000181225 001__ 181225
000181225 005__ 20181205220145.0
000181225 0247_ $$2doi$$a10.5075/epfl-thesis-5494
000181225 02470 $$2urn$$aurn:nbn:ch:bel-epfl-thesis5494-6
000181225 02471 $$2nebis$$a7353147
000181225 037__ $$aTHESIS
000181225 041__ $$aeng
000181225 088__ $$a5494
000181225 245__ $$aElucidating the role of post-translational modifications of alpha-synuclein using semisynthesis$$bphosphorylation at Tyrosine 125 and monoubiquitination at Lysine 6
000181225 269__ $$a2012
000181225 260__ $$aLausanne$$bEPFL$$c2012
000181225 336__ $$aTheses
000181225 520__ $$aAlpha-synuclein (α-syn) is a natively unfolded protein that is closely linked to Parkinson’s disease (PD) by genetic, neuropathologic and biochemical evidence. Aggregated and fibrillar forms of α-syn are the main components of intracellular protein inclusions found in PD patients’ brains, termed Lewy Bodies (LB). Both in animal models and in vitro, α-syn forms fibrillar aggregates that resemble those observed in PD brain tissues. Although disease-associated mutations have been shown to promote the fibrillization of α-syn, the exact mechanisms responsible for triggering α-syn aggregation and toxicity in sporadic PD remain unknown. Addressing this gap of knowledge is crucial for understanding the molecular basis of the disease and developing effective therapies for the treatment of PD and other synucleinopathies. This project was initiated on the basis of the working hypothesis that post-translational modifications (PTM) may play important roles in modulating α-syn function and/or regulating its aggregation and toxicity. More specifically, α-syn is ubiquitously N-terminally acetylated, and phosphorylated (serines 87 and S129), ubiquitinated (lysines 12, 21 and 23) and truncated forms of α-syn have been observed in association with wild-type α-syn in LB and in brain tissues from PD patients and transgenic animals. Other modifications, such as phosphorylation at tyrosine 125 (Y125), were significantly reduced in diseased brains. Despite the discovery of candidate enzymes that mediate α-syn phosphorylation, ubiquitination and truncation, little is known about how each of these modifications alters α-syn structure, function, aggregation and toxicity in vivo. This is primarily due to the lack of tools that allow site-specific introduction of these modifications and the lack of natural mutations that can mimic the effect of these modifications, including phosphorylation. The primary focus of this thesis was to develop strategies to overcome these limitations so as to elucidate the effect of phosphorylation at Y125 and monoubiquitination at lysine 6 (K6) on α-syn structure, fibril formation, membrane binding and subcellular localization. Towards this goal, we developed two semisynthetic strategies that allow site-specific introduction of PTM in α-syn based on the ligation of synthetic peptides containing the desired modified amino acid with recombinantly expressed proteins using Expressed Protein Ligation. This approach enables the introduction of single or multiple PTM in the N- or C-terminal regions of α-syn, and the preparation of modified α-syn in milligram quantities. Using these approaches, we were able to show for the first time that ubiquitination stabilizes the monomeric form of the protein and inhibits, rather than promotes, α-syn aggregation, while phosphorylation at Y125 does not significantly change the structure and aggregation propensity of α-syn. With the semisynthetic pY125 α-syn in our hands, we were also able to investigate for the first time the sub-cellular localization of pY125 α-syn through its microinjection into primary neurons. This was not previously possible due to technical limitations related to the absence of appropriate antibodies against pY125 α-syn for immunocytochemical studies and difficulties in generating sitespecifically modified α-syn. Furthermore, we were able to investigate the effect of α-syn phosphorylation on its interactions with other proteins, by probing the effect of pS129 and pY125 on the binding to a nanobody that was specifically selected by phage-display to tightly bind to the C-terminal domain of α-syn. Accordingly, we demonstrated that phosphorylation at a single residue is capable of disrupting the binding of full-length α-syn to another protein. These results have wide-ranging implications for the potential role of phosphorylation and other PTM in regulating α-syn’s function(s). We also exploited our ability to combine semisynthetic and enzymatic approaches to investigate potential cross-talk between different PTM, namely phosphorylation at Y125 and S129 and monoubiquitination at K6. These advances would eventually allow investigating the effect of cross-talk between other N and C-terminal modifications on α-syn’s properties and to investigate PTM-dependent protein-protein and protein-ligand interactions in vitro and in cellular models of synucleinopathies. Together, our semisynthetic approaches provide novel means for the introduction of site-specific modifications in the N- and C-termini of α-syn. Current efforts in our laboratory are focused on extending these approaches to investigate the dynamics of PTM through the use of photocaged modified amino acids and to prepare novel fluorescently labeled α-syn variants to investigate its folding at the single-molecule level in living cells. The results presented in this thesis and preliminary studies from our group demonstrate that semisynthetic α-syn can provide unique opportunities to investigate structure-function of α-syn and the role of PTM in the biology of α-syn in health and disease.
000181225 6531_ $$aParkinson’s disease (PD)
000181225 6531_ $$aalphα-synuclein (α-syn)
000181225 6531_ $$aLewy Bodies (LB)
000181225 6531_ $$aAggregation
000181225 6531_ $$aAmyloid
000181225 6531_ $$aOligomers
000181225 6531_ $$aDopamine (DA)
000181225 6531_ $$aSolid-phase peptide synthesis (SPPS)
000181225 6531_ $$aNative Chemical Ligation (NCL)
000181225 6531_ $$aExpressed Protein Ligation (EPL)
000181225 6531_ $$aIntein
000181225 6531_ $$aCircular Dichroism (CD)
000181225 6531_ $$aNuclear Magnetic Resonance (NMR)
000181225 6531_ $$aTransmission Electron Microscopy (TEM)
000181225 6531_ $$aMicroinjection
000181225 6531_ $$aImmunocytochemistry
000181225 6531_ $$aFluorescent Microscopy
000181225 6531_ $$aNanobodies
000181225 6531_ $$aIsothermal Titration Calorimetry (ITC)
000181225 700__ $$0243213$$aHejjaoui, Mirva$$g174344
000181225 720_2 $$0240880$$aLashuel, Hilal$$edir.$$g167337
000181225 8564_ $$s14932603$$uhttps://infoscience.epfl.ch/record/181225/files/EPFL_TH5494.pdf$$yn/a$$zn/a
000181225 909C0 $$0252159$$pLMNN$$xU11104
000181225 909CO $$ooai:infoscience.tind.io:181225$$pthesis$$pthesis-bn2018$$pDOI$$pSV$$qDOI2$$qGLOBAL_SET
000181225 917Z8 $$x108898
000181225 917Z8 $$x108898
000181225 917Z8 $$x108898
000181225 918__ $$aSV$$cBMI$$dEDBB
000181225 919__ $$aLMNN
000181225 920__ $$a2012-9-21$$b2012
000181225 970__ $$a5494/THESES
000181225 973__ $$aEPFL$$sPUBLISHED
000181225 980__ $$aTHESIS