Residue-by-residue details of FUS protein phase separation and aggregation

Brown University, Providence RI

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Abstract

Project Summary RNA-binding protein aggregates and inclusions are hallmarks of numerous neurodegenerative diseases including frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). These nuclear proteins facilitate fundamental processes including transcription, splicing, and DNA damage repair. While many possess low-complexity, disordered regions that are critical to their ability to form liquid-like, protein rich condensates that carry out normal RNA-processing, these same disordered regions enable the proteins to form neural inclusions. Fused in Sarcoma (FUS) is one of twenty-nine human RNA-binding proteins that contains both a disordered low- complexity domain (LC) with an unusual composition low in charged residues and high in aromatic residues, as well as several RGG motif regions. Inclusions comprised of FUS as well as TAF15 and EWS are found in approximately 10% of FTD cases and dominant mutations in the coding region of FUS cause aggressive forms of familial ALS. The current hypothesis is that these ALS-associated mutations, many of which are localized in the disordered domains of FUS, promote contacts that accelerate the liquid to solid transition (LST) of FUS and ultimately enhance the protein’s aggregation propensity. Several of these aggregated FUS structures, including aggregated short peptide microcrystals formed in vitro and aggregated full-length protein directly extracted from patient tissue, are being visualized with techniques such as cryo-electron microscopy (cryo-EM). However, the self-interactions that drive the formation of FUS assemblies and the mechanism by which physiological FUS converts to these are currently unknown because the self-interactions of FUS are invisible to traditional structural biology techniques. To address this gap, we will apply our tools to visualize the dynamic aggregation of FUS with residue-level resolution. This project will develop advanced nuclear magnetic resonance (NMR) techniques to identify self-contacts that are critical to the LST pathway. In particular, this project will employ NMR-based hydrogen-deuterium exchange (HDX), a methodology that has never been applied to the structural details of FUS, to elucidate FUS residues and regions that are essential to aggregate core formation. Moreover, through several well-established collaborations, this study will utilize molecular simulations and cellular models involving FUS aggregates to complement in vitro structural data revealed via NMR-based HDX experiments. These studies of the FUS aggregation pathway in vitro and in cells will provide essential structure/function information on future pharmacological targets for inhibiting pathological protein associations in types of ALS. Furthermore, because FUS is only one of many essential RNA-binding proteins containing aggregation-prone low complexity domains, the results of the project will serve as a foundation for understanding an entire class of proteins and for correcting their dysfunctions in disease. These studies in this two-year project will establish new structural and cellular biology methods applied to disease aggregation and lay the foundation for unraveling how FUS aggregates form in ALS.