The Proteasome-Interacting Ecm29 Protein Disassembles the 26S Proteasome in Response to Oxidative Stress

J Biol Chem. 2017 Sep 29;292(39):16310-16320. doi: 10.1074/jbc.M117.803619. Epub 2017 Aug 15.

Abstract

Oxidative stress has been implicated in multiple human neurological and other disorders. Proteasomes are multi-subunit proteases critical for the removal of oxidatively damaged proteins. To understand stress-associated human pathologies, it is important to uncover the molecular events underlying the regulation of proteasomes upon oxidative stress. To this end, we investigated H2O2 stress-induced molecular changes of the human 26S proteasome and determined that stress-induced 26S proteasome disassembly is conserved from yeast to human. Moreover, we developed and employed a new proteomic approach, XAP (in vivo cross-linking-assisted affinity purification), coupled with stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative MS, to capture and quantify several weakly bound proteasome-interacting proteins and examine their roles in stress-mediated proteasomal remodeling. Our results indicate that the adapter protein Ecm29 is the main proteasome-interacting protein responsible for stress-triggered remodeling of the 26S proteasome in human cells. Importantly, using a disuccinimidyl sulfoxide-based cross-linking MS platform, we mapped the interactions of Ecm29 within itself and with proteasome subunits and determined the architecture of the Ecm29-proteasome complex with integrative structure modeling. These results enabled us to propose a structural model in which Ecm29 intrudes on the interaction between the 20S core particle and the 19S regulatory particle in the 26S proteasome, disrupting the proteasome structure in response to oxidative stress.

Keywords: MS; oxidative stress; proteasome; protein cross-linking; protein purification; protein–protein interaction; structural model.

MeSH terms

  • ATPases Associated with Diverse Cellular Activities
  • Adaptor Proteins, Signal Transducing / chemistry
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Affinity Labels
  • Cross-Linking Reagents / pharmacology
  • HEK293 Cells
  • Humans
  • Isotope Labeling
  • LIM Domain Proteins / chemistry
  • LIM Domain Proteins / genetics
  • LIM Domain Proteins / metabolism
  • Models, Molecular*
  • Oxidative Stress*
  • Proteasome Endopeptidase Complex / chemistry
  • Proteasome Endopeptidase Complex / genetics
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping
  • Protein Multimerization
  • Proteolysis
  • RNA Interference
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Tandem Mass Spectrometry
  • Transcription Factors / chemistry
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Ubiquitins / chemistry
  • Ubiquitins / genetics
  • Ubiquitins / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Affinity Labels
  • Cross-Linking Reagents
  • ECPAS protein, human
  • LIM Domain Proteins
  • PSMC5 protein, human
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Ubiquitins
  • Ubl4A protein, human
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease
  • ATPases Associated with Diverse Cellular Activities

Associated data

  • PDB/5GJR