Di- and poly-ubiquitin deubiquitinase substrates and uses thereof
Inventors
Chelur, Dattananda • Eddins, Michael • Leach, Craig • Orcutt, Steven J. • Peroutka, Raymond J. • Strickler, James • Yan, Yilin • Longenecker, Kathryn
Assignees
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Publication Number
US-9382574-B2
Publication Date
2016-07-05
Expiration Date
Abstract
Methods for detection of the activity of proteolytic enzymes, particularly isopeptidases, are disclosed.
Core Innovation
The invention provides improved fluorescently labeled diubiquitin and ubiquitin-like substrates for measuring the isopeptidase activity of a deubiquitinase. The substrates use internally quenched fluorescence in which energy transfer pairs convert cleavage of an isopeptide bond into a measurable fluorescent signal, including IQF/FRET-based gain-of-signal approaches.
The core substrate architecture links a first ubiquitin or ubiquitin-like molecule to a second ubiquitin or ubiquitin-like molecule by an isopeptide bond from the C-terminus of the first molecule to the side chain of a lysine residue of the second molecule. Both the first and second molecules are operably linked to at least one energy transfer pair member, where the first and second energy transfer pair members are individually either a fluorescent group or a quenching group, supporting linkage-selective diubiquitin substrates such as K63-linked and K48-linked diubiquitin.
The first and second ubiquitin or ubiquitin-like molecules further comprise a cysteine residue introduced at a position corresponding to amino acid residues at positions between 8 and 70 of the amino acid sequence of SEQ ID NO: 1, with the energy transfer pair members attached at the introduced cysteine residues. The substrate additionally comprises at least one additional ubiquitin or ubiquitin-like molecule linked to the first or second molecule.
Claims Coverage
The independent claim defines a substrate for measuring isopeptidase activity with one independently defined inventive concept and related refinements in the provided excerpt.
Isopeptide-bond linked first and second ubiquitin or ubiquitin-like molecules for deubiquitinase isopeptidase activity measurement
A first ubiquitin or ubiquitin-like molecule operably linked to at least one first energy transfer pair member, and a second ubiquitin or ubiquitin-like molecule operably linked to at least one second energy transfer pair member, wherein the first and second molecules are linked by an isopeptide bond from the C-terminus of the first ubiquitin or ubiquitin-like molecule to the side chain of a lysine residue of the second ubiquitin or ubiquitin-like molecule.
Cysteine-introduced labeling at positions corresponding to residues 8 to 70 of SEQ ID NO: 1
The first and second ubiquitin or ubiquitin-like molecules comprise a cysteine residue introduced at a position corresponding to amino acid residues at positions between 8 and 70 of SEQ ID NO: 1.
Energy transfer pair members as fluorescent group and quenching group
The first and second energy transfer pair members are individually either a fluorescent group or a quenching group, and are attached to the first and second ubiquitin or ubiquitin-like molecules at the introduced cysteine residues.
Additional ubiquitin or ubiquitin-like molecule linked to first or second molecule
The substrate further comprises at least one additional ubiquitin or ubiquitin-like molecule linked to the first or second ubiquitin or ubiquitin-like molecule.
The central claim coverage is the internally quenched fluorescence substrate concept for measuring deubiquitinase isopeptidase activity. The inventive features are the isopeptide bond connectivity, operable attachment of fluorescent and quenching energy transfer pair members, cysteine-introduced labeling, and inclusion of at least one additional ubiquitin or ubiquitin-like molecule.
Stated Advantages
Enables measurement of true isopeptidase activity using physiologically relevant diubiquitin substrates.
Produces a gain-of-signal upon cleavage of the isopeptide bond in internally quenched fluorescence and FRET-based readouts.
Supports linkage-selective cleavage by deubiquitinases, including K63-linked and K48-linked diubiquitin formats.
Allows determination of Michaelis-Menten kinetics, including Km and kcat, using fluorescence-based kinetic assays.
Suitable for high-throughput screening and microarray, array, and kit implementations.
Documented Applications
Measuring deubiquitinase or isopeptidase activity in fluorescence assays by contacting a sample with the substrate and detecting fluorescence change indicative of activity.
High-throughput screening using the fluorescence-based substrate formats.
Use in microarray, arrays, and kits for assay implementation.
Kinetic fluorescence monitoring to determine Michaelis-Menten kinetics, including Km and kcat, for isopeptidase activity.
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