Abstract
Alzheimer’s disease is the most common type of dementia which effects millions of people worldwide. The disease is still not very well understood however two main hallmarks of the disease have been identified, those are senile plaques and neurofibrillary tangles. The hallmarks are caused by amyloid-beta peptide and tau protein respectively.Amyloid-beta peptide exists in three different forms, monomer, oligomers and fibrils. There is growing evidence which suggests that with age uncontrolled accumulation of amyloid-beta oligomers/fibrils due to reduced levels of enzymatic degradation is one of the main causes of toxicity in the brain leading to Alzheimer’s disease.
The notion of slowing down disease progression by clearing amyloid-beta peptide from the brain has been present for a long time. However, only recently treatments have been developed which support this hypothesis. Recent antibody treatments (such as Lecanemab and Donanemab) which target the clearance of amyloid-beta peptide have shown significant reduction in the rate of Alzheimer’s disease progression. Unfortunately, these treatments can result in severe side effects, showing that research into more novel amyloid-beta clearing methods is necessary.
One possible alternative to antibodies for amyloid-beta clearance is enzyme therapy by amyloid-beta degrading enzymes. Some of the more common amyloid-beta degrading enzymes are zinc metalloproteases: neprilysin, endothelin-converting enzyme, insulin degrading enzyme, and angiotensin-converting enzyme. Increasing the activity or expression of these enzymes could compensate for the observed reduction in amyloid-beta clearance. Studies on mice have found that upregulation of amyloid-beta degrading enzymes showed reduction in amyloid-beta levels in the mice brain, leading to improved cognitive function in some subjects.
The research described in this thesis specifically focuses on neprilysin as the target enzyme. Neprilysin is a promiscuous enzyme involved in many physiological processes within the human body. In addition to amyloid-beta clearance, neprilysin has also been linked to other processes such as blood pressure regulation. Imbalances in neprilysin activity could have a positive effect on amyloid-beta clearance, but a negative effect on blood pressure regulation and vice versa. One of the methods which could help tackle the problem of neprilysin’s promiscuity is enzyme engineering. Successful engineering of neprilysin could increase its specificity and affinity for the amyloid-beta peptide, resulting in more efficient peptide clearance without the side effects of disrupting blood pressure regulation.
In this research, neprilysin was recombinantly expressed and purified from yeast cells, with two different cell lines explored. The two cell lines (GS115 and SMD1168H) were tested alongside each other to assess differences in the yield of recombinant protein expression. High expression yield of neprilysin is necessary for future experiments (i.e. X-ray crystallography). Additionally, to exploring different Pichia pastoris cell lines, the yeast cell pellet was analysed for non-secreted protein. Neprilysin is expressed with the utilisation of an alpha secretion signal, however certain complications (such as hyper-glycosylation) can have negative effects on protein secretion. In parallel to trials of improving protein expression yield, expressed neprilysin was used for X-ray crystallography studies to further characterise the protein structure and active site residues responsible for ligand recognition. Through co-crystallisation studies of neprilysin with peptide-like inhibitors, neprilysin’s exosite and glycosylation was further explored within this study. Several mutagenesis studies were approached with the aim of increasing neprilysin’s specificity and affinity for amyloid-beta peptide. Native and mutant neprilysin were tested on a fluorescence activity assay to observe any differences in enzymatic activity. Differences in activity can be used as an indicator of whether a mutation will have an effect on neprilysin’s specificity. In the future successful neprilysin mutants could be tested on neprilysin’s physiological substrates such as the amyloid-beta peptide. Successful engineering of enzymes such as neprilysin could aid the development of enzyme therapeutics for Alzheimer’s disease.
Overall, research presented in this thesis should aid with the understanding of neprilysin’s recombinant protein expression in yeast cell lines. Moreover, the novel structures of neprilysin in complex with inhibitors help further characterise neprilysin’s glycosylation and exosite. Finally, mutagenesis of neprilysin active site residues shows potential candidates for future activity studies of neprilysin against its physiological substrates.
| Date of Award | 18 Feb 2026 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Ravi Acharya (Supervisor) & Vasanta Subramanian (Supervisor) |
Cite this
- Standard