• Bile Acids as Biomarkers and Evolutionary Phenotypes

      Shiffka, Stephanie; Swaan, Peter W.; Kane, Maureen A.; 0000-0002-3571-1836 (2021)
      Bile acids (BAs) are the amphipathic end products of cholesterol metabolism and represent a critical means of cholesterol excretion. BAs have a plethora of functions, including digestive roles, homeostatic feedback loops, energy metabolism, regulation of the microbiome, inflammation, and more. These effects implicate BAs in physiological and pathological processes throughout the body, not just within the enterohepatic circuit. To date, BAs have been linked to the pathogenesis of multiple types of cancer, type 2 diabetes mellitus, metabolic syndrome, and neurological disorders, among others. In health, BA homeostasis is precisely regulated by a process termed enterohepatic circulation (EHC). Several transport proteins are instrumental to this process, and disruptions in any of these transport systems lead to dysregulation of BA homeostasis, further leading to complications such as cholestasis and liver disease. BA metabolism and the EHC are conserved throughout vertebrate evolution, but the BA pool of more modern species has been modified to be more hydrophilic while still retaining properties of digestive surfactants. Though EHC is well-characterized, the understanding of eukaryotic transporters in this process is lacking, especially at the molecular level. Despite the recognition of bile acids as signaling molecules involved in disease progression, there remain numerous BAs that are poorly characterized. This is especially important because BAs are an extremely diverse group of molecules that represent the effects of host and microbiome metabolism. Furthermore, the unique physicochemical properties of these variations confer these molecules with differential levels of cytotoxicity and divergent, sometimes opposing, activation of cell signaling pathways. Thus, the scope of this dissertation is two-fold: first, to further characterize the BA pool in health and injury using cell and animal models; secondly, to use this information in order to probe the transporter responsible for the first step of the enterohepatic circulation, ASBT (SLC10A2). Completion of the first objective yielded improved understanding of BA metabolism in cell culture models and non-human primate laboratory models, as well as in radiation injury in the latter model. Accomplishment of the second objective returned insight into ASBT and BA evolution through the use of multiple vertebrate orthologs.
    • Effects of Static Growth on P. aeruginosa Iron Homeostasis and Virulence

      Brewer, Luke; Oglesby, Amanda G.; Kane, Maureen A. (2020)
      Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogens that causes life-threatening, antimicrobial resistant infections in vulnerable patient populations, including patients with cystic fibrosis, cancer, and chronic wounds. During infection, P. aeruginosa requires iron to maintain critical aspects of its metabolism, and possesses numerous virulence factors and iron uptake mechanisms that allow it to compete for this essential metallo-nutrient in the iron-limiting host environment. These systems are tightly regulated by iron-responsive regulatory mechanisms, which ensure adequate uptake while preventing iron toxicity. Because of the essential role of these regulatory mechanisms in maintaining iron homeostasis, they are considered a promising approach for treating P. aeruginosa infections. One prominent regulator of P. aeruginosa iron homeostasis is the PrrF small RNA (sRNA) regulator, which is essential for virulence in acute murine lung infection. Unfortunately, the exact contribution of PrrF to P. aeruginosa pathogenesis has not yet been elucidated. Moreover, our current understanding of PrrF and other iron regulatory mechanisms is largely based on studies using shaking and highly aerated cultures, which are not likely representative of microbial communities in vivo. To address these gaps, the work in this thesis utilizes proteomic, metabolic, and genetic approaches to determine the impact of static growth on iron-responsive regulatory mechanisms in P. aeruginosa, including PrrF sRNAs. We demonstrate that iron regulation paradigms in P. aeruginosa are dramatically altered in static conditions, due in part to changes in PrrF activity. Notably, we identify type 6 secretion systems (T6SS) as a target of enhanced iron regulation in P. aeruginosa in static conditions, and demonstrate that this altered regulation is caused by changes in the production and activity of the PrrF-regulated quorum signaling molecules, 2-alkyl-4(1H)-quinolones (AQs). Furthermore, we demonstrate that altered AQ activity may modulate clinically-significant interactions with other opportunistic pathogens, such as S. aureus, In turn, the work described herein has broad implications for the study of P. aeruginosa infections, and highlights the need to further probe essential P. aeruginosa iron homeostasis mechanisms in static conditions.
    • Impact of cellular retinol-binding protein, type I on retinoic acid biosynthesis and homeostasis

      Pierzchalski, Keely A.; Kane, Maureen A.; 0000-0002-2456-0382 (2015)
      Statement: A global Rbp1 knock out (Rbp1-/-) mouse model was used to correlate direct retinoid measurements with vitamin A metabolizing and atRA biosynthesizing enzyme activities, Crbp function and tissue microenvironment for the first time. Methods: atRA was quantified by LC-MRM3 and ROL/RE/RAL was quantified by HPLC-UV. Enzyme activities were measured from enzymes present in subcellular fractions isolated from WT and Rbp1-/- tissues. Mouse CrbpI and CrbpIII were purified from transformed Escherichia coli for functional comparative studies. Tissue were formalin fixed for histological examination. Relative gene expression was analyzed using quantitative PCR. Results: Reduced atRA was consistently quantified in extrahepatic tissues with elevated ROL/RE. Relative gene expression showed altered expression in retinoid pathway proteins and atRA loss preceded expression changes in some cases. Tissue microenvironments also consistently showed a loss of structure and organization along with accumulation of extracellular matrix and hyperplasia without apparent disease. Functional studies showed that CrbpIII binds retinol with less affinity than CrbpI and does not function equivalently to CrbpI in regulation of atRA biosynthesis. Also, metabolizing enzymes had altered activities in the Rbp1-/- tissues with reduced atRA biosynthesis. Conclusions: Loss of CrbpI results in altered regulation of enzyme activity and atRA homeostasis cannot be maintained by other Crbp homologs in extrahepatic tissues. Dysfunctional atRA biosynthesis due to loss of CrbpI results in altered tissue microenvironment characteristic of dietary vitamin A deficiency and precancerous dysfunction associated with cancers that are observed to have silenced CrbpI.
    • Targeting the Activator Protein-1 Complex to Inhibit Airway Smooth Muscle Cell Hyperproliferation in Asthma

      Defnet, Amy Elizabeth; Shapiro, Paul, Ph.D.; Kane, Maureen A. (2021)
      Hyperproliferation of airway smooth muscle (ASM) cells leads to increased ASM mass causing airway obstruction in inflammatory diseases such as asthma. Currently, there are no effective therapies to modulate ASM cell proliferation that contributes to debilitating bronchoconstriction in severe asthmatics. Previous studies suggest that activator protein-1 (AP-1) transcription factor expression is upregulated in airway cells in asthma and inhibition of AP-1 could mitigate the hyperproliferation of ASM cells. AP-1 activity has been shown to be enhanced by upstream extracellular signal-regulated kinase (ERK1/2) signaling or antagonized by retinoic acid receptor (RAR)-mediated signaling. The overall goal of the current study was to evaluate the therapeutic potential of a combination therapy of an ERK1/2 inhibitor and RAR agonist to modulate AP-1 complex formation and activation. Aim 1 studies tested the hypothesis that a novel function-selective ERK1/2 inhibitor, referred to as SF-3-030, would mitigate off-target toxicity while regulating platelet-derived growth factor (PDGF) induced AP-1 activity and ASM cell proliferation. In Aim 2 studies we evaluated the role of retinoids in controlling AP-1 complex formation and identified a RARγ isoform-specific agonist, CD1530, as a potential therapeutic option for inhibition of AP-1 activity and ASM cell hyperproliferation. Aim 3 studies determined whether a polypharmacological approach of combining ERK1/2 inhibition and RAR agonism to target two different aspects of the AP-1 complex activation and formation would have an additive effect in preventing ASM hyperproliferation. Overall, these studies help further our understanding of how AP-1 signaling causes the hyperproliferation of ASM cells while elucidating possible therapeutic treatment options through ERK1/2 inhibition and RAR agonism.
    • "Zoom in" on protein functions through integrated mass spectrometry

      Li, Wenjing; Kane, Maureen A.; 0000-0002-5387-7004 (2018)
      Mass spectrometry (MS) has been a powerful tool in cracking the protein codes for human biology regarding to their structure and function. Rich information can be collected through MS either at protein level (native/intact, top-down) or peptide level (middle-down and bottom-up), however integration of these approaches in order to generate a comprehensive view of the protein(s) has been underutilized. In this thesis, strategic integration of MS platforms was developed for two protein systems with the aim of elucidating the fundamental molecular function related to protein-ligand or protein-protein interactions toward fulfilling the potential of MS-based platforms for application in drug discovery. In the first study, the integration of native top down mass spectrometry coupled with ion mobility analysis provided extensive structural information to understand gold finger protein complex that formed with the exchange of zinc. Native top down analysis identified the stoichiometry, binding residues and preferable binding sites when gold replaces zinc in the parent nonclassical zinc finger protein tristetraprolin (TTP). The subtle difference in conformation were monitored by ion mobility simultaneously. The heterogeneity of gold fingers that were reflected by MS-based assays was not obvious by other conventional assays, suggesting the unique analytical power of MS for in-depth drug-target investigations. In the second study, bottom-up and native MS were applied on interrogation of the role of cellular retinol binding protein, type I (CrbpI) in maintaining retinoid homeostasis. The endogenous level of CrbpI was confidently quantified through customized bottom-up assays, which demonstrated a positive correlation with active metabolite retinoic acid (RA). Further investigation focused on the interaction between CrbpI and its biological ligands (retinol and retinal), and the RA-producing enzyme retinal dehydrogenase 1(Raldh1), using native mass spectrometry. These studies contributed fundamental information toward elucidating the role of CrbpI in facilitating RA biosynthesis, and also suggested its potential as a disease marker.