Protein Semisynthesis Provides Access to Tau Disease-Associated Post-translational Modifications (PTMs) and Paves the Way to Deciphering the Tau PTM Code in Health and Diseased States

The microtubule-associated protein Tau plays a central role in neurodegeneration and is a leading therapeutic target for the treatment of Alzheimer’s disease (AD). Several lines of evidence suggest that post-translational modifications (PTMs) regulate the function(s) of Tau, including its subcellular localization, clearance, aggregation, toxicity, and pathology spreading. However, the lack of tools and methodologies that allow site-specific introduction of PTMs in Tau have limited our ability to dissect the role of PTMs in regulating Tau functions in health and disease. To facilitate deciphering the Tau PTM code, we have developed, for the first time, semisynthetic strategies that allow for the site-specific introduction of single or multiple physiological or disease-associated PTMs that occur within residues 246-441 of Tau, which includes the microtubule-binding domain (MTBD). As a proof of concept, we produced unmodified Tau and three Tau variants with single or multiple disease-associated PTMs that were not previously accessible as homogeneously modified proteins; AcK280, pY310, and pS396/pS404. We then focused on investigating the effect of acetylation at lysine280 (AcK280) on the structure, aggregation, and microtubule binding properties of Tau. Our results show that site-specific acetylation at K280 significantly enhances the aggregation rate of Tau and impairs microtubule assembly. Surprisingly, compared with unmodified Tau, which forms long and flexible filaments, AcK280 Tau forms predominantly globular oligomers and short fibrils (< 200 nm) that exhibit a reduced propensity to assemble into long filaments. These findings are consistent with the increased aggregation propensity and pathogenicity of this mutant in animal models of AD and suggest that acetylation at this residue might enhance the seeding capacity or formation of toxic Tau species in vivo. Beyond acetylation and phosphorylation, the development of this semisynthetic strategy provides new opportunities to investigate other types of Tau PTMs and to study the cross-talk between PTMs that occurs within residues 246-441, which were previously inaccessible, thereby paving the way to deciphering the Tau PTM code in health and disease.

Elucidating the role of post-translational modifications of alpha-synuclein using semisynthesis

Mirva Hejjaoui

Alpha-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.

EPFL2012

Reversible attachment of Cell-Penetrating Peptides for the efficient delivery of α-synuclein to HeLa cells and primary cortical neurons

Carole Desobry

The discovery of alpha-synuclein (a-syn) as the main component of Lewy Bodies (LBs), the pathological hallmarks of Parkinson's disease (PD), and the identification of mutations in the gene coding for a-syn in familial form of PD have reinforced the central role of a-syn in the etiology of PD. Nevertheless, the molecular mechanisms by which a-syn is involved in neurodegeneration are still not fully understood. Many studies have raised the potential role of post-translational modifications (PTMs) in regulating a-syn physiological properties but also its pathogenicity in PD development. However, a-syn can be modified simultaneously by several PTMs making it extremely challenging to understand which of those PTMs is responsible in regulating the cellular properties of a-syn. Therefore, our laboratory developed a strategy to study the role of individual PTMs using semi-synthesis, enabling the generation of site-specifically modified a-syn. However, in the absence of an efficient system to internalize these semi-synthetic proteins in cells, those proteins have been limited to biophysical and structural studies. Therefore, we proposed to use cell penetrating peptides (CPPs) to deliver site-specifically modified a-syn inside cells. In a first step, we compared CPPs to deliver a-syn inside the cells. Strikingly, only N-terminal CPP fusions were efficient in delivering it to primary cortical neurons. In order to explain the differences, we performed biophysical assays and established that C-terminal CPPs formed a hairpin with the C-terminus of a-syn, preventing penetration of the plasma membrane. N-terminal fusions displayed a more transient interaction that allowed uptake. In addition, we showed that the CPPs were enhancing the aggregation of a-syn. Among the CPPs tested, TAT fused at the N-terminus was one of the most efficient. Therefore, we evaluated its capacity to deliver a-syn in different cellular models, its subcellular localization and its stability in the cells. We started by evaluating the uptake and molecular properties of TAT- a-syn in HeLa cells and in primary cortical neurons. We found that it was rapidly and efficiently internalized and distributed in the cytosol as punctuated structures. Once internalized, TAT-a-syn was cleared from the cells through autophagy and the proteasome without promoting any cellular toxicity. Finally, TAT-a-syn phosphorylated on Serine 129 or fibrillar TAT-a-syn were also successfully delivered to cells thereby showing that our tool could be used to deliver modified a-syn in cells. However, our data suggested that TAT could influence the properties of a-syn. Thus, we aimed at improving our delivery tool by developing two reversible approaches to relieve a-syn from the influence of TAT after its uptake: 1) a photocleavable linker to release a-syn from TAT upon UV irradiation or 2) a disulfide bond which can be cleaved once in the cell. Both approaches were successful in delivering a-syn to neurons. While the photocleavable version did not allow full release, TAT-S-S-a-syn was efficiently reduced and efficient in delivering pS129 a-syn. In summary, our study allowed developing a versatile tool capable of carrying a-syn inside the cells and allowing its study in the absence of constraints originating from the presence of the CPP sequence in fusion with a-syn. We believe that this tool will be useful for the study of a-syn's PTMs, interactions and cross-talk between modifications.

EPFL2014

Elucidating the role of C-terminal post-translational modifications using protein semisynthesis strategies: α-synuclein phosphorylation at tyrosine 125

Bruno Claude Daniel Fauvet, Mirva Hejjaoui, Hilal Lashuel, Michel Prudent, Filip Vercruysse, Yan Zhang

Despite increasing evidence that supports the role of different post-translational modifications (PTMs) in modulating α-synuclein (α-syn) aggregation and toxicity, relatively little is known about the functional consequences of each modification and whether or not these modifications are regulated by each other. This lack of knowledge arises primarily from the current lack of tools and methodologies for the site-specific introduction of PTMs in α-syn. More specifically, the kinases that mediate selective and efficient phosphorylation of C-terminal tyrosine residues of α-syn remain to be identified. Unlike phospho-serine and phospho-threonine residues, which in some cases can be mimicked by serine/threonine → glutamate or aspartate substitutions, there are no natural amino acids that can mimic phospho-tyrosine. To address these challenges, we developed a general and efficient semisynthetic strategy that enables the site-specific introduction of single or multiple PTMs and the preparation of homogeneously C-terminal modified forms of α-syn in milligram quantities. These advances have allowed us to investigate, for the first time, the effects of selective phosphorylation at Y125 on the structure, aggregation, membrane binding, and subcellular localization of α-syn. The development of semisynthetic methods for the site-specific introduction of single or PTMs represents an important advance toward determining the roles of such modifications in α-syn structure, aggregation, and functions in heath and disease.