Study of Human Circadian Protein (hRORγ) and Lipid-Protein Interaction in Giant Virus (PBCV-1)
Abstract
Project 1: Circadian rhythm is a 24-hour cycle that regulates physical and behavioral changes such as sleep-wake patterns in humans, tailoring the daily light and dark changes. Long-term disruption in circadian rhythms can cause sleep disorders such as sleep apnea, insomnia, et al. Limited research has been done on potential drugs to treat against circadian related sleep disorders. Inside the cell at molecular level, the circadian rhythm is regulated by interlocked time-delayed feedback loops, which involve positive and negative transcriptional regulators. Experimental results showed transcriptional factors Retinoic Acid Receptor-Related Orphan Receptors (RORs) improve the stability and functionality of the circadian rhythm. Nobiletin is a natural flavonoid that has been reported to enhance the circadian amplitude by binding to human ROR gamma (hRORγ) with high affinity. However, the molecular interaction between Nobiletin and hRORγ remains unknown, which limits the future application of Nobiletin as a potential circadian interfering medicine to treat related diseases. The final goal of the project is to determine the atomic three-dimensional structure of the hRORγ/Nobiletin complex, with a focus on comprehending their interaction. The hRORγ gene was cloned and expressed in bacteria to obtain substantial quantities of purified recombinant hRORγ. The expression and purification protocols have been intensively optimized. The affinity tag has been removed by enzymatic digestion, providing homogenous charge-neutral recombinant hRORγ to be ready to co-crystalized with Nobiletin for atomic structural determination by X-ray crystallography. The resultant structure will illustrate the mechanism of Nobiletin’s impact on circadian rhythm, paving the way for potential drug development.Project 2: Giant viruses are characterized by their remarkable size, complex genetic content, and unique replication mechanisms. Most giant viruses have icosahedral shell called capsid assembled mainly from the major capsid proteins (MCPs). Closely beneath this MCP layer is a lipid membrane. The goal of the project is to study the interaction between MCP and lipid using the giant virus Paramecium bursaria chlorella virus 1 (PBCV-1). By employing mass spectrometry (MS) analyses and the following lipidomic analyses, various lipids have been identified within the virion. To investigate the specific interaction between MCP and lipids, Viral protein 54 (Vp54), the MCP of PBCV-1, was purified through multiple steps, allowing for an in-depth study of its affinity to lipids. The following liposome pull-down assay revealed Vp54 specifically binds to cardiolipin, a lipid found in mitochondria. This research contributes to the broader knowledge of the mechanisms underlying giant virus assembly, shedding light on the critical role of lipid-protein interactions in this process.
Subject Area
Chemistry|Virology|Biochemistry
Recommended Citation
Noor, Laila, "Study of Human Circadian Protein (hRORγ) and Lipid-Protein Interaction in Giant Virus (PBCV-1)" (2023). ETD Collection for University of Texas, El Paso. AAI30818218.
https://scholarworks.utep.edu/dissertations/AAI30818218