Abstract
Enzymatic cleavage of Nedd8 from its cullin conjugates plays a critical role in ubiquitin-dependent proteolysis by regulating the activity of the cullin-based RING-H2 E3 ubiquitin ligases. This chapter provides methods for the preparation of two Nedd8 isopeptidases: the COP9 signalosome and human deneddylase 1. It also describes the development of cell-free systems for cleavage of the Nedd8-cullin 1 isopeptide bond formed in vitro or in vivo.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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COP9 Signalosome Complex
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Carrier Proteins / biosynthesis
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Carrier Proteins / genetics
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Carrier Proteins / isolation & purification
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Cell Cycle Proteins / metabolism*
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Cell Line
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Chromatography, Gel / methods
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Cullin Proteins / metabolism*
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Endopeptidases / biosynthesis
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Endopeptidases / genetics
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Endopeptidases / isolation & purification
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Endopeptidases / metabolism*
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Humans
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Hydrolysis
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Multiprotein Complexes / isolation & purification
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Multiprotein Complexes / metabolism*
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NEDD8 Protein
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Oligopeptides
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Peptide Hydrolases / isolation & purification
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Peptide Hydrolases / metabolism*
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Peptides / genetics
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Peptides / isolation & purification
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Peptides / metabolism
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Phosphorus Radioisotopes
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Ubiquitins / metabolism*
Substances
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Carrier Proteins
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Cell Cycle Proteins
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Cullin 1
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Cullin Proteins
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Multiprotein Complexes
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NEDD8 Protein
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NEDD8 protein, human
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Oligopeptides
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Peptides
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Phosphorus Radioisotopes
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RBX1 protein, human
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Recombinant Fusion Proteins
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Ubiquitins
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FLAG peptide
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Endopeptidases
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Peptide Hydrolases
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COP9 Signalosome Complex
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SENP8 protein, human