Fluorescence Resonance Energy Transfer (FRET) peptides are convenient tools for the study of peptidase specificity and enzymatic activity. The Insulysin FRET substrate [Mca]/[Dnp] is a synthetic insulysin peptide substrate that contains an N-terminal fluorescent 7-methoxycoumarin (Mca) group and a C-terminal 2, 4-dinitrophenyl (Dnp) quencher; the FRET peptide exhibits internal fluorescence quenching when intact. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence, enabling the quantitative measure of enzymatic activity.
Insulysin, also known as Insulin-degrading enzyme (IDE), is a highly conserved zinc metallopeptidase that contains an inverted zincin motif (HXXEH), characteristic of the inverzincin family of proteases. The two histidine (H) residues chelate with the zinc ion, and the glutamate (E) is involved in catalytic water activation. Insulysin functions as a dimer and exhibits allosteric kinetics, whereby small substrate peptides and polyphosphates such as ATP can act as enzyme activators. The endopeptidase cleaves a wide variety of substrates that are diverse in length, structure and sequence and exhibits a preference for basic or bulky hydrophobic residues.
Insulysin was initially discovered as the enzyme responsible for insulin catabolism and is ubiquitously expressed; studies have shown that it is involved in the degradation of many bioactive peptide substrates including glucagon, TGF-α, β-endorphin, dynorphins and atrial natriuretic peptide. Insulysin also selectively degrades amylin and β-amyloid amyloidogenic peptides at multiple sites. The imbalance between synthesis and clearance of β-amyloid peptides can lead to the accumulation and formation of amyloid plaques in the brain, which is one of the hallmarks of Alzheimer’s disease. Insulysin is a key component in the maintenance of proteostasis in many cellular processes, therefore the identification of novel potent and selective insulysin modulators can help advance the development of insulysin-based therapeutics for diseases such as type 2 diabetes and Alzheimer’s Disease.
Unravelling the effects of insulysin modulators on the multiplicity of insulysin targets will provide an insight of its pivotal role in interconnecting several cellular processes.
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Insulysin FRET substrate [Mca]/[Dnp]
catalogue number: crb1100406