The Relationship Between UBQLN2-mediated Mitochondrial Quality Control And The Pathogenesis Of Amyotrophic Lateral Sclerosis

Aim:We aim to study if and how ALS-related protein UBQLN2 contributes to mitochondrial quality control,and to understand the molecular basis of ALS which will provide a theoretical basis for the development of target drugs.Methods:(1)Molecular biology experiment:we obtained mt-Keima,UBQLN2 and Parkin plasmids.We constructed ubiquitin,HSP70 and the mutans what we need.(2)Cell biology experiment:We used Lipofectamine 2000 to transfect plasmids into HEK293 cells and RNAiMAX to silence related proteins.Biochemistry experiment:Cells were lysed in the cell lysis buffer.Immunoblot analysis was performed with the primary antibodies,and secondary antibodies.The proteins were visualized with an ECL detection kit.Intracellular binding of proteins was identified by immunoprecipitation.Results:(1)UBQLN2 tanslocated onto damaged mitochondria via its UBA domain upon Parkin-mediated ubiquitination,and then these mitochondria were degraded.(2)mt-Keima experiments suggested that UBQLN2 participated in PINK1/Parkin-medited mitophagy,and both K48 and K63-linked poly-ubiquitin chains played important roles in UBQLN2 recruitment during mitophagy.(3)In terms of molecular mechanisms,we identified that UBQLN2 acted with HSP70 chaperone machinery and promoted OMM rupture in a proteasome-dependent way,which led to sufficient autophagic clearance of entire mitochondria.(4)ALS-linked mutants impaired the tanslocation of UBQLN2 onto damaged mitochondria and inhibited the degradation of these mitochondria by lowering the combination between UBQLN2 and HSP70.(5)Neuronal experiments also confirmed the important role of UBQLN2 in mitochondrial quality control.Conclution:UBQLN2-HSP70 complex is guided by PD-linked PINK1 and Parkin,and may play in proteasome-dependent "melt" of OMM,which will lead to sufficient autophagic clearance of entire mitochondria.Pathogenic mutations will block this process and prevent the damaged mitochondria from being eliminated.