Semisynthesis | EPFL Graph Search

Semisynthesis, or partial chemical synthesis, is a type of chemical synthesis that uses chemical compounds isolated from natural sources (such as microbial cell cultures or plant material) as the starting materials to produce novel compounds with distinct chemical and medicinal properties. The novel compounds generally have a high molecular weight or a complex molecular structure, more so than those produced by total synthesis from simple starting materials. Semisynthesis is a means of preparing many medicines more cheaply than by total synthesis since fewer chemical steps are necessary. Drugs derived from natural sources are usually produced by isolation from the natural source or, as described here, by semisynthesis from such an isolated agent. From the viewpoint of chemical synthesis, living organisms are remarkable chemical factories that can easily produce structurally-complex chemical compounds by biosynthesis. In contrast, engineered chemical synthesis is necessarily simpler, with a lower chemical diversity in each reaction, than the incredibly-diverse biosynthesis pathways that are crucial to life. As a result, certain functional groups are much easier to prepare by engineered synthesis than others, such as acetylation, in which certain biosynthetic pathways can generate groups and structures with minimal economic input that would be prohibitive via total synthesis. Plants, animals, fungi, and bacteria are all used as sources for those tricky precursor molecules, including the use of bioreactors at the meeting point between engineered and biological chemical synthesis. Semisynthesis, when it is used in drug discovery, aims to retain the sought-after medicinal activity while other molecule characteristics are altered, such as those that affect its adverse events or its oral bioavailability in a few chemical steps. In that regard, semisynthesis stands in contrast with the approach of total synthesis, whose aim is to arrive at a target molecule from low-molecular-weight, inexpensive starting materials, often petrochemicals or minerals.

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

Sophie Vieweg

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.

EPFL2017

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