| Summary: | Alzheimer's disease (AD) is the most common cause of dementia, and the deposition of amyloid β (Aβ) peptide in the AD brains is a hallmark of the disease. Other amyloidogenic proteins like Transthyretin (TTR) and human Cystatin C (hCC) can modulate the aggregation of Aβ. These two proteins are proposed to play a role in the pathophysiology of AD as they are found co-deposited in amyloid deposits in the brains of AD patients, most notably at the cell surface. Animal models and cell line assays showed protective roles for TTR and hCC against Aβ-induced toxicity, and Aβ fibril formation is inhibited through interaction with TTR and hCC. This study investigated the mechanism of in vitro interaction of TTR with Aβ. The ability of WT TTR to inhibit Aβ fibrillisation in the presence of Aβ binding surfaces was higher than in the presence of non-binding surfaces. Then, the interaction between different TTR mutants with different multimeric stabilities and different alloforms of Aβ showed that TTRs with less stable tetramers and unfolded monomers are the best inhibitors of Aβ fibrillisation. Analysing the thioflavin T curves of Aβ aggregation in the presence of TTRs and the interaction of TTRs with different forms of Aβ showed that the interaction between the two proteins occurs mainly through binding to early nucleating species of Aβ rather than to the monomer. The dose-dependent inhibition of Aβ fibrillisation and the promotion of amorphous aggregates by hCC were validated. However, the previous suggestion of simple monomer to monomer interaction between Aβ and hCC was not confirmed. Instead, a proposed hCC binding to oligomeric species of Aβ was supported by the observed hCC interaction with different aggregated species of Aβ. The dimeric form of hCC was found to be a less effective inhibitor compared to WT monomer, indicating that the active site could be the hydrophobic loops involved in dimerisation and protease inhibition. This interpretation was supported, as mutation of hydrophobic residues in the active site significantly reduced the intensity of hCC to inhibit Aβ fibrillisation. We showed that these two proteins are mainly inhibit Aβ fibrillisation through interaction with the early aggregated structures of Aβ (some form of oligomers). As it is known that oligomers are responsible for Aβ toxicity in vivo, the potential of these two proteins to be used as natural modulators is supported by this study.
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