title: Impaired assembly and post-translational regulation of 26S proteasome in human end-stage heart failure.
abstract: BACKGROUND
  This study examined the hypothesis that 26S proteasome dysfunction in human end-stage heart failure is associated with decreased docking of the 19S regulatory particle to the 20S proteasome. Previous studies have demonstrated that 26S proteasome activity is diminished in human end-stage heart failure associated with oxidation of the 19S regulatory particle Rpt5 subunit. Docking of the 19S regulatory particle to the 20S proteasome requires functional Rpt subunit ATPase activity and phosphorylation of the α-type subunits.
  
  
  RESULTS
  An enriched proteasome fraction was prepared from 7 human nonfailing and 10 failing heart explants. Native gel electrophoresis assessed docking of 19S to 20S proteasome revealing 3 proteasome populations (20S, 26S, and 30S proteasomes). In failing hearts, 30S proteasomes were significantly lower (P=0.048) by 37% suggesting diminished docking. Mass spectrometry-based phosphopeptide analysis demonstrated that the relative ratio of phosphorylated:non phosphorylated α7 subunit (serine250) of the 20S proteasome was significantly less (P=0.011) by almost 80% in failing hearts. Rpt ATPase activity was determined in the enriched fraction and after immunoprecipitation with an Rpt6 antibody. ATPase activity (ρmol PO4/μg protein per hour) of the total fraction was lowered from 291±97 to 194±27 and in the immunoprecipitated fraction from 42±12 to 3±2 (P=0.005) in failing hearts.
  
  
  CONCLUSIONS
  These studies suggest that diminished 26S activity in failing human hearts may be related to impaired docking of the 19S to the 20S as a result of decreased Rpt subunit ATPase activity and α7 subunit phosphorylation.
authors.affiliation: Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
authors.forename: Sharlene M
authors.initials: SM
authors.lastname: Day
authors.forename: Andras
authors.initials: A
authors.lastname: Divald
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.forename: Frank
authors.initials: F
authors.lastname: Davis
authors.forename: Sarah
authors.initials: S
authors.lastname: Bartolone
authors.forename: Richard
authors.initials: R
authors.lastname: Jones
authors.forename: Saul R
authors.initials: SR
authors.lastname: Powell
mesh_terms: D047390:Casein Kinase II / Q000378:metabolism; D005260:Female; D006333:Heart Failure / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D008297:Male; D008875:Middle Aged; D009206:Myocardium / Q000201:enzymology*; D063347:Native Polyacrylamide Gel Electrophoresis; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism* / Q000502:physiology; D011499:Protein Processing, Post-Translational
chemical_list: D047390:Casein Kinase II; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C119011:26S proteasome non-ATPase regulatory subunit 13
keywords: heart failure; human; phosphorylation; ubiquitin proteasome system