Fusion of Hsp70 to GFP Impairs Its Function and Causes Formation of Misfolded Protein Deposits under Mild Stress in Yeast.

Protein misfolding is a common feature of aging, various diseases and stresses. Recent work has revealed that misfolded proteins can be gathered into specific compartments, which can limit their deleterious effects. Chaperones play a central role in the formation of these misfolded protein deposits and can also be used to mark them. While studying chimeric yeast Hsp70 (Ssa1-GFP), we discovered that this protein was prone to the formation of large insoluble deposits during growth on non-fermentable carbon sources under mild heat stress. This was mitigated by the addition of antioxidants, suggesting that either Ssa1 itself or some other proteins were affected by oxidative damage. The protein deposits colocalized with a number of other chaperones, as well as model misfolded proteins, and could be disassembled by the Hsp104 chaperone. Notably, the wild-type protein, as well as a fusion protein of Ssa1 to the fluorescent protein Dendra2, were much less prone to forming similar foci, indicating that this phenomenon was related to the perturbation of Ssa1 function by fusion to GFP. This was also confirmed by monitoring Hsp104-GFP aggregates in the presence of known Ssa1 point mutants. Our data indicate that impaired Ssa1 function can favor the formation of large misfolded protein deposits under various conditions.

Structural Bases of Prion Variation in Yeast.

Amyloids are protein aggregates with a specific filamentous structure that are related to a number of human diseases, and also to some important physiological processes in animals and other kingdoms of life. Amyloids in yeast can stably propagate as heritable units, prions. Yeast prions are of interest both on their own and as a model for amyloids and prions in general. In this review, we consider the structure of yeast prions and its variation, how such structures determine the balance of aggregated and soluble prion protein through interaction with chaperones and how the aggregated state affects the non-prion functions of these proteins.

Amyloid Fragmentation and Disaggregation in Yeast and Animals.

Amyloids are filamentous protein aggregates that are associated with a number of incurable diseases, termed amyloidoses. Amyloids can also manifest as infectious or heritable particles, known as prions. While just one prion is known in humans and animals, more than ten prion amyloids have been discovered in fungi. The propagation of fungal prion amyloids requires the chaperone Hsp104, though in excess it can eliminate some prions. Even though Hsp104 acts to disassemble prion fibrils, at normal levels it fragments them into multiple smaller pieces, which ensures prion propagation and accelerates prion conversion. Animals lack Hsp104, but disaggregation is performed by the same complement of chaperones that assist Hsp104 in yeast-Hsp40, Hsp70, and Hsp110. Exogenous Hsp104 can efficiently cooperate with these chaperones in animals and promotes disaggregation, especially of large amyloid aggregates, which indicates its potential as a treatment for amyloid diseases. However, despite the significant effects, Hsp104 and its potentiated variants may be insufficient to fully dissolve amyloid. In this review, we consider chaperone mechanisms acting to disassemble heritable protein aggregates in yeast and animals, and their potential use in the therapy of human amyloid diseases.

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

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.

Proteinase K resistant cores of prions and amyloids.

Amyloids and their infectious subset, prions, represent fibrillary aggregates with regular structure. They are formed by proteins that are soluble in their normal state. In amyloid form, all or part of the polypeptide sequence of the protein is resistant to treatment with proteinase K (PK). Amyloids can have structural variants, which can be distinguished by the patterns of their digestion by PK. In this review, we describe and compare studies of the resistant cores of various amyloids from different organisms. These data provide insight into the fine structure of amyloids and their variants as well as raise interesting questions, such as those concerning the differences between amyloids obtained ex vivo and in vitro, as well as the manner in which folding of one region of the amyloid can affect other regions.

Analysis of novel hyperosmotic shock response suggests 'beads in liquid' cytosol structure.

Proteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci, revealed by super-resolution microscopy, indicated that the foci were compressed between other entities. Based on our findings, we suggest a new model of cytosol architecture as a collection of numerous gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions.

Yeast Sup35 Prion Structure: Two Types, Four Parts, Many Variants.

The yeast [PSI+] prion, formed by the Sup35 (eRF3) protein, has multiple structural variants differing in the strength of nonsense suppressor phenotype. Structure of [PSI+] and its variation are characterized poorly. Here, we mapped Sup35 amyloid cores of 26 [PSI+] ex vivo prions of different origin using proteinase K digestion and mass spectrometric identification of resistant peptides. In all [PSI+] variants the Sup35 amino acid residues 2-32 were fully resistant and the region up to residue 72 was partially resistant. Proteinase K-resistant structures were also found within regions 73-124, 125-153, and 154-221, but their presence differed between [PSI+] isolates. Two distinct digestion patterns were observed for region 2-72, which always correlated with the "strong" and "weak" [PSI+] nonsense suppressor phenotypes. Also, all [PSI+] with a weak pattern were eliminated by multicopy HSP104 gene and were not toxic when combined with multicopy SUP35. [PSI+] with a strong pattern showed opposite properties, being resistant to multicopy HSP104 and lethal with multicopy SUP35. Thus, Sup35 prion cores can be composed of up to four elements. [PSI+] variants can be divided into two classes reliably distinguishable basing on structure of the first element and the described assays.

Increasing throughput of manual microscopy of cell suspensions using solid medium pads.

Microscopy of multiple samples using slides and coverslips is time consuming and good images are sometimes difficult to obtain due to cell movement. Our method involves manual spotting of multiple cell samples onto solid-medium pads, which creates the following benefits: •Rapid, high-quality imaging of multiple samples (hundreds per day) by visible and fluorescence microscopy.•No need for expensive automated equipment, multi-well plates or large amounts of consumables.•Wide range of working cell concentrations. The method was implemented for S. cerevisiae yeast, however it is likely to be applicable to all types of microorganisms and possibly other microscopic samples such as pollen. The lack of need for automated equipment may also make this method useful for field work.

The Pub1 and Upf1 Proteins Act in Concert to Protect Yeast from Toxicity of the [PSI⁺] Prion.

The [PSI⁺] nonsense-suppressor determinant of Saccharomyces cerevisiae is based on the formation of heritable amyloids of the Sup35 (eRF3) translation termination factor. [PSI⁺] amyloids have variants differing in amyloid structure and in the strength of the suppressor phenotype. The appearance of [PSI⁺], its propagation and manifestation depend primarily on chaperones. Besides chaperones, the Upf1/2/3, Siw14 and Arg82 proteins restrict [PSI⁺] formation, while Sla2 can prevent [PSI⁺] toxicity. Here, we identify two more non-chaperone proteins involved in [PSI⁺] detoxification. We show that simultaneous lack of the Pub1 and Upf1 proteins is lethal to cells harboring [PSI⁺] variants with a strong, but not with a weak, suppressor phenotype. This lethality is caused by excessive depletion of the Sup45 (eRF1) termination factor due to its sequestration into Sup35 polymers. We also show that Pub1 acts to restrict excessive Sup35 prion polymerization, while Upf1 interferes with Sup45 binding to Sup35 polymers. These data allow consideration of the Pub1 and Upf1 proteins as a novel [PSI⁺] detoxification system.

Proteomic screening for amyloid proteins.

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.

Carnosine prevents necrotic and apoptotic death of rat thymocytes via ouabain-sensitive Na/K-ATPase.

It is known that ouabain, a selective inhibitor of Na/K-ATPase, not only can cause the activation of signal cascades, which regulate the cell viability, but also can cause the accumulation of free radicals, which can evoke the oxidative stress. We have shown that the nanomolar concentrations of ouabain result in the temporary increase in the level of intracellular free radicals, but the millimolar concentration of ouabain induces a stable intracellular accumulation of free radicals in rat thymocytes. The increasing level of free radicals resulting from both low and high concentrations of ouabain can be attenuated by the antioxidant, carnosine. Moreover, the long-term incubation with ouabain leads to the cell death by necrosis and apoptosis. Ouabain-mediated apoptosis and necrosis were also abolished by carnosine.