Mutations in phosphodiesterase 6 (PDE6) underlie photoreceptor degeneration through cyclic guanosine monophosphate (cGMP) accumulation, triggering a series of down-stream processes, which eventually kill photoreceptors. We hypothesize that knocking out inosine monophosphate dehydrogenase 1 (IMPDH1), the rate-limiting enzyme in de novo guanine synthesis, will rescue cell death caused by PDE6 mutations. Supporting evidence from a mouse mutant model (rd10) suggests that inhibiting IMPDH1 pharmacologically delays photoreceptor degeneration (Yang, 2020). Our procedure for this experiment is as follows. Fish heterozygous for impdh1a and pde6c mutations are mated to produce fish that are double homozygotes. Fish homozygous for mutations in both the cone-specific pde6c and the impdh1a genes are genotyped and embedded for histological analysis of the retina. Histology is examined on days 3,5, and 7 post-fertilization (dpf) for cone degeneration. To date, we have genotyped our two mutant lines. Normally, pde6c^-/- fish have severe cone photoreceptor degeneration at 5 dpf and impdh1a^-/- fish show no signs of photoreceptor degeneration even as adults. If degeneration is rescued, the double knockout larvae should retain similar photoreceptor nuclei counts to wildtype fish at all time points. Demonstrating that IMPDH1 inhibition rescues PDE6 deficiency would provide proof-of-concept for the therapeutic potential of IMPDH1 targeted inhibition for the treatment of photoreceptor degeneration due to cGMP imbalance.

One of the most common types of vaccines used today are subunit vaccines. Subunit vaccines consist of an antigen that triggers the adaptive immune system to create antibodies but also require a separately added adjuvant, which is a substance that induces longer-term immunity by stimulating the immune system to pay attention to the antigen. Current adjuvants are non-specific - often things like oil-water emulsions that irritate the immune system and cause inflammation in unknown ways to draw attention to the antigen. This project aims to create a more specific adjuvant by directly stimulating B cells. In order for B cells to replicate antibodies, they need a primary signal from the antigen and a secondary signal that certain ligands on T-cells can initiate. We decided to investigate whether CD40Ligand (CD40L), an immune protein present on T cells that works to signal B cells to either replicate or create antibodies, could be used to achieve this goal. The idea behind the project is to co-display CD40L with antigen on the nanoparticle in hopes of creating a more specific adjuvant. We designed 10 different versions of this nanoparticle, where we tested two versions of CD40L, the placement of CD40L, and the linker length between CD40L and the nanoparticle surface. Our preliminary results also show that our cages retain their ability to bind both antibodies and CD40 as well as activate NFkB transcription - a proxy for B cell activation. We expect CD40L-displaying nanoparticles will promote B-cell proliferation to a greater extent than the nanoparticle vaccine displaying only hemagglutinin (flu) antigen accompanied with an adjuvant like Addavax. Ultimately, we hope to examine how co-display of CD40L with antigen will change the quality of immune response and memory in-vivo in comparison to currently used vaccine adjuvants, and begin testing in-vivo in the coming quarters.

DNA-binding proteins (DBPs) capable of targeting specific DNA sequences play key roles in genetic regulation and manipulation in both natural and synthetic contexts. Aided by advances in machine learning and protein engineering, the design of entirely new DBPs is now possible. However, early attempts yielded small, single-chain proteins that were unable to induce changes in genetic expression despite successfully binding to their target DNA sequence.  It is thought that contacts made by these early de novo DBPs are not sufficient to maintain binding in the presence of other proteins involved in DNA transcription, preventing the DBPs from altering gene expression. In contrast, many natural DBPs consist of protein complexes composed of two identical subunits called homodimers. These complexes have increased intermolecular contacts with DNA, enhancing their DNA-binding affinity. In hopes of improving the affinity of current de novo DBPs, machine learning tools like RFdiffusion, ProteinMPNN, and AlphaFold2, were invoked to engineer homodimerization domains onto these monomers. Promising designs were filtered, ordered, synthesized, and tested for efficacy as genetic inhibitors in E. coli. Homodimers that consistently and specifically bind to their target DNA sequences and induce genetic repression in E. coli have promise as tools for genetic engineering and manipulation. In future research, successful designs could also have applications in synthetic gene circuits and serve as foundations for de novo allosteric transcription factors for use as biosensors.

Over the past few years, patients have been identified with debilitating phenotypes due to mutations in MSTO1, a nuclear gene. These patients often have distal muscle loss and weakness leaving patients incapable of walking but to date there is no known treatment. One barrier to progress is that virtually nothing is known about MSTO1 function, making the development of therapeutics for these patients extremely challenging. The goal of this project is to use an unbiased approach to discover functions of MSTO1. To do this, I will find genetic interactors utilizing yeast to perform an unbiased screen. Yeast DML1 is the homolog to MSTO1 and is required to keep the yeast cells alive. This screen will identify genes in the yeast genome that support survival of cells lacking DML1 when the gene is overexpressed. We utilize an auxin-degron system that targets DML1 for degradation when the yeast are grown with auxin. To find genes from the yeast genome that keep the cells alive when DML1 is degraded, I express random fragments of genomic DNA. Those genes must, therefore, be linked to DML1 function in some way, thus providing insight into what MSTO1 does, how it works, and how to help MSTO1 defective patients. I have obtained hundreds of yeast colonies that survive without DML1 when other genes are overexpressed. Currently, I am extracting these overexpressed DNAs to determine the gene(s). This work is an essential step toward fully understanding MSTO1 function in cells and we plan to characterize these connections in yeast and human cells.

We use genes to survive and reproduce, but this means that genes can hold our survival hostage to ensure their own propagation, without providing any benefit to us. Selfish genes are a brutal, and poorly-characterized, demonstration of this concept. Instead of producing beneficial proteins, they produce nonessential proteins that prevent individuals who carry the selfish gene from successfully reproducing with non-carriers. One such example, the PEEL-1/ZEEL-1 system, is natively found in C. elegans. In this system, PEEL-1 is a toxin protein that kills cells when it is expressed without the antitoxin protein ZEEL-1. My aim is to determine the mechanism of toxicity employed by PEEL-1. AlphaFold predictions suggest that PEEL-1 contains an amphipathic helix. The amphipathic property of this region is hypothesized to play a critical role in PEEL-1 toxicity. To test this hypothesis, I am conducting a Deep Mutational Scanning (DMS) on the amphipathic helix of PEEL-1, with the goal of identifying key polar or nonpolar residues in this region that are essential to PEEL-1 toxicity. First, I generate a library of single-residue PEEL-1 mutants. Second, I transfect these constructs into HEK293T cells. I sample this initial pool of cells for sequencing, to identify which PEEL-1 mutants the pool carries, and in what proportions. Third, I induce expression, exposing each cell to the effects of the PEEL-1 mutant they carry. Only the cells expressing loss-of-function mutants survive. Now, I sequence this final surviving pool of cells, similarly to the initial pool. Mutations that drastically alter the polarity of the residue and thus break the overall amphipathic structure of the region are expected to be overrepresented in the surviving pool. This result would provide a broader understanding of the various methods of cell death in nature and provide novel insight into how animal-derived selfish genetic systems function.

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.

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.

Damage to the spinal cord causes one of the most debilitating injuries to the human body. The challenge of promoting the regeneration of this dense network of neurons and glia after spinal cord injury has been seen as insurmountable. However, new techniques emerging from the field of regenerative medicine have illustrated the possibility of encouraging the body to repair these injuries on its own. In the Wills Lab, we study the model organism Xenopus tropicalis, or the Western clawed frog, which has the ability to regenerate its spinal cord and associated tissue following amputation. My project focuses on how X. tropicalis uses the developmental morphogen Sonic Hedgehog (Shh) to re-establish the dorsal-ventral (DV) patterning of the spinal cord during regeneration. I have used cyclopamine, a Shh inhibitor, and SAG, an agonist, in order to perturb Shh signaling during regeneration. I then monitored the effect on DV patterning via immunohistochemical labeling of dorsal and ventral markers. Work so far has shown that Shh signaling is in fact necessary to the establishment of proper DV domains in the regenerate spinal cord. However, my research has also hinted that this specification is complex. Shh appears to have a more proliferative role early on, with patterning effects coming later. In addition, there appears to be an interaction between Shh and other signals that specify anterior-posterior polarity. Overall, my research so far has generated new evidence for how developmental signals are repurposed in the context of regeneration. 

In response to acute genotoxic stress, such as chemoradiation therapy, stem cells undergo temporary cell cycle arrest at the G1/S phase transition. This state, called quiescence, is reversible once stress-free conditions allow reentry into the cell cycle. We have previously identified the underlying mechanism behind quiescence in Drosophila Germline Stem Cells (GSCs) and human-induced pluripotent stem cells (hiPSCs). Mitophagy, or autophagy of the mitochondria, is required to enter quiescence. Surprisingly, we have observed a reserve of cyclin E (CycE) associated with the outer mitochondrial membrane that’s present in normal GSCs and hiPSCs but is reduced in quiescent stem cells. The role of CycE in quiescence remains unclear. Previously we have shown that reduced levels of CycE via inhibition of mTOR have driven cells toward mitophagy-dependent quiescence. This reveals that mitophagy serves as an alternative mechanism of CycE inhibition in contrast to the typical p21-mediated inhibition. Additionally, Parkin, a ubiquitin ligase activated by a serine/threonine kinase PINK1, is a key protein involved in mitophagy required for quiescence, and it has been found that CycE is a degradation target of this protein complex. Our hypothesis is that CycE degradation is necessary for entry into quiescence. To test this we upregulated CycE with a deleted portion of its PEST domain, which is a target for ubiquitination, under UAS-GAL4 control and used the GSC spectrosome morphology to observe quiescence. We observed a six-fold reduction of quiescent GSCs with overexpressed CycE, and hence concluded that CycE degradation is necessary for entry into quiescence. Determining the mechanism of CycE in stem cell quiescence is critical to understanding how cancer stem cells can avoid chemoradiation therapy. This project allows us to characterize the role of CycE within mitophagy and strengthen our understanding of the mechanisms that govern the cell cycle and quiescence.

Cancer is characterized by uncontrolled cell proliferation, and its potential to affect almost anyone as they age poses a significant threat. Extracellular vesicles (EVs) are lipid-bilayer membrane-enclosed structures that cancer cells produce and use for intercellular communication. EVs are typically loaded with a variety of proteins, nucleic acids, and other cargo that can be delivered to recipient cells. Tumor-derived EVs aid in the progression of various cancers by enhancing malignant cell survival, proliferation, and invasion. Working with our graduate mentor, we conducted an 866 chemical screen and found kinase inhibitors that altered EV production by cancer cells via luminescence assay. From the hits, we chose to study kinases from the JNK and p38 MAPK pathways, which both promote cancer progression. Reactive oxygen species (ROS), which damage cells through oxidative stress, can activate both of these pathways. Based on this, we proposed the question: what role do ROS play in EV biogenesis and cancer development in living organisms? To answer this question, our research utilizes Drosophila melanogaster, an ideal in vivo model due to its vast genetic toolbox and brief generation times. We used Drosophila with the Ras^V12, scrib^-/- tumor model to study EV biogenesis, and crossed them with flies that have knocked down homologs of JNK and p38 MAPK. We then selected specific progenies and dissected the imaginal discs and placed them in media to allow for EV biogenesis, and quantification was done by live imaging EV production from tumor discs, fluorescence assays, and qPCR. Our preliminary results show that imaginal discs from Ras^V12, scrib^-/- flies produce a large amount of EVs. We anticipate that in organisms, both JNK and p38 MAPK knockdowns will lead to a decrease in EV production. Future work could be done to implement our findings in humans to potentially develop novel cancer therapeutics.

Signaling of fibroblast growth factor receptors (FGFR) is critical for the development of vascular cell types. FGFR exists as two alternative splice variants: the b and c isoforms. Previous experiments have shown that activation of the c isoform leads to arterial endothelial cell development and inhibition of the c isoform is critical to perivascular development. These results were found using a c isoform-specific computationally designed protein. The goal of my project is to replicate these isoform specific results in an endogenous context. Our hypothesis is that induced pluripotent stem cells (IPSCs) overexpressing the b isoform will develop into pericytes and IPSCs overexpressing the c isoform will develop into arterial endothelial cells. I used the Gibson assembly method to create b/c isoform overexpression plasmids that can be inserted into the AAVS safe harbor site and used bacterial transformation to increase the amount of DNA. I am using stable transfection to create IPSC overexpression cell lines and adapting a previously verified 14-day protocol for creating endothelial cells from IPSCs to monitor each cell line’s differentiation. I am performing assays such as qPCRs, Western Blots, and immunofluorescence to quantify perivascular and endothelial markers in the cell lineages. Our findings should agree with our isoform specific hypothesis. In future experiments, we plan to engraft the overexpression cell lines into immunodeficient mice and assay how varying ratios of the two cell types affect their regenerative potential in vivo.

When a cell undergoes stress conditions, such as oxidation or aging, an increase in protein instability can occur and prevent proper cell functions. Small Heat Shock Proteins (sHSPs) are molecular chaperones that work to maintain a healthy proteome by associating with misfolded “client” proteins to delay aggregation under such conditions. HSPB5, a human sHSP, is ubiquitously expressed throughout the body. HSPB5’s disease mutant, R120G, is a defective chaperone associated with cataracts and desmin-related myopathy. It is still unknown how this mutation is detrimental despite many years of research. My research aims to understand how this mutation retunes the electrostatic properties of HSPB5, affecting its chaperone activity. Residue R120 is part of an electrostatic network that helps create an important structural feature in the folded region of HSPB5, the alpha-crystallin domain (ACD). In the unmutated (WT) protein, the ACD surface is overall positively charged. Substitution of the positive R120 to glycine alters both ACD’s structure and electrostatics. I generated two mutants, R120K (retaining positive charge) and R120D (switching to negative charge) to investigate how R120 plays a role in ACD’s conformation. Using a negatively-charged molecule, ATP, as an “electrostatic” probe in 2D NMR, I observed differences between its binding affinity to my R120 variants. I found that only R120K ACD behaves similar to WT ACD, suggesting a possible correlation between charge potential and ACD’s interactions with ATP. Currently, I am investigating if charge potential affects chaperone activity through aggregation assays with a client protein, human γD-crystallin, found in the lens and implicated in cataracts. I predict that WT and R120K, with similar electrostatic properties, will have similar chaperone activity. R120G and R120D, prevalently in an “active” state, will have higher chaperone activity. Understanding how such mutations affect HSPB5’s conformations and chaperone activity is a step forward in understanding sHSPs’ chaperone mechanism.

Small heat shock proteins (sHSP) are a family of molecular chaperones whose function is to delay the harmful aggregation of other proteins. Protein aggregation is associated with neurological disorders such as Alzheimer's disease and Parkinson's disease. In many tissues, multiple sHSPs are coexpressed and tend to assemble into hetero-oligomers. Hetero-oligomers are complexes of two or more different protein species. The extent and mechanism by which these hetero-oligomeric complexes form is yet to be fully understood. The goal of my discovery-driven research is to assess how the properties of sHSP hetero-oligomers differ from the properties of homo-oligomers. In my project, I focus on three sHSPs that are highly expressed in muscle: HSPB1, HSPB5, and HSPB6. Each of these proteins exhibit different behavior when on their own. HSPB1 and HSPB5 form a distribution of large homo-oligomers, whereas HSPB6 forms a small homo-dimer. One of the most characteristic properties of the small heat shock proteins is formation of oligomers that span different sizes. Thus I am primarily determining the sizes and composition of the sHSP hetero-oligomers. I performed a comprehensive study to characterize the sizes of the hetero-oligomers using three complementary methods: analytical size exclusion chromatography, mass photometry, and native gel electrophoresis. I have found that HSPB6 is able to readily incorporate into hetero-oligomers as the concentration of the other sHSP is increased, and that the complexes are formed in a distribution of intermediate sizes. I am currently working on assessing the ability of the hetero-oligomers to act as molecular chaperones by aggregation assays. I predict the hetero-oligomers will delay protein aggregation more efficiently than HSPB6 on its own. The findings of my project give insight into why sHSPs are coexpressed and form hetero-oligomers in cells. Understanding these hetero-oligomers sheds light into the complex pathways of sHSP function. 

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.

The effective treatment of individuals with HIV relies on maintaining therapeutic drug concentrations, necessitating accurate measurement of antiretroviral (ARV) drug levels. Current methods, such as liquid chromatography tandem mass spectrometry (LC-MS/MS), are limited by cost and accessibility. Our research addresses this gap by developing the INTEGRase activITY (INTEGRITY) assay for measuring integrase strand transfer inhibitors (INSTIs), a leading class of ARV drugs. This 2-step assay quantifies INSTIs using a DNA strand transfer reaction and quantitative polymerase chain reaction (qPCR). The presence of INSTI drugs disrupts the strand transfer reaction, inhibiting full-length target DNA formation, which is then measured through real-time qPCR. My work focused on optimizing the limit of detection of INTEGRITY by altering the strand transfer reaction conditions and protocol. Specifically, I conducted experiments altering INSTI drug concentrations and optimizing pre-incubation times of integrase with the drug to enhance the LOD. I observed that preliminary incubation of integrase and INSTI drugs for 5 minutes at 37 degrees Celsius improved the LOD of INTEGRITY by an order of magnitude. The simplicity of the INTEGRITY assay, utilizing standard laboratory equipment, holds immense promise for broadening access to routine clinic-based ARV drug level monitoring. This advancement has the potential to significantly enhance HIV care on a global scale by offering a cost-effective and accessible solution for monitoring therapeutic drug concentrations.

Angiogenesis, or the formation of new blood vessels, is crucial for normal bodily function but is especially important in diseases that cause blood vessel breakdown such as diabetic vasculopathy. Angiogenesis is regulated by activation of the Tie2 receptors in endothelial cells, which have two main ligands: angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 binding has been shown to stabilize blood vessels and inhibit vascular leakage, while Ang2 antagonizes these effects. We have previously shown that a computationally designed Tie2 super-agonist which presents eight copies of the Ang1 F-domain strongly activates Ang1-like signaling in human umbilical vascular endothelial cells (HUVECs). In this project, we hope to assess the Tie2 super-agonist’s ability to rescue diabetes induced blood vessel defects in a diabetic blood vessel organoid (BVO) model. To model diabetic conditions, a three-dimensional blood vessel organoid model has been cultured in a high glucose media along with inflammatory cytokines associated with the diabetic phenotype. Western blotting and immunofluorescence staining will be used to assess the relative quantities and localization of proteins involved in vascular stability and inflammations upon treatment with the Tie2 super-agonist. Vascular degeneration is a very harmful condition associated with many prevalent diseases including diabetes, so the Tie2 super-agonist could potentially be a new therapeutic drug candidate for treating blood vessel dysfunction in patients with these conditions in the future.

Two common craniofacial anomalies are cleft lip and palate, where the lip and roof of the mouth do not form completely, and craniosynostosis, where the soft spots of the skull fuse prematurely. Despite the prevalence of these birth defects, the genetic mechanisms by which they occur are still widely unknown. Hedgehog (Hh) signaling is a core developmental pathway that plays many roles in skull development including functioning as a guidance cue for cranial neural crest cells (cells that provide the foundation for bone and cartilage within the head) and regulating bone ossification (the hardening of the bone by osteoblasts developing into osteocytes). I am exploring the mechanism by which elevated Hh signaling changes cell fates, either through neural crest cells or osteoblasts, to influence craniofacial development. I investigated a mutant embryonic mouse model to identify regions where overexpression of Hh correlates to abnormal craniofacial phenotypes. I explored and measured these phenotypes via imaging, utilizing a genetic Hh reporter mouse line and skeletal stained embryos of various ages. My early qualitative and quantitative observations show patterns of widening midline structure that are seen by the increase in eye spacing and incomplete formation of the nasal and palate regions, as well as regions of premature ossification in the parietal regions of the skull. These findings suggest that elevated Hh signals result in abnormal development of the craniofacial region similar to cleft lip and palate and craniosynostosis. Deriving from these findings, I’m continuing to explore how Hh signaling plays a role in craniofacial development as well as gaining insight into the mechanism in which craniofacial anomalies arise.

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.

Transportation between the Endoplasmic Reticulum (ER) and Golgi is the first step of the secretory pathway, essential for correctly localizing intracellular proteins. USO1 is a long tethering protein between the ER and Golgi. USO1 acts as the first contact between transport vesicles and the Golgi initiating the transport process. Deletion of USO1 is lethal. YPT1 is a GTPase that is needed to recruit USO1 to these membranes. It is believed that physical interaction between YPT1 and USO1 is required for this transport function. Using AlphaFold2 predictions, we identified potential binding sites of YPT1 on USO1. If those sites are mutated, how would it affect the cell? I mutated sites proposed to bind to YPT1 on USO1 by Alphafold2 in order to break this interaction. Using yeast, I plan to determine if these mutation sites break the physical interaction. The resulting mutants cannot grow at elevated temperature. Further experiments will test how important these sites are in the overall process of protein secretion.

An important and universal aspect of cancer cells is the ability to proliferate rapidly. Rapid proliferation imposes specific metabolic demands which are often targeted for cancer therapies, and yet these demands are not well understood. A crucial aspect of cell metabolism is from the Tricarboxylic Acid (TCA) cycle. The TCA cycle is amphibolic, both catabolic and anabolic, and disruptions in the cycle are implicated in the onset and progression of various human cancers. Succinate Dehydrogenase (SDH) and Fumarate Hydratase (FH) are two TCA cycle enzymes that are tumor suppressors, proteins that when lost contribute to the malignant phenotype. In the TCA cycle, SDH catalyzes the conversion of succinate to fumarate, and FH catalyzes the subsequent step of fumarate to malate. Due to this proximity, one may predict SDH and FH mutations would have similar metabolic effects. However, this prediction, surprisingly, does not hold true. Paradoxically, loss of SDH generally impairs cell proliferation by disrupting synthesis of the amino acid Aspartate, a crucial output of mitochondrial respiration. Our lab recently discovered that SDH-deficient cancer cells adapt to overcome this metabolic deficiency by downregulating Complex I of the Electron Transport Chain (ETC). By downregulating Complex I, SDH-null cells increase the capacity of alternative aspartate synthesis pathways aside from the usual TCA cycle dependent pathway to enable faster proliferation. For SDH-null cells, treatment of a Complex I inhibitor improves proliferation for reasons discussed above. However, when FH-null cells are treated with the same Complex I inhibitor, there is a decrease in proliferation rate. It is not well understood why this difference exists, but characterizing it can provide insights on the roles of these enzymes and could inform better treatments for SDH and FH linked cancers.

One of the most prevalent issues in regenerative medicine is the impact of spinal cord injuries, as it can lead to an irreparable buildup of inhibitory scar tissue and, thus, paralysis. However, organisms such as Xenopus tropicalis tadpoles are able to regenerate their tails as soon as one week post-injury. By studying how they successfully regenerate, we can start to generate effective therapies for spinal cord medicine. I specifically want to know how quickly neurons populate the regenerating spinal cord and how this repopulation leads to functional motor recovery. To do this, I used the process of immunohistochemistry, where a fluorescent marker antibody binds to specific cells to create a fluorescent image for visualization purposes. First, I amputated around â…“ of their tail and created clutches of tadpoles stained for a neuron-specific protein. From my imaging, I noticed that the neurons populated the regenerating spinal cord by five days post-amputation (dpa). I became curious about how this regeneration rate impacted their ability to swim. To test this question, we set up a camera with a lightbox to set up Petri dishes of tadpoles. Then, I uploaded recordings of their swimming into a platform called ImageJ to use particle tracking to quantify the paths of each tadpole into measures such as distance, displacement, and velocity. Currently, we are trying to find other antibody markers that can provide more specific staining of neurons so the program can count them. With more specific staining, I hope to count the number of neurons over a set of zero, three, five, and seven dpa tadpoles. This project will help us answer foundational questions about how Xenopus tropicalis tadpoles regenerate functional neurons after injury.

During mitosis, the kinetochore plays a central role in ensuring the proper segregation of chromosomes by connecting them to spindle microtubules, which facilitate the equal distribution of chromosomes to daughter cells. In budding yeast, the Dam1 complex is an essential protein complex that binds the kinetochore to spindle microtubules. The Dam1 complex strengthens the kinetochore-microtubule attachment by self-assembling into a sliding ring around microtubules. This self-assembly occurs at nanomolar concentrations of the complex in the presence of microtubules, but in their absence, appreciable oligomerization occurs at concentrations in the micromolar range. Dimers of the complex predominate in high salt concentrations (500 mM NaCl). This is thought to be due to hydrophobic interactions between the monomers. Yeast strains relying on human histones in place of their yeast histones grow slowly. This slow-growth phenotype is rescued by several different mutations in the Dam1 complex. Preliminary characterization of the mutant Dam1 complexes led to the hypothesis that the mutations that allow the yeast cells to adapt to the humanized histones changed the monomer-dimer equilibrium for the Dam1 complex. To measure the affinity of Dam1 complex monomers for each other, I purified the wild-type and mutant protein complexes and used size-exclusion chromatography and mass photometry to determine the different quaternary structures that arise at different concentrations of the complex. I found that each mutation that enhances growth of yeast strains with humanized histones decreased the affinity of the Dam1 complex monomers for each other. The results of this investigation yielded a greater understanding of the requirements for accurate chromosome segregation.

Protein nanoparticles are useful for the design of novel vaccines. We can use these nanomaterials for display of antigens; however, antigens tested thus far have been homooligomers—consisting of a single unique component, and existing protein nanoparticle assemblies are not well-suited for the display of heterooligomeric antigens (such as HCV E1E2). We attempt to solve this problem through designing uniformly symmetric icosahedral nanoparticles that contain two distinct protein chains within their fundamental geometric component—referred to as the asymmetric unit; this allows us to retain the particle symmetry and double the number of accessible linkage points, or chain termini. This doubling of termini could allow us to fuse heterodimeric antigens to our cages. To accomplish this design goal, I utilized RFdiffusion—a generative machine learning model—to generate two-component icosahedral protein backbones, which were then filtered by evaluating subunit packing through a set of contact distance calculations. ProteinMPNN, a DL-based sequence design method, was used to assign candidate sequences to each of the filtered backbones. Finally, complete designs were filtered by using AlphaFold2 to evaluate fidelity to the original design model. I expressed the top 96 designs in E. coli, but saw minimal protein with no indication of assembly. In an attempt to maximize my chances of forming successful cages, I have elected to conditionally generate backbones that favor alpha-helical secondary structure. In this new design round, I hope to see favorable improvements in inter-chain packing; this will lead to an increase in passing design candidates and hopefully allow my computationally generated structures to have a higher chance of assembly in lab. This work serves to streamline the development of a therapeutic platform that can display multi-component antigens, which could enable the creation of new vaccines.

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.