The rate of the insulin-degrading enzyme (IDE)-catalyzed hydrolysis of the fluorogenic substrate 2-aminobenzoyl-GGFLRKHGQ-ethylenediamine-2,4-dinitrophenyl is increased 2-7-fold by other peptide substrates but not by peptide non-substrates. This increased rate is attributed to a decrease in K m with little effect on V max . An ϳ2.5-fold increase in the rate of amyloid ␤ peptide hydrolysis is produced by dynorphin B-9. However, with insulin as substrate, dynorphin B-9 is inhibitory. Immunoprecipitation of differentially tagged IDE and gel filtration analysis were used to show that IDE exists as a mixture of dimers and tetramers. The equilibrium between dimer and tetramer is concentration-dependent, with the dimer the more active form. Bradykinin shifted the equilibrium toward dimer. Activation of substrate hydrolysis is not seen with a mixed dimer of IDE containing one active subunit and one subunit that is catalytically inactive and deficient in substrate binding. On the other hand, a mixed dimer containing one active subunit and one subunit that is catalytically inactive but binds substrate with normal affinity is activated by peptides. These findings suggest that peptides bind to one subunit of IDE and induce a conformational change that shifts the equilibrium to the more active dimer as well as activates the adjacent subunit. The selective activation of IDE toward amyloid ␤ peptide relative to insulin suggests the potential for development of compounds that increase IDE activity toward amyloid ␤ peptide as a therapeutic intervention for the treatment of Alzheimer's disease.

Insulysin (EC. 3.4.22.11) has been implicated in the clearance of beta amyloid peptides through hydrolytic cleavage. To further study the action of insulysin on Abeta peptides recombinant rat insulysin was used. Cleavage of both Abeta(1-40) and Abeta(1-42) by the recombinant enzyme was shown to initially occur at the His(13)-His(14), His(14)-Gln(15), and Phe(19)-Phe(20) bonds. This was followed by a slower cleavage at the Lys(28)-Gly(29), Val(18)-Phe(19), and Phe(20)-Ala(21) positions. None of the products appeared to be further metabolized by insulysin. Using a rat cortical cell system, the action of insulysin on Abeta(1-40) and Abeta(1-42) was shown to eliminate the neurotoxic effects of these peptides. Insulysin was further shown to prevent the deposition of Abeta(1-40) onto a synthetic amyloid. Taken together these results suggest that the use of insulysin to hydrolyze Abeta peptides represents an alternative gene therapeutic approach to the treatment of Alzheimer's disease.

Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K m of 13 M and a V max of 2.6 mol min ؊1 mg ؊1 . Derivatives of this peptide in which the P 2 leucine or the P 2 ' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P 2 residue produced a 4-fold range in K m and a 7-fold range in k cat . The nature of the P 2 residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond. Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the R-K bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and R-K were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P 2 ' residues produce less of a change in both K m and k cat and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the R-K bond, produce significant cleavage at the L-R bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site.Insulysin (insulin-degrading enzyme; EC 3.4.24.56) was first described as a proteolytic enzyme capable of degrading insulin (1). The enzyme is primarily located in the cytosol and peroxisomes (2), although its presence on the cell membrane (3, 4) and its secretion (5) have recently been reported. Although insulysin has the highest affinity for insulin, the enzyme has been shown to cleave a number of other physiological peptides in vitro including glucagon (6), insulin-like growth factors I and II (7), atrial natriuretic peptide (8), and transforming growth factor-␣ (9). The finding of a variety of substrates for the enzyme, as well as its presence at high levels in insulin-insensitive cells, suggest that insulysin has a variety of physiological functions. Those proposed include processing of insulin for antigen recognition (10), regulation of the multicatalytic proteinase (11), and modulation of steroid receptor action (12).Insulysin was shown to be identical to an enzymatic activity referred to as ␥-endorphin-generating enzyme, an enzyme that converts ␤-endorphin to ␥-endorphin (␤Ϫendorphin 1-17) and ␤Ϫendorphin 1-18 (13). In that study GRF, dynorphin B 1-13, dynorphin A 1-17, and pancreastatin 1-49 were shown also to be substrates. Recent interest in insulysin stems from its ability to degrade the amyloid peptides A␤ 1Ϫ40 and A␤ 1-42 (5, 14 -16) and its possible role as an enzyme involved in the clearance of amyloid peptides in the brain. Decreases in insulysin activity in the brains...

Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP). In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro. In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis. In contrast, overexpression of pitrilysin protects insulinoma cells from human islet amyloid polypeptide-induced apoptosis. Since pitrilysin is a mitochondrial protein, we used immunofluorescence staining of pancreases from human IAPP transgenic mice and Western blot analysis of IAPP in isolated mitochondria from insulinoma cells to provide evidence for a putative intramitochondrial pool of IAPP. These results suggest that pitrilysin regulates islet amyloid polypeptide in beta cells and suggest the presence of an intramitochondrial pool of islet amyloid polypeptide involved in beta-cell apoptosis.

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