Biochemistry & Molecular Biology Department Theses and Dissertations

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Characterization of Missense Variants in the RNA Exosome Associated with Pontocerebellar Hypoplasia
(2025-05) Runnebohm, Avery Morgan; Mosley, Amber; Cornett, Evan; Vilseck, Jonah; Angus, Steve
High-throughput sequencing has been essential in identifying genetic variants associated with disease. However, protein-level characterization of identified genetic variants is lacking, especially for rare genetic diseases. RNA sequencing and proteomics techniques were used to characterize missense variants associated with the rare neurodegenerative disorder pontocerebellar hypoplasia 1b (PCH1b). Several variants of EXOSC3, a subunit of the RNA exosome, have been identified in patients with PCH1b. We investigated three missense variants of EXOSC3, including one variant of uncertain significance (VUS). We compared the VUS with two pathogenic variants, one of which is the most common in the general population by allele frequency. The RNA exosome complex consists of 9 core subunits and 2 catalytic subunits and cooperates with several cofactors to guide its function. The RNA exosome is involved in diverse cellular functions including general RNA turnover, rRNA processing, and regulation of RNA splicing. We focused on variants in the EXOSC3 core subunit, identifying differences in protein abundance and stability changes within missense variant cell lines generated by CRISPR. While overall RNA-level changes were limited, we observed differences in rRNA processing and RNA abundance. Proteomics revealed significant decreases in RNA exosome subunit protein abundance, and interestingly, four spatially proximal subunits displayed a greater degree of reduced abundance suggesting altered interactions between / assembly of these subunits. We see potential compensation with increased protein abundance of the catalytic subunit DIS3 and several rRNA processing proteins. Comparison of the VUS with the known pathogenic variants highlights the similarities in the molecular phenotypes but determined that the effects are not as pronounced in the VUS. Ongoing experiments seek to rescue the phenotypes caused by the missense variants by proteasome inhibition and exogenous overexpression of reference or variant EXOSC3. Overall, these experiments illustrate that disease associated EXOSC3 variants have a continuum of functional changes that could explain the range of severity observed in PCH1b patients. By obtaining protein-level insights into exosome complex dynamics, we can design rescue experiments to alleviate dysfunction.
The Role of Mutant P53 in the Pathogenesis of Myelodysplastic Syndromes
(2025-05) Barajas, Sergio; Mayo, Lindsey; Liu, Yan; Dong, Charlie; Liu, Yunlong; Nakshatri, Harikrishna
Human aging is associated with the development of clonal hematopoiesis of indeterminate potential (CHIP), which increases the risk of hematologic neoplasms such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Somatic mutations in the tumor suppressor gene TP53 are found in 10-15% of MDS patients and are associated with poor prognosis and reduced survival. TP53 mutations promote clonal expansion and drive MDS pathogenesis during aging. However, extrinsic factors contributing to the progression of p53 mutant clonal hematopoiesis to MDS remain unknown. We discovered that chronic inflammation provides a competitive growth advantage to p53 mutant hematopoietic stem and progenitor cells (HSPCs) through activating the NLRP1 inflammasome, leading to increased secretion of pro-inflammatory cytokines such as IL-1β and IL-6 from p53 mutant HSPCs, thereby generating a pro-inflammatory microenvironment that negatively affects the fitness of wild-type HSPCs in a paracrine manner. Furthermore, we found that approximately 60% of p53R248W/+ mice develop MDS with age and exhibit elevated levels of IL-1β and IL-6 in their bone marrow (BM). Similarly, increased levels of IL-1β and IL-6 were observed in the BM of MDS or AML patients with TP53 mutations. Mechanistically, mutant p53 dysregulates pre-mRNA splicing in key regulators of the inflammatory response in HSPCs, such as IKBKE and USP15, leading to enhanced NF-κB activation and increased secretion of pro-inflammatory cytokines in the BM of middle-aged p53 mutant mice. Moreover, we discovered that TP53 and SRSF2 mutations cooperate in accelerating the development of hematologic malignancies, possibly through convergent effects on the NF-κB pathway. Thus, we demonstrate that chronic inflammation and aberrant pre-mRNA splicing contribute to the progression of p53 mutant clonal hematopoiesis to MDS. Notably, blocking IL-1β or inhibiting gasdermin D (GSDMD) maturation reduces the fitness of mutant p53 HSPCs, suggesting that IL-1β and GSDMD are potential therapeutic targets for TP53 mutated CHIP and myeloid neoplasms.
Role of Polycomb Group Protein Mel18 in Hematopoietic Stem Cell Maintenance
(2025-05) Cai, Wenjie; Wek, Ronald; Zhang, Ji; Capitano, Maegan; Wan, Jun; Ren, Hongxia; Liu, Yan
Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1). PRC2 complex deposits the mono-, di-, and tri- methylation on histone 3 lysine 27, whereas PRC1 introduces mono-ubiquitination on histone 2A lysine 119 (H2AK119ub1). PRC1 can also regulate 3D chromatin structure. Bmi1 (PCGF4) and Mel18 (PCGF2) are two major homologs of the PCGF subunit within the canonical PRC1 complex. While Bmi1 is a positive regulator of hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) self-renewal, the role of Mel18 in normal and malignant hematopoiesis is not fully understood. Based upon our previous studies and the literature, I hypothesized that Mel18 inhibits HSC self-renewal and proliferation but promotes HSC senescence. To test my hypothesis, I examined HSC behavior in Mel18 conditional knockout mice (Mel18f/f-Mx1Cre+). I found that acute deletion of Mel18 enhances the repopulating potential of HSCs and increases the number of functional HSCs, without affecting HSC homing. Loss of Mel18 decreased hematopoietic stem and progenitor cell (HSPCs) senescence. In addition, loss of Mel18 promotes cell cycle progression in HSPCs. Therefore, I demonstrated that loss of Mel18 enhances the repopulating potential of HSCs, promotes cell cycle progression in HSCs, but reduces HSC senescence. Mechanistically, loss of Mel18 increases the chromatin accessibility to genes important for HSC self-renewal and ex vivo expansion such as homeobox gene Hoxb4. CUT&RUN sequencing assays revealed that loss of Mel18 reduces the H2AK119ub1 enrichment at the promoter regions of Cdk4 and Cdk6, leading to their enhanced expression in HSPCs. Furthermore, I identified a Mel18-specific chromatin loop at the S100a9 locus, a gene important for senescence and inflammation, using Hi-C assays, Collectively, these findings demonstrated that Mel18 plays an important role in hematopoietic stem cell maintenance. Mel18 inhibits HSC self-renewal and proliferation but promotes HSC senescence through modulating histone modifications, chromatin accessibility, and 3D chromatin structure in HSCs.
Genetic and Functional Dissection of Neurodegeneration: Multiomic Analysis of Genetic Risk Variants in Pontocerebellar Hypoplasia 1B and Alzheimer's Disease
(2025-05) Wijeratne, H. R. Sagara; Mosley, Amber; Kim, Jungsu; Baucum, A. J.; Vilseck, Jonah
Each neurodegenerative diseases have a combination of features such as neuronal death, protein aggregation, inflammation, cytoskeletal abnormalities, altered proteostasis, synaptic defects, RNA/DNA defects, and altered energetics. While genetic variants are crucial to disease pathogenesis, the molecular mechanisms linking the genotype to these hallmarks remain unclear. We investigate two risk genes linked to different neurodegenerative diseases, pontocerebellar Hypoplasia 1B (PCH1b) and Alzheimer’s disease (AD), using a multiomic approach to uncover their genetic and functional underpinnings. PCH1b is caused by variants in an RNA exosome complex subunit EXOSC3. These variants disrupt RNA processing, leading to neuronal dysfunction and progressive neurodegeneration. By analyzing cell lines carrying a variant in between two RNA-binding domains of EXOSC3, we found significant changes in RNA abundance across multiple RNA classes and showing enrichment of transcripts containing AU-rich elements. Molecular dynamics simulations of EXOSC3 indicate that the variants produce an unstructured EXOSC3 isoform, possibly prone to degradation. Proteomics reveals altered protein abundance and thermal stability in specific RNA exosome subunits, particularly affecting the MPP6 cofactor. Apolipoprotein E (APOE) is the strongest genetic risk factor for sporadic AD though its exact molecular effects are not fully understood. Thermal proteome profiling of astrocytes from APOE knockout mice showed that APOE deficiency alters the thermal stability of mitochondrial proteins, particularly in Complex I of the electron transport chain. The functional consequences of this stability change is an increased ATP-linked respiration in APOE-deficient astrocytes, suggesting a shift in mitochondrial activity. These findings provide new insights into how APOE impacts mitochondrial function and protein stability, emphasizing thermal proteomic profiling as a powerful tool for studying neurodegenerative diseases. Multiomics analyses effectively link genotype to phenotype in neurodegenerative diseases by uncovering molecular alterations that define disease-specific hallmarks, such as RNA homeostasis changes in PCH1b and mitochondrial alteration in AD, offering valuable insights into their pathophysiology and therapeutic targets.
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.

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