Some mutations which occur in the α/β-discordant region (resides 15 to 23) of β-amyloid peptide (Aβ) lead to familial Alzheimer's disease (FAD). In vitro studies have shown that these genetic mutations could accelerate Aβ aggregation. We recently showed that mutations in this region could alter the structural propensity, resulting in a different aggregative propensity of Aβ. Whether these genetic mutations display similar effects remains largely unknown. Here, we characterized the structural propensity and aggregation kinetics of Dutch-type Aβ 40 (Aβ 40 (E22Q)) and its L17A/F19A-substituted mutant (Aβ 40 (L17A/F19A/E22Q)) using circular dichroism spectroscopy, nuclear magnetic spectroscopy, and thioflavin T fluorescence assay. In comparison with wild-type Aβ 40 , we found that Dutch-type mutation, unlike Artic-type mutation (E22G), does not reduce the α-helical propensity of the α/β-discordant region in sodium dodecyl sulfate micellar solution. Moreover, we found that Aβ 40 (L17A/F19A/E22Q) displays a higher α-helical propensity of the α/β-discordant region and a slower aggregation rate than Aβ 40 (E22Q), suggesting that the inhibition of aggregation might be via increasing the α-helical propensity of the α/β-discordant region, similar to that observed in wild-type and Artic-type Aβ 40 . Taken together, Dutch-type and Artic-type mutations adopt different mechanisms to promote Aβ aggregation, however, the L17A/F19A mutation could increase the α-helical propensities of both Dutch-type and Artic-type Aβ 40 and inhibit their aggregation.
Inhibitor-1 is converted into a potent inhibitor of native protein phosphatase-1 (PP1) when Thr35 is phosphorylated by cAMP-dependent protein kinase (PKA). However, PKA-phosphorylated form of inhibitor-1 displayed a weak activity in inhibition of recombinant PP1. The mechanism for the impaired activity of PKA-phosphorylated inhibitor-1 toward inhibition of recombinant PP1 remained elusive. By using NMR spectroscopy in combination with site-directed mutagenesis and inhibitory assay, we found that the interaction between recombinant PP1 and the consensus PP1-binding motif of PKA-thiophosphorylated form of inhibitor-1 was unexpectedly weak. Unlike binding to native PP1, the subdomains 1 (residues around and including the phosphorylated Thr35) and 2 (the consensus PP1-binding motif) of PKA-thiophosphorylated form of inhibitor-1 do not exhibit a synergistic effect in inhibition of recombinant PP1. This finding implied that a slight structural discrepancy exists between native and recombinant PP1, resulting in PKA-thiophosphorylated form of inhibitor-1 displaying a different affinity to native and recombinant enzyme.
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