Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family Borodovsky A, Ovaa H, Kolli N, Gan-Erdene T, Wilkinson KD, Ploegh HL, Kessler BM Chemistry and Biology 2002, 9,

Ubiquitination Proteins don’t just “go away” Labeling with ubiquitin (a marker protein) delivers proteins to the proteasome for degradation ubiquitin the proteasome

The ubiquitin proteasome system (UPS) Proteins destined for proteasomal degradation are tagged with a chain of ubiquitin (Ub) proteins via multiple reactions catalyzed by ubiquitin activating (E1), conjugating (E2) and ligating enzymes (E3) Ubiquitination reactions are reversed by the action of deubiquitinating enzymes (DUBs)

Ubiquitination and deubiquitination DUB Ub gly 76 ubiquitination deubiquitination

Deubiquitinating enzymes (DUBs) can rescue key cell-cycle proteins The tumor suppressor p53 is deubiquitinated by USP7 (aka HAUSP) and cell growth is repressed Ubiquitin (Ub) is removed from histones during chromatin condensation Ub is removed from membrane receptors during endocytosis Expression of many DUBs is tissue specific Each DUB may be dedicated to a specific substrate

What do we know? Sequencing of open reading frames (ORFs) suggests > 40 DUBs in mammalian genomes DUBs belong to 2 families of cysteine proteases No sequence homology DUBs are specific for the hydrolysis of a peptide bond at the Ub C-terminus

Genetics doesn’t tell us… How many of the 40 DUBs are active Whether activity is tissue specific Whether any are constitutively inactive Evolution of different function Co-expressed with an inhibitor Proenzymes with an inhibitory domain

What do we want to know? How many DUBs are active cysteine proteases? How specific are DUBs for their substrates? Can we selectively inhibit a single DUB in the presence of other active cysteine proteases? activity-based profiling activity-based profiling

DUB-Ub Michael adduct Active DUB is tagged by modified Ub Inactive DUBs do not react Other cysteine proteases do not react Ub required for specificity If Ub is labeled we can identify active DUBs

Suicide substrates * *Used in immunoprecipitation studies HA (hemagglutinin) tag allows detection using fluorescent anti-HA antibody

Incubation with whole-cell lysate 8% SDS PAGE Visualized by immunoblotting with anti-HA If Ub is labeled we can identify active DUBs 1. Michael acceptors label with equal efficiency 2. Michael acceptors do not label with equal efficiency 3. Michael acceptors do not label with equal efficiency Alkyl halides label a subset of those proteins labeled by Michael acceptors

DUBs and their partners can be identified Anti-HA antibody covalently attached to agarose beads pulls down HA- labeled Ub-DUB conjugates Some reactive proteins are associated with unknown partners These non-covalently associated proteins can be retained by using a native gel to separate the proteins Protein identification is by tryptic digest and MS/MS

DUBs and their partners can be identified