Peptide Self-assembly Tools for Detecting Sirt5 Activity in Living Cells
DescriptionSirt5 is a mitochondria-localized enzyme that belongs to a family of NAD+-dependent HDACs. It can modulate protein activity through desuccinylation, demalonylation and deglutarylation mechanism. Biological studies have shown that Sirt5 is involved in regulating diverse biological processes, including tricarboxylic acid cycle, reactive oxygen defense, fatty acid metabolism and apoptosis. Recently a number of studies have shown Sirt5 is associated with promoting cancer formation. Due to the important roles of Sirt5 in biology and cancer, it will be highly useful to develop chemical tools for detecting Sirt5 activity in living cells. In this proposal, we will employ molecular self-assembly approach to develop peptide-based tools to detect Sirt5 activity in living cells. Molecular self-assembly has found wide applications in cellular activity control, biomarker imaging, drug delivery and other areas. Mimicking nature’s ability to orchestrate a high level of self-assembly in living organisms is fundamentally important and technically challenging. The engineering of self-assembly in living systems demands precise control over molecular forces in complex cellular environment. Our approach is based on designing peptide precursors that can selectively respond to Sirt5 and self-assemble to form nanofiber. We will first develop a succinylated peptide microarray to investigate the substrate specificity of Sirt5. After identifying the peptide substrates with the strongest activity, we will synthesize the corresponding NBD-linked peptide precursor for Sirt5-triggered self-assembly study. In the presence of Sirt5, we expect the soluble non-assembling peptide precursor to be transformed into self-assembling building block, leading to the formation of nanofibers and hydrogel. At the same time, the environment-sensitive fluorophore NBD in the peptide can produce bright fluorescence due to the hydrophobic environment of nanofibers. After proving Sirt5 can induce nanofiber formation in vitro, we will next investigate whether the peptide precursor can be used to image Sirt5 activity in living cells. Co-localization experiments and SiRNA assay will be conducted to further validate the roles of Sirt5 in catalyzing nanofiber formation. In the last stage, we will adopt fragment-based approach and synthesize a small molecule-based inhibitor library. After obtaining the inhibitors, we will employ hydrogelation assay and cell-based assay to screen the potent hits. Through our interdisciplinary research encompassing organic chemistry, peptide microarray, self-assembly and molecular imaging, we hope to generate new chemical tools to facilitate research on Sirt5 and generate useful inhibitors to aid in drug discovery.
|Effective start/end date||1/09/19 → …|