Protein Aggregation, Fragmentation and its role in Neurodegenerative Diseases
Open Access
- Author:
- Bhide, Ashlesha
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 27, 2023
- Committee Members:
- Christine Keating, Major Field Member
Igor Aronson, Major Field Member
James Adair, Outside Unit & Field Member
Ayusman Sen, Chair & Dissertation Advisor
Philip Bevilacqua, Program Head/Chair - Keywords:
- Protein Aggregation
Protein Fragmentation
Alzheimer's disease
Liposome Aggregation
Alkaline Phosphatase - Abstract:
- Aggregation refers to the accumulation or clumping together of proteins. Aggregates can be characterized based on the size of aggregates, reversibility, structure of the aggregate and the conformation of proteins within aggregates. Protein aggregation plays an important role in neurodegenerative diseases and it is important to understand it so that we can find cures to these diseases. It could also cause errors in experimental measurements if assumptions are made about the size of the protein. The work presented here clarifies various aspects of protein aggregation and its role in neurodegenerative diseases as well as its application in targeted drug delivery. In Chapter 2, I investigate the aggregation and fragmentation of enzymes. Our lab has previously studied the enhanced diffusion of enzymes. Previous papers have shown that enzymes diffuse faster when the substrate of the enzyme is present. However, if the size of the enzyme changes, the diffusion will change inversely. So, if there is a change in the diameter of enzymes due to aggregation or fragmentation, it could cause artifacts in diffusion measurements. I investigated this phenomenon for enzymes in the presence of chemicals that are relevant to its catalysis such as the substrate of the enzyme, co-factor, product of the reaction etc. Using Fluorescence Resonance Energy Transfer (FRET), I showed that glucose oxidase fragments in the presence of its substrate, D-Glucose but not its non-substrate enantiomer, L-Glucose. I was also able to identify that a minimum of 0.3mM D-Glucose is needed to cause fragmentation. Interestingly, this is lower than the blood glucose concentration (4-6mM). This study showed that we cannot assume that the size of enzymes remains the same during catalysis. It is known that the enzyme, alkaline phosphatase also plays a role in Alzheimer’s disease (AD). Alkaline phosphatase activity increases in Alzheimer’s disease but the cause for this increase in activity was not known. It is important to investigate this activity increase because alkaline phosphatase plays a role in neurodegeneration by dephosphorylating tau protein in the extracellular space which causes death of other neurons and helps the progression of the disease. Using various spectroscopic methods, I show that the activity of alkaline phosphatase increases in the presence of the peptide, amyloid - β and acetylcholinesterase. I also show that the activity increases occur in the presence of high concentrations of acetylcholine and choline. Using FRET, I also demonstrate that there is an interaction between amyloid - β and alkaline phosphatase. These conditions occur during the disease or might occur due to various drugs that are used for treating AD. By investigating this, we aim to clarify the role of alkaline phosphatase in the disease. I also investigate the application of protein aggregation in targeted drug delivery systems in Chapter 4. Lipid-based delivery systems are commonly used for drug delivery due to their spherical compartmentalized structure and biocompatibility. In this work, using dynamic light scattering (DLS) and confocal microscopy, I demonstrate that we can obtain precise control over the aggregation of uncoated and protein-coated liposomes in the presence of salts such as zinc nitrate and calcium nitrate. The proteins we use are streptavidin and avidin. Our results show that liposome aggregation can be controlled selectively based on the protein attached to the outside of liposomes and the concentration of the salt solution added. I also demonstrate that we can control the aggregation of hard particles such as streptavidin-coated microspheres. By precisely controlling their aggregation in the presence of certain metal salts, we can improve the efficiency of lipid-based targeted drug delivery.