🧫 Experiment Protocol
Exploratorychronic kidney diseaseACAT1in vitro protein-ligand binding assaysproposed
Multiple biophysical approaches were used to confirm direct binding between hyperoside and mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1). Cellular thermal shift assays demonstrated thermal stabilization of ACAT1 upon hyperoside binding. Surface plasmon resonance provided quantitative binding kinetics and affinity measurements. Molecular docking studies predicted the binding mode and interaction sites. These complementary approaches established that hyperoside directly binds to ACAT1, stabilizes the protein, and enhances its enzymatic activity while suppressing ubiquitination-mediated degradation.
PRIMARY OUTCOME
confirmation of direct hyperoside-ACAT1 binding
EXPECTED OUTCOMES
hyperoside would directly bind to and stabilize ACAT1
SUCCESS CRITERIA
detectable binding affinity and thermal stabilization of ACAT1
PROTOCOL
cellular thermal shift assays, surface plasmon resonance, molecular docking, ubiquitination assays