Biochemistry & Molecular Biology Department Theses and Dissertations

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Bioengineering Against Fibrils and Hypoglycemia: Studies on Insulin, Glucagon and the Fusion of Both
(2024-12) Molina, Nicolas Varas; Weiss, Michael; Georgiadis, Millie; Hurley, Thomas; Wells, Clark; Dahlem, Andrew
Insulin drugs are vital for blood glucose control in type 1 and late-stage type 2 diabetes mellitus. Unfortunately, however, they have two notable problems: (1) an intrinsic propensity to physical degradation (amyloid-like fibrillation), which reduces potency and can lead to occlusion of insulin pumps’ catheters, impairing timely drug administration; and (2) an ever-present risk for iatrogenic hypoglycemia with potential acute (or even fatal) consequences and chronic sequelae. The risk of hypoglycemia, its immediate and long-term complications, and associated anxiety can confound efforts to achieve effective glycemic control. Further, insulin’s physical instability impacts worldwide distribution by imposing a refrigeration requirement—often a barrier to global access. A combined solution to these two problems could benefit patients worldwide. To circumvent these limitations, glucose-responsive technologies have been sought to reduce hypoglycemic risk; diverse strategies have focused on novel devices, delivery modes, or protein engineering. In the present doctoral work, we describe an alternative glucose-responsive approach that exploits an endogenous glucose-dependent switch in hepatic physiology: preferential insulin signaling (under hyperglycemic conditions) versus preferential counter-regulatory glucagon signaling (under hypoglycemic conditions). Glucagon, traditionally regarded as a counter-regulatory hormone, has been underutilized in routine glucose control due to a marked propensity to fibrillation. Motivated by the pilot success of a counterintuitive strategy—co-infusion of insulin and glucagon—we have bioengineered and tested a fibrillation-resistant insulin-glucagon fusion protein with favorable relative hormonal activities. The N-terminal glucagon moiety was stabilized as a partial α-helix by Lys13-Glu17 lactam bridge and fused to a C-terminal insulin moiety stabilized as a single chain with a foreshortened C domain. Our in vitro studies demonstrated (a) marked resistance to fibrillation on prolonged agitation at 37 °C and (b) unaffected dual hormonal signaling activity. Glucodynamic responses were monitored in rats relative to control fusion proteins lacking one or the other hormonal activity. Results showed that insulin’s efficacy in hyperglycemia was unaffected, but enhanced endogenous glucose production was observed under hypoglycemic conditions. Together, these findings provide proof of principle for the translational application of a novel glucose-responsive insulin formulation with augmented physical stability, addressing two major problems of insulin replacement therapy in a single molecule.
Mechanisms and Targeted Control of Pancreatic Beta Cell Antioxidant Response
(2024-12) Muncy, Alissa Nicole; Linnemann, Amelia K.; Alves, Nathan J.; Evans-Molina, Carmella; Mastracci, Teresa L.
Type 1 diabetes (T1D) is an autoimmune disorder characterized by the destruction of insulin-producing beta cells in the pancreas, leading to chronic hyperglycemia. The intricate interplay between genetic predispositions, environmental triggers, and immune dysregulation underpins the development of T1D. A commonality between the environmental triggers is their involvement in the generation of reactive oxygen species (ROS). ROS are a byproduct of many cellular reactions and at low levels acts as an important second messenger to regulate proliferation, inflammation, and cell survival. However, excessive ROS accumulation can lead to oxidative stress, which may contribute to the pathogenesis of T1D. Failure to resolve oxidative stress causes damage to DNA, protein, and organelles, and ultimately results in cell death. One of the primary mechanisms to mitigate oxidative stress is through the activation of the transcription factor, nuclear factor erythroid-related 2 factor 2 (NRF2). We hypothesized that rapid activation of the antioxidant response in response to extrinsic stress is essential for proper beta cell function. To investigate the role of the antioxidant response in beta cells, we generated a beta cell specific NRF2 knockout mouse model (NRF2Δβ). These mice do not develop overt diabetes. However, despite these observations, we observed a modest impairment in first-phase insulin secretion. Additionally, the beta cells in NRF2Δβ mice displayed evidence of DNA damage and early signs of apoptosis, which are hallmarks of oxidative stress. To understand the mechanism behind impaired insulin secretion, we investigated mitochondrial structure and function. We observed mitochondria with disrupted morphology. Surprisingly, the mitochondria did not exhibit impaired function. We then asked whether loss of beta cell NRF2 was associated with increased susceptibility to beta cell damage induced by the toxic glucose analog, streptozotocin. We did not observe exacerbated diabetes development in NRF2Δβ mice. Collectively, these data suggest activation of compensatory mechanisms to mitigate beta cell stress and restore homeostasis in the absence of NRF2. RNA sequencing revealed several possible mechanisms such as increased autophagy, upregulation of antioxidants through alternative pathways, increased proteolysis, and increased glycosylation. In summary, this data highlights the importance of redox homeostasis in preserving beta cell function which may play a critical role in preventing or delaying T1D.
Identification and Characterization of TONSL as an Immortalizing Oncogene
(2024-11) Khatpe, Aditi Sanjay; Nakshatri, Harikrishna; Nephew, Kenneth; Motea, Edward; Cornett, Evan; Corson, Timothy
The global issue of exponentially increasing breast cancer cases necessitates investigating the early genomic aberrations leading to tumorigenesis. To address this, we employed our unique biobank of healthy breast tissue to generate an isogeneic cell line model system, comprising primary breast epithelial cells and their immortalized counterparts. By comparing the genetic alterations between these cell types, we discovered that TONSL upregulation is one of the early events during breast tumorigenesis. TONSL is a Tonsoku-like DNA repair protein located on chromosome 8q24.3. Our findings reveal that TONSL is an immortalizing oncogene, capable of transforming primary breast epithelial cells in conjunction with defined oncogenes, resulting in Estrogen Receptor-positive breast adenocarcinomas. Furthermore, we observed that TONSL-amplified breast cancer cells are dependent on TONSL for tumor growth, and these TONSLHigh cells and tumors exhibit an upregulated homologous recombination DNA repair pathway, which may contribute to chemotherapy resistance. It is noteworthy that higher levels of TONSL protein in primary breast cancers, particularly in ER+ breast cancers, are associated with poor outcomes. Approximately 20% of breast cancers display recurring genomic amplification involving chromosome 8q24.3, with TONSL amplification potentially being the initial hit leading to tumorigenesis. In an attempt to target these TONSL/chr. 8q24.3 amplified breast tumors, we observed that breast cancer cells with this amplification are sensitive to CBL0137 – a TONSL-FACT complex inhibitor, both in vitro and in vivo. TONSL interacts with multiple proteins and functions in multiple cellular processes. To study the TONSL specific interactome, we performed immunoprecipitation with a TONSL antibody using protein lysates from TONSL-immortalized primary breast epithelial cells, followed by mass spectrometry analysis of the immunoprecipitates. Our results identified several proteins selectively enriched with the TONSL antibody, with the most significant being ETS variant transcription factor 6 (ETV6). ETV6 is known to play a role as a transcriptional repressor during embryonic development and hematopoiesis. Further studies on TONSL/chromosome 8q24.3 amplification will contribute to our understanding of breast tumor initiation, progression, and metastasis processes, as well as facilitate the development of novel therapeutic agents targeting the TONSL interactome.
The Role of Receptor Interacting Protein Kinases in Diabetogenic Beta-Cell Loss and Hyperglycemia
(2024-11) Mukherjee, Noyonika; Templin, Andrew T.; Dong, X. Charlie; Elmendorf, Jeffrey S.; Evans-Molina, Carmella; Linnemann, Amelia K.
Diabetes is characterized by pancreatic -cell loss, insulin insufficiency, and hyperglycemia. Although major efforts have been made to manage diabetes using pharmacological agents that lower blood glucose levels, less effort has been focused on therapies to prevent the two major forms of diabetes, type 1 (T1D) and type 2 diabetes (T2D). Hence, there is a critical need to understand the mechanisms that underlie -cell demise in these diseases, and to develop therapies targeting such mechanisms. Recent studies in non-islet cell types identified receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3) as mediators of inflammation and programmed cell death. RIPKs are being considered as potential therapeutic target in human diseases including renal, hepatic and neurodegenerative diseases. However, the role of RIPKs in -cell loss in diabetes remains unknown. My thesis work evaluated the roles of RIPK1 and RIPK3 in mediating -cell cytotoxicity and islet inflammation in diabetes pathogenesis. Through the studies, I examined the role of RIPK1 and RIPK3 in -cell loss in response to known inducers of diabetogenic -cell stress, including proinflammatory cytokines, endoplasmic reticulum (ER) stress and islet amyloid deposition. My work revealed roles of RIPK1 and RIPK3 in mediating both caspase-dependent and caspase-independent cell death, kinase activation and transcriptional responses in vitro. Furthermore, I found that RIPK1 and RIPK3 play important roles in regulating glucose homeostasis in mouse models in vivo. The studies revealed a novel role of RIPKs in the pathogenesis of diabetes and suggests that RIPKs might be a potential target to treat or prevent the disease.
Role of Tumor Oxygen Tension in Signaling and Response to Targeted Therapies
(2024-10) Adebayo, Adedeji Kolawole; Nakshatri, Harikrishna; Quilliam, Lawrence; Capitano, Maegan; Kim, Jaeyeon
Most tumor cells in solid tumors are exposed to oxygen levels ranging from 0.5% to 5%, but never to ambient air oxygen levels of about 21%. We developed an approach that allows collection, processing and evaluation of cancer and non-cancer cells under physioxia (3%-5% oxygen), ensuring little to no exposure to ambient air. This approach allowed for comparison of baseline and targeted therapy-induced changes in signaling pathways in cells under physioxia and ambient air and to identify potentially efficacious therapeutic combinations based on signaling pathways uniquely active under physioxia. Using tumor cells from two transgenic models of breast cancer and cells from breast tissues of clinically breast cancer-free women, we demonstrate oxygen level-dependent differences in cell preference for EGFR or PDGFRβ signaling. Physioxia caused PDGFRβ-mediated activation of AKT and ERK that reduced tumor cell sensitivity to EGFR and PIK3CA inhibition and maintained PDGFRβ+ epithelial-mesenchymal hybrid cells with potential cancer stem cell properties. Cells in ambient air displayed differential EGFR activation and were sensitive to EGFR and PIK3CA inhibition. Tumor cells grown under physioxia were sensitive to high affinity PDGFRβ inhibitor sunitinib. Furthermore, significantly higher synergistic growth inhibition and apoptosis was observed with lapatinib (a clinically used dual EGFR and ErbB2/HER2 inhibitor) and sunitinib combination only in tumor cells under physioxia both in vitro and in vivo. Our data emphasize the importance of oxygen considerations in preclinical cancer research to evaluate clinically relevant signaling pathways and identify novel drug targets or combination therapy approaches. We suggest that evaluation of candidate drugs for their efficacy under physiologic oxygen levels in preclinical models, prior to transitioning into clinical trials, would not only accelerate the development of effective drugs but also reduce failure at the clinical trial stage.
Functional and Quantitative Mass Spectrometry-Based Approaches for Mapping the Lysine Methylome
(2024-09) Berryhill, Christine Annette; Cornett, Evan M.; Aoki, Scott T.; Georgiadis, Millie M.; Mosley, Amber L.; Turchi, John J.
Proteins are frequently modified with small chemical tags, or modifications, that play a key role in controlling their functions within the cell. One modification, lysine methylation, is found on thousands of human proteins and is added and removed by lysine methyltransferases (KMTs) and lysine demethylases (KDMs), respectively. Recognition of methylated lysines by specific reader proteins regulates downstream processes. Lysine methylation, KMTs, KDMs, and reader proteins therefore create a signaling network. Components of lysine methylation signaling networks are frequently dysregulated in human disease, but current methods to detect lysine methylation present barriers for understanding the impact an awry signaling network has on lysine methylation. In this study, we investigated the use of mass spectrometry (MS)-based proteomics to better detect and quantify both lysine methylation sites and methyl regulators across multiple samples. We investigated the sequence bias of commercially available pan-methyllysine antibodies using both a lysine-oriented peptide library and immunoprecipitation mass spectrometry. Our results showed that most antibodies have a preference for certain sequences. Furthermore, we observed that unenriched samples obtained the same number of identified lysine methylation sites as enriched samples. Following the establishment of an efficient and quantitative MS-based proteomics approach, we applied it to profile both lysine methylation and KDMs within breast cancer cell lines. Studies have repeatedly shown that components of the lysine methylation signaling network are overexpressed within breast cancer. Indeed, we characterized distinct lysine methylation and KDM patterns across the cell lines, suggesting the existence of different lysine methylation signaling. Given the ability to quantitatively profile lysine methylation, this work also characterized the impact of a compound known to disrupt the lysine methylation signaling network, 3-deazanplanocin A. The observed transcript, protein, and lysine methylation site abundance changes highlight how dysregulation of methyl mediators impacts lysine methylation and cellular signaling. Overall, we developed a reproducible pipeline that promises to enable a deeper understanding of how a dysregulated lysine methylation landscape influences cellular signaling and associated phenotypes.
Investigating the Impact of SND1 and CHD3 ad PDX1 Interacting Partners on β Cell Function
(2024-08) Kanojia, Sukrati; Spaeth, Jason; Molina, Carmella Evans; Linnemann, Amelia K.; Mastracci, Teresa; Elmendorf, Jeffrey S.
Pancreatic β cells are integral in synthesizing, packaging, and secreting insulin, crucial for maintaining blood glucose homeostasis. However, in diabetic conditions, some β cells lose function of transcription factors (TFs), which drive expression of genes critical for insulin secretion. Among these, PDX1 plays a vital role in pancreas development and mature β cell function. The activity of PDX1 is modulated by coregulators like the SWI/SNF chromatin remodeling and Nucleosome Remodeling and Deacetylase (NuRD) complexes. Our study unveils a novel interacting partner of PDX1, the Staphylococcal Nuclease and Tudor domain-containing protein (SND1), known for its role in facilitating protein-protein interactions and transcriptional control across tissues. Confirming PDX1:SND1 interactions in rodent and human β cells, we employed CRISPR-Cas9 to delete Snd1, revealing altered gene expression associated with insulin secretion and cell proliferation, which were confirmed through functional analyses, highlighting the importance of SND1 in β cell function. Notably, PDX1:SND1 interactions were reduced in human β cells from type 2 diabetes (T2D) donors, indicating its role in diabetes pathogenesis. Additionally, our investigation into the NuRD complex discovered interactions between PDX1 and CHD4, a helicase within the complex, are crucial for modulating PDX1 transcriptional activity in β cells. Deletion of Chd4 led to increased CHD3 protein levels, prompting us to explore the role of CHD3 in compensating for loss of CHD4. β cell specific-inducible deletion of Chd3 alone did not impact glucose homeostasis, whereas concurrent deletion of Chd3 and Chd4 resulted in severe glucose intolerance, reduced β cell mass, and compromised insulin release. Loss of both subunits led to β cell dysfunction and loss of identity, emphasizing the compensatory role of CHD3. Future investigations will evaluate gene expression and chromatin accessibility changes in Chd3/Chd4-deficient β cells, along with assessing the impact of diabetes on PDX1:CHD3 interactions in T2D human donor tissues.
MiR-10a as a Modulator of Proliferation and Cell Cycle Progression in Ovarian Clear Cell Carcinoma
(2024-08) Collins, Kaitlyn Elizabeth; Hawkins, Shannon; Kim, Jaeyeon; Mayo, Lindsey; Nephew, Kenneth; Zhang, Chi; Zimmers, Teresa
Endometriosis, a benign inflammatory disease whereby endometrial-like tissue grows outside the uterus, is a significant risk factor for endometriosis-associated ovarian cancers. In particular, ovarian endometriomas, cystic lesions of deeply invasive endometriosis, are a potential precursor lesion for ovarian clear cell carcinoma (OCCC). To explore the transcriptomic landscape, OCCC from women with pathology-proven concurrent endometriosis (n=4) were compared to benign endometriomas (n=4) by bulk RNA and small-RNA sequencing. Analysis of protein-coding genes identified 2449 upregulated and 3131 downregulated protein-coding genes (DESeq2, P<0.05, log2 fold-change>|1|) in OCCC with concurrent endometriosis compared to endometriomas. Gene set enrichment analysis showed upregulation of cell cycle regulation and DNA replication pathways and downregulation in cytokine receptor signaling and matrisome pathways. Analysis of miRNAs revealed 64 upregulated and 61 downregulated mature miRNA molecules (DESeq2, P<0.05, log2 fold-change>|1|). Hsa-miR-10a-5p represented over 21% of the miRNA molecules in OCCC with endometriosis and was significantly upregulated (NGS: log2 fold change=4.37, P=2.43E-18; QPCR: 8.1-fold change, P<0.05). Correlation between miR-10a expression level in OCCC cell lines and IC50 (50% inhibitory concentration) of carboplatin in vitro revealed a positive correlation (R2=0.92). The cellular function of miR-10a was investigated by overexpressing miR-10a in vitro. MiR-10a overexpression revealed a significant decrease in proliferation (n=6; P< 0.05), compared to a non-targeting control. Cell-cycle analysis revealed a significant shift in cells from S and G2 to G1 in (n=6; P<0.0001). MiR-10a overexpression in vitro was correlated with decreased expression of predicted miR-10a target genes critical for proliferation, cell-cycle regulation, and cell survival [SERPINE1 (3.2 downregulated; P<0.05), CDK6 (2.4 downregulated; P<0.05) and, RAP2A (2-3 downregulated; P<0.05)].
Structural Basis of Arrestin Binding to Cell Membranes
(2024-04) Miller, Kyle Warren; Chen, Qiuyan; Takagi, Yuichiro; Georgiadis, Millie M.; Hurley, Thomas D.
Two non-visual arrestins, arrestin2 (Arr2) and arrestin3 (Arr3), selectively interact with activated and phosphorylated G protein-coupled receptors (GPCRs) and play crucial roles in regulating many important physiological processes. Arrestins also engage the lipid bilayer surrounding activated GPCRs, which further potentiates arrestin activation and regulates GPCR trafficking in cells. Because of this, structural and functional understanding of arrestins would provide insight in enhancing arrestin’s GPCR desensitization for various diseases where constitutively active GPCR mutants play a role including congenital endocrine disorders and familial gestational hyperthyroidism. To better understand the membrane binding role of arrestins, we performed in vitro binding assays and demonstrated that Arr2 selectively binds to nanodiscs containing Phosphatidylinositol 4,5-bisphosphate (PIP2) even in the absence of different binding sites. Our cryo-electron microscopy (Cryo-EM) structure of Arr2 in complex with PIP2 nanodisc reveals that multiple structural elements of Arr2, including the finger loop, C domain and C-edge loop, contribute to membrane binding. Eliminating one individual site does not significantly impact Arr2 binding to the nanodisc. Moreover, a preactivated variant of Arr2 shows increased binding to the nanodisc than wildtype. We also labeled four potential membrane binding sites with monobromobimane (mBrB) and detected different levels of fluorescence increase in the presence of nanodisc containing various types of phospholipids. Overall, our study provides detailed structural evidence on how arrestins engage the membrane via multiple contact points and how this can impact arrestin-mediated signaling.
GCN2 eIF2 Kinase Promotes Prostate Cancer by Maintaining Amino Acid Homeostasis
(2024-04) Cordova E., Ricardo A.; Wek, Ronald C.; Staschke, Kirk A.; Pili, Roberto; Mosley, Amber L.; Elmendorf, Jeffrey S.; Zhang, Ji
Activation of the integrated stress response (ISR) contributes to the progression of many cancers, including prostate cancer (PCa). The ISR features a family of protein kinases that phosphorylate the eukaryotic translation initiation factor 2 (eIF2) during different stress conditions, repressing global protein synthesis. In parallel, eIF2 phosphorylation also enhances the translation of select gene transcripts, such as ATF4, which directs the transcription of ISR-target genes critical for stress adaptation. We reported that the eIF2 kinase GCN2 is a critical driver of the ISR in PCa and is crucial to maintaining amino acid (AA) homeostasis. GCN2 is activated in PCa due to AA limitation, resulting in increased expression of key AA transporters which providing nutrient import to fuel protein synthesis and metabolism that drive prostate tumor cell proliferation. Inhibition of GCN2 results in lowered expression of AA transporters, leading to severe depletion of intracellular AA and reduced proliferation in PCa. We identified purine biosynthesis as a key metabolic pathway dependent on GCN2. Inhibition of GCN2 and the accompanying depletion of AAs decreases purine levels in PCa cells, ultimately resulting in reduced ribosome biogenesis leading to the activation of a p53-dependent cell cycle checkpoint, termed the Impaired Ribosome Biogenesis Checkpoint (IRBC). Interestingly, induction of p53 promotes survival of PCa following GCN2 inhibition by halting cell cycle progression and reprogramming metabolism to restore metabolic homeostasis. We found that reductions in select AAs that impact nucleotide pools activate GCN2 and p53 in parallel, and that cooperation of these stress pathways is critical for maintaining AA and purine pools. Of importance, deletion of p53 sensitizes PCa cells to GCN2 inhibition suggesting that loss of p53 creates a dependency for GCN2. Of importance, we demonstrate that a small molecule inhibitor of GCN2 showed robust in vivo efficacy in androgen-sensitive and castrationresistant mouse models of PCa, supporting its therapeutic potential for the treatment of PCa.

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