BEGIN:VCALENDAR VERSION:2.0 X-WR-CALNAME:EventsCalendar PRODID:-//hacksw/handcal//NONSGML v1.0//EN CALSCALE:GREGORIAN BEGIN:VTIMEZONE TZID:America/New_York LAST-MODIFIED:20240422T053451Z TZURL:https://www.tzurl.org/zoneinfo-outlook/America/New_York X-LIC-LOCATION:America/New_York BEGIN:DAYLIGHT TZNAME:EDT TZOFFSETFROM:-0500 TZOFFSETTO:-0400 DTSTART:19700308T020000 RRULE:FREQ=YEARLY;BYMONTH=3;BYDAY=2SU END:DAYLIGHT BEGIN:STANDARD TZNAME:EST TZOFFSETFROM:-0400 TZOFFSETTO:-0500 DTSTART:19701101T020000 RRULE:FREQ=YEARLY;BYMONTH=11;BYDAY=1SU END:STANDARD END:VTIMEZONE BEGIN:VEVENT CATEGORIES:Thesis/Dissertations DESCRIPTION:Advisors: Shuowei Cai, Professor, Department of Chemistry and B iochemistry, Dissertation Advisor. Xiaofei Jia, Associate Professor, Depar tment of Biomedical Sciences, Florida State University College of Medicine , Tallahassee, FL, Dissertation Advisor. Committee members: Catherine Neto , Professor, Department of Chemistry and Biochemistry, Dissertation Commit tee. Tracie Ferreira, Professor, Department of Bioengineering, Dissertatio n 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. T he viral accessory protein Nef contributes substantially to HIV-1 persiste nce: it downregulates multiple host immune receptors and restriction facto rs and thus enables infected cells to evade immune surveillance. Consequen tly, Nef represents an attractive therapeutic target; its inhibition shoul d restore host immune functions and promote the clearance of HIV-1 infecte d cells, which may lead to a functional cure. The development of Nef inhib itor has two major limitations: lack of enzymatic activity and thus a drug gable-site and the great conformational dynamic of the Nef protein. Succes sful development of Nef inhibitors would require competent screening assay (s) that can address these issues. Chapter 2 of this dissertation describe s our work in developing such an assay for the discovery of inhibitors of Nef-mediated MHC-I downregulation. Here, time-resolved fluorescence resona nce energy transfer (TR-FRET) was used, and the assay signal is associated with complex assembly between MHC-I cytoplasmic domain (MHC-ICD), Nef, an d clathrin AP1. The optimized assay is robust, and its suitability for hig h-throughput screening (HTS) was validated through a proof-of-concept scre ening of a medium-sized library. Chapter 3 of this dissertation describes our work in developing cyclic MHC-ICD-mimetic peptides as potential inhibi tors of Nef-mediated MHC-I downregulation. Robust assays were developed to investigate the interactions between cyclic peptides and the Nef/AP1 comp lex and thus help evaluate peptide inhibitors developed by our collaborato rs. The framework established here for evaluating cyclopeptide hits also p aved the way for future structural studies aimed at elucidating the inhibi tor-Nef/AP1 interaction to guide the rational optimization of future inhib itors. 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 Ne f and thus facilitate single-particle cryo-EM analysis. In addition, to ad dress the weak interaction between Nef/AP2 and the intracellular loop 4 (I CL4) 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 SERIN C5 ICL4 has also been observed; however, the poor quality of the density h as prevented successful building of the ICL4 structure. This milestone suc cess has nonetheless provided important guidance for further construct opt imization and subsequent structural studies of Nef-mediated SERINC5 downre gulation. Zoom Meeting ID: 970 8991 3327 https://fsu.zoom.us/j/97089913327 #success \nEvent page: https://www.umassd.edu/events/cms/7-31-26-defense- by-kequan-wang-inhibitors-screening-against-hiv-1-nef.php\nEvent link: htt ps://fsu.zoom.us/j/97089913327#success X-ALT-DESC;FMTTYPE=text/html:

Advisors:

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Shuowei Cai\, Professor\, Department of Chemistry and Biochemistry\, D issertation Advisor.

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Xiaofei Jia\, Associate Professor\, Departmen t of Biomedical Sciences\, Florida State University College of Medicine\, Tallahassee\, FL\, Dissertation Advisor.

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Committee members :

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Catherine Neto\, Professor\, Department of Chemistry an d Biochemistry\, Dissertation Committee.

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Tracie Ferreira\, Profess or\, Department of Bioengineering\, Dissertation Committee.

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Abstract:

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Human immunodeficiency virus type 1 (HIV-1) r emains a major global health challenge. Although current treatments effect ively suppress viral replication\, they are unable to eliminate infected c ells. The viral accessory protein Nef contributes substantially to HIV-1 p ersistence: it downregulates multiple host immune receptors and restrictio n factors and thus enables infected cells to evade immune surveillance. Co nsequently\, Nef represents an attractive therapeutic target\; its inhibit ion should restore host immune functions and promote the clearance of HIV- 1 infected cells\, which may lead to a functional cure.

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The develo pment of Nef inhibitor has two major limitations: lack of enzymatic activi ty and thus a druggable-site and the great conformational dynamic of the N ef protein. Successful development of Nef inhibitors would require compete nt screening assay(s) that can address these issues. Chapter 2 of this dis sertation 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 domai n (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.

\n

Chapter 3 of this dissertation describes our work in developing cyclic MHC-ICD-mimet ic peptides as potential inhibitors of Nef-mediated MHC-I downregulation. Robust assays were developed to investigate the interactions between cycli c peptides and the Nef/AP1 complex and thus help evaluate peptide inhibito rs developed by our collaborators. The framework established here for eval uating cyclopeptide hits also paved the way for future structural studies aimed at elucidating the inhibitor-Nef/AP1 interaction to guide the ration al optimization of future inhibitors.

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Chapter 4 of this dissertati on describes our work in using cryo-EM to study the structural basis of HI V-1 Nef-mediated SERINC5 downregulation. A novel AP2 hemicomplex was desig ned to stabilize the conformation of Nef and thus facilitate single-partic le cryo-EM analysis. In addition\, to address the weak interaction between Nef/AP2 and the intracellular loop 4 (ICL4) of SERINC5\, a chemical cross linking strategy was employed to capture the assembly complex. Prepared sa mples 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 fol ded core domain of Nef. Density for SERINC5 ICL4 has also been observed\; however\, the poor quality of the density has prevented successful buildin g of the ICL4 structure. This milestone success has nonetheless provided i mportant guidance for further construct optimization and subsequent struct ural studies of Nef-mediated SERINC5 downregulation.

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Zoom Meeting ID: 970 8991 3327

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https://fsu.zoom.us/j/97089913327#success 

Event page: https://www.umassd.edu/events/cms/ 7-31-26-defense-by-kequan-wang-inhibitors-screening-against-hiv-1-nef.php< /a>
Event link: htt ps://fsu.zoom.us/j/97089913327#success

DTSTAMP:20260721T084831 DTSTART;TZID=America/New_York:20260731T100000 DTEND;TZID=America/New_York:20260731T120000 LOCATION:Virtual SUMMARY;LANGUAGE=en-us:PhD Dissertation Defense by Kequan Wang, "Inhibitors Screening against HIV-1 Nef-Mediated MHC-I Downregulation and Structural Studies of Nef-Mediated SERINC5 Downregulation" UID:3d79ea842da6c9cc8037286ea6bd4ac6@www.umassd.edu END:VEVENT END:VCALENDAR