Trauma-induced coagulopathy is a severe complication of trauma that alters the normal mechanism of blood clotting through a number of complex factors. If clots are hypercoagulable, there is risk for dangerous vascular blockages. Conversely, if the clotting is hypocoagulable, it can lead to fatal hemorrhaging. Prior research indicates that actin has a major impact on platelet activity and blood clot formation. Actin is a highly abundant cytoskeletal protein that forms long, insoluble filaments. When released into the blood during cellular death, these filaments have complex effects on blood clot formation. Actin filaments can be integrated into the scaffolding of the clot, increasing strength. My experiment aims to investigate the roles of actin and on human blood clotting. Healthy donor whole blood in 3.2% sodium citrate was spiked with either a saline control or recombinant human skeletal muscle-derived actin (final concentration 200 nM) and allowed to incubate for 5 min. Samples were then activated with either 10 mM adenosine diphosphate (ADP) or 2 mg/mL collagen. The platelet aggregation response was then measured by impedance aggregometry. Each pair of control and actin conditions was run simultaneously. The impedance area under the curve (AUC) was compared between control and actin groups under each activation condition using a paired t-test with significance at p<0.05. Preliminary results show ADP was no different between the control and actin groups (p=0.400, n=5). The AUC in response to collagen was significantly higher in the presence of actin compared to control (p=0.005, n=7). Exogenous muscle actin appears to increase platelet aggregation through the collagen but not the ADP activation pathway. Further investigation is required to better characterize this interaction. A better understanding of the mechanisms of actin on hemostasis could direct research into pharmaceuticals and therapies that could yield better outcomes for trauma patients.
 

Unlike humans, Xenopus tropicalis tadpoles are able to successfully regenerate tissue after traumatic injury. Unraveling the mechanisms which permit major tissue regeneration may allow us to develop treatments that enhance wound healing in humans. Proliferating tissues have a high demand for cell division, requiring a constant supply of nucleotides for DNA replication, though it remains unclear how this gross demand is fulfilled. The biosynthetic enzyme inosine-5’-monophosphate dehydrogenase 2 (IMPDH2) catalyzes the rate limiting step of de novo guanosine synthesis. IMPDH2 has been shown to polymerize into filaments when under nucleotide stress in vitro, subsequently increasing the production of guanosine. Given the increased demand for nucleotides during regeneration, I hypothesized IMPDH2 may similarly form enzyme filaments in these tissues to meet the increased demand for guanosine. To address this, I amputated the tails of X. tropicalis tadpoles then treated with the IMPDH2 inhibitor mycophenolic acid (MPA); inhibition of IMPDH2 decreased both regeneration length and quality. IMPDH2 only polymerizes when inhibited, aggregating into larger rod and ring structures, of which seem to preferentially form in some tissues. To better understand the effects of IMPDH2 on normal development, we also carried out micro injections of wild type and patient mutant IMPDH2 mRNA, finding that the latter disrupts some aspects of tadpole motor function. Based on these findings, I hypothesize that differential guanosine requirements across regenerating tissues drives varying sensitivity to IMPDH2 inhibition and subsequent filament assembly throughout the tadpole tail. To test this, I am characterizing the formation and distribution of filaments across various IMPDH2 mutants in regenerating tissues to assess correlation of IMPDH2 polymerization with cell proliferation. Completing these experiments allows me to develop a model of nucleotide stress biochemistry in X. tropicalis to further investigate the metabolic demands of development and regeneration.

The association of eukaryotic DNA with histone proteins serves not only to package entire genomes into the nucleus of a cell, but these histone-DNA functional units, called nucleosomes, are hubs for biochemical signaling that regulates gene expression. In the Chatterjee lab, we are fascinated by the transcriptional biology of the small ubiquitin-like modifier protein 3 (SUMO-3), a posttranslational modification that occurs on histones and has been correlated with reduced gene expression. Previous members have demonstrated that SUMO-3 stimulates the activity of transcriptionally repressive enzymes by binding with a scaffolding protein called CoREST1. Hence, my focus has been to understand the functional details of the SUMO-CoREST interaction, particularly how cancer-associated mutations in the SUMO-interacting motif (SIM) of CoREST1 affect its ability to bind SUMO-3. To answer this question, I started by using solid-phase peptide synthesis to prepare truncated CoREST SIM peptides bearing the mutations of interest. I then utilized these peptides, along with SUMO-3 enriched in nitrogen-15, for two-dimensional nuclear magnetic resonance spectroscopy. By comparing the chemical shifts of [¹⁵N]-SUMO-3 with and without the presence of each peptide, I could assess the effects of mutations on the proportion of bound and unbound species in solution. Of special interest was an acidic residue in the hydrophobic core of the CoREST SIM, which distinguishes it from canonical SIMs found in other proteins. Excitingly, my results indicate that substitution of this amino acid with lysine, a mutation found in gallbladder cancer, ablates binding. I observed a similar effect for other mutations in the hydrophobic core of the CoREST SIM. Using these results to guide studies with full-length CoREST in biochemical assays, my research will identify the effects of these mutations on downstream biochemical pathways that may be misregulated in human cancers.

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.

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. 

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.