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-8518660-B2
Publication Date
2013-08-27
Expiration Date
Abstract
Methods for detection of the activity of proteolytic enzymes, particularly isopeptidases, are disclosed.
Core Innovation
The invention relates to a diubiquitin for measuring the isopeptidase activity of a deubiquitinase. The diubiquitin includes a first ubiquitin molecule and a second ubiquitin molecule operably linked by an isopeptide bond from the C-terminus of the first ubiquitin molecule to the side chain of a lysine residue of the second ubiquitin molecule. The linkage is designed to present the isopeptidase reaction site in a physiologically relevant diubiquitin/polyubiquitin substrate context.
The first and second ubiquitin molecules are each operably linked to energy transfer pair members, where each energy transfer pair member is individually either a fluorescent group or a quenching group. The diubiquitin comprises cysteine residues at any position between residues 8 and 70, and the energy transfer pair members are attached to the first and second ubiquitin molecules at those cysteine residues. This arrangement enables an internally quenched fluorescence/FRET gain-of-signal readout associated with isopeptidase activity.
The disclosed approach addresses limitations of prior linear peptide/amide-bond assays for measuring isopeptidase, especially DUB, activity, by providing a diubiquitin probe that preserves substrate recognition while enabling fluorescence-based detection. The document further describes the use of such internally quenched fluorescence/FRET probes in detection and screening workflows, including high-throughput screening, with optional microarrays and microtiter formats.
Claims Coverage
One independent claim is provided. It defines a diubiquitin probe structured to measure deubiquitinase isopeptidase activity, with two independently specified energy transfer pair systems placed on cysteine residues between residues 8 and 70, and an isopeptide bond connecting the two ubiquitin molecules.
Diubiquitin probe for measuring deubiquitinase isopeptidase activity
A diubiquitin comprising a first ubiquitin molecule operably linked to at least one first energy transfer pair member and a second ubiquitin molecule operably linked to at least one second energy transfer pair member, wherein the first ubiquitin molecule is operably linked to the second ubiquitin molecule by an isopeptide bond from the C-terminus of the first ubiquitin molecule to the side chain of a lysine residue of the second ubiquitin molecule.
Paired fluorescent/quencher energy transfer pairs on engineered cysteine residues
The first and second energy transfer pair members are individually either a fluorescent group or a quenching group, and the first and second ubiquitin molecules comprise a cysteine residue at any position between residues 8 and 70; the first and second energy transfer pair members are attached to the first and second ubiquitin molecules at said cysteine residues.
The independent claim coverage centers on a diubiquitin probe that couples an isopeptide bond between ubiquitin C-terminus and a lysine side chain with internally positioned fluorescence/quenching energy transfer pairs attached to engineered cysteines between residues 8 and 70, thereby enabling measurement of deubiquitinase isopeptidase activity.
Stated Advantages
Provides a diubiquitin probe that enables fluorescence/FRET gain-of-signal readout for measuring isopeptidase activity.
Enables measuring isopeptidase activity using a physiologically relevant diubiquitin/polyubiquitin substrate format rather than linear peptide/amide-bond assays.
Supports high-throughput screening using fluorescence detection, including microarray and microtiter formats.
Documented Applications
Measuring the isopeptidase activity of a deubiquitinase using the disclosed diubiquitin probe.
Screening agents that modulate isopeptidase activity by contacting an isopeptidase with the diubiquitin and detecting fluorescence, where a fluorescence difference indicates modulation.
Use in high-throughput screening formats, including microarray and microtiter plate implementations.
Linkage-selectivity cleavage experiments using specific DUB/isopeptidase targets, with kinetic parameter determination using fluorescence readout in microtiter assays.
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