Biochemistry & Molecular Biology Department Theses and Dissertations

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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.
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.
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.
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.
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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