Lysine 222 in PPAR γ1 functions as the key site of MuRF2-mediated ubiquitination modification

Peroxisome proliferator-activated receptor gamma (PPAR γ) plays key roles in the development, physiology, reproduction, and homeostasis of organisms. Its expression and activity are regulated by various posttranslational modifications. We previously reported that E3 ubiquitin ligase muscle ring finger protein 2 (MuRF2) inhibits cardiac PPAR γ1 protein level and activity, eventually protects heart from diabetic cardiomyopathy; furthermore, by GST-pulldown assay, we found that MuRF2 modifies PPAR γ1 via poly-ubiquitination and accelerates PPAR γ1 proteasomal degradation. However, the key ubiquitination site on PPAR γ that MuRF2 targets for remains unclear. In the present study, we demonstrate that lysine site 222 is the receptor of MuRF2-mediated PPAR γ1 ubiquitination modification, using prediction of computational models, immunoprecipitation, ubiquitination assays, cycloheximide chasing assay and RT-qPCR. Our findings elucidated the underlying details of MuRF2 prevents heart from diabetic cardiomyopathy through the PPAR γ1 regulatory pathway.