Pulling apart DNA during replication induces DNA strands to wrap around each other, producing “positive supercoils” ahead of the replication fork. These positive supercoils present a significant obstacle for further DNA replication. Type II topoisomerases (Top2s), a group of essential DNA replication enzymes, cleave these positive supercoils and relax DNA to a state that is easy to separate. Errors in the resolution of supercoils are implicated in many health conditions including autoimmune diseases and cancer. However, the mechanism by which Top2s localize to positive supercoils is unknown. We recently discovered that GapR, an essential DNA binding protein conserved in alphaproteobacteria, binds positive supercoils and stimulates the activity of bacterial Top2s DNA Gyrase and Topoisomerase IV. Although GapR stimulates Top2s in vitro, we do not know the mechanism by which GapR may recruit Top2s. We hypothesize that GapR recruits Top2s to positive supercoils by direct interaction. We investigated this mechanism by using a Bacterial Two-Hybrid assay to screen for GapR interaction with Top2 subunits, and I formed GapR truncations to probe for interaction in biochemical and genetic assays. We identified an interaction between GapR and the homologous Top2 subunits GyrA of DNA Gyrase and ParC of Topoisomerase IV. I discovered that this interaction terminates with the truncation of the last 13 amino acids of GapR. In the future, we aim to identify the surface that mediates direct interaction between GapR and Top2s aided by structure prediction. This work will reveal a previously unknown mechanism of Top2 recruitment. Because GapR is broadly conserved by alphaproteobacteria, our research could reveal a novel mechanism to inhibit with antibiotics. As GapR is the first identified Top2 recruiter, our work could reveal a novel pathway to target with anticancer therapeutics if conserved, as human Top2 inhibitors are important chemotherapy drugs.

Traditional vaccines use inactivated or live attenuated pathogens to elicit an effective adaptive immune response, but these vaccines can lack safety for immunocompromised individuals. Subunit vaccines–which display characteristic components of pathogens–are safe, stable, and readily engineered, but struggle to elicit a strong immune response. These next-generation vaccines require adjuvants (substances that stimulate the immune system) to increase efficacy. However, many currently used adjuvants lack well-understood mechanisms or wide applicability across vaccines. There is a need for new adjuvant platforms, and protein-based adjuvants are appealing because they are stable, readily engineered, and can be co-delivered with antigens on subunit vaccines. Toll-like Receptor (TLR) proteins are promising adjuvant targets that bind pathogen-associated molecules to activate the innate immune system. Of this family, TLR5 binds the bacterial protein flagellin, but flagellin is not a suitable adjuvant candidate because it is degradation- and aggregation-prone. Therefore, this project aims to design, test, and characterize novel protein-based adjuvants that can bind TLR5 and activate the immune system. Here, I test and characterize de novo mini-proteins that I have computationally designed to bind mouse TLR5 (mTLR5). I use cell-based binding and signaling assays to identify and characterize successful binders. Preliminary results show de novo mini-proteins bind and activate mTLR5 and key knockout mutations at the binder interface decrease signaling. Ultimately, this work hopes to provide a mouse model for these novel protein-based vaccine adjuvants with clinical aims. This project has wide-reaching public health implications, as vaccines offer the potential to improve the health and lives of countless individuals.

Major Histocompatibility Class I (MHC) Molecules serve as a window into the cell, whereby T-cells can use their T-cell receptor to recognize foreign peptides presented on the MHC of a cell and induce apoptosis. Unfortunately, for diseases such as cancer, cancerous mutations may only be a single residue different from the native protein, resulting in similar mutant and wild-type peptide-MHCs. This small difference often results in negative selection of T-cell receptors that recognize mutant peptide-MHCs, leading to an absence of T-cells that can target cancer cells. This issue motivated us to use de novo protein design to generate binders with a high level of specificity between mutant and native peptide-MHCs. Using deep learning based protein design methods such as RFdiffusion and ProteinMPNN, we have generated promising in silico designs against a range of target peptides and MHC alleles. After selecting our top designs, we tested them using yeast surface display against our target MHC molecules with peptides loaded from KRas, PIK3CA, MAGE, and TP53 mutants. We observed binding events to all targets; some designs also had specificity to their respective mutant peptide-MHC over the wild-type peptide-MHC. After further work we have shown that the same design scaffold can bind to multiple peptide-MHC targets after slight redesign, similar to native T-cell receptors, holding promise that we could easily and quickly repurpose these scaffolds for new targets. Following these results we will incorporate our binders into T-cells as chimeric receptors and test for the ability of our binders to activate T-cell signaling and cell killing. This method of targeting peptide-MHC molecules is promising as a novel and rapid way to target cancer.

Tuberculosis remains a global public health threat due to the rising number of multi- and extensively drug resistant strains of the causative pathogen Mycobacterium tuberculosis. Development of novel drugs and an understanding of their resistance mechanisms is urgently needed. Aminothiazoles (AmT) are potent molecules with killing activity against M. tuberculosis; these compounds act as copper ionophores and target a key enzyme (enolase) by displacing its Mg²⁺ co-factor, a substance required for its activity, with Cu²⁺ imported by the compounds. Spontaneous mutations in an essential protein export system (the Esx 3 Type VII secretion system) confers resistance to AmTs. My research focuses on understanding how mutations in the secretion system cause AmT resistance. We hypothesize that copper imported by AmTs could disrupt other metallo-proteins including EccA3, a key ATPase of the of the Esx-3 secretion system that hydrolyzes ATP into ADP and inorganic phosphate, and that resistance mutations (e.g. E237K) reduce Mg²⁺ co-factor displacement by Cu²⁺. To test this hypothesis, I expressed wild-type (WT) EccA3 and mutant EccA3 [E237K] proteins in Escherichia coli BL21(DE3) expression strain and purified the proteins via Ni-NTA His-tag chromatography. Subsequently, I measured the activity of the purified EccA3 (WT) and EccA3 [E237K] proteins via an ATPase assay based on colorimetric detection of free inorganic phosphate released by ATP hydrolysis. I aim to understand whether copper inhibits EccA3 activity through this assay, anticipating that copper reduces EccA3 (WT) ATPase activity while EccA3 [E237K] ATPase activity is unaffected. Thus, my work will provide an avenue for understanding AmT resistance in M. tuberculosis.

How do organs have such consistent and reproducible shape, form, and volume? One factor of this complex phenomena is cell-cell adhesion. Cell-cell adhesion plays a vital role in organ formation, as it is an essential driver of cell shape, cell arrangements, and tissue structure. To determine the role of adhesion in organ formation, I define the role of E-Cadherin, a cell-cell junction projection that adheres neighboring cells. The developing renal system of Drosophila, Malpighian tubules, are an excellent system because I can selectively manipulate expression of E-Cadherin in the organ and can utilize fluorescence microscopy to observe how these changes affect tubule morphogenesis. I observe where the adhesion protein is located during organ growth, and what happens to organ growth when expression of the adhesion protein is reduced. To track the dynamic localization of E-Cadherin, I take measurements of specific location of E-Cadherin between cells and concentration of E-Cadherin throughout organ development. I expect the concentration of E-Cadherin to increase during elongation, and that it will be enriched in more looped parts of the organ. To define the requirement of E-cadherin during organ formation, I use RNA interference to reduce E-Cadherin expression. Because of how vital E-Cadherin is in other developmental morphogenetic processes, I expect a decrease of expression to have profound impacts, leading to severe organ developmental defects. I measure these defects by comparing cell shape change and organ shape in control and E-Cadherin reduced organs. The results of this study will not only help us understand Malpighian tubule morphogenesis, but it will also help us understand organogenesis more generally. Elucidating the precise mechanisms behind cell behavior, shape, and cell-cell interaction has important human health implications and will enable work in many other fields such as cancer, regenerative treatments, tissue growth, and organ synthesis.

Although the relationship between the structure, function, and physiology of organs is well documented, the mechanisms by which cells collectively coordinate into three-dimensional tissues and organ components remains unknown.The countless factors that inform the morphogenesis of mammalian organs poses a challenge to understand organogenesis from first principles. However, the Malpighian tubules of the fruit fly Drosophila offer an excellent model system for investigating this question due to their rapid development, relative simplicity, and the degree to which scientists can manipulate variables that affect their development. These tubules are the renal equivalent of the fruit fly excretory system; further, many of the genes involved in sculpting these tubules are conserved from flies to humans. One conserved gene is the fly homolog of β-catenin, which is known to play an essential role in cell-cell adhesion. The goal of my research is to define how β-catenin impacts organ morphogenesis. To do this, I use fluorescence microscopy and live imaging to compare wildtype Drosophila to those with decreased β-catenin expression. Using tissue-specific fluorescent protein tagging, I can differentiate Malpighian tubule cells from other embryonic cells under the microscope so that their shapes can be analyzed, and I control the level of β-catenin expression specifically in Malpighian tubule cells using RNAi. Due to β-catenin’s integral role in cell-cell adhesion, I expect to find localization of β-catenin to the cell membranes of the tubules, with high concentration along membranes undergoing the greatest adhesion or motion, and interrupted tubule morphogenesis in reduced-expression lines. I also suspect that cells may completely fail to adhere and will be unable to transmit tension effectively along the tissue. The results of this experiment will contribute to our understanding not only of Malpighian tubule morphogenesis, but of one of the components of morphogenesis in general.