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Sex differences exist in the progression of heart disease, as premenopausal women are protected from developing severe hypertension, aortic stenosis, myocardial infarction and hypertrophic cardiomyopathies. The susceptibility and progression of cardiovascular disease increases in post-menopausal women. This is at least partially underlined by a pronounced decrease in circulating estrogen levels. Estradiol (E2), the most abundant estrogen in premenopausal women, is known to be cardioprotective. Recently, AMP-activated protein kinase (AMPK) has emerged as a prominent player in the development of cardiac hypertrophy and heart failure. AMPK is central to the energetic metabolism of the cell and is activated in response to energy deprivation. E2 has been shown to activate AMPK, by yet an unknown mechanism. The first part of this dissertation focuses on describing the molecular mechanism behind this AMPK activation. We found that E2 activates AMPK through a non- genomic pathway and involves direct interaction of classical estrogen receptors (ERα and ERβ) with the α-catalytic subunit of AMPK. These receptors also associate with the upstream kinase LKB1, which is required for E2-dependent activation of AMPK. Furthermore, the two estrogen receptors play opposite roles, where ERα increases AMPK activation, and ERβ acts as a repressor, inhibiting AMPK phosphorylation. To translate our findings to heart disease, the next step was to determine the effect of ovarian failure, underlined by E2 loss, on AMPK signaling during the progression of cardiac hypertrophy. We hypothesized that ovarian failure decreases cardiac AMPK signaling, translating in worsening of hypertrophy. We found that the status of cardiac AMPK signaling depends on the nature of the hypertrophic stimulus and the timing of ovarian failure in relation to the onset of hypertrophy. Furthermore, we did not detect any differences in the development of cardiac hypertrophy between wild type mice and mice in ovarian failu

Proliferating cells have unique metabolic requirements beyond those of quiescent cells. Specifically, blood forming hematopoietic stem cells, during periods of severe blood loss, switch from a quiescent glycolytic state to a state dependent on mitochondrial metabolism during differentiation and proliferation. This dissertation attempts to define some of the signaling details of this switch by using erythropoietin receptor signaling as a model. In cytokine-dependent Ba/F3 cell line expressing the receptor for erythropoietin (EpoR) (Ba/F3-EpoR), chemical inhibition of mitochondrial function by rotenone decreases in erythropoietin(Epo)-stimulated proliferation. This observation led to the examination of whether Epo could stimulate mitochondrial function. To further assess the role of mitochondria in cell proliferation and the metabolic functions of Epo, levels of oxidative phosphorylation markers and signaling molecules important for mitochondrial biogenesis were measured. Western blotting scans showed increased protein levels of cytochrome oxidase subunit IV (CoxIV) and Complex III core protein 2 following 24 hours of Epo treatment. Interestingly, inhibition of Janus Kinase 2 (Jak2), the tyrosine kinase associated with Epo receptor, by AG490 elicited a similar decrease in CoxIV to Epo withdrawal even in the presence of Epo. In addition, Epo increased the levels of the mitochondrial biogenesis regulator AMP-activated protein kinase α (AMPKα) in a Jak2-dependent manner within Ba/F3 cells. Both total and phosphorylated (activated) AMPKα were increased following Epo stimulation. Treatment with the AMPK inhibitor Compound C decreased Epo stimulation of CoxIV, suggesting a linear signaling cascade from Jak2 to mitochondrial biogenesis through AMPKα. Examining potential mechanisms, direct binding of AMPKα to (EpoR) and Jak2 were observed through immunoprecipitations of transfected lysates in a manner exclusive to AMPK regulator subunits β and γ. Furthermore AMPKα was found t

Obesity rates have risen steeply in recent decades. This is accompanied with increased prevalence of several obesity-related disorders including type 2 diabetes. Genetic, environmental, and lifestyle factors (increased caloric intake and/or decreased physical activity) predispose individuals to type 2 diabetes by decreasing the body's responsiveness to the pancreatic hormone insulin, a physiological phenomenon commonly referred to as insulin resistance. Insulin resistance occurs due to induction of inflammation characterized by increased secretion of pro-inflammatory cytokines from enlarged adipose tissue, endoplasmic reticulum stress, and oxidative stress associated with excess blood glucose. There is a strong correlation between insulin resistance and decreased circulating levels of adiponectin (APN), a hormone implicated in promoting insulin-like activities. Further, inflammation negatively affects both insulin and adiponectin levels. It is recognized that folding of APN 18mer-subunits (insulin-sensitizing oligomer) is hindered in obese and type 2 diabetic individuals. Likewise, formation of normal hexameric insulin complex is compromised in type 2 diabetes. Insulin biogenesis, packaging, and assembly are impaired and unable to compensate for high blood glucose levels. As insulin and APN are key metabolic hormones essential for proper glucose regulation, maintaining their correct folding and assembly is required for conserving overall metabolic homeostasis. This dissertation centers on investigating proper assembly pathways of APN and insulin isoforms to form the higher order complexes necessary for their function. The interaction between APN oligomers was studied in the presence and absence of zinc, previously shown to inhibit formation of disulfide bonds in APN. We observed that zinc protects APN from collapse under acidic conditions and likely stabilizes oligomers through high affinity histidine coordination. The interaction between oligomers was further asses

Skeletal muscle is composed of repeating units called sarcomeres which contain distinct sets of thin and thick filaments that slide past each other during contraction. In addition to these proteins a third filament called titin acts as a molecular spring and prevents overstretching of muscle. In skeletal muscle, titin's spring-like elements include the PEVK sequence which elongates upon stretch and immunoglobulin-like (Ig) domains. A fourth myofilament, nebulin, is anchored into the Z-disk and present along the length of the thin filament. Nebulin is proposed to be a regulator of thin filament length. These large sarcomeric proteins can be differentially spliced to yield proteins of various sizes and properties. During my graduate career I sought to elucidate the role of titin and nebulin in skeletal muscle development and disease. Firstly, I studied if differential splicing of titin and nebulin occurred during development. Early post-natal development is a time of rapid isoform switching and growth, and I hypothesized that these large proteins could be affected. In post-natal development of the mouse, large compliant titin molecules are gradually replaced with shorter, stiffer isoforms through removal of PEVK exons. In nebulin, C-terminal exons present in the Z-disk are differentially expressed between muscle types and throughout development which correlates with differences in Z-disk width. My research has shown that titin and nebulin transcripts are tuned during development with changes in titin affecting the I-band region of the molecule and changes in nebulin affecting the Z-disk region. Secondly, I sought to study the effect of specific titin domains on titin elasticity in skeletal muscle. Changes in titin's stiffness occur in various myopathies but whether these are a cause or an effect of the disease is unknown. To test this, a genetically engineered mouse model was created in which part of the constitutively expressed immunoglobulin-like (Ig) domains of tit

Thrombospondin-1 (TSP-1) binding to its membrane receptor CD47 results in an inhibtion of the nitric oxide (NO) receptor soluble guanylate cyclase (sGC) and a decrease in intracellular cGMP levels. This causes physiologic effects such as vasoconstriction and a rise in blood pressure. The mechanism by which TSP-1 binds to CD47 at the membrane to decrease sGC activity is largely unknown. CD47 can physically associate with a number of binding partners, including α(v)β₃ and vascular endothelial growth factor receptor 2 (VEGFR2). Binding of a C-terminal fragment of TSP-1 called E3CaG1 to CD47 leads to a rise in intracellular calcium ([Ca²⁺](i)), which decreases sGC activity via a phosphorylation event. Binding of E3CaG1 is also known to disrupt the interaction between CD47 and VEGFR2, leading to a decrease in endothelial nitric-oxide synthase (eNOS) activity and cGMP levels through an Akt signaling pathway. However, it is not known whether other membrane proteins associated with CD47 are required for E3CaG1 binding and a subsequent [Ca²⁺](i) increase. Plasmon-waveguide resonance (PWR) spectroscopy was employed to elucidate the mechanism of TSP-1 inhibition of sGC activity through membrane complexes involving CD47. Using PWR, I found E3CaG1 can bind specifically to CD47 within native Jurkat membranes with picomolar and nanomolar dissociation constants (K(d)), suggesting multiple CD47 complexes are present. Among these complexes, CD47/VEGFR2 was found to bind E3CaG1 with a picomolar K(d)and CD47/α(v)β₃ was found to bind E3CaG1 with a nanomolar K(d). In addition, the presence of an anti-VEGFR2 antibody inhibited the E3CaG1-induced calcium response, which suggested CD47 in complex with VEGFR2 was responsible for TSP-1 reduction of sGC activity. I show that when both CD47 and VEGFR2 are returned to a HEK 293T cell line that does not contain these receptors, an increase in [Ca²⁺](i) upon E3CaG1 binding is restored. Interestingly, E3CaG1 was also found to bind to VEGFR2 in comp

Glucocorticoid receptor (GR) is known to associate with KATs and KDACs to regulate transcription. The current model of GR-mediated transcription focuses on agonist-dependent recruitment of KATs to acetylate histones and casts KDACs as corepressors in the presence of antagonist. Recent studies have shown KDACs to function as coactivators in the GR-mediated activation of the MMTV promoter and inhibition of KDACs impairs this activation. Nevertheless, the effect of KDAC inhibition on the GR-regulated transcriptome is unknown. Our expression profiling studies in a glucocorticoid (GC) responsive hepatoma-derived cell line, show that the class I-selective KDACi, VPA, has a profound impact on the GR-regulated hepatic transcriptome. VPA treatment alone mimics GC signaling at some GR-target genes and cooperates with GC to activate a small number of genes. However, the predominant effect of VPA, seen in more than 50% of the GR-target genes, is impairment of normal GR-mediated activation. This suggests that KDACs play a significant role in facilitating GR signaling. We have shown that VPA does not impair GR processing and that the inhibitory effects of VPA are due to impaired transcription. We have also determined that apicidin, a structurally distinct class I-selective KDACi, impairs GR-transactivation similar to VPA, while valpromide, a structural analog of VPA without KDACi activity, does not. In addition, siRNA-mediated depletion of KDAC1 fully or partially mimics the effects of VPA at most of the VPA impaired GR-target genes and co-depletion of KDACs 1 and 2 caused full or partial impairment of Dex-activation at a few other genes. Collectively, our results show that class-I KDACs facilitate GR-mediated transcription at most of the GR-target genes and that KDAC1 alone or in co-operation with KDAC2 is required for efficient GR-mediated transactivation. Furthermore, ChIP assays have shown that active KDACs are constitutively present at the gene promoters and that KDAC inhibi

Endoplasmic reticulum (ER) stress plays an important role in the pathogenesis of obesity-related inflammation and insulin resistance in adipose tissue. However, the mechanisms responsible for induction of ER stress are presently unclear. Proper ER redox state is crucial for oxidative protein folding and secretion and impaired protein folding in ER leads to induction of unfolded protein response and ER stress. However, while ER redox state is more oxidizing compared to the rest of the cell, its regulation is poorly understood. In order to determine the effects of ER redox state on development of ER stress and insulin resistance, several fluorescence-based sensors have been developed. However, these sensors have yielded results that are inconsistent with each other and with earlier non-fluorescence-based studies. In this study we attempted to develop and characterize a sensitive tool to study the ER redox state in adipocytes in real-time by targeting a new generation of redox-sensitive green fluorescent protein (roGFP) to ER. The roGFP1-iL sensor targeted to the ER is termed ‘eroGFP1-iL’ by convention. The ER-targeting eroGFP1-iL construct contains the signal peptide from adiponectin and the ER retention motif KDEL and has a midpoint reduction potential of -229 mV in vitro in oxidized and reduced lipoic acid. Despite having a midpoint reduction potential that is 50 mV higher than the previously determined midpoint reduction potential of the ER, eroGFP1-iL was found capable of detecting both oxidizing and reducing changes in the ER. In an attempt to determine the mechanisms by which roGFP1-iL detects oxidizing changes, we found that, first, glutathione mediated the formation of disulfide-bonded roGFP1-iL dimers with an intermediate excitation fluorescence spectrum resembling a mixture of oxidized and reduced monomers. Second, glutathione facilitated dimerization of roGFP1-iL, which in effect shifted the equilibrium from oxidized monomers to dimers, thereby increasing t

Pleckstrin homology (PH) domains are structurally conserved domains, which generally bind to phosphatidylinositol phosphate (PtdInsP) lipids. They are present in a variety of proteins, including those that are upregulated in cancer growth and metastasis, and represent a crucial component of intracellular signaling cascades and membrane translocation. Thus, they may be considered as attractive targets for cancer drug therapy. AKT (protein kinase B), a pleckstrin homology lipid binding domain and a serine/threonine kinase-containing protein, is a key component of the phophatidylinositol-3-kinase (PI3K)/AKT cell survival signaling pathway which is activated in a variety of cancers, including prostate, pancreatic, and skin cancers. In this study, I report the finding of a novel inhibitor of AKT; PH-427. I describe its effects on binding to the PH domain of AKT thus preventing its binding to PtdIns3-P at the plasma membrane and subsequent activation. In vivo testing of the drug led to reduction of tumor size and numbers in a mouse pancreatic cancer model. Additional testing of PH-427 on squamous cell carcinomas revealed that the drug is able to reduce tumor burden and multiplicity in vivo when topically applied. Thus, we demonstrate proof-of-principle in targeting PH domains as a viable cancer drug therapy option. The effects of PH-427 raised the intriguing possibility that targeting PH domains may have beneficial effects in other signaling pathways with PH domain-containing proteins. Guanine exchange factors (GEFs) contain a Dbl homology (DH) domain and a PH domain and have been shown to be involved in the process of metastasis. More specifically, RacGEFs activate Rac1 GTPase by facilitating the exchange of GDP to GTP. Over-expression of certain GEFs has been shown to contribute to increased malignancy in a variety of cancers. T-lymphoma invasion and metastasis-inducing protein-1 (Tiam1) is a highly conserved GEF and contains an N-terminal pleckstrin homology domain (nP

Nitric Oxide (NO) regulates cardiovascular homeostasis by binding to soluble guanylate cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca²⁺]ᵢ) and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, I show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells, and that inhibition requires an increase in [Ca²⁺]ᵢ. Using digital imaging microscopy on live cells, I further show that E3CaG1 binding results in a substantial increase in [Ca²⁺]ᵢ, up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca²⁺]ᵢ, also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca²⁺ remains inhibited, while addition of kinase inhibitors staurosporine, genistein, PP1 or PP2 reverse inhibition, indicating inhibition likely involves a tyrosine kinase, more specifically, a src family kinase. Rat sGC is also inhibited by lysates supplemented with Ca²⁺, suggesting that the site of modification is at an evolutionarily conserved residue. Inhibition is through an increase in K(m) for GTP, which rises to 834 μM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca²⁺]ᵢ in response to NO, inducing vasodilation, but is also inhibited by high [Ca²⁺]ᵢ, providing a fine balance between signals for vasodilation and vasoconstriction.

Microtubule-based mitochondrial transport into dendrites and axons is vital for sustaining neuronal function. Transport along microtubules proceeds in a series of plus- and minus-end directed movements facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective long distance transport remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites. To directly examine Miro's significance for kinesin- and/or dynein-mediated mitochondrial motility, we live imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of either antero- or retrograde mitochondrial transport by selectively impairing kinesin- or dynein-mediated movements, depending on the direction of net transport. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We concluded that dMiro promotes effective antero- and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport. To determine how Miro achieves this control mechanistically, we introduced point mutations that render each GTPase either constitutively active or inactive. Expression of either first GTPase mutant impaired antero- (inactive) or retrograde motor movements (active) in a direction dependent manner. The active state of the second GTPase domain up-regulated the number of consecutive kinesin motions during anterograde transport but impaired kinesin transport biases while the inactive second GTPase state impaired transport in either direction. Together, these data sugge

Melanin pigmentation is a complex trait governed by many genes. Variation in melanin pigmentation within, and between, populations makes it an important trait for assisting in physical identification of an individual in forensic investigations. Utilizing a training sample (n=789) comprised of various ethnicities and SNPs (75) in 24 genes previously implicated in human or animal pigmentation studies, I determined three-SNP multiple linear regression models that accounted for large proportions of pigmentation variation in skin (45.7%), eye color (76.4%), and hair [eumelanin-to-pheomelanin (43.2%) and total melanin (76.3%)], independent of ethnic origin. Rather than implementing stepwise regression, to ascertain the three-SNP predictive models, I devised an algorithm that is likely more robust than stepwise regression. The algorithm consisted of two steps: the first step reduced the pool of 75 SNPs to a pool of 40 by selection of SNPs that were significant (p<0.05) by one-way ANOVA; the second step enabled selection of SNPs for model incorporation based on their frequency in the best-fitted models of all possible combinations of three-SNP models (i.e., 40 choose 3).Prediction models were validated utilizing an independent cohort (n=242, test sample) that was very similar in ethnic composition to the training sample. Relative shrinkage was moderate for skin reflectance (23.4%), eye color (19.4%), and eumelanin-to-pheomelanin (37.3%) of hair, and largest for total melanin (67%) of hair. Additionally, we refined our model-building algorithm, enabling visual comparison of the frequency and co-linearity due to linkage or co-inheritance of SNPs of the best-fitted models. Application of our algorithm to the test sample yielded the same or similar models as the training sample. Two of the three SNPs composing the models were the same, with some variability in the third SNP of the model.

The tumor suppressor Breast Cancer Susceptibility Protein 1 (BRCA1) protects our cells from genomic instability in part by facilitating the efficient repair of DNA double-strand breaks. Other functions of BRCA1 include transcriptional regulation, apoptosis, DNA damage signaling, chromatin remodeling and protein ubiquitination. The major contribution of BRCA1 to maintaining genomic stability is thought to be through its role in DNA repair. BRCA1 promotes the error-free repair of double-strand breaks by homologous recombination, and is also implicated in the regulation of non-homologous end joining repair. Here we investigated the role of BRCA1 in maintaining the fidelity of non-homologous end joining repair following a double-strand break. We also examined the frequency of microhomology-mediated end joining (MMEJ) and the fidelity of double-strand break repair relative to BRCA1 protein levels in both control and tumorigenic breast epithelial cells. In addition to altered BRCA1 protein levels, we tested the effects of cellular exposure to mirin, an inhibitor of Meiotic recombination enzyme 11 (Mre11) 3' to 5' exonuclease activity. Knockdown or loss of BRCA1 protein resulted in an increased frequency of overall plasmid DNA repair mutagenesis and MMEJ following a double-strand break. Inhibition of Mre11 exonuclease activity with mirin significantly decreased the occurrence of MMEJ, but did not considerably affect the overall mutagenic frequency of plasmid double-strand break repair, although some of our data indicate that the size of sequence deletions may be reduced by mirin inhibition. The results suggest that BRCA1 protects DNA from mutagenesis during non-homologous double strand break repair in plasmid-based assays. The increased frequency of double-strand break mutagenesis and MMEJ repair in the absence of BRCA1 suggests a potential mechanism for carcinogenesis.

Cardiovascular disease (CVD) is the leading cause of death in the United States, and the risk and severity of CVD are increased with type 2 diabetes. However, the exact mechanisms that result in the enhanced association between CVD and type 2 diabetes have not been clearly elucidated. Inflammation and oxidative stress are strongly implicated in both diseases. In type 2 diabetes, there is a dysregulation of inflammatory mediators where an anti-inflammatory molecule adiponectin and a pro-inflammatory cytokine TNF-alpha, are reduced and increased, respectively. Even lower plasma adiponectin concentrations are associated in type 2 diabetic patients with cardiovascular disease. Thus, adiponectin may be a significant link between the two diseases. The studies in this dissertation examined the in vivo cardioprotective actions of adiponectin and apocynin, a NADPH oxidase inhibitor, in the Zucker diabetic fatty (ZDF) type 2 diabetic model. A model of coronary artery occlusion was utilized to induce myocardial ischemia-reperfusion (I/R) injury. The mechanisms of protective actions were assessed by measures of inflammation, oxidative stress and DNA damage. Further, in vitro actions of adiponectin in human whole blood were investigated. We found that in vivo treatments of adiponectin and apocynin significantly reduced myocardial infarction in the type 2 diabetic heart by 40% and 68%, respectively. The cardioprotective action of adiponectin was associated with 2 to 4 fold significant attenuations in several inflammatory characteristics, such as neutrophil adhesion molecule CD11b expression, myocardial adhesion molecule ICAM-1 expression, myocardial neutrophil accumulation and plasma TNF-alpha concentration. The cardioprotective action of apocynin was associated with a significant reduction in myocardial oxidative stress by 25%. In vitro adiponectin actions demonstrated the ability of adiponectin to reduce neutrophil ROS production in human whole blood. These studies were the fir

This dissertation presents an investigation into the structure of macromolecular protein assemblies by mass spectrometry. The experiments described within are designed to systematically assess the analytical utility of surface-induced dissociation (SID) tandem mass spectrometry in the characterization of multi-subunit protein complexes. This is accomplished by studying the effects of ion-surface collision on the fragmentation products of protein assemblies that vary by mass, number of subunits, and protein structural features. The dissociation energetics and mechanisms of protein complexes are considered by examining the influence of ion internal energy and sub-oligomeric protein structure on the dissociation process. Conditions are first established for the preservation of “native” protein quaternary structure and applied to previously characterized systems for proof-ofconcept. These conditions are subsequently extended to determine the molecular weight and subunit stoichiometry of several small heat shock proteins. Native mass spectrometry is then combined with limited proteolysis experiments to characterize the subunit interface of a unique small heat shock protein, Hsp18.5 from Arabidopsis thaliana, identifying regions of the protein essential for preservation of the native dimer. The dissociation of non-covalent protein assemblies is then explored on a quadrupole time-of-flight (Q-TOF) mass spectrometer, modified for the study of ion-surface collisions. This instrument allows ions to be dissociated through collisions with a surface or more conventional collisions with gas atoms. The dissociation of protein complexes is explored by both activation methods beginning with specific and non-specific dimers with masses less than 40 kDa. These studies are extended to larger assemblies with as many as 14 subunits weighing over 800 kDa, and are applied to both homo- and hetero-oligomeric protein complexes. Activation of a protein complex with “n” subunits through multip