The role of the polyglutamine and N-terminal domains in regulating the aggregation and structural properties of Huntingtin Exon 1

Huntington Disease (HD) is caused by a CAG repeat expansion in the huntingtin gene leading to the formation of mutant Huntingtin protein (Htt) with an expanded polyglutamine (polyQ) domain (>36Q). Generation of short N-terminal Htt fragments by proteolysis plays a key role in HD pathogenesis and Huntingtin exon1 (Httex1) is one of the most used models to study Htt aggregation in vitro. We developed a new expression and purification strategy to produce tag-free Httex1 and Httex1 fragments with different polyQ-lengths from E.coli for biophysical studies and for optimizing protein semisynthesis in our laboratory. Having these proteins in hand we sought to explore the effect of the polyQ and Nt17 domain on Httex1 aggregation and structure, given their essential role in mediating intra- and intermolecular interactions in Htt. We found that polyQ-length inversely correlates with fibril length and that expanded polyQ tracts increase the Î²-sheet content modulating the roughness and mechanical properties of Httex1 aggregates. This suggests that polyQ-dependent structural differences exist in aggregates formed by wildtype and mutant Httex1. Moreover, using infrared nanospectroscopy we were able to show that Httex1 aggregates are characterized by Î²-turn structures, whereas fibrils formed by Nt17-truncated Httex1 (âNt17-Httex1) contain antiparallel Î²-sheets, indicating that the NT17 domain influences secondary structure within the polyQ domain. The Nt17 effect on Httex1 structure translates into morphological differences between fibrils formed by Httex1 or âNt17-Httex1 as discerned by high-resolution imaging techniques. The presence of the Nt17 domain induces the formation of smooth and regular amyloid-like fibrils, while in absence of the Nt17 domain irregular and broad fibrils are formed due to strong lateral interaction between fibril filaments. Introduction of a helix-breaking proline into the Nt17 domain (M8P-Httex1) revealed that the Nt17 effect on the aggregate morphology is independent of its conformation. Interestingly, we were able to show that the Nt17 domain is not tightly bound within the fibril core of Httex1 fibrils and able to interact with membranes by performing trypsin digests of Httex1 fibrils and comparing the internalization of Httex1, âNt17-Httex1 and M8P-Httex1 fibrils into primary striatal neurons. The Nt17 domain harbors many post-translational modifications, e.g. phosphorylation, SUMOylation, ubiquitination and acetylation, which modulate Htt aggregation and toxicity. Therefore, we aimed at elucidating the effect of SUMOylation on the aggregation and structural properties of Httex1. After production of site-specifically SUMO1-modified Httex1 by semisynthesis, we observed that SUMO1-modification at K6 or K9 increased the compaction and stability of Httex1 inhibiting aggregation. The developed purification strategy enabled an efficient generation of the native Httex1 sequence. Using unmodified and modified (âNt17, M8P, SUMO1) Httex1 variants with varying polyQ lengths we were able to provide novel insights into how the polyQ and Nt17 domains regulate aggregate formation and influence the final aggregate structure. Our results contribute to a better understanding of the molecular determinants of Htt aggregate structure and could facilitate the development of inhibitors or biomarkers of Htt aggregation in vivo.

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

Le concept switch-peptides

Karine Murat

Conformational transitions have found broad interest due to their impact on protein misfolding and self-assembly as key events leading to the development of neurodegenerative diseases. However, investigation of these dynamic events has been limited so far mainly due to the intrinsic tendency of the involved polypeptides for self-association and aggregation. Consequently, the elucidation of β-sheet and fibril-forming processes related to degenerative diseases remains a difficult task, often giving contradictory results. In order to overcome these difficulties and to investigate early steps of misfolding and subsequent fibril and plaque formation, a new concept termed "Switch-peptides" is presented in this thesis, opening new perspectives for the rational design of drugs preventing Alzheimer's disease. Figure. Elaboration of the "Switch-peptides" concept via S to N acyl migration. As shown in the Figure, the switch-peptides represent stable, self-contained folding precursors, in which folding and self-assembly is blocked by the presence of the Ser-, Thr-, Cys- derived switch elements S dissecting the regular peptide backbone by an (thio)ester and a flexible C-C bond (Soff). Removal of the protecting group Y by enzymatic cleavage or by change of pH, triggers X,N acyl migration restoring the native peptide backbone in situ, and setting off the folding process in the nascent state ("in statu nascendi", ISN) of the parent molecule. These resulting conformational changes i.e. from a random coil to a folded state, can be used to study the onset of biological activity (A) onset (B) and disruption (C) of secondary and tertiary structures. The work described in the present thesis elaborates the structural and chemical foundations of the concept, notably in the induction of structural and conformational properties of peptides. Special attention is focused on the S to N acyl migration in elaborating an in situ ligation methodology. For potential applications in vitro and in vivo, we explore a new thioester solubilizing group to realize efficient native chemical ligations at physiological conditions (Figure). Besides the synthetic access of switch-peptides thioesters via solid-phase techniques, the impact of internal (steric hindrance) and external pH factors on the kinetics of the intramolecular acyl migration is evaluated by analytical HPLC. The major applications of the switch-peptides concept are explored via three main axis : In order to settle the foundations of strategy B, our study was based on the possibility of inducing in situ the formation of secondary and tertiary structures. For this purpose, an amphiphilic model peptide is designed to mimic conformational transitions relevant in degenerative disease, i.e. transitions of type random coil to β sheet. The role of the switch element S in the disruption of secondary structures due to the conformational decoupling of σ on P (Soff), is demonstrated by CD.The induced conformational transition to β sheet conformations is accompanied by a significant decrease in peptide solubility. Moreover, the controlled release of nucleophile within the S element can modulate the rate of structure transition over a broad time scale, as demonstrated by kinetic studies. The use of orthogonal protecting groups Y allows the sequential and individual triggering of multiple elements S, localized in strategic positions in more complex polypeptides. With this objective in mind, the concept is expanded to the induction of ββα supersecondary structures such as the Zinc finger domain (Zif). Actually, the simultaneous introduction of a chemically (pH) and enzymatically (DPPIV) cleavable group at the N- and C-terminus in a sequence derived of Zif1 268 permited the consecutive onset of secondary and tertiary structure by a hierarchical triggering of acyl migrations. The second part of the thesis is devoted to the elaboration of a new generation of β breaking switch-peptides (Figure, strategy C). For the design of such inhibitors, the concept of "separating" recognition (σ inactivated) and functional (σ activated) states appears essential. To construct molecules able to switch between recognition and functional state, we assemble different pseudoproline (ψ Pro)-containing building blocks as induction units σ for β-sheet breaking and a target peptide P. The impact of the ψ Pro-moiety is decoupled from the peptide chain via a flexible thioester bond (Soff) and set off after acyl migration (Son). As a consequence, the insertion and association into existing β-sheet templates should be promoted. Preliminary results by EM studies indicate that a controlled enzyme-induced acyl migration results in a significant improvement of the β-sheet breaking potential. For example, one of the designed switch-peptides slowed down the fibrillogenesis of an amyloid model, representing a significant step in the developement of therapeutically relevant compounds in Alzheimer's disease. The third part focuses on the in situ creation of structure and function of potentially bioactive peptide ligands (Figure, strategy A) via a novel ligation method ("Switch-ligation"). For the example of an angiotensin II-derived analogue, we explore the potential of chemoselective ligations for the onset of biological function under physiologically relevant conditions. Receptor (AT1) binding studies of [Cys5]Ang II in collaboration with PD. Dr. E. Grouzmann, CHUV reveal a fast and efficient ligation without the need of additives (IC50= 25 nM). As an alternative example, the covalent self-assembling of two precursors results in the in situ creation of an efficient peptide deformylase inhibitor. The perspective to extend these methodologies for in vivo applications has been tentatively explored as a new concept in prodrug design. In conclusion, the results of this thesis contribute substantially to the proof of concept of switch-peptides, opening new perspectives in drug design as well as in the study of peptide and protein misfolding relevant in degenerative diseases.

EPFL2006

Characterization of semisynthetic and naturally Nα-acetylated α-synuclein in vitro and in intact cells: implications for aggregation and cellular properties of α-synuclein

Mohamed Bilal Fares, Bruno Claude Daniel Fauvet, Hilal Lashuel

N-terminal acetylation is a very common post-translational modification, although its role in regulating protein physical properties and function remains poorly understood. α-Synuclein (α-syn), a protein that has been linked to the pathogenesis of Parkinson disease, is constitutively N(α)-acetylated in vivo. Nevertheless, most of the biochemical and biophysical studies on the structure, aggregation, and function of α-syn in vitro utilize recombinant α-syn from Escherichia coli, which is not N-terminally acetylated. To elucidate the effect of N(α)-acetylation on the biophysical and biological properties of α-syn, we produced N(α)-acetylated α-syn first using a semisynthetic methodology based on expressed protein ligation (Berrade, L., and Camarero, J. A. (2009) Cell. Mol. Life Sci. 66, 3909-3922) and then a recombinant expression strategy, to compare its properties to unacetylated α-syn. We demonstrate that both WT and N(α)-acetylated α-syn share a similar secondary structure and oligomeric state using both purified protein preparations and in-cell NMR on E. coli overexpressing N(α)-acetylated α-syn. The two proteins have very close aggregation propensities as shown by thioflavin T binding and sedimentation assays. Furthermore, both N(α)-acetylated and WT α-syn exhibited similar ability to bind synaptosomal membranes in vitro and in HeLa cells, where both internalized proteins exhibited prominent cytosolic subcellular distribution. We then determined the effect of attenuating N(α)-acetylation in living cells, first by using a nonacetylable mutant and then by silencing the enzyme responsible for α-syn N(α)-acetylation. Both approaches revealed similar subcellular distribution and membrane binding for both the nonacetylable mutant and WT α-syn, suggesting that N-terminal acetylation does not significantly affect its structure in vitro and in intact cells.

Amer Soc Biochemistry Molecular Biology Inc2012