Biochemistry & Molecular Biology Department Theses and Dissertations

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    Cell-Type Specific Function of STAT4 in an Animal Model of Multiple Sclerosis
    (2023-12) Alakhras, Nada S.; Kaplan, Mark H.; Cook-Mills, Joan; Dong, X. Charlie; Quilliam, Lawrence A.
    Signal transducer and activator of transcription 4 (STAT4) is a critical regulator of inflammation. STAT4 promotes protective immunity and autoimmunity downstream of pro-inflammatory cytokines including IL-12 and IL-23. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), germ-line deletion of STAT4 in mice results in resistance to the development of inflammation and paralysis. In parallel, genome-wide association studies (GWAS) have identified polymorphisms in the STAT4 gene associated with susceptibility to several autoimmune diseases including MS demonstrating a potential role for STAT4 in human autoimmunity. Here, we examined cell-type requirements for STAT4 in EAE. Using conditional Stat4 mutant mice, we found that mice lacking Stat4 in T cells and CD11c+-expressing cells are resistant to EAE, while mice lacking Stat4 in Lyz2+-expressing cells are susceptible to EAE. STAT4 is expressed and activated in CD11c+ dendritic cells (DCs) in the CNS during peak disease severity. Stat4fl/flCD11cCre mice exhibit significantly decreased classical dendritic cell (cDC) expansion in the CNS and this correlates with diminished numbers of infiltrated T cells in the CNS and decreased inflammatory cytokine production. Adoptive transfer of wild type but not Stat4-/- or Il23r-/- DCs into Stat4fl/flCD11cCre rescues the development of EAE. Transferred Il23r-/- DCs were retained in the lymph nodes suggesting that IL-23-STAT4 signaling promotes their migration to and expansion in the CNS. Single-cell RNA-seq analyses of CNS DCs from WT and Stat4fl/flCD11cCre mice identified cDC populations with STAT4-dependent gene expression and migratory phenotypes. Collectively, our results demonstrate that STAT4 in cDCs is required for expansion in the CNS, the development of encephalitogenic T cells, and the clinical symptoms of EAE. Thus, our study reveals previously unrecognized functions of STAT4 in cDCs that provide mechanistic insight into CNS autoimmunity and provide a foundation for identifying new therapeutic targets for the disease.
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    Endoplasmic Reticulum Calcium in the Pathogenesis of Type 1 Diabetes
    (2023-12) Weaver, Staci Anne; Evans-Molina, Carmella; Roh, Hyun Cheol; Sims, Emily K.; Wek, Ronald C.
    Type 1 diabetes (T1D) accounts for 5-10% of all diabetes cases and results from immune-mediated destruction of pancreatic β-cells. Individuals with Darier Disease, which is caused by loss of function germline mutation(s) in the sarcoendoplasmic reticulum Ca2+-ATPase pump (SERCA2) gene, have an elevated risk of being diagnosed with T1D (risk ratio, 1.74; 95% CI, 1.13-2.69), suggesting a potential mechanistic relationship between SERCA2 and T1D pathogenesis. To determine the impact of reduced SERCA2 expression on T1D pathogenesis, we generated SERCA2 haploinsufficient mice by backcrossing C57BL6/J-S2+/- mice onto the non-obese diabetic (NOD) background (NOD-S2+/- mice). Female NOD-S2+/- mice showed accelerated T1D onset (14wks vs. 18wks, p<0.0001), elevated circulating anti-insulin antibodies, and increased immune cell infiltration into the islets compared to NOD-WT mice. Single-cell RNA sequencing (scRNA-seq) on islets and spatial proteomics on pancreatic lymph node (PLN) and spleen at 6 wks of age revealed increased immune cell presence in islets and enhanced B and T cell activation in PLN and spleen of NOD-S2+/- mice. Furthermore, scRNA-seq on isolated islets revealed temporal alterations in pathways related to mitochondria function in β cells, and mechanistic studies revealed decreased glucose-stimulated ATP production, reduced mitochondrial membrane potential, decreased islet expression of ATP synthase/mitochondrial complex III, increased mitochondrial Ca2+, and altered mitochondrial ultrastructure in NOD-S2+/- islets at 10 wks of age. In co-culture experiments, NOD-S2+/- B cells showed increased activation and NOD-S2+/- T cells showed increased proliferation and activation when cultured with NOD-WT islets. Interestingly, NOD-S2+/- islets induced B and T cell proliferation and T cell activation when cultured with NOD-WT immune cells. Lastly, administration of a small molecule SERCA activator in NOD-S2+/- mice decreased immune cell infiltration into the islet and delayed T1D onset. In summary, our results demonstrate a novel pathway whereby modulation of SERCA2 impacts islet mitochondrial function, islet immunogenicity, and immune cell proliferation and activation which fuel progression to T1D.
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    Examining the Potential of Targeting the HSP60 Chaperonin System as a Broadly Applicable Chemotherapeutic Strategy
    (2023-12) Liechty, Hope Lauren; Johnson, Steven M.; Motea, Edward A.; Turchi, John J.; Vilseck, Jonah Z.
    This study methodically examined our diversity set of GroEL and HSP60 inhibitors to identify lead candidates that exhibited the most potent and selective cytotoxicity to colon cancer cells over non-cancer cells in vitro. While several structurally distinct candidates were identified, we found that our nitrofuran and hydroxyquinoline-containing N-acylhydrazone series (NF-NAH and HQ-NAH, respectively) were among the most potent and selective. Subsequent screenings across an NCI panel of cancer cell lines of different origins revealed the superior efficacy of the NF-NAH and HQ-NAH series as chemotherapeutic candidates, in contrast to the ABK-based inhibitors we previously reported, which showed poor efficacy across the panel. Given the emerging evidence of the role of mis-localized HSP60 in cancer cell survival, this study also compared the structure and function of naive cHSP60 (the aberrant form presumed to be in the cytosol) with that of mHSP60 (the processed and mature form that is in mitochondria). Analytical size exclusion chromatography revealed cHSP60 is a more stable oligomer consisting of both single and double-ring complexes. Intriguingly, cryoEM analyses revealed that cHSP60 formed a unique face-to-face double-ring complex, as opposed to the structures of other double-ring GroEL and HSP60 chaperonins where their rings stack back-to-back with one another. Subsequent assays demonstrated similar ATPase activities for both mHSP60 and cHSP60, with stimulatory effects observed in the presence of HSP10 for both. Despite the apparent engagement of HSP10, cHSP60 was unable to refold the denatured MDH client protein efficiently, suggesting potential functional divergence in vivo. These enticing results offer novel insights into the physiological importance of the cHSP60 complex and its possible role in cancer progression. As our previous studies examined inhibitors that were developed as GroEL-targeting antibacterial candidates, and given the unique structural/functional differences of cHSP60 compared to GroEL and other chaperonins, including mHSP60, the findings from this study underscore the need for future to identify and optimize inhibitors specifically for targeting cHSP60 to enhance chemotherapeutic effectiveness.
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    Characterizing the Unfolded Protein Response by Changes in Protein Thermal Stability
    (2023-09) McCracken, Neil Andrew; Mosley, Amber; Wek, Ron; Evans-Molina, Carmella; Georgiadis, Millie; Quinney, Sara
    The Unfolded Protein Response (UPR) protects eukaryotic cells from the threat of excessive protein flux into the Endoplasmic Reticulum (ER). UPR sentries PERK, Ire1 and ATF6 detect unfolded protein in the ER and alert the cell of the condition. Downstream pathways increase translation of select responders while simultaneously decreasing the global protein load in order that toxic protein aggregates do not form in the cell. While this warning system has been characterized over several decades through extensive reporting of UPR impact on transcript and protein abundance, little is known about the biophysical changes that occur to proteins as part of the UPR in the context of the cellular environment. An understanding of how the UPR affects the folding, stability and protein oligomerization is vital for describing subtle but important changes that occur and contribute to maladaptive physiology in diseases including diabetes, cancer, and neurodegeneration. I propose that deficiencies in characterizing the UPR can be overcome by using thermal shifts assays (TSA) that quantify changes in protein stability post stimuli. Findings described herein show the utility of the biophysical thermal shift assay in characterizing the UPR. Thermal shift assays (TSA) measure susceptibility of proteins to denature upon heat treatment and consequently detect changes in protein structure, modification, and interactions in the cellular environment. Previously unobserved protein relationships related to the UPR were detected using TSA. These workflows were improved through more strategic upstream sampling and downstream data analysis through creation of the publicly available InflectSSP program. Observed UPR phenomena during N-linked glycosylation inhibition and UPR induction include protein degradation, changes in stability of N-linked glycosylation enzymes, and transcriptional targets canonical to the UPR. Stability changes in proteins downstream of PERK were also observed in experiments where PERK genetic ablation was combined with UPR induction. Finally, the thermal shift assay was used to develop a “signature” for the UPR that holistically describes the ER stress response. Results described in this dissertation provide an improved perspective of the UPR along with an approach that can be used to identify novel targets for therapeutic intervention of the UPR.
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    Sex-dependent differential chromatin state changes in adipocytes across different fat depots during obesity
    (2023-08) Chaisakhon, Natthamon; Roh, Hyun Cheol; Mosley, Amber L.; Cornett, Evan M.
    Adipose tissue exhibits a high degree of plasticity and undergoes significant cellular remodeling in response to nutritional availability. While adipose tissue remodeling displays sexual dimorphism, its underlying molecular mechanisms remain poorly understood. In this study, we investigate the role of epigenetic modifications and gene expression in sexual dimorphism of adipose tissue in mice during obesity. We perform CUT&Tag (Cleavage Under Targets and Tagmentation) technique in conjunction with RNA-sequencing with female adipocytes from different fat depots and compare them to male mice. We find significant changes in H3K27ac, H3K27me3, H3K9me3 and RNA-seq after high fat diet (HFD) feeding. The changes are functionally characterized by a reduction in cellular metabolism and an elevation in cytoskeletal organization. Interestingly, these changes commonly occur in both visceral and subcutaneous fat depots with minor differences in females, which distinguishes them from males that exhibit differential responses between depots. Surprisingly, the chromatin states and expression profiles of female subcutaneous fat depots closely resemble those of male visceral adipose tissues during obesity. These results suggest that distinct responses across fat depots in male and females may contribute to different susceptibilities to metabolic diseases between the sexes. These findings enhance our understanding of the molecular mechanisms underlying adipocyte dysfunction during obesity and highlights the interplay between sex, adipose tissue depots, and metabolic responses.
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    Mass Spectrometry Based Elucidation of Transcriptional and Protein Folding Stress
    (2023-08) Baldwin, Dominique Adom; Mosley, Amber L.; Wek, Ronald C.; Motea, Edward A.; Linnemann, Amelia K.
    RNA Polymerase II (RNAPII) plays a pivotal role in cellular homeostasis due to its role of RNA transcription and gene expression. Many protein-protein interactions (PPIs) are required to assist RNAPII during RNA generation and these interactions can be directed by the addition of many post-translational modifications (PTMs) of proteins. Notably, the largest subunit of RNAPII is dynamically phosphorylated to regulate progression through transcription. As such, the coordination of many protein kinases and phosphatases is required to regulate signaling that occurs throughout transcription by RNAPII. These transcriptional regulators play additional roles in other cellular pathways as well such as the unfolded protein response (UPR). To further understand the regulation and dysregulation of RNAPII phosphorylation and the regulatory roles RNAPII kinases and phosphatases play, especially from a disease perspective, it is important to develop and utilize workflows to monitor changes surrounding proteins and their modifications. One method is quantitative mass spectrometry (MS), which allows biomolecules to be directly quantified, thus serving as a powerful tool to better understand the regulation of transcription with high accuracy. This work explores the development and experimental application of various OMICS workflows to answer long-standing questions surrounding transcription biology and onward.
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    The Mechanisms by Which Small Molecules Modulate the HSP60/10 Chaperonin System to Elicit Antimicrobial Effects
    (2023-06) Stevens, Mckayla Marie; Johnson, Steven; Turchi, John; Hoang, Quyen; Wek, Ronald; Absalon, Sabrina
    Heat Shock Protein 60/10 (HSP60/10, or GroEL/ES in bacteria) chaperonin systems play a critical role in protein homeostasis through facilitating proper folding of misfolded or partially folded polypeptides that are otherwise prone to aggregation. HSP60 chaperonins are highly conserved and essential in nearly all organisms studied thus far, making them a promising target for antibiotic development. Early high-throughput screens in the Johnson lab have identified five main scaffolds that, though hit-to-lead development, have been optimized for chaperonin inhibition and antimicrobial effects. While these initial studies have shown promising evidence to support the viability of a chaperonin-targeting antibiotic strategy, it was unclear whether the conservation of human HSP60 (48% identity to bacterial GroEL) would hinder this therapeutic strategy from advancing due to potential toxicity associated with off-target inhibition of the human homolog. Additionally, while chaperonin inhibition often correlated with cytotoxicity to the various pathogens studied, there was a clear need to investigate inhibitor mechanisms to 1) verify on-target effects, and 2) guide future development of more potent and selective chaperonin-targeting antibiotic candidates. Herein, we conduct a medium-throughput screening of known bioactive molecules, approved drugs, and natural products against both bacterial GroEL and human HSP60, demonstrating that most molecules exhibited low-to-no toxicity to human cells in culture, despite being near equipotent inhibitors of human HSP60 and E. coli GroEL in our refolding assays. Thus, sequence conservation between human HSP60 and bacterial GroELs does not necessarily predict toxicity in vivo. We then investigate inhibitory mechanisms of our most well-established inhibitor series, the phenylbenzoxazole (PBZ) series, identifying three binding sites whereby PBZ molecules modulate GroEL folding and ATPase functions in a site-specific manner, predominately through its ability to interact with its co-chaperone GroES. Finally, we demonstrate that two standard of care drugs for T. brucei infections, suramin and nifurtimox, may elicit their trypanocidal effects through inhibiting HSP60. Due to structural similarities, we then screened our N-acylhydrazone (NAH) and α,β-unsaturated ketone (ABK) series of HSP60 inhibitors against T. brucei, finding that they are highly potent and selective trypanocidal agents. Together, these studies further support HSP60 as a viable drug target for antibiotic development.
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    The Essential Role of the Non-Essential Amino Acid Asparagine in Lymphoid Malignancies
    (2023-05) Srivastava, Sankalp; Zhang, Ji; Dong, X. Charlie; Mosley, Amber L.; Wek, Ronald C.
    Cancer cells display increased metabolic demands to support their proliferation and biosynthetic needs. It has been extensively shown in cancers, that amino acids have functions beyond the role of mRNA translation. The breadth of functions makes amino acid restriction an effective strategy for cancer therapy; hence an important line of research involves targeting amino acid acquisition and metabolism therapeutically. Currently, asparagine depletion via L-Asparaginase in acute lymphoblastic leukemia (ALL) remains the only clinically approved therapy to date. In the first project, we showed that ALL cells are auxotrophic for asparagine and rely on exogenous sources for this non-essential amino acid. However, sensitivity to L-Asparaginase therapy is mitigated by the expression of the enzyme asparagine synthetase (ASNS), involved in de novo asparagine biosynthesis. We showed that this adaptive response requires two essential steps; demethylation of the ASNS promoter and recruitment of activating transcription factor 4 (ATF4) to the promoter to drive ASNS transcription. Our follow-up study in ALL cells showed that asparagine bioavailability (through de novo biosynthesis or exogenous sources) is essential to maintain the expression of the critical oncogene c-MYC. c-MYC is a potent transcription factor and is dysregulated in over 60% of cancers, including hematopoietic malignancies. We showed that this regulation by asparagine is primarily at the translation level and c-MYC expression is rescued only when exogenous asparagine is available or when cells can undertake de novo biosynthesis. At the biochemical level, asparagine depletion also causes an induction of ATF4 mediated stress response and suppression of global translation mediated by decreased mammalian target of rapamycin complex 1 (mTORC1) activity. However, we found that neither inhibition of the stress response or rescuing global translation rescued c-MYC protein expression. We also extended this observation to c-MYC-driven lymphomas using cell lines and orthotopic in vivo models. We showed that genetic inhibition of ASNS or pharmacological inhibition of asparagine production can significantly limit c-MYC protein and tumor growth when environmental asparagine is limiting. Overall, our work shows an essential role for asparagine in lymphoid cancers and has expanded on the usage of L-Asparaginase to resistant leukemias and lymphomas.
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    Elucidating the Role of Biliary Senescence and Mast Cell-Mediated Therapy in Non-Alcoholic Fatty Liver Disease
    (2023-05) Kundu, Debjyoti; Francis, Heather; Dong, Charlie X.; Alpini, Gianfranco; Linnemann, Amelia; Ekser, Burcin
    Non-alcoholic fatty liver disease, or NAFLD, is characterized by excess fat deposition in the liver. Cellular senescence is a critical hallmark of NAFLD. Cholangiocytes in the liver plays a significant role in the progression of fatty liver by contributing to senescence. p16 is the main senescent protein expressed by cholangiocytes in primary sclerosing cholangitis (PSC). Thus, we aimed to downregulate p16 by vivo-morpholino and evaluate the disease phenotypes and signaling mechanisms in a murine model of NAFLD. We found that downregulation of p16 reduced i) steatosis), ii) inflammation, iii) fibrosis, and cholangiocyte proliferation in HFD mice compared to the HFD-fed, control vivo-morpholino injected mice. Moreover, the downregulation of p16 reduced insulin-like growth factor-1 (IGF-1) in cholangiocytes, previously identified by our laboratory as a principal SASP factor secreted from cholangiocytes during NAFLD. By ingenuity pathway analysis, we found that p16 might regulates IGF-1 expression via the E2F1/FOXO1axis. Further analyses indicate that p16 downregulation reduces E2F1 mRNA transcription, inhibiting FOXO1 and subsequent IGF-1 expression in cholangiocytes. The presence of mast cells in the liver has been implicated in multiple cholangiopathies. Our lab demonstrated that mast cell stabilization by cromolyn sodium treatment reduced histamine secretion, fibrosis, and biliary proliferation in Mdr2-/- mice, a model of PSC. Thus, we aimed to determine mast cell stabilization as a therapeutic approach to managing NAFLD and its more advanced form, NASH. We found that cromolyn sodium ameliorated i) serum histamine levels, ii) intrahepatic mast cells, iii) inflammation, iv) fibrosis, v) steatosis, and cholangiocyte proliferation in methionine choline deficient diet-fed mice compared to the saline controls. Overall, we report that amelioration of senescence is a critical factor in improving the disease phenotypes in NAFLD. Biliary senescence plays a crucial role in modulating the disease progression in NAFLD, and mast cell stabilization can be used as a therapeutic approach to reduce pathological hallmarks of fatty liver.
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    Melatonin-MT1 Signaling Axis Ameliorates the Phenotypes of Primary Sclerosing Cholangitis
    (2023-03) Ceci, Ludovica; Alpini, Gianfranco; Francis, Heather; Ekser, Burcin; Dong, Charlie X.; Maiers, Jessica L.
    Background: Primary Sclerosing Cholangitis (PSC) is characterized by hepatic fibrosis and portal inflammation. Melatonin is synthesized by arylalkylamine N-acetyltransferase (AANAT) in cholangiocytes. We found that: (i) the MT1 receptor is primarily expressed in cholangiocytes; (ii) melatonin reduces biliary proliferation via MT1 receptor signaling; and (iii) melatonin treatment for 1 wk decreases ductular reaction (DR) and liver fibrosis in cholestatic rats by downregulation of MT1 and clock genes. Melatonin administration to male Mdr2-/- mice (PSC model) reduces angiogenesis and portal inflammation via decreased miR-200b. Downregulation of maspin triggers angiogenesis during tumorigenesis by interaction with glutathione S-transferase (GST). We aimed to evaluate the effects of long-term melatonin treatment and MT1 signaling on PSC phenotypes in Mdr2-/- mice. Methods: Male FVB/NJ and Mdr2-/- mice had access ad libitum to drinking water with/without melatonin for 3 months. Immortilized-SV40-cholangiocytes isolated from human liver samples (control and PSC) were treated with melatonin (10-3 mol/L) for 24 hr. Male C3H-Hej (WT for MT1-/-), FVB/NJ (WT for Mdr2-/-), MT1-/-, Mdr2-/- mice and MT1-/-/Mdr2-/- mice were euthanized at 12 wk. We analyzed liver damage, PSC phenotypes, angiogenesis and AANAT, melatonin receptors and clock genes by immunohistochemistry, immunofluorescence, ELISA and western blots in liver samples and isolated cholangiocytes. Melatonin signaling was evaluated in human control and PSC samples. Results: Long-term melatonin treatment and inhibition of MT1 receptor ameliorates cholestatic liver phenotypes in Mdr2-/-mice by decreasing the immunoreactivity of melatonin enzymes and clock genes. GST activity and maspin expression decreased in Mdr2-/- mice and human PSC samples compared to controls; the phenotypes were reversed by melatonin. Conclusion: Chronic melatonin treatment improves liver histology and restores biliary circadian rhythm by interaction with MT1. Suppression of MT1 ameliorates biliary/liver phenotypes through changes in clock genes and melatonin enzymes. Restoration of the circadian rhythm by modulation of melatonin/MT1 signaling may be key for PSC management.