Inhibitor of Nrf2 (INrf2 or Keap1) degrades Bcl-xL via Phosphoglycerate mutase 5 and controls cellular apoptosis

Inhibitor of Nrf2 (INrf2 or Keap1) degrades Bcl-xL via Phosphoglycerate mutase 5 and controls cellular apoptosis Suryakant K. Niture and Anil K. Jaiswal Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, MD 21201 Running title: INrf2 degradation of PGMA5-Bcl-xL Address correspondence to: Dr. Anil K. Jaiswal, Professor, Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201, Tel. # 410 706-2285, Email: [email protected] SUMMARY INrf2 (Keap1) is an adaptor protein that facilitates INrf2/Cul3-Rbx1-mediated ubiquitination/degradation of Nrf2, a master regulator of cytoprotective gene expression. Here, we present evidence that members of the phosphoglycerate mutase family 5 (PGAM5) proteins are involved in the INrf2mediated ubiquitination/degradation of antiapoptotic factor Bcl-xL. Mass spectrometry and co-immunoprecipitation assays revealed that INrf2, through its DGR domain interacts with PGAM5 which in turn interacts with anti-apoptotic Bcl-xL protein. INrf2/Cul3Rbx1 complex facilitates ubiquitination and degradation of both PGAM5 and Bcl-xL. Overexpression of PGAM5 protein increased INrf2-mediated degradation of Bcl-xL, whereas knocking down PGAM5 by siRNA decreased INrf2 degradation of Bcl-xL resulting in increased stability of Bcl-xL. Mutation of PGMA5E79AS80A abolished INrf2/PGAM5-Bcl-xL interaction. Therefore, PGAM5 protein acts as a bridge between INrf2 and Bcl-xL interaction. Further studies showed that overexpression of INrf2 enhanced degradation of PGAM5-Bcl-xL complex, led to etoposide mediated accumulation of Bax, increased release of cytochrome C from mitochondria, activated caspase-3/7, and enhanced DNA fragmentation and apoptosis. In addition, antioxidant (tBHQ) treatment destabilized the Nrf2-INrf2/PGAM5-Bcl-xL complex, which resulted in release of Nrf2 in cytosol and mitochondria, release of Bcl-xL in mitochondria, increase in Bcl-xL heterodimerization with Bax in mitochondria and reduced cellular apoptosis. These data provide the first evidence that INrf2 controls Bcl-xL via PGAM5 and controls cellular apoptosis.