Abstract
There are several diseases which are caused by amyloid, a deposit of aggregated protein. Examples of these diseases are Alzheimer’s disease, caused by the aggregation of the peptide Aβ, and Diabetes type 2, caused by hIAPP aggregates. A large number of proteins interact with these amyloid fibrils, some preventing or
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inhibiting toxic effects whereas others enhance toxicity. Two groups of amyloid binding proteins, which reduce cytotoxicity, were studies: 1. single domain antibodies (VHH) and 2. Finger domain containing proteins, like tissue type plasminogen activator (tPA). Our initial hypothesis for the VHHs was that the VHH sequestered monomeric Aβ and by doing so preventing aggregation and reducing toxicity. However, solution state NMR experiments showed that these VHHs only bound to Aβ in fibrillar form. Based on crystal structures that were determined for these VHHs several residues that might be important for the binding were mutated. Subsequent experiments with these mutants showed important of these positively charged residues. A stretch of positively charged resides can interact with a stretch of negatively charged residues on the surface of Aβ fibrils. The finger domains were thought to be the amyloid binding domain of a family of proteins. After several unsuccessful attempts to gain more insight in the binding mechanism, truncated forms of tPA, which lacked the finger domain, were made to determine the importance of this domain for the binding. A number of techniques, like SPR, immuno-EM and pull down assays, were used to assess the binding capacities of the truncated constructs with full length tPA. Surprisingly, no differences in binding were observed, indicating that the finger domain is not the essential domain for amyloid binding. We propose that tPA interacts with amyloid fibrils using several domains. In contrast to the VHHs this interaction is not dominated by electrostatic interaction, which implies that hydrophobic interactions are important for the binding of tPA with amyloid. In our view proteins interact with amyloid aggregates through the repetitive amino acid side chains that are exposed on the surface of these highly structured aggregates. The interaction can be mediated through electrostatic interactions as we have showed for the VHHs or the binding can be via hydrophobic interactions
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