Solution nuclear magnetic resonance studies of proteins involved in Alzheimer's disease

Abstract

As an intrinsically disordered protein (IDP), the Aß monomer is unsuitable for traditional, experimental structure determination methods. This has excluded Aß from molecular docking studies that are routinely performed with NMR and crystal structures of proteins to determine novel interactions with large databases of compounds. There are REMD simulations of Aß that are supported by experimental data in the literature that present a possible means of determining novel Aß drug interactions using molecular docking. We have aimed to determine if such screening was possible using centroid structures of Aß from these REMD simulations, verifying the results with solution NMR and amyloid aggregation assays. This screening positively identified a novel binder, the anticancer drug tranilast, as well as known binders found in the literature.

Alzheimer's disease (AD) accounts for 60-80 percent of all dementia cases, affecting ~5.3 million people in the US alone. According to the amyloid cascade hypothesis, AD pathology is due to the aggregation of amyloid-ß; peptide (Aß). We have developed a novel disaggregation treatment for the E22 deletion FAD mutant of Aß40, allowing for the observation and studies of the monomer of this mutant by solution NMR for the first time. Isotopically labeled monomer samples of E22 deleted Aß40, supported by REMD simulations of the peptide, have demonstrated distinct differences from WT Aß40 in hydrogen bond propensities that may explain the unique aggregation properties of E22 deleted Aß40. Our similar NMR studies of E22G Aß40 have revealed key differences in hydrogen bond propensities from WT Aß40, as well as differences in backbone dynamics on the ps-ns time scale.

Mitochondrial dysfunction is a hallmark of Alzheimer's disease (AD) pathology. This dysfunction is accompanied by oxidative stress that some believe to be a direct cause of Aß toxicity and is the trigger for increased Aß production. However, there exists more evidence that Aß is the cause of mitochondrial dysfunction. Cyclophilin D (CypD) is one protein thought to have a major role in Aß oligomer toxicity in mitochondria. The toxicity associated with this interaction is inhibited in APP transgenic mice by cyclosporine A (CsA). We have determined that the CsA binding site is disrupted in the R55G mutant of CypD, which does not bind Aß oligomers. We also determined that the R55G mutant does not bind CsA. These results indicate that CsA and Aß oligomers have the same or similar binding sites on CypD.