Prion diseases are caused by a structural rearrangement of the cellular prion protein, PrPC, into a disease-associated conformation, PrPSc, that is β-sheet rich and can form amyloid deposits in the brain. PrPSc formation induces neuronal death and an invariably fatal neurodegenerative disease. The pathology of prion diseases is among the best understood of a group of neurodegenerative diseases that show similar features including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease because it is experimentally infectious. Samples containing PrPSc when introduced into a host bind to native PrPC and promote conversion to PrPSc in a seeding or templating manner. In this work we seek an understanding of how particular amino acids contribute to prion disease pathogenesis and ultimately how this information can be translated to the production of more efficient therapies.
We developed a high-throughput screening method to determine the amino acid specific effects of the PrP sequence contributing to the interaction with anti-prion antibodies and alternative PrP conformations. A library of mouse PrP mutants expressed on the surface of yeast cells was screened for their binding interactions with anti-prion antibodies and beta-sheet rich PrP conformations. Those substitutions in PrP that prevented these interactions were identified by single molecule real-time (SMRT) sequencing of the screened population, providing greater than 10,000 full-length nucleotide sequences. The sequences were then aligned to the wild-type PrP gene to identify mutations. We found that optimization of the alignment scoring parameters for the Needleman-Wunsch algorithm and rejecting the lowest 10% of sequences in terms of sequence quality reduced the substitution error rate of sequencing from 7.90 x 10-5 to 2.19 x 10-5 and improves the statistical power of the method. By examining the entire gene sequences correlated to the protein function, we were able to obtain residue-level resolution of conformational protein-protein interaction interfaces that are critical for binding, as well as a quantitative measure of the impact of mutations on binding affinity.
When the library was screened against anti-prion antibodies we found that they made contacts with discontinuous residues that are brought into close proximity when PrP adopts an alpha-helix rich and PrPC like structure. When the library was screened against different conformations of PrP conformation specific interactions were observed. We found that antibodies ICSM18 and D18 binding was influenced by discontinuous residues in helix 1 of PrP, brought into close proximity to one another only when the alpha helix was intact, while full affinity of the 6H4 antibody was dependent on the negative charge on the genetically distal but conformationally adjacent D201 residue. Furthermore, the relative enrichment of mutants correlated to the magnitude of the change in binding affinity, demonstrating how residues such as W144 were essential for binding for all three antibodies, while residues such as D201 only modestly contributed to 6H4 affinity. We observed that high affinity PrP-PrP interactions with yeast surface displayed PrP were consistently achieved when unbound PrP was folded into beta-sheet rich structures. These interactions persisted over a wide range of solution conditions and blocking conditions, and were facilitated predominantly by residues 101-111, though other regions throughout the entire protein such as residues 28-33 and 203-206 also appeared to contribute to binding. These findings reinforce the conformational importance of PrP-PrP interactions and suggest potential mechanisms by which existing and new therapeutics may act by inhibiting interactions at these sites.
In the final portion of this work we develop anti-prion antibodies for increased therapy. By yeast surface display affinity maturation, we isolated ICSM18 mutants with a greater than 300 fold increase in affinity for both recombinant PrP and for native PrP expressed by a mouse nueroblastoma cell line. When these antibodies were expressed by cells persistently infected with prions the improved affinity antibody fragments showed reduced levels of PrP in the disease conformation compared to the cells expressing the parental antibody fragment. We also developed new lead candidate antibody fragments that bind to the helix2-helix3 region that may play a role in PrPC to PrPSc conversion, and are useful for structural characterization and as potential therapeutics.
Overall, a method was developed for amino-acid level characterization of protein-protein interactions and this method was applied to understand factors that contribute to PrP self-associations relevant to disease pathology and to identify the mechanism by which antibodies recognize PrP relevant to disease treatment.
|Advisor:||Lenhoff, Abraham M.|
|Commitee:||Bahnson, Brian J., Chen, Wilfred, Lee, Kelvin H., Papoutsakis, Eleftherious T.|
|School:||University of Delaware|
|School Location:||United States -- Delaware|
|Source:||DAI-B 77/02(E), Dissertation Abstracts International|
|Keywords:||Conformation, Mutational scanning, Prions, Protein engineering, Protein-protein onteractions, Single molecule real-time seqeunecing|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be