Chromatin plays a key role in determining gene expression and cellular states. The basic unit of chromatin is a nucleosome composed of ~147bp of DNA wrapped around a histone core with exposed N-terminal tails that help regulate gene expression programs through their post-translational modifications (PTMs). Facultative heterochromatin inheritance, a type of chromatin containing the repressive trimethylation of histone H3 at lysine 27 (H3K27me3) PTM, is necessary for maintaining cell fate, identity, and plasticity. The parental facultative heterochromatin gene repression is maintained during the S-phase by recycling parental nucleosomes containing H3K27me3 onto the daughter strands of DNA and spreading their PTM onto newly synthesized nucleosomes by the polycomb repressive complex 2 (PRC2). Dr. Escobar found that Nucleophosmin 1 (NPM1), a histone chaperone, assists in facultative heterochromatin domain inheritance through interactions with PRC2 in mouse embryonic stem cells. NPM1 is found mutated in roughly 30% of cases of acute myeloid leukemia (AML). Preliminary data finds a significant presence of cleaved NPM1 product at 20 kDa (p20) within the nucleus of normal hematopoietic stem cells (HSCs), but a lack of this NPM1 modification in AML cell lines. Cathepsin B has been shown to cleave normal NPM1 to produce a fragment at p20. I hypothesize that the lack of cleaved NPM1 product factors into leukemogenesis. To test this hypothesis, I have three aims; Aim 1 identifies the Cathepsin B cleavage site of NPM1 using in vitro cleaving assays; Aim 2 assesses the phenotype of HSCs containing a non-cleavable NPM1 mutant; and Aim 3 monitors the phenotype of AML cell lines when inducibly expressing Cathepsin B. This project allows for future investigations into how NPM1 modifications impact facultative heterochromatin inheritance of HSCs and AML cancer cells.

Activins are a class of growth factors belonging to the Transforming Growth Factor-Beta (TGF-β) superfamily that recruit a tetrameric complex of type-I (ACTRI) and type-II (ACTRII) transmembrane serine/threonine kinase receptors to cause downstream signaling in various critical cell-signaling pathways. Inhibition and supplementation of the signaling molecules offer new possibilities in many therapeutic and clinical applications. The overexpression of Activin A has been shown to be a major driver of diseases such as pulmonary hypertension, chronic kidney disease, and cerebrovascular disease. Sotatercept, one of the few existing activin-A-inhibiting therapeutics, is in clinical trials for treating pulmonary arterial hypertension, but, like many ligand traps, it is designed by fusing a large antibody domain to a native activin receptor domain, making it expensive to produce and unstable due to its large size. There is a need for therapeutics that can be produced in bacteria, replicate high efficacy and strong affinity, and minimize side effects. These therapeutics can be developed through de novo protein design, which involves designing proteins from scratch, with a new, unique sequence predicted to fold into its specific corresponding structure or by grafting native protein-protein interfaces onto novel scaffolds. This project aims to use computational de novo protein design methods to develop a therapeutic ligand trap for Activin A. The design pipeline involves scaffolding functional motifs, optimizing protein sequences, predicting protein structures, and filtering designs using metrics calculated by Rosetta, a protein design tool. With this pipeline, we have tested several design strategies, providing knowledge-based guidance and analyzing outputs to inform strategies for future design iterations. Recent designs have passed Rosetta filters, and we have ordered the corresponding genes to express and purify the designed proteins. Once purified, they can be experimentally characterized, testing binding and stability in vitro, and then further optimized to create stable Activin A ligand traps.

Automatically, we consider genes as existing solely to serve the host. Aiding in host survival allows the host to reproduce, causing these genes to propagate in the next generation. However, the existence of selfish genes, which ensure their own survival at the cost of their host, brings this conventional wisdom into question. One such type of gene is called a toxin-antidote (TA) system, which express a molecular toxin and its antidote, the latter of which prevents death, but actively kills hosts that express toxin without co-expressing antidote. The aim of this project is to develop a mechanistic understanding of one invertebrate TA system called PEEL-1/ZEEL-1, which expresses an evolutionarily novel, transmembrane toxin protein PEEL-1, and a transmembrane antidote protein ZEEL-1. Data shows that PEEL-1 co-opts a protein called PMPL-1 to kill animal cells; however, PEEL-1+PMPL-1 do not kill yeast. By identifying why PEEL-1 doesn’t kill yeast, we gain insight into PEEL-1’s toxin mechanism. One hypothesis is that it kills by inducing osmotic stress, which yeast may be invulnerable to due to their cell wall designed to resist osmotic stress. To test this, we’ve expressed PEEL-1, PMPL-1, and PEEL-1+PMPL-1 in separate yeast cultures, chemically degraded their cell walls via Zymolyase treatment, and screened for growth. Preliminary data showing that the digested experimental culture (co-expressing) experiences little post-treatment growth compared to the digested controls (non co-expressing) supports the osmotic stress-related mechanism of PEEL-1 toxicity. Our work in identifying PEEL-1 toxicity will define the first mechanism of an animal TA system and guide our understanding of the different ways that protein-driven cell death can develop in nature.

Chromatin, the complex of DNA-wrapped histone octamers that make up our chromosomes, is decorated with post-translational histone modifications (PTMs) that either increase or decrease transcriptional accessibility. Most regions have either predominantly active or repressive modifications that shape chromatin into euchromatin or heterochromatin, respectively. In addition to euchromatin and heterochromatin, some cells have poised chromatin that is decorated with both permissive and repressive modifications. While much is still unknown about a poised chromatin state, it is thought to permit swift changes in gene expression, which is a feature common in stem cells and lymphoid memory cells. Ultimately, the Escobar lab aims to determine the epigenetic mechanisms involved in maintaining the poised chromatin state of memory CD8+ T cells, and in line with this aim, plans to use a CRISPR-Cas12a biotinylation system to tag and precipitate poised chromatin regions for protein analysis and mechanistic studies. This project details the development and proof of concept of this CRISPR-Cas12a biotinylation system. Using traditional cloning techniques and a one-pot strategy to assemble CRISPR arrays, we will express and purify dCas12a-BirA+gRNA ribonucleoproteins (RNPs), introduce these CRISPR RNPs to mouse embryonic stem cells (mESCs), and perform CUT&RUN to verify effective biotinylation at specific chromatin loci. Preliminary results have demonstrated the successful purification of 5 CRISPR RNPs and a dCas12a-BirA control, as well as verified the presence of these CRISPR RNPs and the biotinylation of H3 upon delivery of this system to mESCs. Upon CUT&RUN analysis, we expect to see biotinylation of H3 at our targeted loci of interest. The completion of this validation step will allow us to apply this technology to any cell of interest, particularly CD8+ T cells, which may support significant insights to the mechanistic determinants of poised chromatin.

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, TLR3 binds viral double-stranded RNA (dsRNA) and TLR5 binds the bacterial protein flagellin. Neither native agonist is a suitable adjuvant candidate; dsRNA is unstable and nonspecific and flagellin is degradation and aggregation-prone. Therefore, this project aims to design, test, and characterize novel protein-based adjuvants that can bind TLRs 3 and 5 and activate the immune system. Here, I test and characterize de novo mini-proteins that I have computationally designed to bind mouse TLR3 (mTLR3) and mouse TLR5 (mTLR5). I use yeast surface display, biolayer interferometry, and cell-surface binding assays to identify and characterize successful binders. Preliminary results show de novo mini-proteins specifically bind mTLR3 and mTLR5. 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.

The Ubiquitin Proteasome System (UPS) is a molecular recycling machine, responsible for  proteolysis and protein activity regulation. The components of the UPS attach a small protein ubiquitin onto other proteins which directly affects their activity or serves as a signal calling for modification or lysis. Ubiquitin-conjugating enzymes (E2) and ubiquitin ligases (E3) are two classes of proteins that determine which proteins are tagged. Ube2W is an E2 with a unique function—it is the only E2 that places ubiquitin onto disordered N termini and amino acylated side chains. In this study we aim to elucidate the mechanism of reactivity and specificity of the enigmatic Ube2W. What structural and chemical features are responsible for its one-of-a-kind functionality? What does this imply about the role of this E2 on the cellular level? We designed a set of Ube2W mutants that had various putatively important features removed or changed to analogs from different E2s. We performed mutagenesis PCR followed by reactivity assays in the presence of known Ube2W substrates. We plan to collect NMR data for the Ube2W-substrate and Ube2W-ubiquitin interactions. We hope to determine which features are critical for this unique E2’s function by following the changes in reactivity when they are removed or altered. The interactions of substrates with these critical residues will help draft an outline for the precise mechanism. Improving our mechanistic understanding of Ube2W will pave the way for being able to control when and under what circumstances this unique biochemical reaction is used by the cell. This work aims to expand the current understanding of the UPS and aid in taming UPS-related diseases.

Deep learning methods for protein sequence and structure generation have shown remarkable success in many design scenarios when combined with structure prediction networks such as AlphaFold2. Despite this advance, many design challenges such as de novo binder design still haven’t been fully solved. Diffusion-based models have demonstrated considerable success in image and language generation yet their application in protein design has not yet been fully explored. Recently, the development of a protein diffusion model called RoseTTAFold Diffusion (RFdiffusion) has shown significant success in protein design and enabled us to explore the challenging problem of designing protein binders. Here I demonstrate utilization of RFdiffusion towards generation of de novo binders to disordered major histocompatibility complex (MHC) peptides. Specifically, we took an MHC peptide from KrasG12D and used RFdiffusion to generate a diverse range of structures that can bind this peptide. To optimize the sequence of these structures we used ProteinMPNN. We used AlphaFold2 to predict the structures of these optimized binders in complex with the peptide and saw promising interaction metrics. Further, structure prediction of the designs in complex with Kras wild type (WT) peptide resulted in lower AlphaFold2 confidence metrics of the interaction occurring. This is a promising preliminary result that RFdiffusion can generate fully de novo MHC-mimics, which can differentiate between neoantigens and WT peptide. Many cancers are caused by a single point mutation such as KrasG12D, thus, designing protein binders with point mutant specificity is exciting as it allows for targeting of disease causing proteins over healthy WT proteins. 

Interstrand crosslinks (ICLs), commonly induced by chemotherapeutic drugs, are a form of DNA damage that results in stalled replication forks during DNA replication. When stalled replication forks occur, Ataxia telangiectasia and Rad3 related (ATR) kinase activates the Checkpoint Kinase 1 protein (Chk1) through phosphorylation. This initiates a signaling cascade that leads to cell cycle arrest. Therefore, checkpoint activation via Chk1 phosphorylation is crucial for the cellular DNA damage response (DDR) and overall genomic stability. Mre11 is another protein involved in DNA damage repair. Alongside two other proteins, Rad50 and Nbs1, it forms the MRN complex. As with ATR, the MRN complex is activated in response to DNA damage mainly in response to double stranded break formation. In our lab, we have now found that inhibition of Mre11 using mirin, a potent Mre11 inhibitor, surprisingly led to decreased phosphorylation of Chk1 (p-Chk1). In this work, we are exploring the relationship between Mre11 and checkpoint activation during replication stress. To study this, we made us of cisplatin (a crosslinking agent used in chemotherapy) as well as other replication stress-inducing drugs. Using western blotting to visualize the phosphorylation levels of Chk1 we are examining how Mre11 inhibition affects Chk1 activation. Results showed increased p-Chk1 in response to DNA damage in the cisplatin treatment compared to the control, but a decreased levels of phospho-Chk1 upon Mirin co-treatment. This suggests that MRE11 plays a role in phosphorylating Chk1 and activating the DDR pathway. Our results provide evidence for MRE11 inhibition as a method of decreasing the DNA damage repair response. It also helps to shed light on a potential mechanism of treatment for cancer patients. Future studies will further examine the nature of this relationship by investigating activation of Chk1 using time-course experiments and MRN inhibition through siRNA transfections.

When the cell undergoes stress, it leads to an increase in protein instability and misfolded states that prevents proper cell functions. Small Heat Shock Proteins (sHSPs) are molecular chaperones that work to maintain a healthy proteome by associating with misfolded proteins to delay aggregation under stress conditions. Other chaperones and co-factors will then refold or degrade the misfolded protein client. HSPB5 is a human sHSP ubiquitously expressed throughout the body. A HSPB5 disease mutant, where arginine is mutated to glycine at residue 120 (R120G), is a defective chaperone associated with cataracts and desmin-related myopathy. HSPB5 comprises three domains but only one domain—the alpha-crystallin domain (ACD)—is folded. My research aims to understand the effect of the R120G mutation on the folded ACD’s structure. HSPB5 creates a dimer through electrostatic and hydrophobic interactions between ACD at the dimer interface. This network of interactions causes the dimer interface to be highly sensitive to electrostatics, working like a sensor for environmental charges. Wild-type HSPB5 is more positive at its dimer interface, likely facilitating interactions with negatively charged compounds. In the R120G mutant, the loss of arginines at the dimer interface site causes it to be less positive, hypothetically lowering HSPB5’s affinity for these compounds. Through site-directed mutagenesis, I obtained HIS-tagged cleavable constructs for wild type and R120G B5 ACD that allowed for easier purification. Using these constructs, I grew isotopically labelled N15 B5 ACD in minimal media and purified my protein sample through nickel affinity, size exclusion and anion exchange chromatography. Through NMR titration experimentation, I investigate how amino acid identity at the R120 site will affect ACD interactions with charged molecules in R120 mutant of HSPB5. Learning how mutations at the R120 site affect protein dynamics and client interactions will be a step forward in understanding the sHSPs’ overall chaperone mechanism.

The emergence of the SARS-CoV-2 vaccine helped shine a light on protein subunit vaccines that use fragments of infectious protein from viruses to trigger protective immune responses. Normally in subunit vaccines, the immune system stimulation is triggered by a separately provided adjuvant. These adjuvants elicit immune responses in a broad and indeterminate manner. We hope to replace this with a molecule that can provide a more specific immune stimulation: CD40Ligand (CD40L). CD40L is an immune protein present on T cells and works to signal B cells to either replicate or create antibodies. For B cells to replicate antibodies, they need a primary signal from the antigen and a secondary signal, which CD40L initiates. To achieve this, we are using the I53-dn5 nanoparticle, which has the ability to display different ligands on its two components by putting both the antigen and adjuvant on either the pentamer or trimer component. We designed 8 different constructs where we tested two versions of CD40L, the placement of CD40L, and the linker length between CD40L and the nanoparticle surface. Out of the five stages of the project - designing, expressing, purifying, assembling, and evaluating - we have completed the first three. The designs that have expressed the best throughout each stage have been those with the CD40L truncated sequence instead of the full sequence. Furthermore, we have seen a trend with the pentamer subunit being more amenable to the addition of CD40L. Future in vitro studies and re-expressions will determine if the particle will retain stability and native CD40L function. We expect CD40L-displaying nanoparticles will promote B-cell proliferation to a greater extent than the nanoparticle vaccine displaying only hemagglutinin antigen. Ultimately, we hope to examine how co-display of CD40L with antigen will change the quality of immune response and memory in vivo.

In the ubiquitin-proteasome system, the E2 enzymes are involved in the second step of transferring the ubiquitin (Ub) to a substrate. Specifically, an E2 enzyme receives the ubiquitin from an E1-Ubiquitin(E1-Ub) conjugate and becomes an E2-Ub conjugate. Then, an E3 enzyme can deliver the ubiquitin from the E2-Ub conjugate to a substrate to form a substrate-Ub conjugate. Most of the E2s attach Ub to a substrate lysine residue, but Ubc6, an E2 from yeast, also seems to react with substrate hydroxyl groups on serine/threonine/tyrosine. This project is divided into two parts: first, validate the reactivity of Ubc6 in different amino acid (serine/threonine/lysine/cysteine/tyrosine) conditions. Second, apply a proper machine model to predict the reactivity of Ube2J2-Ub, a mammalian homolog of the Ubc6-Ub conjugate. The quantification analysis on the Ubc6 charge/discharge assays can reveal the rate of the reactivity of the Ubc6 in different amino acid conditions. After validation, three types of E2s with known reactivities: Ubc2D(1/2/3/4)-Ub, Ube2L3-Ub, and Ubc6-Ub, can be used as training sets for the machine learning model. Once the model predicts the reactivity of Ube2J2, the prediction can be validated by performing assays on Ube2J2. We expect that Ubc6 reacts fastest with Cysteine, followed by Threonine, Lysine, Tyrosine, and Serine. Since Ube2J2 is a human homolog of Ubc6, we predict that Ube2J2 has the same reactivity as Ubc6. The implication of this project is whether machine learning can assist with finding the reactivity of a protein enzyme.

Under cellular stress conditions, proteins are destabilized, often causing them to misfold, and in certain cases aggregate. Without protective systems in place, significant cellular dysfunction occurs, often resulting in cell death. One mechanism of molecular stress response is increased expression of small heat shock proteins (sHSPs). sHSPs are ATP-independent chaperones that are thought to keep proteins in a refolding-competent state during stress and work with other chaperone proteins to rescue proteins from irreversible aggregation. Members of the sHSP family are defined by their shared structure: a conserved α-crystallin domain (ACD) flanked by unstructured N-terminal (NTR) and C-terminal (CTR) regions. Transient interactions between the three domains are important regulators in oligomerization. The structured ACD is the building block of sHSP oligomers and provides a framework for studying oligomerization. The ACD dimer contains three grooves into which the NTR and CTR can bind. Though it is clear the ACD plays a major role, the regulation of these domain interactions underlying oligomer formation is not well-defined. HSPB5 and HSPB1 are two human sHSPs that are ubiquitously expressed throughout tissues and can form homo- and hetero-oligomers. My experiments were aimed at studying the effects of mixing the isolated ACD with the full-length protein. To analyze the domain interactions and oligomer size changes upon mixing, I used size-exclusion chromatography (SEC). In these mixing experiments, addition of the ACD to different oligomers increased the number of available grooves with each titration point. The experiments presented here provide glimpses into the hierarchical organization of homo- and hetero-oligomers of HSPB1 and HSPB5. My results indicate that not only does the number of grooves matter, but also their identity. My working hypothesis is that the identity dictated the strength of the interactions between the different binding components and led to differences in the propensity for subunit exchange in oligomers.

In response to acute genotoxic insult, cancer stem cells undergo quiescence, a state of temporary cell cycle arrest, to avoid apoptosis (programmed cell death) and later re-enter the cycle to generate daughter cells under suitable conditions. This event is also observed in the irradiated germline stem cells (GSCs) of female Drosophila melanogaster. Previous studies have shown that quiescence is regulated by various upstream components, including gene silencing by polycomb repressive complex 2 (PRC2) and activation of mitophagy, the selective degradation of damaged mitochondria. Activation of PRC2 is shown to be upstream of mitophagy in regulating quiescence. However, the PRC2 target genes that get silenced in this process remain largely unknown. In humans, a downstream target of PRC2 is microRNA-200 (miR-200), which can enhance mitochondrial elongation by downregulating mitochondrial fission factor (MFF). Here, we hypothesize that the miR-8 gene, the Drosophila ortholog of the human miR-200 family, is a downstream target of PRC2 and is required for stress-induced quiescence to take place. To investigate the role of miR-8 in governing quiescence following stress, we overexpress miR-8 in female Drosophila GSCs under UAS-GAL4 control and study the spectrosome and mitochondrial morphology of the immunostained GSCs. We predict that miR-8 overexpression will prevent mitophagy and entry into quiescence after irradiation. We anticipate that our findings will characterize the role of miR-8 and strengthen our understanding of mechanisms that govern the cell cycle and quiescence. This study is critical as our proposed mechanism of quiescence can be applied to other stem cell types, and may present new therapeutic strategies for cell cycle-related diseases.

Glutamine synthetase (GS) is a highly regulated enzyme that catalytically converts glutamate to glutamine which is associated with ammonia assimilation. One of the effects of dysregulation in the GS inter-conversion process is hyperammonemia, which can cause death or brain damage. GS is conserved across all prokaryotes and eukaryotes. Among enzymes, glutamine synthetase's ability to polymerize is still a structural mystery and the functional characteristics of its self-assembling filaments remain unknown. The aim is to understand the occurrence of filament formation in GS and the effects on enzyme activity. We hypothesized that filaments may influence the association of GS substrates or allosterically regulate GS. I purified the GS of Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Helicobacter pylori by using Ni-column and size exclusion chromatography (SEC). Then, I examined the GS of pseudomonas under different buffer conditions (Mg2+, Co2+) using negative staining. Under Magnesium (10mM) conditions, the known dodecamer structure of GS was observed. Under Cobalt (10mM) conditions, the filament was being induced. To better investigate the structural mechanism of filament formation we turned to cryogenic electron microscopy (Cryo-EM). The next step is to create a model of the filament interface of GS and identify the residues involved. This research has broad implications in the field of metabolic engineering, as understanding the structure and the role of filament formation in GS could help develop new therapeutic targets in metabolism.

Intermediate metabolism in cells has generally been studied without considering the arrangement of enzymes within the cell. However, recent developments have shown that many metabolic enzymes form organization systems that are made up of oligomers stacking linearly into filaments. The enzyme phosphoribosyl pyrophosphate synthetase (PRPS) makes a precursor required for all de novo nucleotide synthesis in cells, and therefore plays an important role in cellular metabolism. This project aims to characterize the PRPS protein in Xenopus tropicalis and Giardia lamblia. We hypothesize that PRPS from X. tropicalis will have similar biochemical and structural properties as compared to human PRPS, while PRPS from G. lamblia will have different biochemical and structural properties. This hypothesis is supported by the small evolutionary difference between PRPS from humans and X. tropicalis as compared to the large evolutionary difference between PRPS from humans and G. lamblia. This difference would be especially interesting to examine from the perspective of filament formations in the PRPS protein. So far, we have created the Xenopus tropicalis and Giardia lamblia plasmids by cloning. Test expressions of the X. tropicalis yielded protein expression in E. coli cell strains C43 and RIL, while test expressions for G. lamblia have been successful in C43, BL21, pLysS, and Rosetta cell strains. This demonstrates that both X. tropicalis and G. lamblia PRPS can be expressed in E. Coli strains. An analysis of X. tropicalis will allow us to test how filament formation changes with only small evolutionary differences in PRPS. It could also be used for further research in vivo using frog eggs that act as a singular cell system. If it is confirmed that the G. lamblia protein is different from human PRPS, PRPS in G. lamblia could serve as an antibiotic target since current methods of treatment for the organism are very harmful to the human microbiome.

Treatment of individuals with HIV using antiretroviral therapy (ART) is highly effective, but effective clinical management depends on maintaining therapeutic drug concentrations. Antiretroviral (ARV) drug concentrations in patients with HIV can vary due to differences in drug metabolism, medication adherence, or interactions between multiple drugs. These individuals may have subtherapeutic or supratherapeutic drug concentrations, putting them at risk of treatment failure, acquisition of drug resistance, and risk of hospitalization or death. Current measurement of ARV concentration is done through liquid chromatography tandem mass spectrometry, which requires expensive equipment and requires a labor-intensive protocol. This restricts accessibility to specialized laboratories, making it difficult for persons with HIV to have routine measurements of ARV drug concentrations. The goal of the project is to develop an assay that is simple to perform and uses standard equipment to increase access to routine clinic-based drug level monitoring to improve HIV care. We designed an assay using a 2-step process of DNA strand transfer and quantitative polymerase chain reaction (qPCR) to quantify integrase strand transfer inhibitors (INSTIs). We tested for dolutegravir (DTG) and cabotegravir (CAB) in both buffer and plasma -- the latter to simulate patient blood samples. We were able to demonstrate that the assay could quantify clinically relevant drug concentrations of DTG and CAB. By developing an assay that can be readily integrated into most clinical laboratories, we will contribute to increasing access to routine HIV drug level monitoring to improve clinical HIV care and maintaining viral suppression in persons with HIV.

Injuries to the spinal cord are among the most debilitating injuries to the human body. The task of repairing this dense network of nerve cells and fibers 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 incredible ability to regenerate its spinal cord and associated tissue following amputation. My project focuses on how Xenopus uses the classic developmental morphogen Sonic Hedgehog (Shh) to recapitulate the dorsal-ventral patterning of the spinal cord during regeneration. Previous research in a closely related organism showed that a switch to non-canonical mode of Shh signaling, not involving the Gli family of transcription factors, was important during regeneration. In order to investigate this further, I used chemical inhibitors of the canonical Shh signaling pathway to confirm this pathway’s limited importance in terms of gross regeneration. Next I used in-situ hybridization to visualize the expression of Shh and its associated genes along a regenerative time-course. It is anticipated that inhibition of the canonical pathway will decrease these expression levels and confirm the selectivity of our inhibitors. An understanding of the role of key biological signals like Shh will be integral in the development of regenerative therapies for spinal cord injury.

Pathogenic bacteria often promote their growth by introducing proteins into host cells they are infecting. Some of these introduced proteins hijack host cell ubiquitin signaling pathways. Ubiquitin (Ub) is a small protein that is involved in many signaling pathways within eukaryotic cells. In ubiquitin-mediated degradation, the degradation of a target protein is promoted by the attachment of chains of ubiquitin onto the target protein. The attachment of poly-ubiquitin chains is facilitated by a class of proteins called E3 ubiquitin ligases. Salmonella Typhimurium secretes into host cells an IpaH/SspH class E3 ubiquitin ligase called SspH1. My research investigates the mechanism for how SspH1 modifies its target protein, Protein Kinase N1 (PKN1), with poly-ubiquitin chains. I am investigating whether ubiquitin is transferred sequentially onto PKN1 or if a long poly-ubiquitin chain is first formed on SspH1 and then transferred en-bloc onto PKN1. Using biochemical methods, I will compare whether the rate of poly-ubiquitin chain synthesis is faster starting with unmodified PKN1 or mono-Ubiquitinated PKN1, an intermediate in the sequential addition pathway. I hypothesize that the formation of poly-ubiquitin chains onto mono-Ub-PKN1 is slower than the formation of poly-ubiquitin chains onto free PKN1. This would suggest that the mechanism does not occur through the sequential addition of ubiquitin. Learning more about the mechanism of SspH1 will allow us to both better understand the IpaH/SspH class of proteins and better understand how ubiquitin can be transferred onto protein substrates.

Cancer cells display dysregulated metabolism to meet the increased metabolic demands of rapid cell proliferation. While cancer cells enact metabolic changes to glucose metabolism, known as the Warburg effect, it is also important to consider the metabolic pathways involved in biomass synthesis which support cellular divisions. For example, the amino acid aspartate is a central facet of proliferating cell metabolism because it is a precursor to both purine and pyrimidine nucleotide synthesis, and essential for asparagine and arginine biosynthesis. Based on research from the Sullivan Lab and others, aspartate biosynthesis is essential for tumor cells to proliferate. However, the cellular mechanisms by which aspartate levels impact the proliferation rate of tumor cells remain unknown. Based on data collected in Sullivan lab, my mentor and I hypothesize that SDH inhibition blocks the production of a metabolic intermediate between succinate and aspartate that has an unknown function in sensing aspartate limitation and therefore dictating cell proliferation. Therefore, my project seeks to investigate how aspartate levels as well as several aspartate precursor metabolites in the TCA cycle govern the proliferation rate of tumor cells. In the first portion of the project, I measure proliferation rates of WT and GOT1/2 (converts oxaloacetate to aspartate) double knock-out (DKO) cells treated with or without the SDH inhibitor Atpenin A5 (AA5) in the presence and absence of aspartate. In the second portion of the project, I examine if modulating fumarate, malate, and OAA levels in the TCA cycle impact the proliferation rate of GOT1/2 DKO cells. I anticipate seeing decreased proliferation rate along with decreased levels of fumarate, malate, and OAA in DKO cells treated with AA5. This research project will contribute to the lab and cancer treatment by providing new insight into aspartate metabolism.

Unlike mammals, western clawed frog (Xenopus tropicalis) tadpoles are able to completely regenerate their spinal cord after tail amputation. This complete spinal cord regeneration is due to the ability of their neural progenitor cells (NPCs) to differentiate into neurons successfully. Our research focuses on two transcription factors—Meis1 and Pbx3– that are upregulated by regenerating neurons and are necessary for successful regeneration. We aim to elucidate how these two proteins are working together to guide successful spinal cord regeneration in X. tropicalis tadpoles. I am specifically investigating Meis1 and Pbx3 splice variant expression during neural regeneration. Previous work in mice found that different known splice variants of Pbx3 have different expression patterns. While X. tropicalis has two predicted splice variants each of Meis1 and Pbx3, nothing is known about their individual expression or function. I sought to fill in this gap by looking at Meis1 and Pbx3 splice variant expression in different tissues and over regenerative time. Based on previous research in mice, I hypothesize that both splice variants of Meis1 and Pbx3 have different gene expression patterns in different cell types over regenerative time. I aimed to investigate this hypothesis by doing two experiments. My first experiment was to study the expression of each splice variant over regenerative time by performing qPCRs in order to look at the presence of splice variant mRNA in uninjured, 24, and 72 hours post-amputation. For my second experiment, I made in situ hybridization probes specific for each splice variant to identify their tissue-specific expression patterns. Based on previous literature, we expect to see differential amounts of expression from each splie variants as spatial expression centralized on the spinal cord. Understanding the transcriptional network that is behind the regenerative mechanism of X. tropicalis can help us develop therapeutic tools to address spinal cord injury in humans.