Two-Step Bioorthogonal Activity-Based Protein Profiling of Individual Human Proteasome Catalytic Sites.
Chembiochem
; 21(1-2): 248-255, 2020 01 15.
Article
in En
| MEDLINE
| ID: mdl-31597011
ABSTRACT
Bioorthogonal chemistry allows the selective modification of biomolecules in complex biological samples. One application of this methodology is in two-step activity-based protein profiling (ABPP), a methodology that is particularly attractive where direct ABPP using fluorescent or biotinylated probes is ineffective. Herein we describe a set of norbornene-modified, mechanism-based proteasome inhibitors aimed to be selective for each of the six catalytic sites of human constitutive proteasomes and immunoproteasomes. The probes developed for ß1i, ß2i, ß5c, and ß5i proved to be useful two-step ABPs that effectively label their developed proteasome subunits in both Raji cell extracts and living Raji cells through inverse-electron-demand Diels-Alder (iEDDA) ligation. The compound developed for ß1c proved incapable of penetrating the cell membrane, but effectively labels ß1c in vitro. The compound developed for ß2c proved not selective, but its azide-containing analogue LU-002c proved effective in labeling of ß2c via azide-alkyne click ligation chemistry both in vitro and inâ
situ. In total, our results contribute to the growing list of proteasome activity tools to include five subunit-selective activity-based proteasome probes, four of which report on proteasome activities in living cells.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Proteasome Endopeptidase Complex
Limits:
Humans
Language:
En
Journal:
Chembiochem
Journal subject:
BIOQUIMICA
Year:
2020
Document type:
Article
Affiliation country:
Netherlands