Profiling proteasome activity in tissue with fluorescent probes.
With proteasome inhibitors in use in the clinic for the treatment of multiple myeloma and with clinical trials in progress investigating the treatment of a variety of hematologic and solid malignancies, accurate methods that allow profiling of proteasome inhibitor specificity and efficacy in patients are in demand. Here, we describe the development, full biochemical validation, and comparison of fluorescent proteasome activity reporters that can be used to profile proteasome activities in living cells with high sensitivity. Seven of the synthesized probes tested label proteasomes in lysates, although the fluorescent dye used affects their specificity. Two differentially labeled probes tested are suitable for studying proteasome activity in living cells by gel-based assays, by confocal laser scanning microscopy, and by flow cytometry. We established methods using these fluorescent reporters to profile proteasome activity in different mouse tissues, carefully avoiding postlysis artifacts, and we show that proteasome subunit activity is regulated in an organ-specific manner. The techniques described here could be used to study in vivo pharmacological properties of proteasome inhibitors.
Berkers
Celia R
CR
Division of Cellular Biochemistry, Tumor Biology, and Immunology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
van Leeuwen
Fijs W B
FW
Groothuis
Tom A
TA
Peperzak
Victor
V
van Tilburg
Erica W
EW
Borst
Jannie
J
Neefjes
Jacques J
JJ
Ovaa
Huib
H
D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D051379:Mice; D015394:Molecular Structure; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism
D005456:Fluorescent Dyes; D021122:Protein Subunits; D046988:Proteasome Endopeptidase Complex