Intra-condensate demixing of TDP-43 inside stress granules generates pathological aggregates.

Cytosolic aggregation of the nuclear protein TDP-43 is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43 enriched phase within stress granules, which subsequently transitions into pathological aggregates. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We conclude that up-concentration inside condensates and simultaneous exposure to environmental stress could be a general pathway for protein aggregation, with intra-condensate demixing constituting a key intermediate step.

Direct proof of the amyloid nature of yeast prions [PSI+] and [PIN+] by the method of immunoprecipitation of native fibrils.

Prions are proteins that can exist in several structurally and functionally distinct states, one or more of which is transmissible. Yeast proteins Sup35 and Rnq1 in prion state ([PSI+] and [PIN+], respectively) form oligomers and aggregates, which are transmitted from parents to offspring in a series of generations. Several pieces of indirect evidence indicate that these aggregates also possess amyloid properties, but their binding to amyloid-specific dyes has not been shown in vivo. Meanwhile, it is the specific binding to the Congo Red dye and birefringence in polarized light after such staining that is considered the gold standard for proving the amyloid properties of a protein. Here, we used immunoprecipitation to extract native fibrils of the Sup35 and Rnq1 proteins from yeast strains with different prion status. These fibrils are detected by electron microscopy, stained with Congo Red and exhibit yellow-green birefringence after such staining. All these data show that the Sup35 and Rnq1 proteins in prion state form amyloid fibrils in vivo. The technology of fibrils extraction in combination with standard cytological methods can be used to identify new pathological and functional amyloids in any organism and to analyze the structural features of native amyloid fibrils.

Functional amyloids of eukaryotes: criteria, classification, and biological significance.

Amyloids cause incurable diseases in humans and animals and regulate vital processes in bacteria and eukaryotes. Amyloid fibrils have unique properties, such as amazing resistance to a variety of agents, mechanical strength, and elasticity, and it is not surprising that in the course of evolution eukaryotes have learned to employ amyloid structures to regulate various vital processes. Proteins exhibiting amyloid properties have been detected in lower eukaryotes and in diverse cell lines of arthropods and vertebrates. A growing number of studies of eukaryotic proteins that demonstrate certain amyloid-like properties require clear criteria to systematize modern knowledge about the functional amyloids. In this review, we propose to separate eukaryotic proteins, whose amyloid properties are clearly proven, and proteins, which show some amyloid characteristics in vivo or in vitro. In order to assert that a protein is a functional amyloid, it is necessary to prove that it has a cross-ß structure in vivo. Here, we consider the advantages and disadvantages of various methods for the analysis of the amyloid properties of a protein. Analysis of the current data shows that amyloids play an important role in the regulation of vital processes in eukaryotes, and new functional amyloids should be searched primarily among structural, protective, and storage proteins. A systematic search for functional amyloids in eukaryotes is only beginning, and the use of novel proteomic methods opens up great prospects for identification of amyloids in any organs and tissues of various organisms.

Screening for amyloid proteins in the yeast proteome.

The search for novel pathological and functional amyloids represents one of the most important tasks of contemporary biomedicine. Formation of pathological amyloid fibrils in the aging brain causes incurable neurodegenerative disorders such as Alzheimer's, Parkinson's Huntington's diseases. At the same time, a set of amyloids regulates vital processes in archaea, prokaryotes and eukaryotes. Our knowledge of the prevalence and biological significance of amyloids is limited due to the lack of universal methods for their identification. Here, using our original method of proteomic screening PSIA-LC-MALDI, we identified a number of proteins that form amyloid-like detergent-resistant aggregates in Saccharomyces cerevisiae. We revealed in yeast strains of different origin known yeast prions, prion-associated proteins, and a set of proteins whose amyloid properties were not shown before. A substantial number of the identified proteins are cell wall components, suggesting that amyloids may play important roles in the formation of this extracellular protective sheath. Two proteins identified in our screen, Gas1 and Ygp1, involved in biogenesis of the yeast cell wall, were selected for detailed analysis of amyloid properties. We show that Gas1 and Ygp1 demonstrate amyloid properties both in vivo in yeast cells and using the bacteria-based system C-DAG. Taken together, our data show that this proteomic approach is very useful for identification of novel amyloids.