Biochemistry & Molecular Biology Department Theses and Dissertations
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Item The mRNA Elements Directing Preferential Translation in the Integrated Stress Response(2022-09) Amin, Parth Hitenbhai; Wek, Ronald C.; Dong, X. Charlie; Elmendorf, Jeffrey S.; Mosley, Amber L.In response to environmental and physiological stresses, cells impose translational control to reprogram adaptive gene expression and conserve energy and nutrients. A central mechanism regulating translation involves phosphorylation of the a-subunit of the eukaryotic initiation factor -2 (p-eIF2a), which reduces delivery of initiator tRNA to ribosomes and represses global protein synthesis. The pathway featuring p-eIF2a is called the integrated stress response because it involves multiple related eIF2a kinases, each responding to different stress arrangements. While p-eIF2a limits global protein synthesis, a subset of mRNAs are preferentially translated in response to p-eIF2a. Preferential translation of stress adaptive mRNAs is regulated by upstream opening reading frames (uORFs) present in the 5’-leader of these transcripts. In most cases uORFs are inhibitory in nature, but in some case uORFs can instead promote the translation of the downstream CDS. This study is focused on preferential translation of the gene Inhibitor of Bruton’s Tyrosine Kinase-alpha (IBTKa) in response to endoplasmic reticulum stress. The human IBTKa gene encodes a 1353 amino acid residue protein, along with a 5’-leader featuring predicted canonical uORFs. Among the four predicted uORFs, the 5'-proximal uORF1 and uORF2 are phylogenetically conserved among mammals and are well translated as judged by reporter assays, whereas uORF3 and uORF4 are not conserved and are poorly translated. In addition to the uORFs in the IBTKa mRNA, a phylogenetically conserved stem-loop (SL) of moderate stability is present 11 nucleotides downstream of uORF2. Using luciferase reporter assay, the uORF2 and SL were shown to function together to repress the translation of human IBTKa. In non-stressed conditions, the SL combined with uORF2 are critical for reducing ribosomes from reinitiating at the IBTKa coding sequence (CDS), thus repressing IBTKa expression. Upon ER stress and induced p-eIF2a, the more modestly translated uORF1 facilitates the bypass of the inhibitory uORF2/SL to enhance the translation of main CDS of IBTKa. This study demonstrates that uORFs in conjunction with RNA secondary structures can be critical elements that serve as a “bar code” by which scanning ribosomes decide which mRNAs are preferentially translated in the integrated stress response.Item Using Chemical Probes to Define the Role of Aldehyde Dehydrogenase 1A in a Breast Cancer Model(2022-09) Takahashi, Cyrus; Hurley, Thomas; Georgiadis, Millie; Harrington, Maureen; Hawkins, Shannon; Wek, RonaldThe aldehyde dehydrogenase (ALDH) superfamily comprises a group of NAD(P)+-dependent enzymes that catalyze the conversion of aldehydes to their corresponding carboxylic acids. Of the nineteen human ALDH enzymes, members of the ALDH1A subfamily consisting of ALDH1A1, ALDH1A2, and ALDH1A3 have attracted interest as markers of cancer stem cells (CSCs) in several cancer types including lung, breast, and ovarian. CSCs represent a distinct subpopulation of highly tumorigenic cells that promote metastasis, recurrence, and resistance to conventional cancer therapies. The increased expression and activity of ALDH1A in CSCs is well-documented, as is the correlation between ALDH1A and a more aggressive cancer phenotype with poorer treatment outcomes. However, the actual functional role of ALDH1A in the context of CSCs has yet to be clearly defined. Elucidating this role will lead to a greater understanding of CSC biology and evaluate ALDH1A as a potential anti-CSC therapeutic target. In this study, previously developed and characterized selective small-molecule inhibitors of ALDH1A were used in conjunction with global transcriptomic, proteomic, and metabolomic analyses to identify pathways that could potentially establish a link between ALDH1A activity and early events in CSC formation in a triple-negative breast cancer (TNBC) model. These approaches revealed that ALDH1A inhibition is associated with mitochondrial and metabolic dysfunction and perturbation of the electron transport chain. ALDH1A inhibition also resulted in an increase in markers of endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), specifically mediated through the Protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway. These effects appear to occur independently of both the canonical function of ALDH1A in detoxifying reactive aldehydes as well as its potential metabolic contribution through the generation of NADH. Together, these results suggest a separate role for ALDH1A in TNBC CSCs in protecting against ER stress that warrants further study.Item Disruption-Compensation (DisCo) Network Analysis of the RNA Polymerase II Interactome(2022-08) Burriss, Katlyn Hughes; Mosley, Amber L.; Georgiadis, Millie M.; Goebl, Mark G.; Turchi, John J.During RNA Polymerase II (RNAPII) transcription, a dynamic network of protein-protein interactions (PPIs) coordinates the regulation of initiation, elongation, and termination. Taking a proteomics approach to study RNAPII transcription can offer a comprehensive view of the regulatory mechanisms mediated by PPIs within the transcription complex. However, traditional affinity purification mass spectrometry (APMS) methods have struggled to quantitatively capture many of the more dynamic, less abundant interactions within the elaborate RNAPII transcription interactome. To combat this challenge, we have developed and optimized a quantitative AP-MS based method termed Disruption-Compensation (DisCo) Network Analysis that we coupled with Tandem Mass Tag (TMT) labeling. In this application, TMT-DisCo was applied to investigate the PPIs that regulate RNAPII transcription. In the first study, TMT-DisCo network analysis was used to analyze how perturbation of subunits of four major transcription elongation regulators —Spt6, Spt5 (DSIF), Cdc73 (PAF-Complex), and Spt16 (FACT)— affect the RNAPII PPI network. TMT-DisCo was able to measure specific alterations of RNAPII PPIs that provide insight into the normal functions of Spt6/Spt5/Cdc73/Spt16 proteins within the RNAPII elongation complex. The observed changes in the RNAPII interactome also reveal the distinct mechanisms behind the phenotypes of each perturbation. Application of TMTDisCo provides in vivo, protein-level insights into synthetic genetic interaction data and in vitro structural data, aiding in the understanding of how dynamic PPIs regulate complex processes. The second study focused on the essential RNAPII CTD phosphatases, Ssu72 and Fcp1. TMT-DisCo captures how the ssu72-2 allele affects the ability of RNAPII to proceed through elongation, resulting in more arrested RNAPII that requires proteasomal degradation. Reduction of Ssu72 phosphatase activity shifts cells away from RNAPII reinitiation/ recycling and toward de novo expression and newly assembled RNAPII, aided by chaperones. RNAPII in fcp1-1 cells was observed to increase in interaction with the 26S proteasome, as well as TFIID and mRNA capping enzyme. These data support a model of the nuclear proteasome functioning as a chaperone during transcription initiation, as the fcp1-1 allele leads to inefficient formation of a pre-initiation complex with a hyperphosphorylated RNAPII CTD.Item Small Molecule Inhibitors of GroEL That Disrupt Active Replication of Mycobacterium Tuberculosis and ESKAPE Bacteria(2022-07) Tepper, Katelyn; Johnson, Steven M.; Georgiadis, Millie; Motea, Edward; Absalon, SabrinaGlobally, millions of people die every year due to complications involving infections from antibiotic-resistant bacteria. Of these infections, the most common organisms are Mycobacterium tuberculosis (Mtb) and a group of bacteria known as the ESKAPE pathogens (an acronym that stands for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter species). Unfortunately, as the need for antibiotics increases, industrial antibiotic development programs are drying up. However, unique antibiotic candidates targeting new pathways may be better for addressing antibacterial resistance. A target that is currently not the focus of any drug on the market is the bacterial GroEL chaperonin system. GroEL chaperonins are complex, oligomeric proteins that are upregulated in the cell under stressful conditions and prevent the misfolding and aggregation of other proteins. All bacteria have one homolog that performs protein folding functions – such is the case for E. coli and the ESKAPE bacteria – while others, like M. tuberculosis, contain additional GroEL isoforms that appear to perform non-canonical functions that are not well understood. The canonical isoforms are essential for survival under all conditions; thus, these chaperonins represent excellent targets for antibiotic development. This study aimed to identify inhibitors of GroEL in the ESKAPE bacteria and Mtb from a library of compounds with known antibiotic properties that was provided by the Medicines for Malaria Venture. Using two orthogonal assays that assess GroEL activity via its refolding of denatured enzymes Malate Dehydrogenase and Rhodanase, 37 inhibitors of E. coli GroEL were identified. Of these, 33 were examined in dose response testing in in vitro biochemical and cell viability assays. Compound 23 stood out in potency for inhibiting GroEL functions and actively-replicating Mtb bacteria, and thus a small panel of analogs were evaluated to develop structure-activity relationships (SAR) and study their mechanism. Two cysteine residues were identified as covalently modified by compound 23 and one of the lead analogs, giving insight into inhibitory sites on GroEL. Another lead analog bearing a nitrofuran moiety exhibited inhibition of actively-replicating E. coli, S. aureus, and Mtb bacteria. Importantly, this study identified new classes of GroEL inhibitors to explore for optimization as antibacterial candidates.Item Musculoskeletal Effects of Oncostatin M in Pancreatic Cancer Cachexia(2022-07) Jengelley, Daenique Heather Andrene; Zimmers, Teresa A.; Goebl, Mark G.; Mayo, Lindsey D.; Nakshatri, Harikrishna; Ostrowski, Michael C.Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancerrelated deaths with a five-year survival rate of 11%. PDAC tumors are characterized by a dense desmoplastic stromal microenvironment, mediated in part through local cytokine production. PDAC tumors also elicit a systemic inflammatory response in the host; this, combined with a loss of body weight due to muscle and fat wasting, is characteristic of cachexia. Understanding the molecular mechanisms that drive malignant inflammation is critical to improve PDAC therapy and increase patient survival. Oncostatin M (OSM) belongs to the IL-6/GP130 family of cytokines, members of which have been shown to promote PDAC tumor development, inflammation, and cachexia. Much less is known of OSM. My central hypothesis was that OSM promotes pancreatic cancer and cachexia by inducing local and systemic inflammation, fibrosis, and wasting via OSM signaling through the receptor, OSM receptor (OSMR). We investigated effects of exogenous OSM administration in wildtype and IL-6 null mice without cancer. OSM induced systemic fibrosis, bone loss, local muscle wasting, and cardiac dysfunction in presence and absence of IL-6. We further defined the roles of OSM/OSMR in the pancreatic cancer microenvironment and macroenvironment. OSM activated genes involved in inflammation, fibrosis, and tumor progression in both tumor cells and fibroblasts and altered the tumor microenvironment, promoting a dense compaction of tumor cells and cancer associated fibroblasts. Loss of systemic OSM signaling altered tumor metabolism and reduced the stromal compartment without affecting tumor size. Loss of OSMR signaling in tumor cells reduced tumor size and promoted survival. However, systemic loss of OSM or OSMR in host cells did not halt effects of cachexia including muscle dysfunction, atrophy, or inflammation/anemia. Overall, OSM/OSMR signaling in the microenvironment is necessary in modulating tumor phenotype and promoting survival in PDAC but may not be necessary for pancreatic cancer cachexia.Item Function of Parkinson's Disease-Associated Protein PINK1(2022-05) Engel, Victoria Alexe'; Hoang, Quyen Q.; Harrington, Maureen A.; Johnson, Steven M.; Wang, Mu; Yamamoto, Bryan K.Mutations in PINK1 (PTEN-induced Kinase 1) are the second most common cause of early-onset Parkinson’s Disease (PD). PINK1 is believed to maintain mitochondrial integrity by orchestrating mitophagy of dysfunctional mitochondria through phosphorylation of its substrate, Parkin. However, the effects of PD-associated mutations remain unclear. To investigate this, a PINK1 orthologue, Tribolium castaneum PINK1 (TcPINK1), was genetically engineered and purified for biochemical studies. Then, TcPINK1 was reacted against the Ubiquitin-like domain (UBL1-76) of Parkin and other proteins with a similar beta-grasp fold including Ubiquitin, ATG8, NEDD8, and SUMO using an in vitro radioisotopic filter-based kinase assay. The data revealed that TcPINK1’s preferred substrate with the highest amount of activity was UBL followed by Ubiquitin, NEDD8, and SUMO, with no activity against ATG8, which lacks a Serine residue equivalent to the phosphorylated residue in UBL. NEDD8 and SUMO were phosphorylated even though they are not substrates which suggests that PINK1 is capable of nonspecific phosphorylation of proteins with a similar fold to UBL. In addition, it is possible that the phosphorylation of Ubiquitin as reported in the literature may be nonspecific as well. TcPINK1 point mutations equivalent to the PD-associated human PINK1 mutations were genetically engineered, purified, and reacted against UBL. The P374L mutant showed a similar activity to wild type, and the A194D, G285D, and S289M mutants showed a significant decrease in activity. Since P374 resides in the C-lobe of the kinase away from the active site, the data suggest that this residue may not be involved with catalysis or with UBL binding. As A194, G285, and S289 all reside in the N-lobe near the active site, the data suggest that these point mutations may be involved with catalysis. In conclusion, the data suggest that PINK1 specificity for Parkin may involve binding outside of the UBL domain.Item Mast Cells Regulate Bile Acid Signaling and Cholestasis via Alteration of Farnesoid X Receptor/Fibroblast Growth Factor 15 Axis in Mice(2022-03) Meadows, Victoria E.; Francis, Heather; Alpini, Gianfranco; Dong, X Charlie; Esker, Burcin; Ren, HongxiaPrimary Sclerosing Cholangitis (PSC) is a rare and slow progressing cholangiopathy characterized by hepatic inflammation, fibrosis and ductular reaction with liver transplantation as the sole therapeutic option. PSC patients are at high risk of auto-immune comorbidities like irritable bowel disease (IBD), found in up to 80% of PSC patients (PSC-IBD). There are indications of genetic and environmental components for auto-immune development in IBD; however, its etiology remains unclear. Mast cells (MCs) infiltrate the liver and can become activated leading to degranulation and release of mediators, like histamine (HA), which result in increased intrahepatic bile duct mass, biliary senescence, hepatic inflammation, and hepatic stellate cell activation. Similarly, MCs infiltrate the intestine and increase inflammation which alters host-microbiome communication. MCs are necessary for successful liver regeneration and the combat of intestinal pathogens; however, chronic HA signaling exacerbates damage in cholangiopathies and IBD. Bile acid synthesis is tightly regulated by Farnesoid X Receptor (FXR) and its downstream mediator, fibroblast growth factor 15 (FGF15, -19 in humans). Cholangiocytes (i) are the target of cholangiopathies, (ii) modify and recycle bile acids through Apical Sodium Bile Acid Transporter (ASBT)-mediated cholehepatic shunting, which functions outside of enterohepatic circulation of bile acids and (iii) are capable of autocrine HA signaling. The complex relationship between hepatic and intestinal MC infiltration and bile acid signaling has not been established; therefore, identifying MC regulation of bile acid pool and FXR/FGF15 signaling pathway will provide insight into therapeutic treatment of PSC-IBD. Under the rationale that (i) cholestatic liver diseases are positively correlated with auto-immune comorbidities like IBD, (ii) during disease, MCs infiltrate the liver and intestine and release signaling factors like HA, and (iii) MCs express FXR and secrete FGF15/19; we propose the central hypothesis that MC activation regulates bile acid signaling and PSC progression through paracrine crosstalk with cholangiocytes in the liver and intestinal inflammation.Item An Integrative Genome-Based Metabolic Network Map of Saccharomyces Cerevisiae on Cytoscape: Toward Developing A Comprehensive Model(2022-03) Hamidi, Aram; Goebl, Mark; Cocklin, Ross; Wells, Clark; Harrington, MaureenMetabolic flux analyses and their more comprehensive forms, genome-scale metabolic networks (GSMNs), have gained tremendous attention in industrial and medical research. Saccharomyces cerevisiae (S. cerevisiae) is one of the organisms that has had its GSMN subjected to multiple frequent updates. The objective of this study is to develop a visualization tool for the GSMN of S. cerevisiae for educational and research purposes. This visualization tool is called the Master Metabolic Map of Saccharomyces cerevisiae (MMMSC). In this study, a metabolic database of S. cerevisiae developed by us was transferred to Cytoscape, a useful and efficient bioinformatics software platform for visualizing molecular networks. After the MMMSC was created, nodes, representing metabolites and enzymes, and edges, representing the chemical reactions that connect the nodes, were curated manually to develop a metabolic visualization map of the whole metabolic system of S. cerevisiae (Figure 4). In the discussion, examples are provided regarding possible applications of MMMSC to predict possible effects of the manipulation of the S. cerevisiae metabolome for industrial and medical purposes. Ultimately, it is concluded that further work is needed to complete the metabolic database of S. cerevisiae and the related MMMSC. In future studies, these tools may be integrated with other omics and other approaches, especially the directed-evolution approach, to increase cost and time efficiency of future research and to find solutions for complex and, thus far, poorly managed environmental and health problems.Item Investigation of Protein – Protein Interactors of Setmar Using Tandem Mass Tag Mass Spectrometry(2022-03) Segizbayeva, Lana; Georgiadis, Millie M.; Mosley, Amber L.; Wells, Clark D.The nuclear protein SETMAR has been reported to be involved in many processes such as non-homologous end joining (NHEJ), di-methylation (arguably) of K36 of histone H3, restart of stalled replication forks, chromosome decatenation, enhancing of TOPII inhibitors which results in resistance to chemotherapeutics in cancer patients, etc. All these purported functions are impossible to execute without interaction with other proteins. It is established that SETMAR binds specifically to DNA at terminal inverted repeat sequences and can loop DNA. This DNA sequence specific pull-down exploits this attribute to identify possible protein interactors of SETMAR. As a result of this experiment several proteins have been identified for further research: BAG2, c12orf45, PPIA, XRCC5/6, and ZBTB43, all of which are found in higher statistical abundances in full length SETMAR samples.Item β-Cell Autophagy in the Pathogenesis of Type 1 Diabetes(2021-12) Muralidharan, Charanya; Linnemann, Amelia K.; Dong, Charlie X.; Sims, Emily K.; Kaplan, Mark H.Type 1 diabetes (T1D) is a multifactorial disease involving genetic and environmental factors. One of the factors implicated in disease pathogenesis is early life viral infection. A typical immune response to viral infection includes production of type 1 interferons (IFN), such as IFN-α, which can induce stress in the pancreatic β-cells. Reactive oxygen species (ROS) accumulation occurs after exposure to other inflammatory cytokines, causing oxidative stress that may be linked to T1D pathogenesis. Therefore, we hypothesized that IFN-α may also elicit β-cell ROS accumulation. Our in vivo and in vitro experiments with human islets showed rapid and heterogenous ROS accumulation with IFN-α. Although T1D is characterized by autoimmune destruction of β-cells, some cells survive this persistent attack. We hypothesized that survival/ death of β-cells could be attributed to the ability to effectively mitigate ROS accumulation. One mechanism to mitigate ROS is autophagy, which degrades and recycles cellular components to promote cellular homeostasis. We observed an impairment in autophagy in β-cells of donors with T1D as well as in islets of diabetic non-obese diabetic (NOD) mouse model of autoimmune diabetes. Autophagic flux was also impaired in diabetic NOD mouse islets, further confirming impairment of autophagy. Interestingly, we observed an induction of autophagy after acute treatment with IFN-α both in vitro and in vivo, suggesting compensatory upregulation of autophagy to restore homeostasis. Similarly, we observed an increase in autophagosomes and telolysosomes in β-cells of normoglycemic autoantibody positive organ donors compared to nondiabetic organ donors. Together, these data implicate a defect in the final degradation step of autophagy involving lysosomes. Therefore, we analyzed the activity and expression of lysosomal cysteine protease Cathepsin H (CTSH, a T1D susceptibility locus), and found both to be increased in islets of pre-diabetic NOD mice. Together, these data support compensatory hyperactivation of lysosomal enzymes prior to overt diabetes, potentially to rid the cell of ROS and degradation-resistant oxidized proteins and lipids. We also observed that C57Bl/6J mice lacking a key autophagy enzyme, ATG7, in their β-cells, spontaneously developed hyperglycemia. Collectively, these data highlight the importance of -phagic degradation process in the pathogenesis of T1D.