Chemical Synthesis of Alpha-Synuclein Proteins via Solid-Phase Peptide Synthesis and Native Chemical Ligation
von Luisa Maria Gatzemeier
Datum der mündl. Prüfung:2024-01-16
Erschienen:2024-03-05
Betreuer:Prof. Dr. Franc Meyer
Gutachter:Prof. Dr. Franc Meyer
Gutachter:Prof. Dr. Tiago Fleming Outeiro
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Name:Dissertation Luisa Gatzemeier revision_final.pdf
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Zusammenfassung
Englisch
Parts of this chapter have already been published and have been adapted from the original publication: L. M. Gatzemeier, F. Meyer, U. Diederichsen, T. F. Outeiro, Chemical Synthesis of Alpha-Synuclein Proteins via Solid-Phase Peptide Synthesis and Native Chemical Ligation, Chem. Eur. J. 2023, 29, e202300649, doi: 10.1002/chem.202300649.[1] α-Synuclein (α-Syn) is a 140-amino acid (AA) protein implicated in neurodegenerative disorders known as synucleinopathies, such as Parkinson’s disease (PD), where it accumulates in proteinaceous inclusions in the brain. In neuronal cells, α-Syn is thought to play a crucial role in neurotransmitter release.[2–4] However, the protein also exists in non-neuronal cells, such as erythrocytes, where it performs other roles, given the distinct function of these cells.[5–8] The propensity of the protein to aggregate is influenced by changes in the native AA sequence of the protein as well as by post-translational modifications (PTMs), implying that mutations and PTMs contribute to its pathogenicity.[9] In the last decades, a lot of research was done aiming for the toxic species of α-Syn. Therefore, numerous types of aggregates, their fibril morphology, aggregation kinetics and cytotoxicity were studied using microscopic techniques, biochemical assays and several in vitro and in vivo models. Currently, oligomeric species of α-Syn are thought to cause neuronal cell death and neurodegeneration, suggesting increasing evidence for this species as the most toxic one, but this is still under controversial debate.[2–4,10,11] To study the physiological functions of α-Syn and its aggregation behavior, sufficient amounts of wild type (WT) and/or modified protein are required. Usually, recombinant protein expression is applied to produce pure α-Syn, but biological expression is limited in the synthesis of modified protein variants. In recombinant protein synthesis, protein modification mostly is performed after protein expression by utilizing enzymes or reagents to introduce different kinds of modifications. This is very efficient, but lacks site-selectivity and control. For example, kinases introduce often more than one single phosphorylation at a specific AA, and glycating agents, such as methylglyoxal (MGO), modify several Lys residues yielding a mixture of glycated protein variants. Although these heterogenous modified proteins may reflect the biological complexity and diversity of naturally occurring α-Syn modifications, they make it impossible to determine effects of specific modifications defined by their kind, number and position in the protein sequence. However, this is required to clarify the structure-function and structure-toxicity relationships of these modifications as well as to investigate effects of defined combined modifications. Thus, strong research efforts are necessary in order to elucidate the role of modifications in the biology and pathobiology of the protein. To overcome the challenges of biological protein expression, total chemical and semi-synthetic approaches have been investigated and facilitate the preparation of site-selectively modified α-Syn variants for biological characterization. Given the tremendous interest in studying α-Syn and the existing limitations in the production of modified forms of the protein, strategies for the chemical synthesis of α-Syn were developed within this thesis. These include the combination of peptide fragment synthesis via automated microwave-assisted solid-phase peptide synthesis (MW-SPPS) and ligation strategies. Based on our working groups expertise in peptide synthesis and ligation, an efficient solid-phase peptide synthesis (SPPS) method was generated to synthesize long α-Syn peptide fragments in sufficient yields. The peptide fragments were purified and used successfully in chemical ligation, enabling the total chemical synthesis of α-Syn and protein variants of interest carrying either mutations, PTMs or both. Further investigations of the effects of the introduced modifications on the structure and aggregation behavior of the proteins were performed by spectroscopic and biochemical techniques. Glycation is a common PTM occurring in α-Syn. α-Syn glycation caused by elevated blood sugar levels, as found in diabetes patients, has been reported to increase the risk of developing PD.[12] Thus, a cooperation project with the group of Prof. Dr. Tiago Fleming Outeiro from the Department of Experimental Neurodegeneration of the University Medical Center Göttingen was started. They are interested in the investigation of biological effects of α-Syn mutations and PTMs, especially glycation. The aim of this side-project was the synthesis of α-Syn peptide fragments carrying defined glycations at their Lys residues for the investigation of antibody specifity against these glycations. Until present, no cure is available for PD treatment. During the last decades, α-Syn has been moved further into the focus as potential target in the development of disease-modifying drugs for treating PD. One strategy is the inhibition of α-Syn aggregation by peptides.[13] Thus, a second side-project together with Dr. Blagovesta Popova from the Institute of Molecular Microbiology and Genetics of the Georg-August University Göttingen was set up, in which short peptides intended to inhibit the aggregation of α-Syn were synthesized. Ultimately, this study forms the foundation for future syntheses and studies of other custom-made α-Syn variants with a single or several modifications, contributing to enlightenment of pathomechanisms in α-Syn-associated neurodegenerative disorders and discovery of potential future therapies.
Keywords: Alpha-Synuclein; Parkinson's Disease; Chemical Protein Synthesis; Solid-Phase Peptide Synthesis; Native Chemical Ligation