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Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family

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, 1149-1159. Ubiquitination. Proteins don’t just “go away”

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Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family

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  1. 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, 1149-1159

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

  3. 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)

  4. Ubiquitination and deubiquitination gly76 gly76 ubiquitination DUB Ub deubiquitination

  5. 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

  6. 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

  7. 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

  8. 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

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

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

  11. Incubation with whole-cell lysate 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 • 8% SDS PAGE • Visualized by immunoblotting with anti-HA • If Ub is labeled we can identify active DUBs

  12. DUBs and their partners can be identified 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 Anti-HA antibody covalently attached to agarose beads pulls down HA-labeled Ub-DUB conjugates Protein identification is by tryptic digest and MS/MS

  13. DUBs and their partners can be identified

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