Glutamine synthetase (GS) is a highly regulated enzyme critical for converting glutamate to glutamine and associated with ammonia assimilation. Dysregulation in the GS interconversion process can lead to hyperammonemia, potentially resulting in death or brain damage. GS is conserved across prokaryotes and eukaryotes. Among enzymes, glutamine synthetase has the ability to polymerize but the functional characteristics of its self-assembling filaments remain unknown. This study aims to elucidate the occurrence of filament formation in GS and its effects on enzyme activity. We hypothesized that filaments might influence the association of GS substrates or allosterically regulate the enzyme. I purified GS from Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Helicobacter pylori using Ni-column and size exclusion chromatography (SEC). The focus was primarily on Pseudomonas GS, examining it under various buffer conditions (Mg²⁺, Co²⁺) through negative staining. Under magnesium conditions (10 mM), dodecamer strcture of GS was observed and filaments was induced under cobalt conditions (10 mM). To investigate the structural mechanism of filament formation further, we utilized cryogenic electron microscopy (Cryo-EM) to create a model of the GS filament interface and identifying involved residues. Additionally, I am conducting mutagenesis on key residues of Pseudomonas GS to disrupt filament formation. This research holds significant implications for metabolic engineering, as understanding the structure and role of filament formation in GS could lead to new therapeutic targets in metabolism.

Phosphoribosyl Pyrophosphate Synthetase (PRPS1) is an enzyme in the nucleotide biosynthesis pathway that makes a molecule necessary for de novo nucleotide synthesis. It is known that PRPS1 protein hexamers can stack into linear filaments in the presence of ADP and phosphate. When these filaments are broken, catalytic activity is lost, and it is hypothesized that enzyme inhibition is lost as well. Mutations in PRPS1 lead to a wide spectrum of diseases in humans. In addition, changes in cell regulation of the enzyme have been linked to cancer. Motivated by research that connects PRPS1 phosphorylation to increased cancer proliferation, my project investigates the effects of phosphorylation on PRPS1 structure, enzyme activity, and inhibition properties. I have transformed plasmid DNA containing the PRPS1 phosphomimetic mutations S47E, S103D, and S308E into E. coli strains BL21 and pLysS. I then grew overnight bacterial cultures and induced protein expression using IPTG. After verifying protein expression with gel electrophoresis, I purified the protein from bacteria using nickel resin affinity and size exclusion chromatography. Having made and purified protein mutations that mimic phosphorylation, I conducted a negative stain screen to analyze filament formation trends. This has yielded preliminary findings that S47E and S103D phosphorylation mutations of PRPS1 break enzyme filament formation. Variation in filament formation between mutations points to the importance of phosphorylation location and its potential impact on enzyme activity and inhibition. To assess the catalysis of the phosphomimetic mutations in PRPS1, I will conduct biochemical assays which measure the activity and inhibition of the enzyme. Through these ongoing experiments we will learn how phosphorylation modifies PRPS assembly and activity and the implications of PRPS1 dysregulation in cancer proliferation.

Merkel cell carcinoma (MCC) is a rare and aggressive cancer of the skin with a mortality rate of ~30%. In the US, most MCC tumors arise from integration of the Merkel cell polyomavirus (MCPyV) DNA into a host chromosome, leading to expression of viral T-Antigen (T-Ag) oncoproteins that drive tumorigenesis. Though current treatment options have significantly improved MCC prognosis, new therapies are needed to address recurrent/resistant disease. While T-Ag-specific antibodies are usually detected in the blood of patients with virus-driven MCC, the role of these antibodies in tumor immunity remains unclear. Here, we analyzed blood samples from 100 MCC patients prior to definitive treatment, 51 of whom had high titers of antibodies recognizing the T-Ags. These 51 high titer samples were assessed for binding across two domains of the T-Ag. Suprisingly, we found that patients who had high titers of antibodies binding both regions of the T-Ag had worse MCC control than patients whose antibodies predominantly bound one region (median PFS 5.5 vs. 14.2 months, p=0.003). These data suggest that careful mapping of circulating antibody reactivity to different regions of T-Ag can serve as a biomarker to identify high-risk patients for which a more aggressive treatment regimen is needed. Future work is also focused on understanding the immune response resulting in differential response to T-Ag domains.

Computational protein design has successfully designed nanoparticle cages that self-assemble and effectively deliver encapsulated therapeutics to cells. These nanoparticle cages are readily taken up by the cell via receptor-mediated endocytosis. Despite the promise of these cages, one of the greatest challenges that remain is the successful endosomal escape of the encapsulated biologics and their precise delivery to the cytosol. To address this, we have engineered a high throughput complementation assay, based on split green fluorescent protein (GFP) construct, that helps screen and quantify cytoplasmic delivery of therapeutics through fluorescence intensity. Split-GFP is a protein complementation assay in which the normally monomeric GFP is made of two fragments: the larger non-fluorescent beta barrel and a 15 amino acid (a.a) peptide. When these two components unite, the GFP fluoresces. In this project, I created a stable HeLa cell line expressing the beta barrel of split GFP using lentiviral transduction under antibiotic selection. The cell line has been further validated, through transient transfection of the complementary 15 a.a peptide to test the assay performance. I propose to test endosomal escape, through introduction of endolytic peptides (EEPs) into model proteins, which force early endosomal membrane fusion and destabilization. Future research will explore adapted designs of nanoparticle cages, incorporating the EEPs and the split-GFP complementary strand in the HeLa cell line, to quantify the endosomal escape of our designs. The outlook of this project has transformative implications for targeted therapeutic delivery. By creating a screening assay that can quantify targeted delivery into cytosol, we can expedite refinement of protein designs for therapeutic delivery, thus accelerating the timeline for developing novel protein-based therapeutics.