Distinguishing aggregate formation and aggregate clearance using cell-based assays

J Cell Sci. 2016 Mar 15;129(6):1260-70. doi: 10.1242/jcs.179978. Epub 2016 Jan 27.

Abstract

The accumulation of ubiquitylated proteinaceous inclusions represents a complex process, reflecting the disequilibrium between aggregate formation and aggregate clearance. Although decreasing aggregate formation or augmenting aggregate clearance will ultimately lead to a diminished aggregate burden, in terms of disease pathogenesis, the different approaches can have distinct outcomes. Using a novel cell-based assay that can distinguish newly formed versus preformed inclusions, we demonstrate that two proteins previously implicated in the autophagic clearance of expanded polyglutamine inclusions, HspB7 and Alfy (also known as WDFY3), actually affect very distinct cellular processes to affect aggregate burden. Using this cell-based assay, we also establish that constitutive expression of the aggregation-prone protein can measurably slow the elimination of protein aggregates, given that not all aggregates appear to be available for degradation. This new assay can therefore not only determine at what step a modifier might influence aggregate burden, but also can be used to provide new insights into how protein aggregates are targeted for degradation.

Keywords: Autophagy; Chaperone; Polyglutamine protein; Protein aggregation.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Animals
  • Autophagy
  • Autophagy-Related Proteins
  • Biochemistry / methods*
  • HSP27 Heat-Shock Proteins / genetics
  • HSP27 Heat-Shock Proteins / metabolism*
  • HeLa Cells
  • Humans
  • Inclusion Bodies
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Protein Aggregates*
  • Proteolysis
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Autophagy-Related Proteins
  • HSP27 Heat-Shock Proteins
  • HSPB7 protein, human
  • Membrane Proteins
  • Protein Aggregates
  • Transcription Factors
  • WDFY3 protein, human