Mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins: Synthesis of active site-directed probes

Huib Ovaa, Paul J. Galardy, Hidde L. Ploegh

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

The families of ubiquitin and ubiquitin-like modifiers are involved in the regulation of many biochemical pathways. The steady-state level of polypeptide modification with these molecules depends on the opposing activity of conjugating and deconjugating enzymes. Here we describe the generation of mechanism-dependent active site-directed probes that target the large family of ubiquitin/ubiquitin-like isopeptidases. To maintain substrate specificity for these enzymes, we have based the development of these probes on full-length sequences of ubiquitin and several ubiquitin-like molecules. For their construction, this approach necessitates the use of a combination of organic synthesis and expressed protein ligation. These probes have been used in the isolation and identification of active isopeptidases from crude cell extracts and have been instrumental in the discovery of novel ubiquitin and ubiquitin-like deconjugating enzymes. These probes may be generated with or without an epitope tag that enables activity profiling of proteases from cell extracts. In addition, we have used a ubiquitin-based probe in the structural analysis of the ligand-bound form of the enzyme UCH-L3 by x-ray crystallography. Together, these probes greatly facilitate the study of ubiquitin and ubiquitin-like proteases.

Original languageEnglish (US)
Article number32
Pages (from-to)468-478
Number of pages11
JournalMethods in Enzymology
Volume399
DOIs
StatePublished - 2005
Externally publishedYes

Fingerprint

Ubiquitins
Ubiquitin
Proteomics
Catalytic Domain
Enzymes
Cell Extracts
Peptide Hydrolases
Synthetic Chemistry Techniques
Crystallography
Molecules
Substrate Specificity
Complex Mixtures
Ligation
Structural analysis
Epitopes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins : Synthesis of active site-directed probes. / Ovaa, Huib; Galardy, Paul J.; Ploegh, Hidde L.

In: Methods in Enzymology, Vol. 399, 32, 2005, p. 468-478.

Research output: Contribution to journalArticle

@article{8d13ef33d67a45dd9b6f247680478997,
title = "Mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins: Synthesis of active site-directed probes",
abstract = "The families of ubiquitin and ubiquitin-like modifiers are involved in the regulation of many biochemical pathways. The steady-state level of polypeptide modification with these molecules depends on the opposing activity of conjugating and deconjugating enzymes. Here we describe the generation of mechanism-dependent active site-directed probes that target the large family of ubiquitin/ubiquitin-like isopeptidases. To maintain substrate specificity for these enzymes, we have based the development of these probes on full-length sequences of ubiquitin and several ubiquitin-like molecules. For their construction, this approach necessitates the use of a combination of organic synthesis and expressed protein ligation. These probes have been used in the isolation and identification of active isopeptidases from crude cell extracts and have been instrumental in the discovery of novel ubiquitin and ubiquitin-like deconjugating enzymes. These probes may be generated with or without an epitope tag that enables activity profiling of proteases from cell extracts. In addition, we have used a ubiquitin-based probe in the structural analysis of the ligand-bound form of the enzyme UCH-L3 by x-ray crystallography. Together, these probes greatly facilitate the study of ubiquitin and ubiquitin-like proteases.",
author = "Huib Ovaa and Galardy, {Paul J.} and Ploegh, {Hidde L.}",
year = "2005",
doi = "10.1016/S0076-6879(05)99032-0",
language = "English (US)",
volume = "399",
pages = "468--478",
journal = "ImmunoMethods",
issn = "1046-2023",
publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins

T2 - Synthesis of active site-directed probes

AU - Ovaa, Huib

AU - Galardy, Paul J.

AU - Ploegh, Hidde L.

PY - 2005

Y1 - 2005

N2 - The families of ubiquitin and ubiquitin-like modifiers are involved in the regulation of many biochemical pathways. The steady-state level of polypeptide modification with these molecules depends on the opposing activity of conjugating and deconjugating enzymes. Here we describe the generation of mechanism-dependent active site-directed probes that target the large family of ubiquitin/ubiquitin-like isopeptidases. To maintain substrate specificity for these enzymes, we have based the development of these probes on full-length sequences of ubiquitin and several ubiquitin-like molecules. For their construction, this approach necessitates the use of a combination of organic synthesis and expressed protein ligation. These probes have been used in the isolation and identification of active isopeptidases from crude cell extracts and have been instrumental in the discovery of novel ubiquitin and ubiquitin-like deconjugating enzymes. These probes may be generated with or without an epitope tag that enables activity profiling of proteases from cell extracts. In addition, we have used a ubiquitin-based probe in the structural analysis of the ligand-bound form of the enzyme UCH-L3 by x-ray crystallography. Together, these probes greatly facilitate the study of ubiquitin and ubiquitin-like proteases.

AB - The families of ubiquitin and ubiquitin-like modifiers are involved in the regulation of many biochemical pathways. The steady-state level of polypeptide modification with these molecules depends on the opposing activity of conjugating and deconjugating enzymes. Here we describe the generation of mechanism-dependent active site-directed probes that target the large family of ubiquitin/ubiquitin-like isopeptidases. To maintain substrate specificity for these enzymes, we have based the development of these probes on full-length sequences of ubiquitin and several ubiquitin-like molecules. For their construction, this approach necessitates the use of a combination of organic synthesis and expressed protein ligation. These probes have been used in the isolation and identification of active isopeptidases from crude cell extracts and have been instrumental in the discovery of novel ubiquitin and ubiquitin-like deconjugating enzymes. These probes may be generated with or without an epitope tag that enables activity profiling of proteases from cell extracts. In addition, we have used a ubiquitin-based probe in the structural analysis of the ligand-bound form of the enzyme UCH-L3 by x-ray crystallography. Together, these probes greatly facilitate the study of ubiquitin and ubiquitin-like proteases.

UR - http://www.scopus.com/inward/record.url?scp=28844453248&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=28844453248&partnerID=8YFLogxK

U2 - 10.1016/S0076-6879(05)99032-0

DO - 10.1016/S0076-6879(05)99032-0

M3 - Article

C2 - 16338376

AN - SCOPUS:28844453248

VL - 399

SP - 468

EP - 478

JO - ImmunoMethods

JF - ImmunoMethods

SN - 1046-2023

M1 - 32

ER -