Aggregation and catabolism of disease-associated intra-Aβ mutations: reduced proteolysis of AβA21G by neprilysin☆
Introduction
Convergent evidence suggests that accumulation of the amyloid β-protein (Aβ) and its subsequent aggregation initiates a complex cascade that culminates in Alzheimer's disease (AD) (LaFerla and Oddo, 2005, Walsh and Selkoe, 2004). Five point mutations within the Aβ sequence that are associated with hereditary diseases similar or identical to AD are clustered around the central hydrophobic core of Aβ and include: the A21G Flemish mutation, E22K Italian mutation, E22G Arctic mutation, E22Q Dutch mutation and the D23N Iowa mutation (Fig. 1). These mutations have the potential to impact upon all factors known to regulate Aβ monomer levels, namely production, degradation and aggregation. To date, no simple correlation between Aβ production or aggregation and disease phenotype has emerged, but it, was suggested that the pathogenic effect of the Dutch, Flemish, Italian and Arctic mutations arise as a consequence of their resistance to proteolysis (Tsubuki et al., 2003).
A large number of proteases have been implicated in the catabolism of Aβ, but of these neprilysin (NEP, EC 3.4.24.11) and insulin-degrading enzyme (IDE, EC 3.4.22.11) are the most studied (Eckman and Eckman, 2005, Selkoe, 2001, Turner et al., 2004). NEP is a membrane-bound zinc-metallopeptidase that exists as an ectoenzyme preferentially hydrolysing extracellular oligopeptides on the amino side of hydrophobic residues (Carson and Turner, 2002). NEP has been shown by numerous investigators to be capable of degrading Aβ both in vivo (Iwata et al., 2004) and in vitro (Howell et al., 1995, Kanemitsu et al., 2003, Liu et al., 2007, Shirotani et al., 2001), and its physiologic role has been demonstrated by the finding that genetic ablation of NEP causes elevation of endogenous Aβ (Iwata et al., 2001), whereas transgenic or viral expression of NEP causes a lowering of cerebral Aβ (El-Amouri et al., 2007, Hemming et al., 2007, Iwata et al., 2004, Leissring et al., 2003, Marr et al., 2003). IDE is a zinc-metalloprotease which shows no obvious primary amino acid sequence specificity but has been proposed to recognize a conformation that is prone to conversion to β-sheet structure (Kurochkin, 1998). As with NEP, support for the physiological importance of IDE in regulating Aβ levels comes from the findings that genetic deletion of IDE in mice (Farris et al., 2003, Miller et al., 2003) leads to elevated levels of cerebral Aβ, whereas transgenic over-expression of IDE causes a decrease in brain Aβ levels (Leissring et al., 2003). Unlike NEP and IDE, plasmin (EC 3.4.21.7) does not appear to contribute significantly to the normal catabolism of Aβ (Tucker et al., 2004), but rather seems to play a role in the diseased brain. Plasmin is a serine protease that is produced from its inactive precursor, plasminogen, by the action of tissue-type plasminogen activator (tPA), and mice lacking either tPA or plasminogen clear injected Aβ much less well than wild type mice (Melchor et al., 2003). Moreover, recent studies indicate that high levels of Aβ cause a decrease in tPA activity suggesting that excessive accumulation of Aβ results, in part, due to a loss of plasmin (Cacquevel et al., 2007).
In order to investigate the possibility that intra-Aβ mutations mediate their effect through a common pathogenic mechanism, namely resistance to proteolysis, we examined the ability of NEP, IDE and plasmin to degrade both wild type (wt) Aβ(1–40) and Aβ(1–40) bearing disease-associated single amino acid substitutions (Fig. 1). Here we show that the ability of these proteases to degrade Aβ is strongly retarded by aggregation but that when Aβ peptides are presented in their unaggregated, monomeric state, they are efficiently degraded by NEP, IDE and plasmin, with one exception. The Aβ(1–40)A21G peptide is degraded more slowly by NEP than wt Aβ(1–40) or any of the other mutant peptides studied. This resistance to NEP-mediated proteolysis may represent one mechanism by which the A21G mutation causes increased cerebral accumulation of Aβ. On the other hand, our data suggest that resistance to proteolysis by the major known Aβ-degrading enzymes cannot explain the effects of the other four disease-associated intra-Aβ mutations.
Section snippets
Chemicals and reagents
Unless otherwise stated all chemicals were purchased from Sigma-Aldrich, St. Louis, MO, USA and were of the highest purity available.
Synthetic Aβ peptides
The six peptide sequences shown (Fig. 1) were synthesized and purified at the Bioploymer Laboratory in UCLA. Peptide mass and quantity were determined by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Table 1), and quantitative amino acid analysis (Walsh et al., 1997). In all cases peptide purity as determined by
All known disease-associated intra-Aβ mutations except the A21G substitution increase aggregation propensity
To our knowledge, this is the first report to directly compare the aggregation kinetics of wt Aβ(1–40) and Aβ(1–40) peptides bearing each of the five point mutations associated with familial disorders linked to AD. For this study, we used 2 independent methods to monitor the aggregation process and electron microscopy to confirm the presence of amyloid fibrils. Initially, aggregation was monitored by incubating peptides at 37 °C and measuring the loss of peptide from the supernate following
Discussion
This is the first study to simultaneously compare the relative aggregation rates and the susceptibility to degradation of Aβ peptides containing amino acid substitutions corresponding to all the known intra-Aβ mutations linked to AD-like disorders. Here we report that Aβ(1–40)E22G shows the highest propensity for aggregation (i.e., both rapid nucleation and fibril extension) and that Aβ(1–40)E22Q and Aβ(1–40)D23N show slightly slower, but similar aggregation kinetics. Aggregation of
Acknowledgments
We are grateful to Drs. Zuhair Nasrallah (UCD) Francesca Paradisi (UCD) and Kevin O’Connor (UCD) for their assistance using the HPLC system and Ann Molloy (UCD) for use of the micro-balance. We also thank Dr. Mark Findeis (Satori Pharmaceuticals Inc, Boston, MA, USA) for advice on the Thioflavin T binding assay and Drs. Celine Adessi and Fiona Grueninger (F. Hoffman-La Roche Ltd, Basel, Switzerland) for the gift of soluble NEP.
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This work was supported by Wellcome Trust grant 067660 (DMW), NIH grant AG027443 (DMW and DJS) and the Alzheimer's Association (RW). The mass spectrometry study was in part supported by an NIH/NCRR Shared Instrumentation Grant.