaling pathways involved in the regulation of this symbiotic interaction, and in particular, phosphorylation-mediated protein signaling. Phosphorylation is indeed a major post-translational modification that mediates signal transduction within and across cells. To investigate if protein phosphorylation regulates the complex intercellular signaling that occurs between symbiotic partners, a mass spectrometry-based phosphoproteomic approach was employed. Given the novelty of this application in the field of coral reef biology, the first research chapter details the development and optimization of a protocol that allows quantification of protein phosphorylation in the sea anemone Aiptasia. This chapter includes mass spectrometric analysis in 1) data-dependent acquisition (or shotgun proteomics) for the generation of a so-called assay (spectral) library, a reference dataset that servers for 2) accurate and reproducible label-free quantification of protein phosphorylation in data-independent acquisition (DIA/SWATH-MS). In the second research chapter, the developed protocol was employed to generate a phosphopeptide assay (spectral) library for aposymbiotic and symbiotic Aiptasia, which would allow further quantification of protein phosphorylation across symbiotic conditions. We consistently quantified more than 3,000 phosphopeptides, totaling more than 1,600 phosphoproteins, across biological replicates and symbiotic conditions. Characteristic phosphoproteomic profile distinguished the two symbiotic groups and differential phosphorylation targeted biological processes that have not been previously described in the context of cnidarian-algal symbiosis, namely the phospholipase D signaling pathway and protein processing in the endoplasmic reticulum. We suggest that changes in the phosphorylation status of these signaling pathways may have a potentially relevant role in the control of an established cnidarian-algal association.