Effect of DNAJB6 on α-synuclein amyloid fibril aggregation and dis-aggregation
(2023) KFKM05 20231Biophysical Chemistry
- Abstract
- Amyloid proteins are a group of proteins with the ability to form large fibrillar aggregates, identifiable
by their cross-β core structure. These aggregates are linked to several different neurodegenerative
diseases such as Parkinson’s disease and Alzheimer’s disease and due to the growing rate of people
diagnosed, there is a need to combat these diseases. Aggregation of amyloid proteins is caused by the
misfolding of nascent protein. The human body, however, has a machinery to prevent this, through
a protein class known as chaperones. They are defined as protein which can prevent misfolding or
aggregation of other protein. One chaperone from the Hsp40 class, DNAJB6, has been shown to prevent aggregation of amyloid protein. An... (More) - Amyloid proteins are a group of proteins with the ability to form large fibrillar aggregates, identifiable
by their cross-β core structure. These aggregates are linked to several different neurodegenerative
diseases such as Parkinson’s disease and Alzheimer’s disease and due to the growing rate of people
diagnosed, there is a need to combat these diseases. Aggregation of amyloid proteins is caused by the
misfolding of nascent protein. The human body, however, has a machinery to prevent this, through
a protein class known as chaperones. They are defined as protein which can prevent misfolding or
aggregation of other protein. One chaperone from the Hsp40 class, DNAJB6, has been shown to prevent aggregation of amyloid protein. An attempt to describe the thermodynamic background behind
this phenomena has been done by Linse et al. (2021), with the ”unhappy chaperone” hypothesis [1].
Through the formation of co-aggregates between chaperones and amyloid protein, there is a lowering of
the free energy of the system, leading to an increase in solubility for the amyloid protein. An increase
in solubility is only possible through an increase in free energy for the amyloid protein, however this
is offset by a larger decrease for the chaperone. The decrease in free energy for the chaperone when
forming co-aggregates explains the ”unhappiness” of chaperones to be free in solution.
The aim of this project was to exhibit this increase in solubility for the amyloid protein α-synuclein,
whose aggregation is the cause of Parkinson’s disease. DNAJB6 was the chosen chaperone, and the goal
was to determine if an increase in solubility is a true thermodynamic equilibrium, which means this
effect should lead to the same solubility both from a system consisting of 100% monomeric α-synuclein
or 100% fibrillar α-synuclein. Also, there was prior method development for how to best measure the
monomer concentration.
α-synuclein aggregation at 37◦ C and pH 5.5, was measured both without DNAJB6 and with 1%
DNAJB6, this was done through a developed HPLC absorbance protocol and OPA fluorescence. Possible α-synuclein disaggregation was also investigated on samples with aggregated α-synuclein, where
1% and 10% DNAJB6 was added, these also measured with HPLC and OPA. α-synuclein solubility
were furthermore also measured with NMR, and DNAJB6 was also added here, at the end of fibrillization to detect possible dis-aggregation.
From the results, the following conclusions could be determined. α-synuclein solubility was determined
to be 0.39 ±0.016 µM measured with OPA, and 0.11 ±0.013 µM, measured with HPLC. Aggregation
in the presence of 1% DNAJB6 lead to delayed aggregation and possible increase in solubility. With
SDS-PAGE and MALDI, the formation of aggregates consisting of both α-synuclein and DNAJB6 was
identified. Dis-aggregation was not observed and could be attributed to the low solubility of DNAJB6
at pH 5.5. HPLC was determined to be a sensitive and accurate system for measuring α-synuclein
monomer concentration, but further development should be done to improve robustness and reproducibility. Lastly, NMR was determined as a viable method for measuring α-synuclein solubility at pH
5.5, although it is not yet dependable for discontinuous measurements. (Less) - Popular Abstract (Swedish)
- Chaperoner – kroppens egna försvarssystem mot neurodegenerativa
sjukdomar
Parkinsons sjukdom och Alzheimers sjukdom är exempel på neurodegenerativa sjukdomar
vars orsak attribueras till aggregeringen av amyloida proteiner. Chaperoner som DNAJB6
har visat sig vara effektiva i att förhindra denna aggregering, och i detta arbete har deras
termodynamiska beskrivning undersökts.
Proteiner är kroppens byggstenar, men för att ge dem funktionalitet krävs det att de lyckas
veckas i sin korrekta tredimensionella struktur. Vid den chans att proteinveckningen blir fel,
finns det ett system för att korrigera misstaget. Genom proteinfamiljen, chaperoner, som
definieras som protein som kan korrigera felveckning eller bildandet av aggregat hos... (More) - Chaperoner – kroppens egna försvarssystem mot neurodegenerativa
sjukdomar
Parkinsons sjukdom och Alzheimers sjukdom är exempel på neurodegenerativa sjukdomar
vars orsak attribueras till aggregeringen av amyloida proteiner. Chaperoner som DNAJB6
har visat sig vara effektiva i att förhindra denna aggregering, och i detta arbete har deras
termodynamiska beskrivning undersökts.
Proteiner är kroppens byggstenar, men för att ge dem funktionalitet krävs det att de lyckas
veckas i sin korrekta tredimensionella struktur. Vid den chans att proteinveckningen blir fel,
finns det ett system för att korrigera misstaget. Genom proteinfamiljen, chaperoner, som
definieras som protein som kan korrigera felveckning eller bildandet av aggregat hos andra
protein, lyckas proteostas uppehållas. α-synuclein är ett protein som vid felveckning bildar
fibrilla aggregat, vars konsekvens resulterar i sjukdomar som Parkinsons sjukdom och
Lewykroppsdemens. Målet med detta projekt har varit att öka förståelsen för hur
chaperoner hindrar proteiner från att aggregera och missveckas, med ett fokus på den
termodynamiska beskrivningen för chaperoner.
I en vetenskaplig artikel, skriven av Linse et al. (2021), presenterar de hypotesen ”the
unhappy chaperone”, som beskriver hur chaperoner lyckas öka lösligheten hos amyloida
protein, vilket innebär att de hindrar dessa protein från att bilda aggregat. Hypotesen bygger
på att chaperoner bildar co-aggregat med amyloida proteiner, som tillåter de att befinna sig i
sina icke-aggregerade tillstånd i högre utsträckning.
Chaperonet DNAJB6, som är fokuset i detta arbete, har visat sig vara effektiv i att hindra
aggregering hos andra amyloida protein. Just därför är detta protein intressant att testa
hypotesen med. Med hjälp av 3 olika oberoende metoder, testades DNAJB6 förmåga att
hindra aggregering hos det amyloida proteinet α-synuclein. Resultatet var en signifikant
fördröjning av aggregeringen och en potentiell ökning av lösligheten hos α-synuclein. Vidare
identifierades även bildandet av ett aggregat, bestående av både α-synuclein och DNAJB6.
Sammanfattningsvis har detta projekt funnit spännande upptäckter och skapat en grund för
ett flertal högt intressanta experiment för framtiden. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9132860
- author
- Getachew, Josef LU
- supervisor
-
- Sara Linse LU
- Emil Axell LU
- Max Lindberg LU
- Lei Ortigosa LU
- organization
- course
- KFKM05 20231
- year
- 2023
- type
- H3 - Professional qualifications (4 Years - )
- subject
- keywords
- α-synuclein, alpha-synuclein, chaperones, DNAJB6, the unhappy chaperone, parkinsons disease, biophysical chemistry, biochemistry
- language
- English
- id
- 9132860
- date added to LUP
- 2023-08-07 11:20:04
- date last changed
- 2023-08-07 11:20:04
@misc{9132860,
abstract = {{Amyloid proteins are a group of proteins with the ability to form large fibrillar aggregates, identifiable
by their cross-β core structure. These aggregates are linked to several different neurodegenerative
diseases such as Parkinson’s disease and Alzheimer’s disease and due to the growing rate of people
diagnosed, there is a need to combat these diseases. Aggregation of amyloid proteins is caused by the
misfolding of nascent protein. The human body, however, has a machinery to prevent this, through
a protein class known as chaperones. They are defined as protein which can prevent misfolding or
aggregation of other protein. One chaperone from the Hsp40 class, DNAJB6, has been shown to prevent aggregation of amyloid protein. An attempt to describe the thermodynamic background behind
this phenomena has been done by Linse et al. (2021), with the ”unhappy chaperone” hypothesis [1].
Through the formation of co-aggregates between chaperones and amyloid protein, there is a lowering of
the free energy of the system, leading to an increase in solubility for the amyloid protein. An increase
in solubility is only possible through an increase in free energy for the amyloid protein, however this
is offset by a larger decrease for the chaperone. The decrease in free energy for the chaperone when
forming co-aggregates explains the ”unhappiness” of chaperones to be free in solution.
The aim of this project was to exhibit this increase in solubility for the amyloid protein α-synuclein,
whose aggregation is the cause of Parkinson’s disease. DNAJB6 was the chosen chaperone, and the goal
was to determine if an increase in solubility is a true thermodynamic equilibrium, which means this
effect should lead to the same solubility both from a system consisting of 100% monomeric α-synuclein
or 100% fibrillar α-synuclein. Also, there was prior method development for how to best measure the
monomer concentration.
α-synuclein aggregation at 37◦ C and pH 5.5, was measured both without DNAJB6 and with 1%
DNAJB6, this was done through a developed HPLC absorbance protocol and OPA fluorescence. Possible α-synuclein disaggregation was also investigated on samples with aggregated α-synuclein, where
1% and 10% DNAJB6 was added, these also measured with HPLC and OPA. α-synuclein solubility
were furthermore also measured with NMR, and DNAJB6 was also added here, at the end of fibrillization to detect possible dis-aggregation.
From the results, the following conclusions could be determined. α-synuclein solubility was determined
to be 0.39 ±0.016 µM measured with OPA, and 0.11 ±0.013 µM, measured with HPLC. Aggregation
in the presence of 1% DNAJB6 lead to delayed aggregation and possible increase in solubility. With
SDS-PAGE and MALDI, the formation of aggregates consisting of both α-synuclein and DNAJB6 was
identified. Dis-aggregation was not observed and could be attributed to the low solubility of DNAJB6
at pH 5.5. HPLC was determined to be a sensitive and accurate system for measuring α-synuclein
monomer concentration, but further development should be done to improve robustness and reproducibility. Lastly, NMR was determined as a viable method for measuring α-synuclein solubility at pH
5.5, although it is not yet dependable for discontinuous measurements.}},
author = {{Getachew, Josef}},
language = {{eng}},
note = {{Student Paper}},
title = {{Effect of DNAJB6 on α-synuclein amyloid fibril aggregation and dis-aggregation}},
year = {{2023}},
}