Alzheimer's disease (AD) has been linked to the deposition of proteinaceous plaques in certain regions of the brain and cerebrovasculature. Although plaques contain many proteins, the major component is a 42 amino acid peptide named the amyloid beta peptide (AB), which is a fragment of a membrane protein known as the amyloid precursor protein (APP). The function of APP is not known, but under normal conditions, APP goes through a degredation cycle generating a large fragment containing the AB peptide which can then be further degraded by the cell. In certain genetic backgrounds or after other poorly understood changes, the degradation pathway is altered and proteolytic cleavages produce AB itself, which can spontaneously form large, insoluble fibrils that initiate the formation of plaques. Current research suggests that, during the progression of AB to fibrils, intermediates are formed which are neurotoxic, causing neurons to degenerate through apoptosis. The loss of neurons results in the dementia of AD.
Many drug companies are hoping to find ways to inhibit AB from forming fibrils, thus alleviating the formation of plaques and subsequently AD. Chemically synthesized AB is being used for these studies but it is expensive to synthesize and impure due to the process itself. Purity is an important issue for the inhibition studies. Dr. Przybyla's group has developed a vector to recombinantly express large amounts or AB and the proceedures needed to isolate the peptide to very high levels of purity. The procecedures are difficult, as the peptide is extremely hydrophobic, and chromatography steps (HPLC) must be performed at 80ºC. The vector and processes are currently under patent.
The group is also using recombinant peptides to determine the NMR structure of AB. Uniformly 15N and 13C-labelled AB have been expressed and purified in milligram quantities for this purpose. NMR will give us the first structure of AB, as crystallization of the peptide has failed. NMR should also allow us to determine the structure of AB bound to inhibitors discovered by pharmaceutical companies, so that more efficient inhibitors can be modelled and synthesized.
The group is also using site-directed and random mutagenesis to define which of the 42 residues of AB are responsible for aggregation and fibril formation. It is hoped that mutations will be defined that will allow AB monomers to aggregate, but not to form fibrils. These studies could lead to the development of small peptides capable of inhibiting wild type AB monomers from forming fibrils and plaques.
This reasearch is supported by grants from The Alzheimer's Association, Parke-Davis/Warner Lambert Pharmaceutical Company and Yamanuchi Pharmaceuticals.
