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- Jonson, Maria
(författare)
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Investigating Amyloid β toxicity in Drosophila melanogaster
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis Drosophila melanogaster (the fruit fly) has been used as a model organism to study the aggregation and toxic properties of the human amyloid β (Aβ) peptide involved in the onset of Alzheimer's disease (AD). AD is one of many misfolding diseases where the important event of a protein to adopt its’ specific three-dimensional structure has failed, leading to aggregation and formation of characteristic amyloid fibrils. AD has a complex pathology and probably reflects a variety of related molecular and cellular abnormalities, however, the most apparent common denominator so far is abnormal Amyloid-β precursor protein (APP) processing, resulting in a pool of various Aβ-peptides. In AD, the Aβ peptide misfolds, aggregates and forms amyloid plaques in the brain of patients, resulting in progressive neurodegeneration that eventually leads to death.By expressing the human Aβ protein in the fly, we have studied the mechanisms and toxicity of the aggregation in detail and how different cell types in the fly are affected. We have also used this model to investigate the effect of potential drugs that can have a positive impact on disease progression. In the first and second work in this thesis, we have, in a systematic way, proved that the length of the Aβ-peptide is essential for its toxicity and propensity to aggregate. If the peptide expressed ends at amino acid 42 it is extremely toxic to the fly nervous system. However, this toxicity can be completely abolished by expressing a variant that is shorter than 42 amino acids (1-37 to 1-41 aa), or be significantly reduced by expressing a longer variant (1-43 aa). Toxicity can be partly mitigated in trans by co-expressing the 1-42 variant with a 1-38 variant. This supports the theory that the disease progression could be inhibited if the formation of Aβ 1-42 is decreased. In the third work we demonstrate that amyloid aggregates can be found in various cell types of Drosophila, however, the toxicity seem to be selective to neurons. Our results indicate that the aggregates of glial expressing flies have a more mature structure, which appear to be less toxic. This also suggests that glial cells might spread Aβ aggregates without being harmed. The last work in this thesis investigates how curcumin (turmeric) can affect Aβ aggregation and toxicity. Curcumin appears to shift the equilibrium between the less stableaggregates and mature fibers toward the final stage resulting in an improved lifespan for treated flies.In summary, this thesis demonstrates that the toxicity of Aβ in Drosophila is highly dependent on the Aβ variant expressed, the structure of the protein aggregates and which cell type that expresses the protein. We have also shed light on the potential of using Drosophila when it comes to examining possible therapeutic substances as a tool for drug discovery.
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| 2. |
- Lendel, Christofer
(författare)
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Molecular principles of protein stability and protein-protein interactions
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z domain variants with 13 randomised positions at the immunoglobulin Fc-binding surface. This thesis aims to describe the principles for molecular recognition in two protein-protein complexes involving affibody proteins. The first complex is formed by the ZSPA-1 affibody binding to its own ancestor, the Z domain (Kd ~1 μM). The second complex consists of two affibodies: ZTaq, originally selected to bind Taq DNA polymerase, and anti-ZTaq, an anti-idiotypic binder to ZTaq with a Kd ~0.1 μM. The basis for the study is the determination of the three-dimensional structures using NMR spectroscopy supported by biophysical characterization of the uncomplexed proteins and investigation of binding thermodynamics using isothermal titration calorimetry. The free ZSPA-1 affibody is a molten globule-like protein with reduced stability compared to the original scaffold. However, upon target binding it folds into a well-defined structure with an interface topology resembling that displayed by the immunoglobulin Fc fragment when bound to the Z domain. At the same time, structural rearrangements occur in the Z domain in a similar way as in the Fc-binding process. The complex interface buries 1632 Å2 total surface area and 10 out of 13 varied residues in ZSPA-1 are directly involved in inter-molecular contacts. Further characterization of the molten globule state of ZSPA-1 revealed a native-like overall structure with increased dynamics in the randomised regions (helices 1 and 2). These features were reduced when replacing some of the mutated residues with the corresponding wild-type Z domain residues. The nature of the free ZSPA-1 affects the thermodynamics of the complex formation. The contribution from the unfolding equilibrium of the molten globule was successfully separated from the binding thermodynamics. Further decomposition of the binding entropy suggests that the conformational entropy penalty associated with stabilizing the molten globule state of ZSPA-1 upon binding seriously reduces the binding affinity. The ZTaq:anti-ZTaq complex buries in total 1672 Å2 surface area and all varied positions in anti-ZTaq are directly involved in binding. The main differences between the Z:ZSPA-1 and the ZTaq:anti-ZTaq complexes are the relative subunit orientation and certain specific interactions. However, there are also similarities, such as the hydrophobic interface character and the role of certain key residues, which are also found in the SPA:Fc interaction. Structural rearrangements upon binding are also common features of these complexes. Even though neither ZTaq nor anti-ZTaq shows the molten globule behaviour seen for ZSPA-1, there are indications of dynamic events that might affect the binding affinity. This study provides not only a molecular basis for affibody-target recognition, but also contributions to the understanding of the mechanisms regulating protein stability and protein-protein interactions in general.
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