Dangerous Stops: Nonsense Mutations Can Dramatically Increase Frequency of Prion Conversion.

Valery N. Urakov,Alexander A. Dergalev,Vitaly V. Kushnirov,Michael O. Agaphonov,Alexander I. Alexandrov

doi:10.3390/ijms22041542

Abstract

Amyloid formation is associated with many incurable diseases. For some of these, sporadic cases are much more common than familial ones. Some reports point to the role of somatic cell mosaicism in these cases via origination of amyloids in a limited number of cells, which can then spread through tissues. However, specific types of sporadic mutations responsible for such effects are unknown. In order to identify mutations capable of increasing the de novo appearance of amyloids, we searched for such mutants in the yeast prionogenic protein Sup35. We introduced to yeast cells an additional copy of the SUP35 gene with mutated amyloidogenic domain and observed that some nonsense mutations increased the incidence of prions by several orders of magnitude. This effect was related to exposure at the C-terminus of an internal amyloidogenic region of Sup35. We also discovered that SUP35 mRNA could undergo splicing, although inefficiently, causing appearance of a shortened Sup35 isoform lacking its functional domain, which was also highly prionogenic. Our data suggest that truncated forms of amyloidogenic proteins, resulting from nonsense mutations or alternative splicing in rare somatic cells, might initiate spontaneous localized formation of amyloids, which can then spread, resulting in sporadic amyloid disease.

Highlights

Amyloids are filamentous protein aggregates with regular cross-beta structure
Domain amplified bySup35 mutagenic and inWe searched for mutations in the prion was (N) domain of the proteinPCR, that would serted the centromeric homologous recombination yield to frequent
We studied the structure of Sup35(1–112) amyloid, which causes the highest frequency of prion conversion

Summary

Introduction

Amyloids are filamentous protein aggregates with regular cross-beta structure. Amyloids are a hallmark of many degenerative diseases, such as prion diseases, Alzheimer’s, Parkinson’s and Huntington’s diseases, Amyotrophic Lateral Sclerosis (ALS), type 2 diabetes and many less common disorders [1]. Sup includes the essential GTP-ase C domain and non-essential intrinsically disordered NM region, which can drive the formation and inheritance of multiple distinct amyloid folds, that underlie different variants of [PSI+ ] phenotype [13,14,15]. These variants can be divided into two major classes, “weak” and “strong”. [PSI+ ] reduces the level of functional Sup and impairs translation termination, causing readthrough of ade and partial restoration of adenine synthesis Such cells became adenine independent and form white or pink colonies. Our data suggest that truncated forms of amyloidogenic proteins originating from spontaneous nonsense mutations or splicing can exhibit highly increased amyloidogenic potential and might play an important role in sporadic cases of amyloid diseases

Results

Isolation

Frequency

Structure of the Inducer Fragment

Dependence

Proteinase

SUP35 Splicing

Discussion

Sup35 Splicing and Its Effect on Prionogenesis

Support for the “Somatic Mutation” Hypothesis of Sporadic Amyloidoses

Yeast Strains and Media

Plasmids