Chemistry-based functional Chemistry-based functional proteomics reveals novel proteomics reveals novel

members of the deubiquitinating members of the deubiquitinating enzyme familyenzyme family

Borodovsky A, Ovaa H, Kolli N, Gan-Erdene T, Wilkinson KD, Ploegh HL, Kessler BM

Chemistry and Biology 2002, 9, 1149-1159

UbiquitinationUbiquitination

Proteins don’t just “go away” Labeling with ubiquitin (a marker

protein) delivers proteins to the proteasome for degradation

ubiquitin

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the proteasome

The ubiquitin proteasome system (UPS)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 deubiquitinationUbiquitination and deubiquitination

S H N NH

R

O

HN

R'

S HN NHR

O

NR'

H

SN NH

R O

SN NH

R O

R' NH2

H2OSH N NH

DUB

Ub

gly76

gly76

ubiquitination

deubiquitination

Deubiquitinating enzymes (DUBs) can Deubiquitinating enzymes (DUBs) can rescue key cell-cycle proteinsrescue 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?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…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?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 profilingactivity-based profiling

DUB-Ub Michael adduct

Active DUB is tagged by modified UbActive 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

S HN

NH

Ub

HN SO2R

O

gly75

"gly76"

S

HNNH

Ub

HN SO2R

O

gly75

"gly76"H+

DUB DUB

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Suicide substratesSuicide substrates

**Used in immunoprecipitation studies

HA (hemagglutinin) tag allows detection using fluorescent anti-HA antibody

Incubation with whole-cell lysateIncubation with whole-cell lysate

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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 identifiedDUBs and their partners can be identified

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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 identifiedDUBs and their partners can be identified

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