Mutational study of the N-terminus in Aβ42 fibril-catalyzed nucleation
(2020) KEMR45 20201Department of Chemistry
- Abstract
- Proteins are made up by amino acids linked through peptide bonds. They carry out many important tasks in our bodies and the folding of proteins is a crucial process. The folding of proteins determines the function of it and sometimes proteins get misfolded. The misfolded protein can get degraded, refold back or form protein aggregates. These aggregating proteins are related to several diseases for instance Alzheimer’s disease. Alzheimer’s disease, AD, is a neurodegenerative disease and the most common cause of dementia. AD is related to specific protein aggregates called amyloids that are characterised by fibrillar morphology, extracellularly deposition in tissues and by the beta-pleated sheet structure, which form fibrils.
One amyloid... (More) - Proteins are made up by amino acids linked through peptide bonds. They carry out many important tasks in our bodies and the folding of proteins is a crucial process. The folding of proteins determines the function of it and sometimes proteins get misfolded. The misfolded protein can get degraded, refold back or form protein aggregates. These aggregating proteins are related to several diseases for instance Alzheimer’s disease. Alzheimer’s disease, AD, is a neurodegenerative disease and the most common cause of dementia. AD is related to specific protein aggregates called amyloids that are characterised by fibrillar morphology, extracellularly deposition in tissues and by the beta-pleated sheet structure, which form fibrils.
One amyloid protein that is strongly related to the onset and progression of AD is the amyloid β peptide, Aβ, which is the main component in amyloid plaques which is found in increased amount in the brain of Alzheimer’s patients. Aβ exists in various lengths where the 42 amino acid residues peptide, Aβ42, is one of the most aggregation-prone Aβ peptides and thus one of the most important isoforms.
The amyloid hypothesis says that the self-assembly of Aβ peptide is the primary cause of AD and the last 20 years research of AD has been based on this hypothesis. The degradation of the Aβ peptide is normally rather fast but the ability to degrade the Aβ peptide decreases during aging and during the progression of AD.
Monomeric Aβ is unstructured but during the pathology of Alzheimer´s they self-assembly into highly structured aggregates of low solubility. The self-assembly process include three different microscopic mechanisms: primary nucleation, elongation and secondary nucleation. Secondary nucleation dominates in Aβ aggregation and involves both monomers and already existing aggregates. During the aggregation of the Aβ peptide, toxic oligomers are created. More specific, they are formed during the secondary nucleation. These oligomers are neurotoxic and are hence involved in the pathology of AD.
Previous research has discovered that the specific sequence of the N-terminus is important in the aggregation process of Aβ42. Reduced rates of the secondary nucleation have been observed when the residues in the N-terminus were randomly scrambled.
This work has focused on different hydrophobic residues the N-terminus of Aβ42. The four residues A2, F4, Y10 and V12 are all more or less hydrophobic and mutations to amino acids of different size or more hydrophilic amino acids will change the properties of the peptide. Different experiments were performed to investigate wether these four residues are involved in the secondary nucleation process and in turn also in the oligomer formation process. The fluorescent dye Thioflavin T was used both in concentration and time dependence aggregation kinetics and in seeding experiments to follow the aggregation process of Aβ42. The concentration dependent aggregation kinetic experiment of six mutants (A2S, A2T, Y10A, Y10F, V12A and V12S) showed that all aggregated similar to Aβ42 wild type. Some mutants aggregated a little slower than WT and some faster. All curves had a sigmoidal-like appearance indicating that secondary nucleation is dominating.
V12A had the most similar aggregation behavior to WT when looking at a half time plot.
When looking at the seeding experiment results of the same six mutants as mentioned before, all showed concentration dependence for both the self-seeding and cross-seeding. (Less) - Popular Abstract (Swedish)
- Protein består av byggstenar och de har flera livsviktiga funktioner i våra kroppar. Byggstenarna är tjugo olika aminosyror som kan kopplas ihop på många olika sätt. Ett protein som är mindre än 50 aminosyraenheter kallas för en peptid.
Proteinveckning är en spontan process där ett protein får sin tredimensionella form, som oftast är ett biologiskt aktiv tillstånd. Proteinveckningen kan ibland gå fel vilket kan leda till att så kallade proteinaggregat skapas. Dessa proteinaggregat är ofta kopplade till sjukdomar. Amyloid är ett specifikt proteinaggregat som är kopplat till bland annat Alzheimers sjukdom. Alzheimers är en neurodegenerativ sjukdom vilket innebär att neuroner, hjärnans nervceller, skadas. Skadade och döda neuroner är inte... (More) - Protein består av byggstenar och de har flera livsviktiga funktioner i våra kroppar. Byggstenarna är tjugo olika aminosyror som kan kopplas ihop på många olika sätt. Ett protein som är mindre än 50 aminosyraenheter kallas för en peptid.
Proteinveckning är en spontan process där ett protein får sin tredimensionella form, som oftast är ett biologiskt aktiv tillstånd. Proteinveckningen kan ibland gå fel vilket kan leda till att så kallade proteinaggregat skapas. Dessa proteinaggregat är ofta kopplade till sjukdomar. Amyloid är ett specifikt proteinaggregat som är kopplat till bland annat Alzheimers sjukdom. Alzheimers är en neurodegenerativ sjukdom vilket innebär att neuroner, hjärnans nervceller, skadas. Skadade och döda neuroner är inte ersättningsbara. Alzheimers är i nuläget obotligt och är den vanligaste orsaken till demens.
Ett specifikt amyloidprotein med namnet amyloid beta, Aβ, är starkt kopplat till Alzheimers. Aβ förekommer i flera olika längder där den 42 aminosyraenheter-peptiden, Aβ42, är en av de mest aggregationsbenägna Aβ peptiderna och därför också en av de viktigaste. Aβ42 deposition finns naturligt i hjärnan och nedbrytningen sker relativt fort, men kapaciteten att bryta ner Aβ42 minskar under ökad ålder och vid Alzheimers.
Monomerisk Aβ är ostrukturerat men genomgår självsammansättning till amyloidaggregat. Självsammansättningen består av tre olika mekanismer: primär kärnbildning, förlängning och sekundär kärnbildning. Under den sekundära kärnbildningen skapas giftiga oligomerer som är neurotoxiska och därför involverad i patologin av Alzheimers.
Tidigare forskning har upptäckt att den specifika sekvensen av N-terminalen, det vill säga ena änden av proteinet, är viktig för aggregationen av Aβ42. Denna studie undersöker därför olika aminosyror i N-terminalen. Mutationer av de fyra aminosyrorna A2, F4, Y10 och V12 till andra aminosyror som är större, mindre samt mer eller mindre hydrofila studeras. Mutation av en aminosyra, det vill säga en förändring från en aminosyra till en annan, ändrar peptidens egenskaper. Olika experiment genomförs för att undersöka vilka aminosyror som är involverade i sekundär kärnbildning och då också i skapandet av oligomerer. Koncentrations- och tidsberoende aggregationskinetik-experiment samt seeding-experiment utförs med hjälp av färgämnet Tioflavin T.
Koncentrations- och tidsberoende aggregationskinetik-experiment av sex olika mutanter (A2S, A2T, Y10A, Y10F, V12A och V12S) visade att alla aggregerade likt Aβ42 ”wild type”.
Seeding experiment av samma sex mutanter tydde på att alla verkade vara koncentrationsberoende. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9027868
- author
- Willas, Amanda LU
- supervisor
-
- Sara Linse LU
- organization
- course
- KEMR45 20201
- year
- 2020
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Biochemistry, Biokemi
- language
- English
- id
- 9027868
- date added to LUP
- 2020-09-16 13:19:53
- date last changed
- 2020-09-16 13:19:53
@misc{9027868,
abstract = {{Proteins are made up by amino acids linked through peptide bonds. They carry out many important tasks in our bodies and the folding of proteins is a crucial process. The folding of proteins determines the function of it and sometimes proteins get misfolded. The misfolded protein can get degraded, refold back or form protein aggregates. These aggregating proteins are related to several diseases for instance Alzheimer’s disease. Alzheimer’s disease, AD, is a neurodegenerative disease and the most common cause of dementia. AD is related to specific protein aggregates called amyloids that are characterised by fibrillar morphology, extracellularly deposition in tissues and by the beta-pleated sheet structure, which form fibrils.
One amyloid protein that is strongly related to the onset and progression of AD is the amyloid β peptide, Aβ, which is the main component in amyloid plaques which is found in increased amount in the brain of Alzheimer’s patients. Aβ exists in various lengths where the 42 amino acid residues peptide, Aβ42, is one of the most aggregation-prone Aβ peptides and thus one of the most important isoforms.
The amyloid hypothesis says that the self-assembly of Aβ peptide is the primary cause of AD and the last 20 years research of AD has been based on this hypothesis. The degradation of the Aβ peptide is normally rather fast but the ability to degrade the Aβ peptide decreases during aging and during the progression of AD.
Monomeric Aβ is unstructured but during the pathology of Alzheimer´s they self-assembly into highly structured aggregates of low solubility. The self-assembly process include three different microscopic mechanisms: primary nucleation, elongation and secondary nucleation. Secondary nucleation dominates in Aβ aggregation and involves both monomers and already existing aggregates. During the aggregation of the Aβ peptide, toxic oligomers are created. More specific, they are formed during the secondary nucleation. These oligomers are neurotoxic and are hence involved in the pathology of AD.
Previous research has discovered that the specific sequence of the N-terminus is important in the aggregation process of Aβ42. Reduced rates of the secondary nucleation have been observed when the residues in the N-terminus were randomly scrambled.
This work has focused on different hydrophobic residues the N-terminus of Aβ42. The four residues A2, F4, Y10 and V12 are all more or less hydrophobic and mutations to amino acids of different size or more hydrophilic amino acids will change the properties of the peptide. Different experiments were performed to investigate wether these four residues are involved in the secondary nucleation process and in turn also in the oligomer formation process. The fluorescent dye Thioflavin T was used both in concentration and time dependence aggregation kinetics and in seeding experiments to follow the aggregation process of Aβ42. The concentration dependent aggregation kinetic experiment of six mutants (A2S, A2T, Y10A, Y10F, V12A and V12S) showed that all aggregated similar to Aβ42 wild type. Some mutants aggregated a little slower than WT and some faster. All curves had a sigmoidal-like appearance indicating that secondary nucleation is dominating.
V12A had the most similar aggregation behavior to WT when looking at a half time plot.
When looking at the seeding experiment results of the same six mutants as mentioned before, all showed concentration dependence for both the self-seeding and cross-seeding.}},
author = {{Willas, Amanda}},
language = {{eng}},
note = {{Student Paper}},
title = {{Mutational study of the N-terminus in Aβ42 fibril-catalyzed nucleation}},
year = {{2020}},
}