Monitoring proteasome activity in cellulo and in living animals by bioluminescent imaging: technical considerations for design and use of genetically encoded reporters

Methods Enzymol. 2005;399:512-30. doi: 10.1016/S0076-6879(05)99035-6.

Abstract

The ubiquitin-proteasome pathway is the central mediator of regulated proteolysis, instrumental for switching on and off a variety of signaling cascades. Deregulation of proteasomal activity or improper substrate recognition and processing by the ubiquitin-proteasome machinery may lead to cancer, stroke, chronic inflammation, and neurodegenerative diseases. Quantifying total and substrate-specific proteasome activity in intact cells and living animals would enable analysis in vivo of proteasomal regulation and facilitate the screening and validation of potential modulators of the proteasome or its substrates. We discuss examples of tetra-ubiquitin or IkappaBalpha fused to firefly luciferase as genetically encoded reporters for monitoring total and IkappaBalpha-specific proteasomal activity by bioluminescence imaging. Such technology enables repetitive, temporally resolved, and regionally targeted assessment of proteasomal activity in vivo.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Base Sequence
  • DNA Primers
  • Genes, Reporter*
  • I-kappa B Proteins / metabolism
  • Luciferases / genetics
  • Luciferases / metabolism
  • Luminescence
  • Mice
  • NF-KappaB Inhibitor alpha
  • Proteasome Endopeptidase Complex / metabolism*
  • Ubiquitin / metabolism

Substances

  • DNA Primers
  • I-kappa B Proteins
  • Nfkbia protein, mouse
  • Ubiquitin
  • NF-KappaB Inhibitor alpha
  • Luciferases
  • Proteasome Endopeptidase Complex