Proteomic Screening for Amyloid Proteins

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.

Interaction of Prions Causes Heritable Traits in Saccharomyces cerevisiae

“…Proteins present only in the test sample ([ NSI + ]) are pseudocolored in red, proteins present only in the control sample ([ nsi - ]) are pseudocolored in green and yellow spots correspond to proteins that were present in both samples. Such yellow spots were identified as the aminopeptidases Ape1 and Ape4 (Fig 1, S1 and S2 Figs) that we had detected in our previous study in different yeast strains [22]. Red spots specific to the [ NSI + ] sample (Fig 1) were identified as Rnq1 (S3 Fig), which is the structural protein of [ PIN + ] prion [10,23].…”

“…To identify the proteins whose prion conversion determines the [ NSI + ] phenotype, we used a method for proteomic screening and identification of amyloid proteins (PSIA) which we had previously developed and successfully applied for identification of yeast prions [22]. This method is based on the resistance of prion aggregates to treatment with ionic detergents such as sodium dodecyl sulfate (SDS).…”

“…We previously used large-scale overexpression screens to reveal the genes affecting [ NSI + ] manifestation, but we did not identify the structural gene of this factor [19,21]. Recently we developed the proteomic method for identification of yeast prion proteins that form amyloid-like aggregates resistant to treatment with ionic detergents [22]. Here, using this method we demonstrated that the nonsense suppression in the [ NSI + ] strain is a result of interaction between [ PIN + ] and [ SWI + ] prions.…”

Prions, amyloids, and RNA: Pieces of a puzzle

“…However, several methods have been recently developed allowing to screen proteomes for amyloids. Such methods, TAPI 176 and PSIA, 177 facilitate rapid identification of amyloid proteins. PSIA uses two-dimensional difference gel electrophoresis (2D-DIGE) for the separation of proteins.…”

“…The limitation of this method is that it does not allow the identification of proteins with extreme isoelectric points. 177 TAPI employs very efficient highperformance liquid chromatography to separate proteins, but utilizes resistance to sodium dodecyl sulfate, to which not all amyloids are resistant, as a screening criterion. 114,176 Despite the limitations, these methods were validated by detection of a wide spectrum of known amyloids and are promising for the identification of novel amyloid-forming and amyloid-associated proteins in different organisms at the proteomic level.…”