Reversible post-translational modifications are widely used to dynamically regulate protein activities in vivo. The most well known example of polypeptide modifiers is ubiquitin and ubiquitin like proteins (Ubl). Ubiquitin and Ubiquitin like proteins (Ubls), such as NEDD8 and SUMO, play critical roles in various physiological and pathological processes, ranging from signal transduction, cell cycle to tumorgenesis. The Ubls are conjugated to their respective substrates by similar but distinct multi enzyme cascades that involve sequential actions of the E1 activating enzymes, E2 conjugating enzymes and E3 ligases, requiring multiple protein-protein interactions between NEDD8 and NEDD8's E1, E2, E3 enzymes and NEDD8 substrates. NEDD8 specifically is known regulate many critical targets, which play crucial role in cellular processes such as, DNA transcription and repair, signal transduction and cell cycle regulation. And misregulation of NEDD8 cascade is observed in various cancers and neurodegenerative disorders.
Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a valuable tool for elucidating molecular interactions in vitro and in vivo. Förster Resonance Energy Transfer (FRET) is a distance dependent energy transfer phenomenon. Quantitative FRET analysis is a powerful method for determining biochemical parameters and molecular distances at nanometer levels. The goal of my research is to develop FRET based protein assays to dissect the mechanisms of NEDDylation cascade and develop a HTS platform to identify bioactive chemical compounds, which can inhibit the activity of NEDD8 pathway. Using peptide linker-engineering strategy, I developed a high sensitive fluorescent-tagged NEDD8 for a trans FRET assay, which allowed us to follow the covalent conjugation of NEDD8 to its E2 ligase in the presence of E1 and ATP, which was difficult to determine without linker. The understanding of the covalent attachment of NEDD8 to target proteins and its kinetics of conjugation cascade will provide greater insights into this process in normal and pathological conditions, such as cancers. To understand the system of NEDDylation cascade, I develop a series of novel and quantitative FRET-based assays to dissect reaction intermediates and dynamics of NEDD8 to its E1 activating enzyme and its E2 conjugating enzyme. We were able to follow the thioester intermediates of NEDD8 with its E1 and E2 for the first time. We are also applying this technology to determine the specificity of NEDD8 with other ligases of Ubiquitin-like protein pathways.
The Ubls are conjugated to their respective substrates by similar but distinct multi enzyme cascades that involve sequential actions of the E1 activating enzymes, E2 conjugating enzymes and E3 ligases. Although all the Ubls are conjugated to their substrates through a similar enzymatic cascade, the requirement for either single or heterodimer of E1 activating enzymes in various Ubl conjugation cascade is still a mystery. Here, by employing our high sensitive engineered trans-FRET assay we report a significant discovery of the requirements of each subunit of E1 heterodimer for NEDD8 activation. We, for the first time, systematically dissect the NEDD8 activation step in real time by employing our new quantitative FRET technology. These studies show that APPBP1 (a subunit of NEDD8 E1 heterodimer) is not absolutely required for the NEDD8 activation. These findings provide not only detail molecular mechanisms of Ubl activation, but also potential evolution process at molecular level. I further employed by linker-engineering strategy for engineering Cullin (NEDD8's well studied target protein) tagged with YPet, to aid in development of FRET based HTS platform for drug discovery. I was able to successfully engineer YPet-Cullin1 with linker, to drastically improve fluorescence emission. In future this engineered YPet-Cullin1 will be employed for development of FRET based HTS platform for drug discovery for NEDD8 conjugation cascade.