Lysine acetyltransferases (KATs) regulate gene expression and other cellular functions via the deposition of acetyl groups onto lysine residues, most often present on histone tails. These genes are critical for normal development as germline mutations in KAT6A and KAT6B cause various human neurodevelopmental disorders, characterized by cognitive and physiological disabilities. We aim to optimize procedures for expressing HaloTag fusion proteins consisting of a versatile epitope tag and cDNA encoding KAT6A or KAT6B. Tagging our genes of interest with the HaloTag enables detection of the fusion protein via either a fluorescent ligand that binds to the HaloTag protein or by using monoclonal antibodies against the HaloTag protein. Plasmids containing either a KAT6A or KAT6B HaloTag gene fusion were verified through Sanger sequencing, then introduced into HeLa and HEK293FT cells by lipofection to overexpress each gene fusion. After transfection of cells, protein was harvested via cellular fractionation to produce cytoplasmic extract (CE) and nuclear extract (NE). Western blots were then performed on CE/NE from these cells to detect the HaloTag protein using anti-HaloTag antibodies. Additionally, the fluorescence of synthetic ligands covalently bonded to the HaloTag protein was detected. Thus, our results indicate that the KAT6A and KAT6B HaloTag gene fusions were successfully transfected and expressed in both HEK293FT and HeLa cells. By studying the molecular processes involved in the regulation of gene expression by KAT6A and KAT6B, we hope to gain insight into the role of these two genes in developmental disorders upon translating this optimized system into disease-relevant cell types.

Across multiple neurodegenerative diseases, such as Alzheimer's disease (AD), abnormal aggregation of the neuronal microtubule associated protein tau (MAPT) has been observed. However, the Tau protein’s role in disease progression is unclear. In this experiment, organoids were generated using human induced pluripotent stem cells containing the R406W mutation in the MAPT gene which affects translation and the normal function of the Tau protein. Cortex-like (Cx) and ganglionic eminence-like (GE) organoids (Cx+GE) were fused together to recapitulate the mix of excitatory and inhibitory neurons in vivo. Cx+GE fusion organoids were studied due to their ability to recapitulate key aspects of human brain cytoarchitecture and cellular composition, as compared to commonly used rodent models. The cortical fusion organoids were transfected with an adeno-associated virus (AAV) vector containing the genetically encoded calcium indicator (GcAMP) in order to assess calcium activity. Calcium imaging on a multiphoton microscope was conducted to observe the neural activity of the Cx+GE organoids and Matlab based algorithms were used to extract activity patterns. Since tissue hyperexcitability and hypersynchrony is a pathological hallmark of AD, we hypothesized that the R406W mutation will result in increased hypersynchronous bursts of calcium activity in p.R406W Cx+GE organoids, as compared to isogenically matched control Cx+GE.

Given the importance of structurally analyzing ligands binding to proteins, we set out to compare our ability to detect bound ligands via X-ray and electron diffraction. We relied on two model protein systems: Proteinase K, a serine protease that cleaves peptide bonds and covalently binds to AEBSF, an irreversible serine protease inhibitor, and Lysozyme, which breaks down sugars and binds inhibitor N-acetyl-D-glucosamine 6-phosphate sodium salt (GlcNAc-6P). Both proteinase K and lysozyme have been previously characterized by 3D electron diffraction (3DED), but their ligand-bound structures have not yet been determined by 3DED. With the goal of assessing the binding of each ligand to its respective protein and examining corresponding structural differences compared to their native structure, we specifically sought the appearance of density corresponding to bound ligands in structural maps. To generate crystals of enzyme-inhibitor complexes, we relied on crystallization conditions identified from the literature, where ligand binding was favored. We used cryoEM techniques to freeze crystals and collect high-resolution 3DED data for structure determination and evaluation of ligand presence. We collected data from bundles of needle-like proteinase K crystals and prismatic lysozyme crystals but did not observe the presence of bound ligands. Supporting this observation, our X-ray data showed that the lysozyme-GlcNAc-6P complex had not yet crystallized. Once successful, this research will provide further insight into these proteins and their interactions with the respective ligands. The solving of these novel ligand-protein structures will also further demonstrate the potential of electron diffraction.