Estrogen receptor alpha (ERα) and BRCA1 play an important role in the development of breast cancer, and multiple pathways link these two proteins together. Previous studies have identified the ligand binding domain (LBD) of ERα and residues 1 through 258 of BRCA1 as important in the direct physical interaction between these two proteins. This study aimed to characterize the binding kinetics of this interaction in the presence and absence of 17β-estradiol (estrogen) with a shortened BRCA1 construct (residues 177-258); however, binding between ERα LBD and this BRCA1 construct could not be detected through fluorescence emission spectroscopy or isothermal titration calorimetry (ITC). Synthesizing ERα LBD presented challenges with low yield, so the purification protocol was refined to cool bacterial cultures at an OD600 of 0.2 during growth and add dithiothreitol during lysis for improved yield. A 24% decrease in fluorescence intensity upon addition of estrogen to ERα LBD confirmed the ligand-binding functionality of the protein. Additionally, Stern-Volmer studies verified that the estrogen binding site on ERα LBD is located in close vicinity to the tryptophan residues in the protein since fluorescence quenching was more efficient in the absence of estrogen. Finally, factors contributing to the absence of ERα-BRCA1 binding are discussed, including the length of the BRCA1 construct used or the potential necessity of an additional protein, BARD1.

BRCA1 and PALB2 proteins suppress tumor formation by promoting homologous recombination when DNA damage has occurred. Mutations in BRCA1 and PALB2 are associated with a higher prevalence of breast and ovarian cancers. Phosphorylation of BRCA1 and PALB2 occurs upon DNA damage and is vital for maintaining genomic integrity. The molecular mechanism of how phosphorylation directs the activation of these proteins is unknown. It is established that phosphorylation of BRCA1 and PALB2 occurs in or near the coiled-coil regions of both proteins. The proteins use this domain to heterodimerize, so we hypothesize that the phosphorylation events could promote efficient BRCA1/PALB2 interactions. Our study aims to determine the effect of phosphorylation on the BRCA1/PALB2 binding affinity. The serine and threonine residues that are phosphorylated on BRCA1 or PALB2 were mutated to a glutamic acid to mimic phosphorylation. Glutamic acid carries a negative charge and thus mimics the negative charge added to the protein upon phosphorylation. We overexpressed and purified the protein using a bacterial expression system and measured their heterodimerization affinity with isothermal titration calorimetry (ITC). We will share ITC data suggesting phosphorylation of PALB2 does not affect its binding affinity to BRCA1. The phosophomimicking mutations in BRCA1 have also been generated, both individually and in tandem, and we will share results from these binding studies that are ongoing and hypotheses generated from our results regarding phosphorylation as an activation switch to control BRCA1/PALB2 interactions.

The Identification of Novel Genes Related to Iron Acquisition in Bacillus Anthracis Sterne

Jessica Guilhas, Kyle Gallegos, Julio Manceras, Mariah Green, Jacob Malmquist, Shauna M. McGillivray

Bacillus anthracis, the causative agent of anthrax, is a spore-forming, gram-positive bacterium. Its virulence mechanisms are of interest due to its potential use as a biological weapon and high lethality. For B. anthracis to survive and reproduce in a host, it must evade the host's immune response and acquire nutrients. One important nutrient B. anthracis must acquire is iron. Iron is a limiting nutrient in the host because it is usually found sequestered to hemoglobin or bound to host proteins such as transferrin. To acquire iron, pathogens must strip it from the host proteins. To find genes important for iron acquisition from hemoglobin, we screened genetic mutants created through transposon mutagenesis. Media was chelated to remove all divalent cations, including iron, and then hemoglobin was added as the sole iron source. The mutants that were unable to grow were chosen to be tested in a larger volume hemoglobin assay. We confirmed the phenotype of several mutants using this larger volume assay and we are working to confirm the site of transposon disruption via PCR. The mutants thus identified include a mutation in a dUTPase gene and an L-aspartate oxidase gene, neither of which has been previously linked to iron acquisition from hemoglobin. Future directions include making independent mutations and/or complement the disrupted genes to confirm the gene disruption is linked to loss of iron acquisition from hemoglobin. This study allows for a further understanding of how B. anthracis acquires iron and sheds new light on potentially novel virulence mechanisms.

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the formation of amyloid beta (Aβ) plaques in the brain and is the seventh leading cause of death in the United States. Chronic inflammation and oxidative stress associated with AD leads to neuronal cell death. A cellular protective mechanism against oxidative stress involves the Nuclear factor erythroid 2-related factor (Nrf2) pathway. Nrf2 is responsive to the reactive oxygen species (ROS) produced when the cell is under oxidative stress, leading to its translocation into the nucleus where it activates transcription of genes that produce antioxidant enzymes like heme oxygenase-1 (HO-1). To study this pathway in neurons, our lab chose to use the mouse hippocampal HT-22 neuronal cell line. Our previous attempts to grow these cells in culture proved difficult, leading us to hypothesize that providing a growth-enhancing surface of collagen would provide a more stable surface in which to propagate these cells. Here we show that HT-22 cells grown on rat tail collagen provide a model system to investigate the Nrf2 pathway. We also demonstrate that HT-22 cells are viable on tissue culture plastics without the need for collagen.

Exploring EncT Efflux Pump Functionality and their Role in Lipid Signaling
Gabby Linares, Sawyer Diaz, Natalia Castro-Lopez, Floyd Wormley Jr.
Department of Biology, Texas Christian University

Cryptococcus neoformans, a fungal pathogen mainly affecting immunocompromised individuals, has sparked interest in lipid signaling research due to its role in pathogenesis. Eicosanoids, derived from fatty acids, are crucial in virulence and immune modulation; with C. neoformans lacking human enzyme homologs for eicosanoids biosynthesis, we want to identify the enzymes involved in the biosynthesis of cryptococcal eicosanoids and test their potential as antifungal targets. This project is focused on the EncT gene, encoding an efflux pump, which we observed to be upregulated in response to lipid precursors. Using CRISPR technology, we produced an EncT knockout (KO) strain and the corresponding reconstituted strain, aiming to discern shifts in virulence factors like melanin production, capsule formation, and urea production, among others, comparing the knockout, wild-type, and reconstituted strains and, subsequently, employing a mouse model of pulmonary cryptococcosis to delve deeper into virulence dynamics. Our initial results show early production of melanin EncT KO compared to the WT strain and no changes in the capsule formation or growth at 37°C.