Nanobodies as Novel Tools to Target and Study Protein-Protein Interactions of Core Circadian Proteins

Presenter

John Joseph Ferriera

Campus

UMass Amherst

Sponsor

Michelle Farkas, Department of Chemistry, UMass Amherst

Schedule

Session 5, 3:30 PM - 4:15 PM [Schedule by Time][Poster Grid for Time/Location]

Location

Poster Board C34, Poster Showcase Room (163), Row 4 (C31-C40) [Poster Location Map]

Abstract

Circadian rhythms are synchronized biological processes that oscillate on a roughly 24-hour cycle, governing cellular functions such as sleep-wake cycles, metabolism, and feeding schedules. The molecular clock is controlled by a transcription-translation feedback loop regulated by activators CLOCK and BMAL1, which form the CLOCK:BMAL1 transcription factor complex, and repressors CRY1/2 and PER1/2. By day, CLOCK:BMAL1 promotes the transcription of CRY and PER, which translocate out of the nucleus and participate in cellular pathways. By night, CRY and PER heterodimerize and translocate into the nucleus to repress their transcription by interacting with CLOCK:BMAL1. While genetic methods have been used to study circadian protein roles, they often exhibit adverse effects such as network compensation and redundancy. Small molecules have also been used to investigate protein function and modulate circadian rhythms, but they are limited in number, targets, and binding specificity. Therefore, there is a need to develop novel molecular tools that can selectively target core circadian proteins. To this end, we hypothesize that single-domain antibodies called nanobodies can be used as specific targeting agents and disrupt the CRY:PER repressor complex. Here, we describe the selection of candidate nanobodies using in-house expressed and modified CRY2 protein using a nanobody yeast display library combined with screening and cell sorting methods. Candidate nanobody affinity and selectivity towards the PER2 binding site are determined via fluorescence polarization assay. In the future, we will evaluate the effects of the nanobodies in cellular models of circadian rhythms and select candidate nanobodies against CRY1 and other core clock proteins. 

Keywords

Circadian rhythms, Chemical biology, Proteins

Research Area

Chemistry and Materials Science

SIMILAR ABSTRACTS (BY KEYWORD)