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Author Spotlight: Advancements in Glycosomal pH Monitoring in Trypanosoma brucei Using pHluorin2 Biosensor
Published on: January 19, 2024
A new reporter cell line for studies with proteasome inhibitors in Trypanosoma brucei
Danielle M N Moura1, Osvaldo P de Melo Neto2, Mark Carrington3
1Instituto Aggeu Magalhães/FIOCRUZ, Av. Prof. Moraes Rego s/n, Cidade Universitária, Recife, CEP 50740-465, Pernambuco, Brazil; Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, United Kingdom.
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.
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