On the molecular mechanisms of the amyloid β-peptide aggregation

Abstract

The pathogenesis of Alzheimer’s disease is widely believed to be due to production and deposition of the amyloid β-peptide. Several variants of the Aβ peptide are known to exist in in vivo. Variations include mutations or additional functional groups attached to residue side chains and may affect the aggregation process. Early on-set Alzheimer’s is caused by a variety of single amino acid substitutions of the Aβ peptide.

The objectives of this thesis were to find a method to predict the aggregation propensity of Aβ40 variants and to understand the molecular mechanism of Aβ40 or Aβ42 peptide variants. We have extensively used thioflavin T-based fluorescence kinetic experiments to study the aggregation kinetics and the global analysis using the Amylofit platform to understand the molecular mechanism of aggregation of the variants. We showed that the aggregation propensity of Aβ40 variants can be predicted by monitoring the levels of inclusion body formation from peptides that are recombinantly expressed in E.coli cells. We could demonstrate that the net charge of a mutant greatly influences its aggregation propensity. We investigated the influence of various set of mutations on the aggregation mechanism of Aβ peptides. We showed that the aggregation behaviour is greatly modulated by the unstructured N-terminus of the Aβ peptide both in its 40- and 42-residue form. We could establish that specific residues at different positions in the primary sequence of Aβ40 peptide determined different stages of fibril formation. Especially, residues at positons 1, 7 and 13 of the Aβ40 peptide determined the specificity of secondary nucleation process of wild-type monomer. Besides this, we could confirm that the intact sequence of N-terminus is important in the aggregation process of Aβ42 peptide as reduced secondary nucleation rates were observed when residues at the N-terminus were scrambled. We addressed the influence of phosphorylation of two serine residues in the Aβ42 peptide on the aggregation process by designing single amino acid phosphomimic mutants. The rate of secondary nucleation is reduced more significantly for Ser26 substitutions than for Ser8 substitutions. Additionally, we found that Ser26 is a critical residue in the secondary nucleation of the Aβ42 peptide.

Abstract (Swedish)

Proteins are tiny chemical machines that play many critical roles in our body. Some proteins send important messages, some carry crucial supplies, some fight with invaders and protect body from diseases, some clear trash etc. Proteins are strings of hundreds and thousands of small units called amino acids. If the string is built from less than hundred amino acids, it is called a peptide. DNA, a long, double-stranded and spiral-shaped molecule inside living cells provides an instruction manual on how to build the proteins; it is like a recipe telling which amino acids and in what order they should be linked. The human body has a kit of 20 different types of amino acids that can be linked together in countless ways to make variety of proteins. Those 20 amino acids are all built up from the same basic unit, but differ in a specific chemical group sticking out from the basic unit, giving each of them a unique identity. Specific combination of amino acids determines the shape or structure of the protein. The structure of the protein determines its function. Insertion of a wrong amino acid can affect the protein structure, making it impossible for the protein to get the correct structure - then the protein is said to be mis-folded. Mis-folding of proteins can lead to various diseases.
Alzheimer’s in an example of a disease that is caused by mis-folding of a peptide found in the brain. It usually occurs in older adults. Sometimes, the DNA can give a wrong instruction by which the peptide may get a wrong amino acid inserted into the peptide chain. This can make the disease worse by occurring earlier in age. An Alzheimer’s disease patient suffers from loss of memory and thinking abilities. Mis-folding of the Alzheimer´s peptide, results in the single peptide units coming together and forming another kind of structure. The mis-folded structures are referred to as aggregates, more specifically as amyloids. Those big structures are very stable and they look like ropes or spaghetti that are twisted around each other. The formation of those big and stable structures has been found to be toxic and leads to Alzheimer´s disease. How and why those structures form is still not fully understood, which makes it difficult to find a medicine for the disease. This can be related to a simple analogy. If one needs to repair a fridge or any electrical appliance, it is necessary to know and understand the function of each little network or device installed in it. Similarly, in order to find a medicine for the disease, one should understand how and why those big rope-like structures - the aggregates - form.
Understanding how aggregates of a peptide are formed comes with different complexities. In the brain of Alzheimer’s patients, the Alzheimer´s peptide has been found to be varied in different ways: 1) Some peptides are shorter than the original peptide; 2) some are varied by one amino acid; 3) some have some modifications in the chemical group that sticks out from the basic unit of the amino acids. All these variations are speculated to affect the formation of the big and stable aggregates and how fast they form. Variations of the Alzheimer´s peptide, resulting in a faster formation of the aggregates are linked to an earlier onset and a faster progression of the disease. In this thesis we specifically looked at those three peptide variations listed above and compared the behaviour and speed of the aggregation formation from those altered peptides to the original peptide.
In order to get a better understanding of the effects of different variations on the mis-folding of peptide, we performed and designed experiments where we could follow how each and every variation affects the speed of the aggregation formation. We produced various peptides and made pure samples, in order to have a simple system to mimic the aggregation formation. To the pure samples we added a small dye that bind to big stable aggregated structures and emit light. By doing so, we can follow the aggregation rate. Next we analyse the data using mathematical equations, which helps us to understand how the aggregation of the varied peptides is different from the original peptide. Although, we have described the aggregates to being big structures, they are still too small to be seen by the human eye. In order to look at the structures, we need to use big microscope, named electron microscope. Getting an image of the rope-like structures formed by different variations of the peptide, does also add to the understanding of the aggregation formation.
To sum up, this thesis is focused on trying to get a deeper understanding of how different alterations of the Alzheimer´s peptide affect the formation of the large and stable rope-like structures, called aggregates. Getting a better understanding of the formation of these structures is very important as it has been found to be a toxic process, leading to death of cells in the human brain, resulting in Alzheimer’s disease. We believe that the research in this thesis will let us understand properties of peptide that govern aggregation in more detail, which subsequently, can help provide better strategies for developing medicines.

• author: Sanagavarapu, Kalyani
• supervisor: Sara Linse; Derek Logan
• opponent: Professor Jemth, Per, Uppsala universitet
• organization: Biochemistry and Structural Biology; NanoLund: Centre for Nanoscience
• publishing date: 2019-01
• type: Thesis
• publication status: published
• subject: Other Chemistry Topics
• keywords: protein aggregation, amyloid β-peptide, aggregation kinetics, aggregation mechanism N-terminus, mutations, variants, sequence determinants,, cross-seeding
• pages: 64 pages
• defense location: Sal B Kemicentrum, Naturvetarvägen 14, Lund
• defense date: 2019-03-07 09:00:00
• ISBN: 978-91-7422-632-4
• ISBN: 978-91-7422-631-7
• language: English
• LU publication?: yes
• id: 079ae474-ebff-43a1-bc23-dc928b82d272
• date added to LUP: 2019-02-05 16:25:37
• date last changed: 2020-04-15 12:08:24