Walker T. McGraw

Metalloendopeptidase EC 3.4.24.15 is necessary for Alzheimer's amyloid-beta peptide degradation.

We have investigated the functional relationship between metalloendopeptidase EC 3.4.24.15 (MP24.15) and the amyloid precursor protein involved in Alzheimer’s disease (AD) and discovered that the enzyme promotes Aβ degradation. We show here that conditioned medium (CM) of MP24.15 antisense-transfected SKNMC neuroblastoma has significantly higher levels of Aβ. Furthermore, synthetic-Aβ degradation was increased or decreased following incubation with CM of sense or antisense-transfected cells, respectively. Soluble Aβ1–42 was degraded more slowly than soluble Aβ1–40, while aggregated Aβ1–42 showed almost no degradation. Pretreatment of CM with serine proteinase inhibitors 4-(2-aminoethyl)benzenesulfonyl fluoride and diisopropyl fluorophosphate completely inhibited Aβ degradation. Additionally, α1-antichymotrypsin (ACT), a serpin family inhibitor tightly associated with plaques and elevated in AD brain, blocked up to 60% of Aβ degradation. Interestingly, incubation of CM of MP24.15-overexpressing cells with ACT formed an SDS-resistant ACT complex, suggesting an ACT-serine proteinase interaction. Recombinant MP24.15 alone did not degrade Aβ. 14C-Diisopropyl fluorophosphate-radiolabeled CM from MP24.15-overexpressing cells contained increased levels of several active serine proteinases, suggesting that MP24.15 activates one or more Aβ-degrading serine proteases. Thus, ACT may cause Aβ accumulation by inhibiting an Aβ-degrading enzyme or by direct binding to Aβ, rendering it degradation-resistant. Identification of the Aβ-degrading enzyme and MP24.15’s role in its activation is underway. Pharmacological modulation of either enzyme may provide a means of regulating Aβ in the brain.

Heparin-dependent modification of the reactive center arginine of antithrombin and consequent increase in heparin binding affinity.

Antithrombin, the principal plasma inhibitor of coagulation proteinases, circulates in a form with low inhibitory activity due to partial insertion of its reactive site loop into the A-β-sheet of the molecule. Recent crystallographic structures reveal the structural changes that occur when antithrombin is activated by the heparin pentasaccharide, with the exception of the final changes, which take place at the reactive center itself. Here we show that the side chain of the P1 Arg of α-antithrombin is only accessible to modification by the enzyme peptidylarginine deiminase on addition of the heparin pentasaccharide, thereby inactivating the inhibitor, whereas the natural P1 His variant, antithrombin Glasgow, is unaffected, indicating that only the P1 Arg becomes accessible. Furthermore, the deimination of P1 Arg converts antithrombin to a form with 4-fold higher affinity for the heparin pentasaccharide, similar to the affinity found for the P1 His variant, due to a lowered dissociation rate constant for the antithrombin-pentasaccharide complex. The results support the proposal that antithrombin circulates in a constrained conformation, which when released, in this study by perturbation of the bonding of P1 Arg to the body of the molecule, allows the reactive site loop to take up the active inhibitory conformation with exposure of the P1 Arg.

α1-Antichymotrypsin Inhibits Aβ Degradation in Vitro and in Vivo

Abstract: The neuropathology of Alzheimers disease (AD) is characterized by extensive deposition of the toxic amyloid β peptide (Aβ) in selected regions of the brain and brain vasculature (Selkoe, 1999). Thus, lowering the levels of Aβ may be beneficial for AD patients. Aβ is a proteolytic fragment derived from the amyloid precursor protein (APP). The mechanisms of Aβ formation from its precursor have been studied extensively; however, considerably less effort has been invested into studying Aβ clearance. We find that the degradation of Aβ in our system is dependent upon the presence of a metallopeptidase E.C.3.4.24.15 (MP24.15) (Yamin et al., 1999). We have previously purified MP24.15 to homogeneity from AD brain and identified it as an APP‐processing protease in vitro (Papastoitsis, 1994). To confirm its role in cell culture, we transfected SKNMC neuroblastoma cells with sense and antisense cDNAs of MP24.15 and with a mock construct. Compared to mock conditioned media (CM), CM of MP24.15‐overexpressing cells had very high Aβ‐degrading activity. Conversely, CM of antisense‐expressing cells lacked Aβ‐degrading activity. These results suggested that MP24.15 is involved in Aβ degradation. Characterization of the proteolytic activity directly responsible for Aβ degradation using a spectrum of protease inhibitors revealed that only serine protease inhibitors completely blocked Aβ degradation. Therefore, MP24.15 appears to activate a serine protease, which then cleaves Aβ. Interestingly, α1‐antichymotrypsin (ACT) which we discovered to be highly elevated in AD brain (Abraham, et al., 1988) also inhibited Aβ degradation. To our delight, ACT proved to be an inhibitor of Aβ degradation in vivo as well. When we crossed transgenic mice expressing human ACT with plaque‐producing mice expressing human APP, the doubly transgenic mice had twice as many plaques at 20 months of age as the APP mice (Mucke et al., 2000). Successful completion of this study could lead to the design of reagents that would reduce the amyloid load in AD patients.