PhD Dissertation Defense by Kequan Wang, "Inhibitors Screening against HIV-1 Nef-Mediated MHC-I Downregulation and Structural Studies of Nef-Mediated SERINC5 Downregulation"
Virtual
Rachel White
58-999-8232
rwhite@umassd.edu
https://fsu.zoom.us/j/97089913327#success
Advisors:
Shuowei Cai, Professor, Department of Chemistry and Biochemistry, Dissertation Advisor.
Xiaofei Jia, Associate Professor, Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, Dissertation Advisor.
Committee members:
Catherine Neto, Professor, Department of Chemistry and Biochemistry, Dissertation Committee.
Tracie Ferreira, Professor, Department of Bioengineering, Dissertation Committee.
Abstract:
Human immunodeficiency virus type 1 (HIV-1) remains a major global health challenge. Although current treatments effectively suppress viral replication, they are unable to eliminate infected cells. The viral accessory protein Nef contributes substantially to HIV-1 persistence: it downregulates multiple host immune receptors and restriction factors and thus enables infected cells to evade immune surveillance. Consequently, Nef represents an attractive therapeutic target; its inhibition should restore host immune functions and promote the clearance of HIV-1 infected cells, which may lead to a functional cure.
The development of Nef inhibitor has two major limitations: lack of enzymatic activity and thus a druggable-site and the great conformational dynamic of the Nef protein. Successful development of Nef inhibitors would require competent screening assay(s) that can address these issues. Chapter 2 of this dissertation describes our work in developing such an assay for the discovery of inhibitors of Nef-mediated MHC-I downregulation. Here, time-resolved fluorescence resonance energy transfer (TR-FRET) was used, and the assay signal is associated with complex assembly between MHC-I cytoplasmic domain (MHC-ICD), Nef, and clathrin AP1. The optimized assay is robust, and its suitability for high-throughput screening (HTS) was validated through a proof-of-concept screening of a medium-sized library.
Chapter 3 of this dissertation describes our work in developing cyclic MHC-ICD-mimetic peptides as potential inhibitors of Nef-mediated MHC-I downregulation. Robust assays were developed to investigate the interactions between cyclic peptides and the Nef/AP1 complex and thus help evaluate peptide inhibitors developed by our collaborators. The framework established here for evaluating cyclopeptide hits also paved the way for future structural studies aimed at elucidating the inhibitor-Nef/AP1 interaction to guide the rational optimization of future inhibitors.
Chapter 4 of this dissertation describes our work in using cryo-EM to study the structural basis of HIV-1 Nef-mediated SERINC5 downregulation. A novel AP2 hemicomplex was designed to stabilize the conformation of Nef and thus facilitate single-particle cryo-EM analysis. In addition, to address the weak interaction between Nef/AP2 and the intracellular loop 4 (ICL4) of SERINC5, a chemical crosslinking strategy was employed to capture the assembly complex. Prepared samples allowed cryo-EM analysis leading to a 2.88 Å electron density map. Structural solution was achieved for the AP2 components as well as the folded core domain of Nef. Density for SERINC5 ICL4 has also been observed; however, the poor quality of the density has prevented successful building of the ICL4 structure. This milestone success has nonetheless provided important guidance for further construct optimization and subsequent structural studies of Nef-mediated SERINC5 downregulation.
Zoom Meeting ID: 970 8991 3327