HSP70ASYN · Structural studies of the interaction between Hsp70 and alpha-synuclein

Structural studies of the interaction between Hsp70 and alpha-synuclein

Summary description of the project objectives The project was aimed at elucidating the molecular basis of the interaction between Hsp70 and aSyn (alpha-synuclein) and at investigating the structural basis of diversity of binding modes between the two proteins. and their effects on the modulation of amyloidogenesis. One specific novel aspect of this project was the use of a full-length protein substrate instead of a fragment target in a high-resolution investigation of the substrate binding mechanism for Hsp70, which makes this proposal challenging and increases its potential impact. This project was designed to achieve the overall objective through different levels of complexity that can be summarised in the following objectives: Objective 1. To identify chaperone-affine and aggregation-prone peptide fragments of aSyn Affinity and binding properties of putative peptide targets with full-length Hsp70 and isolated SDB are investigated using different techniques: fluorescence spectroscopy, NMR spectroscopy, isothermal titration calorimetry and surface plasmon resonance. Objective 2. To characterise Hsp70/aSyn interactions in vitro using FRET methods The interaction of aSyn in its diverse molecular forms (monomeric, oligomeric at early stages of fibrillisation, prefibrillar large aggregates, amyloid fibrils.) with full-length Hsp70 and its SBD (Substrate binding domain) will be thoroughly investigated, using ensemble and single-molecule fluorescence resonance energy transfer analysis. Objective 3. To characterise the aSyn fibrillisation pathway in the presence of Hsp70 The aggregation behaviour of aSyn will be monitored when interacting with of Hsp70 present in different variants and under different conditions (full-length, SBD, truncated constructs, no nucleotide, ATP, ADP). The outcome of these experiments will give a comprehensive profile of the influence of Hsp70 ATP-hydrolysis cycle and of oligomerisation state of aSyn substrate on the fibrillisation pathway. Objective 4. To obtain resonance assignments of Hsp70 constructs In addition to wild type human Hsp70, different truncated and mutated variants of the full-length protein, the NBD and SBD domains will be tested in order to facilitate the assignment of chemical shifts. For the individual NBD and SBD, as well as for the full-length Hsp70, selective labelling strategies will be employed to achieve the assignment of methyl groups distributed across the protein structure, to be used as sensitive reporters for intra- and inter-molecular interactions. Objective 5. To perform RDC and PRE NMR measurements of Hsp70 constructrs Different sets of residual dipolar couplings (RDCs) (HN, Ha-Ca, Ca-CO) will be collected in multiple alignment media (Pf1, polyacrylamide gels, bicelles, PEG/alcohol mixtures). Paramagnetic relaxation enhancements (PREs) will be measured using the spin-label MTSL attached to distinct sites in single-cysteine aSyn. 15N relaxation data (T1, T2, heteronuclear NOE) will also be recorded. All these NMR parameters contain both structural and dynamical information and thus collection of data in different states of Hsp70 (SBD, nucleotide-free, ADP-bound, ATP-bound) may allow a direct comparison of these diverse conformations when bound to an intact natural substrate. Objective 6. To determine the structure and the dynamics of the different Hsp70 constructs, and of the Hsp70/aSyn complex by incorporating NMR measurements into molecular dynamics simulations Novel methods for determining the structure and dynamics of proteins will be employed in which structural information based primarily on C.S., RDCs and PRE is used to build restraints in molecular dynamics simulations. Description of the work performed Production of proteins The researcher was trained in the production of the proteins (unlabelled and 15N/13C-labelled) needed to carry out the experimental part of the project. Different cycles of production of aSyn were performed allowing the researcher to acquire very good familiarity with this kind of production and with the host laboratory and equipment. The researcher produced three versions of the SBD domain of Hsp70 (SBD556: aa 385-556; SBD641: aa 385-641; SBD DL641:aa 396-641) in sufficient amounts to be preliminarily studied using the HSQC experiment. The NMR quality of these constructs was investigated. NMR of aSyn over time The researcher developed a method to follow the decay of signal upon aSyn fibrillisation in real time and to detect spectral changes over time, using standard 1D and 2D NMR experiments. This approach set the basis to study in real time the effect of Hsp70 on aSyn conformational profile. NMR of interaction between aSyn and SBD The interaction of aSyn (unlabelled) with 15N-labelled constructs of Hsp70 was investigated using proton 1D NMR and 15N-1H HSQC. The proteins that were tested were the whole SDB (SBD641: aa 385-641), a construct of the SBD lacking part of the hydrophobic linker t its N-terminus (SBD DL641: aa 396-641), a truncated version lacking the C-terminal part (SBD556: aa 385-556). Due to the extremely heterogeneous profile in intensity distribution in the SBD641 spectra, different sample preparations were used and tested and the experiments were replicated two to three times on different occasions in order to carry out a detailed analysis on spectral quality and dynamics and to investigate the effect of adding aSyn to these constructs. A titration analysis of aSyn with the NR peptide (NRLLLTG) was also done. Description of the main results achieved The researcher prepared a large amount of protein samples required for the experimental work and the analysis of the resulting measurements for the following part of the project. The researcher set up an NMR approach to study in real time the effect of Hsp70 on aSyn conformational profile. The researcher carried out an NMR analysis of the constructs to be used in the more general aSyn-Hsp70 interaction study, identifying significant drawbacks in the suitability of SDB641 constructs and highlighting the need to use as first approach C-terminal truncated versions of SBD. Expected final results and potential impact and use The overall impact of the project concerns a comprehensive understanding at atomic level of the basis of the interaction between aSyn and Hsp70 and the characterisation of their multiple modes of binding. The work carried out represents an early, fundamental step in developing alternative therapeutic strategies for controlling progression or improving prevention of Parkinson's disease, leading to unknown benefits to patients, society and healthcare systems. In order to enable follow up studies to be carried out in the host laboratory, given the premature termination of the project because of the researcher's personal reasons, the researcher's work has been accurately communicated to other members of the group with whom he collaborated for this project.

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