• 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.
    • Functional genomic approaches to delineate preterm birth and its regulation of placental transporter proteins

      Mason, Clifford W.; Swaan, Peter W. (2008)
      Preterm birth (PTB) affects 12.7% of all pregnancies in the US and causes significant fetal and neonatal morbidity and mortality. Greater than 85% of PTBs (< 28 weeks gestation) have evidence of localized inflammation. Current efforts to prevent prematurity have met with little success and progress to identify effective new therapeutic agents has been modest. This dissertation is aimed at improving the current understanding of the molecular pathways underlying PTB and the effects of PTB and inflammation on drug transport and metabolism. Chapter 1 describes concepts and current understanding of preterm birth and the pharmacological challenges to its prevention. The first part of this chapter includes a discussion of the clinical issues and mechanistic research in PTB and genomic approaches to understand its pathophysiology. The second part focuses on placental transporters and their regulation during pregnancy and disease. A difficult challenge in identifying the mechanisms that control labor has been developing tools for examining whole-genome expression profiles in the context of known biology. Chapter 2 presents a novel methodology in gene array profiling used to extract relevant information from gene networks to elucidate the mechanisms of myometrial quiescence and activation. In Chapter 3, this approach was exercised in human myometrial tissue from term and preterm pregnancies. Results indicate that labor is characterized by muscle contraction, cell adhesion and remodeling gene sets. The initiation of term labor involves proteins mapped to inflammatory-related processes while preterm labor is triggered by multiple regulatory pathways. Chapter 4 addresses the involvement of key drug transporters in pregnancy- and inflammation-mediated changes in drug disposition and fetal susceptibility. Changes in transporter expression were analyzed by real-time PCR and immunoblotting techniques on placentas of women with term, preterm and inflammation-associated preterm pregnancies. MDR1 and BCRP were up-regulated in early gestation and amplified with inflammation but these changes were not mediated by nuclear hormone receptors, regulatory cytokines, or gram-negative bacterial components. In conclusion, inflammatory responses initiate preterm labor and result in an inherent up-regulation of MDR1 and BCRP to protect the fetus from endogenous stress factors. Finally, in Chapter 5, future directions to extend this research are presented.
    • In-vitro Efficacy and Intracellular Mechanism of Riboflavin-Conjugated PEGylated Poly- L-Lysine Dendrimer

      Pak, Yewon; Swaan, Peter W. (2017)
      Chemotherapeutic drugs have advanced using different drug delivery methods to treat breast cancer specifically. This development has arisen because many classical drugs exhibit physicochemical limitations including solubility, specificity, stability, biodistribution, and therapeutic efficacy. There were numerous adverse effects associated with these limitations because chemotherapeutic drugs enter normal tissues. In order to eliminate off-target side-effect,nanoparticles were developed to target anticancer drugs to a specific carcinogenic area. As one of developing nanomedicines, dendrimers possess ability to be utilized in different administration routes and has potential to stay in the blood circulation longer while showing increased accumulation in tumor cells. Commercially available poly (amidoamine) (PAMAM) dendrimers have the potential to cause toxicity in vivo due to lack of biodegradation at sites of accumulation. Poly-L-Lysine (PLL) dendrimers are an alternative class of dendrimers that possess a biodegradable structure. PEGylated poly-l-lysine (PLL) dendrimers are known to be more favorable due to lessened cytotoxicity manifested by masking of cationic charges and avoiding uptake by Reticulo Endothelial System (RES). Using this biodegradable dendrimer, we sought to examine the effect of PEGylation as well as delivering anti-cancer drug, Doxorubicin (DOX), to a targeted internalization pathway in human breast cancer cells effectively. PEGylated PLL dendrimers also have their limitation, in which some tumor cells are not dependent upon enhanced permeability and retention (EPR) effect. As a result, riboflavin receptor, which is found to be upregulated in the exterior of breast and ovarian cancer cells, was utilized by attaching a riboflavin ligand to PEGylated PLL dendrimers in order to be actively uptaken by breast cancer cells. To target chemotherapeutic drug selectively and efficaciously, riboflavin conjugated PLL dendrimers were assessed in-vitro by investigating cytotoxicity, uptake accumulation, and intracellular colocalization. Further investigation on the endocytosis mechanism and detailed intracellular trafficking in different compartments of the cells were analyzed in order to fully understand the machinery behind delivering chemotherapeutic drugs successfully.
    • Mechanisms of Dendrimer-Mediated Oral Drug Delivery

      Avaritt, Brittany; Swaan, Peter W. (2014)
      Oral administration of chemotherapeutics remains a challenge despite the benefits for both the patient and health care system. To overcome the poor solubility and low oral bioavailability of anti-cancer drugs, polymeric delivery systems have been investigated. Dendrimers, a class of highly branched polymers, have proven useful for drug delivery because of their compact, nanoscopic size. Specifically, poly(amidoamine) (PAMAM) dendrimers have been shown to permeate the intestinal epithelium indicating potential as oral drug delivery carriers. While studies in our laboratory have determined the effects of surface modification on dendrimer transport and uptake, a large gap in knowledge exists in the transport and cytotoxicity mechanisms of PAMAM dendrimers. Additionally, alternatives to PAMAM dendrimers such as biodegradable poly-L-lysine (PLL) dendrimers have yet to be investigated for use in oral delivery. In this work we report the mechanisms of tight junction modulation by PAMAM dendrimers. While anionic dendrimers modulated tight junction proteins, cationic dendrimers opened tight junctions through phospholipase C-mediated calcium signaling allowing for paracellular small molecule transport. In comparison, cationic PLL dendrimers also allowed for small molecule transport with similar decreases in transepithelial electrical resistance. Small generation PAMAM and PLL dendrimers (16 and 32 surface amines) activated Caspase-3 and -7 resulting in apoptosis. In contrast, PLL dendrimers showed less long term toxicity compared to PAMAM dendrimers illustrating the benefits of dendrimer biodegradability. We also investigated the mechanisms of PLL dendrimer internalization and subcellular trafficking and the impact conjugation had on these mechanisms. The pH and enzymes present vary within different intracellular vesicles. Knowledge of the environment a drug delivery system will encounter is crucial for proper drug release. While PLL dendrimers were internalized via cholesterol- and dynamin-mediated endocytosis and macropinocytosis, conjugation site impacted uptake and localization. By conjugating a model compound to either the dendrimer core or surface, the uptake and transport properties of the delivery system were modified. Core-conjugated dendrimers had higher uptake and localized to the lysosomes and nucleus while surface conjugation resulted in higher transport and less accumulation in lysosomes. This research provides important knowledge for designing an effective dendrimer-based oral drug delivery system.
    • Molecular Insight into the Structure, Function, and Regulation of Bile Acid Transport

      Czuba, Lindsay Christine; Swaan, Peter W.; 0000-0001-9142-5706 (2017)
      The human Apical Sodium-dependent Bile Acid Transporter (SLC10A2), also known as hASBT, plays an integral role in the enterohepatic circulation of bile acid and cholesterol homeostasis. As a member of the solute carrier family of membrane transporters, it uses the established Na+ electrochemical gradient as an energy source to reclaim bile salts from the ileum. hASBT has been identified as a promising target for the management of hypercholesterolemia, cholestatic pruritis, and as a prodrug-targeting approach for improved bioavailability of drugs. Limiting the development of such therapeutics, is an incomplete understanding of hASBT's structure. Extensive biochemical and mutagenesis studies for hASBT support a seven transmembrane model. Yet conflicting structures have emerged with the elucidation of the crystal structures of two putative homologues from Neisseria meningitidis (nmAsbt) and Yersinia frederiksenii (yfAsbt). In the absence of a physiological context, the use of their structure as models of the human transporter is limited. In addition to the discrepancy in hASBT's fold, there is limited information regarding the specific proteoforms that are relevant to the functional expression of hASBT and in its regulation. In this work we provide novel molecular insight into the structure, function, and regulation of human ASBT. We contrasted the biochemical, inhibitory, and evolutionary attributes of nmAsbt, yfAsbt, and hASBT and identified their critical differences. The fundamental differences in ion dependency, substrate specificity, and evolutionary context imply divergent structure-function relationships and negate the use of the bacterial transporters as suitable models for hASBT. Additionally, we characterized the role of tyrosine phosphorylation in regulating the functional expression and stability of hASBT. We identified Src family kinases as critical modulators and provide support for hASBT's regulation by phosphatases. As the clinical relevance of PTMs is growing, so too are the number of FDA-approved therapeutics that target these modifications. In this regard, we have made critical advances and gained valuable insight into hASBT's regulation. Finally, we have optimized the biological sample preparation methods and have significantly increased the purity of hASBT samples. When coupled with mass spectrometry analysis, these methods will identify critical proteoforms of hASBT and facilitate a global understanding of its structure-function relationship.
    • Molecular Mechanisms of Intestinal Bile Acid Transport and Immunomodulatory Potential of Bile Acids

      Ayewoh, Ebehiremen; Swaan, Peter W.; 0000-0002-8390-1538 (2021-12)
      Bile acids are catabolic products of cholesterol that play an important role in the digestion of dietary facts, lipid soluble vitamins, and drugs, as well as a role in immune regulation and glucose homeostasis. They function as complex signaling molecules to prevent intracellular accumulation of bile acids and modulate bile acid and cholesterol homeostasis via activation of a nuclear receptor, Farnesoid X receptor (FXR), to repress bile acid uptake transporters and enhance bile acid efflux transporters. The human sodium dependent bile acid transporter (ASBT) is a highly regulated intestinal uptake transporter that acts as the rate limiting step in bile acid transport in the enterohepatic circulation. Targeted ASBT inhibition is currently being investigated for use in cholestasis, hyperlipidemia, chronic idiopathic constipation, and type 2 diabetes. While studies on post-translational modifications (PTMs) have revealed N-linked glycosylation and phosphorylation as regulators of ASBT, ASBT regulation is still poorly understood. The lipid-based PTM, S-acylation, is the reversible addition of an acyl chain, via a labile thioester linkage, onto cysteine residues, thereby increasing the affinity of proteins to cellular membranes. In this work, we show that human ASBT is S-acylated and that S-acylation is vital for ASBT function, cell surface expression, substrate transport kinetics, and protein stability. Screening of cysteine mutants in and or near transmembrane domains, some of which are exposed to the cytosol, confirmed Cys314 to be the predominate S-acylated residue. Mutation of cytosolic tyrosine residues resulted in decreased ASBT S-acylation suggestive of crosstalk between both PTMs and the existence of multiple PTM-based proteoforms. Finally, we investigate functional implications of the potential acyl transferases responsible for ASBT acylation. Overall, we have provided valuable insight on human ASBT regulation and highlighted the necessity for further investigation of the impact of PTM proteoform in drug development. While understanding ASBT regulation is vital in addressing intestinal and hepatobiliary disease states, the extent as to which its substrate, bile acids, play in other molecular processes is important in fully understanding the broader relevance of intestinal bile acid transport and bile acid signaling. Bile acids have emerged as complex signaling molecules in glucose homeostasis and immune regulation where they can activate specific receptors to increase insulin secretion and exert anti-inflammatory responses from mucosal immune cells, respectively. Using immunological approaches, we provide preliminary evidence and scientific perspective on the use of bile acids in nanoformulation that aims to exploit the immunomodulatory potential of bile acids.
    • Structural and functional characterization of the human apical sodium-dependent bile acid transporter

      Banerjee, Antara; Swaan, Peter W. (2005)
      The human apical sodium-dependent bile acid transporter (hASBT), an essential component of the enterohepatic circulation (EHC), is responsible for bile acid reabsorption from the lumen of the distal ileum and plays a critical role in bile acid and cholesterol homeostasis. Lack of a crystal structure for ASBT, limits our understanding of the structural and functional determinants of transport. The work in this dissertation was carried out to characterize the structural components and determine their overall role in ASBT function. In particular, the work described here was aimed to (1) elucidate and further understand the topological framework of hASBT by epitope insertion, (2) evaluate the role of N-glycosylation and the N-terminal domain in ASBT function by alanine scanning mutagenesis, (3) probe the role of the endogenous cysteines using thiol modifiers and various bile acid conjugates, (4) and to determine the sodium, and bile acid translocation pathway comprising residues of transmembrane (TM) domain seven and extracellular loop (EL) three using substituted cysteine accessibility method. Due to conflicting experimental evidence, the membrane topography of ASBT, predicted to comprise 7 to 9 putative TM domains, remains unresolved. Our results from epitope insertion clearly, support a 7TM model. The work that followed was aimed at characterizing some of the functionally critical domains of this transporter. Alanine scanning mutagenesis showed that the N-terminal region was vital for function and mutation of the N-glycosylation site was responsible for reduced uptake activity but did not impact trafficking of the protein to the plasma membrane. Evaluation of the endogenous cysteine residues revealed that multiple Cys residues are essential for hASBT function and C270A in combination with methanethiosulfonate (NITS) reagents and bile acid-NITS conjugates can aid in defining the putative ligand binding region(s). Cysteine mutants of EL3 and TM7 were generated using C270A, and in conjunction with thiol-modification it was shown that EL3 forms the primary sodium interaction site while TM7 lines the substrate translocation pathway. Furthermore, critical residues involved in the process were also identified. Overall, the work carried out in this dissertation will aid in the advancement of drug design and improve our understanding of the features that are essential for recognition of effective inhibitors for this transporter.
    • UNDERSTANDING STRUCTURE-FUNCTION RELATIONSHIPS AND PROTEIN STABILITY OF THE HUMAN APICAL SODIUM-DEPENDENT BILE ACID TRANSPORTER (ASBT, SLC10A2)

      Claro da Silva, Tatiana; Swaan, Peter W. (2011)
      The apical sodium-dependent bile acid transporter (ASBT, SLC10A2) is renowned as the major bile acid transporter in the intestine. It utilizes cellular sodium gradient to actively concentrate bile acids in the enterocytes, thereby playing a key role in the enterohepatic circulation of bile acids (EHC). ASBT is a promising target for prodrug approaches aiming at improving drug bioavailability, and for drugs to treat hypercholesterolaemia, since cholesterol metabolism is induced upon bile acid depletion. Moreover, its contributing role in drug-drug interactions is rapidly emerging. Topologically, the human (hASBT) is a glycoprotein that spans the membrane bilayer seven times, oriented with an extracellular N-terminus and a cytoplasmic C-terminus (Nexo/Ccyt). Despite its physiological and pharmacological relevance, ASBT remains to be fully characterized at the molecular level. Here, we summarize advances made by our group and others in the quest to understand ASBT’s structure-function relationships and its complex mechanism of bile acid transport. We also report our novel findings regarding protein regions relevant for function and protein stability in the hASBT. Our observations indicate that residues located at the transmembrane 1 (TM1) play a pivotal role in hASBT function, and that Gly50, placed at the interface of TM1 with the intracellular loop 1 (IL1), is critical for hASBT stability. Expanding our studies to IL1, we identified a cluster of amino acids comprising Cys51 – Lys57, which are likely involved in hASBT protein stability, whereas residues downstream Lys57 appear to be relevant for transport. We have demonstrated that successful mapping of regions implicated in hASBT’s transport cycle can be achieved with a combination of sitedirected mutagenesis, bile acid uptake and kinetics, sodium-activation assays and the substituted-cysteine accessibility method. Moreover, inhibition of the proteasome with MG132, and of prolyl-peptidyl isomerases with cyclosporine A and FK506, are valuable approaches to reveal the contribution of specific amino acids to hASBT stability. Finally, we integrate our data to propose an overall schematic of hASBT transport, which will contribute to a better understanding on ASBT physiology and, potentially, on other proteins in the SLC10 family.
    • Water Soluble Polymer Drug Therapies for Targeted Delivery to Pancreatic Cancer

      Stanton, Joseph D.; Swaan, Peter W. (2014)
      Current therapies of advanced staged pancreatic cancer are limited by poor response and high toxicity of chemotherapeutics. As the molecular basis of pancreatic cancer has become better understood the need for a targeted therapy could help provide an increase in therapeutic response while also limiting side effects. N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer drug conjugates have demonstrated their potential use as carriers of small molecule drugs to improve cancer therapies. The overall goal of this research was to develop a polymer peptide drug conjugate based on HPMA copolymers, which can increase the therapeutic index of pancreatic cancer chemotherapy. Previous investigation of the uMUC1 receptor, which is a glycoprotein overexpressed on the surface of pancreatic tumors, has lead to the development of EPPT1, a small peptide has been found to have a strong binding affinity to uMUC1, (Kd=20uM). Our hypothesis HPMA copolymer with an active target EPPT1 to the uMUC1 receptor will enhance therapeutic action of a cancer chemotherapeutic drug such as gemcitabine. In this study, we were successful in the synthesis and characterization of a series of HPMA copolymer-EPPT1-Gemcitabine conjugates. Using model pancreatic cancer cell lines, the binding efficiency, internalization and mechanisms of cellular uptake were evaluated with polymer EPPT1 conjugates. Polymer gemcitabine conjugates were evaluated for efficacy against free gemcitabine. The optimized polymer peptide drug conjugates were evaluated for efficacy and drug release. Results during synthesis and characterization indicated that copolymer yield, solubility and performance were influenced by each incorporation of peptide and drug. Flow cytomentry determined that polymer peptide conjugates were able to bind with Capan-2 and Panc-1 cell lines. Confocal microscopy verified that polymer peptide conjugates were not only getting internalized into the cytoplasm but also routing to the lysosome. Using endocytosis inhibitors, confirmed that polymer peptide conjugates use clathrin mediated endocytosis pathways when getting internalized into the cell. Drug release studies revealed that gemcitabine will detached from the polymer in lysosomal conditions. Polymer drug conjugates compared to free gemcitabine alone against pancreatic cancer cells in MTT assay had equal efficacy. Attachment of the active targeting moiety EPPT1, exhibited that polymer peptide drug conjugates were superior in killing cells to free gemcitabine alone.