Protein aggregation is a process that involves the assembly of misfolded proteins into soluble intermediate oligomers and insoluble higher-order structures. However, despite the significant interest in protein aggregation diseases, there is an unmet demand for simple methods to directly visualize an aggregation of a protein of interest (POI) in live cells. To address this matter, we recently developed an imaging method named the AggTag (Aggregation Tag) to report on the aggregation of specific POIs within cellular milieu. We demonstrated that the AggTag probe P1, a Halo-tag substrate containing a red fluorescent molecular rotor, can detect the previously invisible soluble oligomers as well as insoluble aggregates in cells. In this work, we present a new library of AggTag probes (P2 ~ P4) to expand the components and application of the AggTag method. The AggTag probe P2, which is a SNAP-tag substrate bearing green solvatochromic fluorophore, can detect aggregation of SNAPf-tagged POIs. Because P1 and P2 possess orthogonal labelling specificities and excitation, P1 and P2 together can be used to simultaneously detect the aggregation of two different POIs with dual-color emission. By co-expressing Halo-tagged huntingtin exon 1 mutant (Htt-Q110) and SNAPf-tagged superoxide dismutase 1 (SOD1) mutants with co-labelling with P1 and P2, we show that the aggregation of SOD1 mutants, but not that of the SOD1 wild type, is induced by the Htt-Q110 aggregates. By swapping each fluorophore on P1 and P2 with one another, additional AggTag probes, P3 and P4, are created and shown to retain similar performance in detecting protein aggregation as the pair of P1 and P2. Given the increasing attention to protein aggregation in neurodegenerative diseases, the presented AggTag system will provide an invaluable tool to unravel complex aggregation mechanisms of POIs in live cells.

Fluorogenic AggTag method based on Halo and SNAP-tags to simultaneously detect aggregation of two proteins in live cells

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.