A new reporter cell line for studies with proteasome inhibitors in Trypanosoma brucei

Mol Biochem Parasitol. 2019 Jan:227:15-18. doi: 10.1016/j.molbiopara.2018.11.001. Epub 2018 Nov 13.

Abstract

A Trypanosoma brucei cell line is described that produces a visual readout of proteasome activity. The cell line contains an integrated transgene encoding an ubiquitin-green fluorescent protein (GFP) fusion polypeptide responsive to the addition of proteasome inhibitors. A modified version of T. brucei ubiquitin unable to be recognized by deubiquitinases (UbG76V) was fused to eGFP and constitutively expressed. The fusion protein is unstable but addition of the proteasome inhibitor lactacystin stabilizes it and leads to visually detectable GFP. This cell line can be widely used to monitor the efficiency of inhibitor treatment through detection of GFP accumulation in studies involving proteasome-mediated proteolysis, screening of proteasome inhibitors or other events related to the ubiquitin-proteasome pathway.

Keywords: Lactacystin; Proteasome; Trypanosoma brucei; Ubiquitin.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line*
  • Drug Evaluation, Preclinical / methods*
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Proteasome Endopeptidase Complex / genetics
  • Proteasome Endopeptidase Complex / metabolism
  • Proteasome Inhibitors / pharmacology*
  • Trypanocidal Agents / pharmacology*
  • Trypanosoma brucei brucei / drug effects*
  • Trypanosoma brucei brucei / enzymology
  • Trypanosoma brucei brucei / genetics
  • Trypanosomiasis, African / parasitology
  • Ubiquitin / metabolism

Substances

  • Proteasome Inhibitors
  • Trypanocidal Agents
  • Ubiquitin
  • Green Fluorescent Proteins
  • Proteasome Endopeptidase Complex