Samenvatting

Alzheimer.s disease (AD) is a neurodegenerative disease. The disease is characterized by the presence of neuropathology hallmarks such as: senile plaques, neurofibrillary tangles and the loss of synapses and neurons in the brain. The senile plaques are mainly composed of amyloid fibrils of the protein amyloid β (Aβ)4,5
Aβ arises naturally in the human brain throughout life as a result of metabolic processing of the amyloid precursor protein (APP)12. There are several Aβ peptides, which originate after cleavage of APP. The 42 residue long peptide, Aβ42 is the major component of the senile plaques in AD15. Alzheimer.s disease is thought to develop through a process called amyloid cascade where an increase of Aβ level results in oligomerization and aggregation of Aβ42 thereby triggering a downstream cascade of pathological events3.
More and more evidence indicates that especially the soluble Aβ oligomers (that are assemblies of the Aβ monomer) are neurotoxic and play an important role in the AD related pathology4,5,23,25.

The ultimate aim of this study was to solve the structure of Aβ42 in an oligomeric state for rational structure based drug design against Alzheimer.s disease. Protein purification and X-ray crystallography techniques were used. Aβ was linked to the apical domain of the chaperonin protein GroEL and the fusion protein ApicalGroEL-Aβ was created. This fusion inhibited the rapid fibrillization of Aβ, thereby forming the possibility to trap Aβ in an oligomeric state. Eleven different constructs were made. They contained a Nterminal His-tag followed by the apical domain of GroEL. In between the apical domain and Aβ different synthetic sequences, the so-called linkers were placed to manipulate the arrangement of Aβ. Also, different size combinations of the apical domain of GroEL and Aβ were tested.

The ApicalGroEL-Aβ constructs were expressed and purified and two different crystallization approaches were used. The first approach was to isolate the protein as a monomer and screen for crystallization. Since Aβ has the propensity to form oligomers when it is at a high enough concentration17, the assumption was made that the protein would oligomerize in the crystal. The second approach was to isolate ApicalGroEL-Aβ in an oligomeric state and to crystallize the protein in this oligomeric state. The protein monomer was purified, brought to high concentration and incubated to see if it would form oligomers in a time dependent manner, which could then be purified and crystallized.

Structures of three different ApicalGroEL-Aβ constructs in a monomeric state were solved. The electron density maps showed electron density for the apical domain of GroEL, up to the last residue. No connected and continuous electron density was present for the linker and Aβ. Further analysis suggested that Aβ is present in the crystals. The missing electron density could have been due to too much flexibility of Aβ in the unit cell. It could also be due to a disordered conformation of Aβ itself.
The ApicalGroEL-Aβ monomer oligomerizes in a time dependent manner. No attempt was made yet to isolate and crystallize these oligomers. The best approach would be to A structural study on the Alzheimer.s disease amyloid β peptide 5 isolate and crystallize the smaller size oligomers (dimers and trimers) since these are stable23,24.

Linking Aβ to the apical domain of GroEL offers advantages; the ApicalGroEL-Aβ protein is soluble and easy to purify and crystallize. In addition the apical domain of GroEL itself does not oligomerize, therefore a stable oligomer formed is due to the presence of Aβ. Unfortunately it was not possible to solve an oligomer structure of Aβ yet. However there are still multiple approaches that can be tried, hopefully one of them will eventually enable us to solve the structure of Aβ in an oligomeric state. Thereby making it possible to design drugs that specifically target Aβ oligomers and help to win the fight against Alzheimer.s disease.