Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery.
Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubl's and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling uses chemical probes that are active-site-directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.
Love
Kerry Routenberg
KR
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02143, USA.
Pandya
Renuka K
RK
Spooner
Eric
E
Ploegh
Hidde L
HL
D000595:Amino Acid Sequence; D000818:Animals; D051379:Mice; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics / Q000378:metabolism*
D015335:Molecular Probes; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D046988:Proteasome Endopeptidase Complex