Cardiac-specific promoters, such as NK2 homeobox 5 (NKX2.5), are essential for driving gene expression during early heart development, making them valuable for studying neonatal congenital heart diseases. However, the large size of the NKX2.5 promoter limits its use in adeno-associated virus (AAV) delivery systems, restricting vector space for therapeutic genes. This study aims to develop a compact hybrid NKX2.5 promoter that retains cardiac specificity while enhancing its efficiency in early-stage cardiac research and gene therapy. To achieve this, we replace the enhancer region of NKX2.5 with a shortened cytomegalovirus (CMV)-derived enhancer, preserving cardiac specificity while reducing promoter size. The hybrid promoter is then cloned into an AAV vector to drive green fluorescent protein (GFP) expression for assessing transcriptional activity and tissue specificity. Following AAV injection into pregnant mice, we will harvest early-stage embryos to evaluate GFP expression in cardiac tissues, comparing the hybrid promoter’s efficiency against the original NKX2.5-driven GFP expression. This study addresses current limitations of cardiac-specific promoters by developing a streamlined version optimized for gene delivery in neonatal models. Our findings strives to enhance gene therapy strategies for congenital heart diseases and provide insights into early cardiac gene regulation.

The small ubiquitin-like modifier protein, SUMO, regulates the activity of many cellular processes through covalent modification of proteins. These modified targets include the protein components of chromatin; histones H2A, H2B, H3, and H4. Chemical modification of histones directly regulates gene expression, necessitating an understanding of the role of each type of modification. The identification and role of histone SUMOylation has been described for H4 in human cells; however, SUMOylation of H2B in human cells has been recently observed but not yet characterized. SUMO is shown to impose a predominantly repressive effect on many cellular processes and proteins that it targets. Therefore, I am working toward identifying the role of H2B SUMOylation to either add to this narrative or describe novel functions of SUMO. To accomplish this, I have purified wild-type histones and SUMO-histone fusions through bacterial expression followed by size-exclusion and affinity chromatography. The purification of several of these proteins has not been described yet; therefore, I designed the purification for these proteins using unique methods, like solubilizing tags, to obtain the product. I reconstituted the purified proteins into octamers, the protein complex that DNA wraps around, and purified the octamers away from other oligomeric forms of the histones via size-exclusion chromatography. I further reconstituted the octamers into mononucleosomes by condensing DNA around them to mimic SUMOylated nucleosomes in chromatin. I hope to then subject the mononucleosomes to in vitro biochemical assays to observe changes in the modifications that regulate other chromatin-associated proteins. A better understanding of the complex dynamics at play during gene expression and repression is needed to identify stronger, safer, and more sustainable therapeutics. Furthermore, SUMO is implicated in a wide array of diseases, such as Alzheimer’s. Therefore, the results of this study will increase our understanding of gene regulation and provide insight towards treating related diseases.

Neuronal and endocrine cells store and secrete molecular cargos like neurotransmitters and metabolic hormones through the regulated secretory pathway. Dense-core vesicles (DCVs) originate from the trans-Golgi network and undergo a maturation process involving peptide processing and cargo sorting before being stimulated to release their cargos outside the cell. Dysregulation of this process leads to a wide range of neurological and metabolic disorders; yet the molecular mechanisms underpinning it remain poorly understood. Vesicular traffic are largely coordinated by the Rab family of GTPase proteins.  Previous work identified the conserved proteins TBC8 and RUND1 as regulators of DCV maturation in Caenorhabditis elegans; and both proteins bind active GTP-bound RAB2. TBC8 is the putative RAB2 GTPase activating protein (GAP) which promotes conversion of GTP-RAB2 into GDP-RAB2, thereby inactivating the Rab. RUND1 interacts with both active GTP-RAB2 and TBC8, yet its precise function remains unknown. This study aims to characterize the biochemical function of TBC8 and RUND1 in regulating RAB2 activity. Using purified proteins, we demonstrate that TBC8 greatly promotes RAB2 GTP hydrolysis, indicating it is the bona fide RAB2 GAP. Additionally, we show that RUND1 strongly inhibits TBC8-stimulated RAB2 GTP hydrolysis, suggesting RUND1 may compete with TBC8 for RAB2 binding. Given this interplay between RUND1 and TBC8 in binding RAB2, we hypothesize that RAB2 exhibits exclusively pairwise interactions with its partners. To test this, we will use mass photometry to study whether RUND1 and TBC8 can bind RAB2 simultaneously or if one complex is preferentially formed. Based on our current findings, we propose a model where TBC8 promotes RAB2 inactivation by stimulating GTP hydrolysis and RUND1 blocks RAB2 inactivation by TBC8, prolonging the activate state of RAB2.

Hydrogen sulfide (H₂S), known for its distinct smell of rotten eggs, is recognized as the third endogenous gaseous signaling molecule, alongside nitric oxide and carbon monoxide. Often described as a double-edged sword, H₂S exhibits both cytoprotective and cytotoxic properties depending on the biological context. A 2018 study suggested that H₂S enhances the efficacy of doxorubicin (Dox), an anticancer drug, by promoting apoptosis and reducing colony formation in HepG2 cells, even restoring drug sensitivity in resistant cells. However, my preliminary experiments indicated a protective role of H₂S in HepG2 cells under stress, particularly when treated with NaSH (an H₂S donor). Rather than inducing apoptosis, H₂S appears to support cell proliferation and regulate reactive oxygen species (ROS) production. My research project aims to identify H₂S -dependent pathways in HepG2 cells under oxidative stress. Using Dox as a stress inducer, I conducted viability and cytotoxicity assays, demonstrating that supplementation with 250 µM NaSH at 0 and 12 hours significantly restored cell survival. To investigate the molecular mechanisms, RNA-seq analysis identified 2,996 differentially expressed genes in the H₂S + Dox group compared to Dox alone. Principal component analysis (PCA) revealed distinct transcriptomic profiles, while KEGG enrichment analysis highlighted significant alterations in genes within the PI3K-Akt pathway. To further validate these findings, I plan to perform flow cytometry and western blot analysis. While the role of H₂S continues to be debated, my data suggest a protective function in liver cells against Dox-induced stress via the PI3K-Akt pathway. Understanding these mechanisms could pave the way for new therapeutic strategies aimed at maintaining or increasing H₂S levels to support cell health in diseases characterized by oxidative stress, such as cancer and diabetes.

Members of the gut microbiota produce an array of bioactive metabolites that impact many aspects of host metabolism, immunity, and behavior. However, the mechanisms by which these metabolites are generated remain poorly understood and the biosynthetic enzymes are largely understudied. Recently, our laboratory discovered a family of gut microbial amidases that were found to affect hunger-related biological pathways in malnourished children. These amidases hydrolyze N-acyl ethanolamines (NAEs), lipid messengers with known roles in satiety, visceral pain, and inflammation. Using one of these family members as a model amidase, I am exploring and defining the catalytic mechanisms that are responsible for NAE hydrolysis and the production of a new class of gut microbial metabolites, N-acyl amino acids (NAAAs). I used computational models to predict specific residues that might be important for the amidase activity. I then cloned mutant enzymes into an E. coli expression vector, induced recombinant protein expression, and tested the ability of the purified mutant enzymes to hydrolyze labeled NAEs using liquid chromatography - tandem mass spectrometry (LC-MS/MS). These analyses have pinpointed residues that are important for substrate recognition and binding. My work is advancing our understanding of the selectivity of these intriguing gut microbial enzymes and the regulation of NAAAs in the gut lumen. These efforts are expected to generate the knowledge required to engineer more selective enzymes that produce metabolites of known bioactivity.

Pulling apart DNA during replication induces DNA strands to wrap around each other, producing positive supercoils ahead of the replication fork. Positive supercoils hinder further DNA replication, and are removed by Type II Topoisomerases (Top2s), a group of essential enzymes that cleave positive supercoils to relax DNA for easy separation. Errors in supercoil resolution are linked to diseases like cancer and autoimmune disorders. A key question in the field is the mechanism by which Top2s locate positive supercoils. We recently discovered that GapR, an essential DNA binding protein conserved across α-proteobacteria, binds positive supercoils and stimulates the activity of bacterial Top2s DNA Gyrase and Topoisomerase IV. We hypothesized 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 subunit and identified an interaction between GapR and the A subunits of DNA Gyrase and Topoisomerase IV. Additionally, we discovered that GapR interacts with Top2 A subunits, and not with Top2 B subunits, in a gel shift assay. In collaboration with the David Baker lab, we generated predictions of the GapR-Top2 interaction which together support a model of interaction between GapR and the Top2 A subunit that is mutually exclusive to the Top2 B subunit. In our current work, we aim to identify the mechanism of direct interaction between GapR and Top2s aided by mass photometry and biochemical experiments to reveal a previously unknown mechanism of Top2 recruitment. Because GapR is conserved by alphaproteobacteria, our research could reveal a target for inhibition by antibiotics. If such Top2 recruiters are more broadly conserved, our work provides a novel pathway to target with anticancer therapeutics as human Top2 inhibitors are important chemotherapy drugs.