Abstract
Despite extensive study, progress in elucidation of biological functions of amyloids and their role in pathology is largely restrained due to the lack of universal and reliable biochemical methods for their discovery. All biochemical methods developed so far allowed only identification of glutamine/asparagine-rich amyloid-forming proteins or proteins comprising amyloids that form large deposits. In this article we present a proteomic approach which may enable identification of a broad range of amyloid-forming proteins independently of specific features of their sequences or levels of expression. This approach is based on the isolation of protein fractions enriched with amyloid aggregates via sedimentation by ultracentrifugation in the presence of strong ionic detergents, such as sarkosyl or SDS. Sedimented proteins are then separated either by 2D difference gel electrophoresis or by SDS-PAGE, if they are insoluble in the buffer used for 2D difference gel electrophoresis, after which they are identified by mass-spectrometry. We validated this approach by detection of known yeast prions and mammalian proteins with established capacity for amyloid formation and also revealed yeast proteins forming detergent-insoluble aggregates in the presence of human huntingtin with expanded polyglutamine domain. Notably, with one exception, all these proteins contained glutamine/asparagine-rich stretches suggesting that their aggregates arose due to polymerization cross-seeding by human huntingtin. Importantly, though the approach was developed in a yeast model, it can easily be applied to any organism thus representing an efficient and universal tool for screening for amyloid proteins.
Highlights
An increasing amount of findings demonstrates that amyloids can be a functional form of a protein in a broad range of organisms from bacteria to mammals
We identified several yeast proteins which form detergent-insoluble aggregates in response to expression of human huntingtin with an expanded polyglutamine domain
We tested the efficiency of our approach for the detection of the Rnq1 protein which is the structural determinant of the yeast prion [PIN+] [7] and for human Amyloid beta peptide (Ab) (1–40 aa) peptide tagged with GFP, which is known to form amyloid-like aggregates in yeast cells both in the presence and absence of [PIN+] [25]
Summary
An increasing amount of findings demonstrates that amyloids can be a functional form of a protein in a broad range of organisms from bacteria to mammals. Interest in such protein aggregates is still mainly caused by their relation to human and animal pathologies. In spite of significant progress in discovering amyloids, most of them were found by analyzing proteins with sequences similar to known amyloid-forming proteins [6], [9] or via timeconsuming and sometimes highly sophisticated genetic screenings for factors that determine non-Mendelian traits [7], [8], [11], [12]. This suggests that development of a reliable and universal biochemical approach for fast identification of novel amyloid-forming proteins is both important and timely
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