Biology subjects

Chappard, A.

Publications and source records attributed to Chappard, A..

3 recordsLinked to original sources

bioRxiv2023

Two-color coincidence single-molecule pull-down for the specific detection of disease-associated protein aggregates

The misfolding and aggregation of protein is a characteristic of many neurodegenerative disorders, including Alzheimers and Parkinsons disease. The wide range of sizes and structures of oligomers and fibrils generated have previously been studied using single-molecule and super-resolution microscopy. These methods, however, tend to rely on the use of either directly labeled protein, or on the addition of non-specific amyloid stains, such as thioflavin-T. This has prevented the characterization of protein aggregate composition in complex biological samples. Here, we have developed a single-molecule two-color aggregate pull-down (STAPull) assay to overcome this challenge by probing immobilized proteins using orthogonally labeled antibodies targeting the same epitope. By looking at colocalized signals, we can eliminate monomeric protein, and specifically quantify aggregated proteins. Using the aggregation-prone alpha-synuclein protein as a model, we demonstrate that this approach can specifically detect aggregates with a limit of detection of 5 pM. Furthermore, we show that STAPull can be used in a range of samples, including in human biofluids. STAPull is generally applicable to protein aggregates from a variety of disorders, and will aid in the identification of biomarkers that are crucial in the effort to diagnose these diseases.

neuroscience↗

bioRxiv2022

Structural conversion of α-synuclein at the mitochondria induces neuronal toxicity

Aggregation of -Synuclein (-Syn) drives Parkinsons disease, although the initial stages of self-assembly and structural conversion have not been captured inside neurons. We track the intracellular conformational states of -Syn utilizing a single-molecule FRET biosensor, and show that -Syn converts from its monomeric state to form two distinct oligomeric states in neurons in a concentration dependent, and sequence specific manner. 3D FRET-CLEM reveals the structural organization, and location of aggregation hotspots inside the cell. Notably multiple intracellular seeding events occur preferentially on membrane surfaces, especially mitochondrial membranes. The mitochondrial lipid, cardiolipin triggers rapid oligomerization of A53T -Syn, and cardiolipin is sequestered within aggregating lipid-protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial ROS generation, which accelerates the oligomerization of A53T -Syn, and ultimately causes permeabilization of mitochondrial membranes, and cell death. Patient iPSC derived neurons harboring A53T mutations exhibit accelerated oligomerization that is dependent on mitochondrial ROS, early mitochondrial permeabilization and neuronal death. Our study highlights a mechanism of de novo oligomerization at the mitochondria and its induction of neuronal toxicity.

neuroscience↗

bioRxiv2021

Selectivity of Protein Interactions along the Aggregation Pathway of a-Synuclein

The aggregation of -SYN follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinsons Disease in neurons. Whilst -synuclein is a major contributor to LB, these dense accumulations of protein aggregates and tangles of fibrils contain over 70 different proteins. However, the potential for interactions between these proteins and the different aggregated species of -SYN is largely unknown. We hypothesized that the proteins present in the Lewy Bodies are trapped or pulled into the aggregates in a hierarchical manner, by binding at specific stages of the aggregation of -SYN. In this study we uncover a map of interactions of a total of 65 proteins, against different species formed by -SYN. We measured binding to monomeric -SYN using AlphaScreen, a sensitive nano-bead assay for detection of protein-protein interactions. To access different oligomeric species, we made use of the pathological mutants of -SYN (A30P, G51D and A53T), which form oligomeric species with distinct properties. Finally, we used bacterially expressed recombinant -SYN to generate amyloid fibrils and measure interactions with a pool of GFP-tagged potential partners. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are selectively recruited to specific steps in the formation of the LB, explaining their presence in the inclusions. Only a few proteins were found to interact with -SYN monomers at detectable levels, and only a subset recognizes the oligomeric -SYN including autophagosomal proteins. We therefore propose a new model for the formation of Lewy Bodies, where selectivity of protein partners at different steps drives the arrangement of these structures, uncovering new ways to modulate aggregation. Significance StatementThe molecular complexity of the Lewy Bodies has been a major hindrance to a bottom-up reconstruction of these inclusions, protein by protein. This work presents an extensive dataset of protein-protein interactions, showing that despite its small size and absence of structure, -SYN binds to specific partners in the LB, and that there is a clear selectivity of interactions between the different -SYN species along the self-assembly pathway. We use single-molecule methods to deconvolute number and size of the co-aggregates, to gain detailed information about the mechanisms of interaction. These observations constitute the basis for the elaboration of a global interactome of -SYN.

biochemistry↗