Controlling the prion propensity of glutamine/asparagine-rich proteins

ABSTRACT

The yeast Saccharomyces cerevisiae can harbor a number of distinct prions. Most of the yeast prion proteins contain a glutamine/asparagine (Q/N) rich region that drives prion formation. Prion-like domains, defined as regions with high compositional similarity to yeast prion domains, are common in eukaryotic proteomes, and mutations in various human proteins containing prion-like domains have been linked to degenerative diseases, including amyotrophic lateral sclerosis. Here, we discuss a recent study in which we utilized two strategies to generate prion activity in non-prion Q/N-rich domains. First, we made targeted mutations in four non-prion Q/N-rich domains, replacing predicted prion-inhibiting amino acids with prion-promoting amino acids. All four mutants formed foci when expressed in yeast, and two acquired bona fide prion activity. Prion activity could be generated with as few as two mutations, suggesting that many non-prion Q/N-rich proteins may be just a small number of mutations from acquiring aggregation or prion activity. Second, we created tandem repeats of short prion-prone segments, and observed length-dependent prion activity. These studies demonstrate the considerable progress that has been made in understanding the sequence basis for aggregation of prion and prion-like domains, and suggest possible mechanisms by which new prion domains could evolve.     

Michael Ter-Avanesyan (1949-2018) - life in science

This commentary describes scientific path and accomplishments of our late colleague, Prof. Michael D. Ter-Avanesyan, who made several seminal contributions into prion research.     

The Hofmeister effect on amyloid formation using yeast prion protein

A variety of proteins are capable of converting from their soluble forms into highly ordered fibrous cross‐β aggregates (amyloids). This conversion is associated with certain pathological conditions in mammals, such as Alzheimer disease, and provides a basis for the infectious or hereditary protein isoforms (prions), causing neurodegenerative disorders in mammals and controlling heritable phenotypes in yeast. The N‐proximal region of the yeast prion protein Sup35 (Sup35NM) is frequently used as a model system for amyloid conversion studies in vitro. Traditionally, amyloids are recognized by their ability to bind Congo Red dye specific to β‐sheet rich structures. However, methods for quantifying amyloid fibril formation thus far were based on measurements linking Congo Red absorbance to concentration of insulin fibrils and may not be directly applicable to other amyloid‐forming proteins. Here, we present a corrected formula for measuring amyloid formation of Sup35NM by Congo Red assay. By utilizing this corrected procedure, we explore the effect of different sodium salts on the lag time and maximum rate of amyloid formation by Sup35NM. We find that increased kosmotropicity promotes amyloid polymerization in accordance with the Hofmeister series. In contrast, chaotropes inhibit polymerization, with the strength of inhibition correlating with the B‐viscosity coefficient of the Jones‐Dole equation, an increasingly accepted measure for the quantification of the Hofmeister series.     

Yeast J-protein Sis1 prevents prion toxicity by moderating depletion of prion protein

[PSI⁺] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI⁺] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI⁺] prions typically affect viability only modestly, so [PSI⁺] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for the propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild-type Sis1. Sis1JGF also supports [PSI⁺] propagation, yet [PSI⁺] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI⁺] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI⁺] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI⁺] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for the propagation of what otherwise would be lethal [PSI⁺] prions.     

酿酒酵母Sup35朊蛋白结构域的自组装机理及其应用的研究进展

Sup35是酿酒酵母的翻译终止因子,其朊蛋白结构域在体内外都能形成淀粉样蛋白纤维。由于其高度有序的交叉β-片层构象与其他物种中的淀粉样蛋白纤维相似,因此,Sup35的分子自组装机理的研究可以作为蛋白质错误折叠性疾病及朊病毒生物学等相关研究的理想模型。而Sup35朊蛋白结构域自组装成纳米线的能力在生物技术和纳米材料等方面已得到广泛的应用。     

Life cycle of cytosolic prions

Prions are self-templating protein aggregates that were originally identified as the causative agent of prion diseases in mammals, but have since been discovered in other kingdoms. Mammalian prions represent a unique class of infectious agents that are composed of misfolded prion protein. Prion proteins usually exist as soluble proteins but can refold and assemble into highly ordered, self-propagating prion polymers. The prion concept is also applicable to a growing number of non-Mendelian elements of inheritance in lower eukaryotes. While prions identified in mammals are clearly pathogens, prions in lower eukaryotes can be either detrimental or beneficial to the host. Prion phenotypes in fungi are transmitted vertically from mother to daughter cells during cell division and horizontally during mating or abortive mating, but extracellular phases have not been reported. Recent findings now demonstrate that in a mammalian cell environment, protein aggregates derived from yeast prion domains exhibit a prion life cycle similar to mammalian prions propagated ex vivo. This life cycle includes a soluble state of the protein, an induction phase by exogenous prion fibrils, stable replication of prion entities, vertical transmission to progeny and natural horizontal transmission to neighboring cells. Our data reveal that mammalian cells contain all co-factors required for cytosolic prion propagation and dissemination. This has important implications for understanding prion-like properties of disease-related protein aggregates. In light of the growing number of identified functional amyloids, cell-to-cell propagation of cytosolic protein conformers might not only be relevant for the spreading of disease-associated proteins, but might also be of more general relevance under non-disease conditions.     

Effects of randomizing the Sup35NM prion domain sequence on formation of amyloid fibrils in vitro

The mechanism by which proteins aggregate and form amyloid fibrils is still elusive. In order to preclude interference by cellular factors and to clarify the role of the primary sequence of Sup35p prion domain in formation of amyloid fibrils, we generated five Sup35NM variants by randomizing amino acid sequences in PrDs without altering the amino acid composition and analyzed the in vitro process of amyloid fibril formation. The results showed that each of the five Sup35NM variants polymerized into amyloid fibrils in vitro under native conditions. Furthermore, the Sup35NM variants showed differences in their aggregation time courses. These findings indicate that specific amino acid sequence features in PrD can modify the rate of conversion of Sup35p into amyloid fibrils in vitro.     

Strain conformation controls the specificity of cross-species prion transmission in the yeast model

Transmissible self-assembled fibrous cross-β polymer infectious proteins (prions) cause neurodegenerative diseases in mammals and control non-Mendelian heritable traits in yeast. Cross-species prion transmission is frequently impaired, due to sequence differences in prion-forming proteins. Recent studies of prion species barrier on the model of closely related yeast species show that colocalization of divergent proteins is not sufficient for the cross-species prion transmission, and that an identity of specific amino acid sequences and a type of prion conformational variant (strain) play a major role in the control of transmission specificity. In contrast, chemical compounds primarily influence transmission specificity via favoring certain strain conformations, while the species origin of the host cell has only a relatively minor input. Strain alterations may occur during cross-species prion conversion in some combinations. The model is discussed which suggests that different recipient proteins can acquire different spectra of prion strain conformations, which could be either compatible or incompatible with a particular donor strain.     

Hsp104, Hsp70 and Hsp40 interplay regulates formation, growth and elimination of Sup35 prions

Self-templating amyloid forms of Sup35 constitute the yeast prion [PSI(+)]. How the protein-remodelling factor, Hsp104, collaborates with other chaperones to regulate [PSI(+)] inheritance remains poorly delineated. Here, we report how the Ssa and Ssb components of the Hsp70 chaperone system directly affect Sup35 prionogenesis and cooperate with Hsp104. We identify the ribosome-associated Ssb1:Zuo1:Ssz1 complex as a potent antagonist of Sup35 prionogenesis. The Hsp40 chaperones, Sis1 and Ydj1, preferentially interact with Sup35 oligomers and fibres compared with monomers, and facilitate Ssa1 and Ssb1 binding. Various Hsp70:Hsp40 pairs block prion nucleation by disassembling molten oligomers and binding mature oligomers. By binding fibres, Hsp70:Hsp40 pairs occlude prion recognition elements and inhibit seeded assembly. These inhibitory activities are partially relieved by the nucleotide exchange factor, Fes1. Low levels of Hsp104 stimulate prionogenesis and alleviate inhibition by some Hsp70:Hsp40 pairs. At high concentrations, Hsp104 eliminates Sup35 prions. This activity is reduced when Ssa1, or enhanced when Ssb1, is incorporated into nascent prions. These findings illuminate several facets of the chaperone interplay that underpins [PSI(+)] inheritance.     

Octapeptide repeat insertions increase the rate of protease-resistant prion protein formation

A central feature of transmissible spongiform encephalopathies (TSE or prion diseases) involves the conversion of a normal, protease-sensitive glycoprotein termed prion protein (PrP-sen) into a pro-tease-resistant form, termed PrP-res. The N terminus of PrP-sen has five copies of a repeating eight amino acid sequence (octapeptide repeat). The presence of one to nine extra copies of this motif is associated with a heritable form of Creutzfeld-Jakob disease (CJD) in humans. An increasing number of octapeptide repeats correlates with earlier CJD onset, suggesting that the rate at which PrP-sen misfolds into PrP-res may be influenced by these mutations. In order to determine if octapeptide repeat insertions influence the rate at which PrP-res is formed, we used a hamster PrP amyloid-forming peptide (residues 23-144) into which two to 10 extra octapeptide repeats were inserted. The spontaneous formation of protease-resistant PrP amyloid from these peptides was more rapid in response to an increased number of octapeptide repeats. Furthermore, experiments using full-length glycosylated hamster PrP-sen demonstrated that PrP-res formation also occurred more rapidly from PrP-sen molecules expressing 10 extra copies of the octapeptide repeat. The rate increase for PrP-res formation did not appear to be due to any influence of the octapeptide repeat region on PrP structure, but rather to more rapid binding between PrP molecules. Our data from both models support the hypothesis that extra octapeptide repeats in PrP increase the rate at which protease resistant PrP is formed which in turn may affect the rate of disease onset in familial forms of CJD.     

GPI anchoring facilitates propagation and spread of misfolded Sup35 aggregates in mammalian cells

Prion diseases differ from other amyloid‐associated protein misfolding diseases (e.g. Alzheimer's) because they are naturally transmitted between individuals and involve spread of protein aggregation between tissues. Factors underlying these features of prion diseases are poorly understood. Of all protein misfolding disorders, only prion diseases involve the misfolding of a glycosylphosphatidylinositol (GPI)‐anchored protein. To test whether GPI anchoring can modulate the propagation and spread of protein aggregates, a GPI‐anchored version of the amyloidogenic yeast protein Sup35NM (Sup35^GPI) was expressed in neuronal cells. Treatment of cells with Sup35NM fibrils induced the GPI anchor‐dependent formation of self‐propagating, detergent‐insoluble, protease‐resistant, prion‐like aggregates of Sup35^GPI. Live‐cell imaging showed intercellular spread of Sup35^GPI aggregation to involve contact between aggregate‐positive and aggregate‐negative cells and transfer of Sup35^GPI from aggregate‐positive cells. These data demonstrate GPI anchoring facilitates the propagation and spread of protein aggregation and thus may enhance the transmissibility and pathogenesis of prion diseases relative to other protein misfolding diseases.     

Amino Acid Proximities in Two Sup35 Prion Strains Revealed by Chemical Cross-linking

Strains of the yeast prion [PSI] are different folding patterns of the same Sup35 protein, which stacks up periodically to form a prion fiber. Chemical cross-linking is employed here to probe different fiber structures assembled with a mutant Sup35 fragment. The photo-reactive cross-linker, p-benzoyl-l-phenylalanine (pBpa), was biosynthetically incorporated into bacterially prepared recombinant Sup(1–61)-GFP, containing the first 61 residues of Sup35, followed by the green fluorescent protein. Four methionine substitutions and two alanine substitutions were introduced at fixed positions in Sup(1–61) to allow cyanogen bromide cleavage to facilitate subsequent mass spectrometry analysis. Amyloid fibers of pBpa and Met/Ala-substituted Sup(1–61)-GFP were nucleated from purified yeast prion particles of two different strains, namely VK and VL, and shown to faithfully transmit specific strain characteristics to yeast expressing the wild type Sup35 protein. Intra- and intermolecular cross-linking were distinguished by tandem mass spectrometry analysis on fibers seeded from solutions containing equal amounts of ¹⁴N- and ¹⁵N-labeled protein. Fibers propagating the VL strain type exhibited intra- and intermolecular cross-linking between amino acid residues 3 and 28, as well as intra- and intermolecular linking between 32 and 55. Inter- and intramolecular cross-linking between residues 32 and 55 were detected in fibers propagating the VK strain type. Adjacencies of amino acid residues in space revealed by cross-linking were used to constrain possible chain folds of different [PSI] strains.     

Probing the role of structural features of mouse PrP in yeast by expression as Sup35-PrP fusions

The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive ‘toolbox’ available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrP^Sc. Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.     

Prion-based memory of heat stress in yeast

Amyloids and amyloid-based prions are self-perpetuating protein aggregates which can spread by converting a normal protein of the same sequence into a prion form. They are associated with diseases in humans and mammals, and control heritable traits in yeast and other fungi. Some amyloids are implicated in biologically beneficial processes. As prion formation generates reproducible memory of a conformational change, prions can be considered as molecular memory devices. We have demonstrated that in yeast, stress-inducible cytoskeleton-associated protein Lsb2 forms a metastable prion in response to high temperature. This prion promotes conversion of other proteins into prions and can persist in a fraction of cells for a significant number of cell generations after stress, thus maintaining the memory of stress in a population of surviving cells. Acquisition of an amino acid substitution required for Lsb2 to form a prion coincides with acquisition of increased thermotolerance in the evolution of Saccharomyces yeast. Thus the ability to form an Lsb2 prion in response to stress coincides with yeast adaptation to growth at higher temperatures. These findings intimately connect prion formation to the cellular response to environmental stresses.     

Amyloid-a state in many guises: survival of the fittest fibril fold

Under appropriate conditions, essentially all proteins are able to aggregate to form long, well-ordered and beta-sheet-rich arrays known as amyloid-like fibrils. These fibrils consist of varying numbers of intertwined protofibrils and can for any given protein exhibit a wealth of different forms at the ultrastructural level. Traditionally, this structural variability or polymorphism has been attributed to differences in the assembly of a common protofibril structure. However, recent work on glucagon, insulin, and the Abeta peptide suggests that this polymorphism can occur at the level of secondary structure. Simple variations in either solvent conditions such as temperature, protein concentration, and ionic strength or external mechanical influences such as agitation can lead to formation of fibrils with markedly different characteristics. In some cases, these characteristics can be passed on to new fibrils in a strain-specific manner, similar to what is known for prions. The preferred structure of fibrils formed can be explained in terms of selective pressure and survival of the fittest; the most populated types of fibrils we observe at the end of an experiment are those that had the fastest overall growth rate under the given conditions. Fibrillar polymorphism is probably a consequence of the lack of structural restraints on a nonfunctional conformational state.     

Spatial sequestration and oligomer remodeling during de novo [PSI+] formation

Prions are misfolded, aggregated, infectious proteins found in a range of organisms from mammals to bacteria. In mammals, prion formation is difficult to study because misfolding and aggregation take place prior to symptom presentation. The study of the yeast prion [PSI⁺], which is the misfolded infectious form of Sup35p, provides a tractable system to monitor prion formation in real time. Recently, we showed that the de novo formation of prion aggregates begins with the appearance of highly mobile cytoplasmic foci, called early foci, which assemble into larger ring or dot structures. We also observed SDS-resistant oligomers during formation, and lysates containing newly formed oligomers can convert [psi^?] cells to the [PSI⁺] state, suggesting that these oligomers have infectious potential. Here, we further characterize two aspects of prion formation: spatial sequestration of early foci and oligomerization of endogenous Sup35p. Our data provides important insights into the process of prion formation and explores the minimal oligomer requirement for infectivity.     

DNA aptamers detecting generic amyloid epitopes

Amyloids are fibrillar protein aggregates resulting from non-covalent autocatalytic polymerization of various structurally and functionally unrelated proteins. Previously we have selected DNA aptamers, which bind specifically to the in vitro assembled amyloid fibrils of the yeast prionogenic protein Sup35. Here we show that such DNA aptamers can be used to detect SDS-insoluble amyloid aggregates of the Sup35 protein, and of some other amyloidogenic proteins, including mouse PrP, formed in yeast cells. The obtained data suggest that these aggregates and the Sup35 amyloid fibrils assembled in vitro possess common conformational epitopes recognizable by aptamers. The described DNA aptamers may be used for detection of various amyloid aggregates in yeast and, presumably, other organisms.     

Characterization and enzymatic degradation of Sup35NM, a yeast prion-like protein

Transmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form of prion protein, PrPSc, is able to aggregate and form amyloid fibrils, very stable and resistant to most disinfecting processes and common proteases. Under specific conditions, PrPSc in bovine spongiform encephalopathy (BSE) brain tissue was found degradable by a bacterial keratinase and some other proteases. Since this disease-causing prion is infectious and dangerous to work with, a model or surrogate protein that is safe is needed for the in vitro degradation study. Here a nonpathogenic yeast prion-like protein, Sup35NM, cloned and overexpressed in E. coli, was purified and characterized for this purpose. Aggregation and deaggregation of Sup35NM were examined by electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting. The degradation of Sup35NM aggregates by keratinase and proteinase K under various conditions was studied and compared. These results will be of value in understanding the mechanism and optimization of the degradation process.     

The story of stolen chaperones

Prions are self-seeding alternate protein conformations. Most yeast prions contain glutamine/asparagine (Q/N)-rich domains that promote the formation of amyloid-like prion aggregates. Chaperones, including Hsp104 and Sis1, are required to continually break these aggregates into smaller “seeds.” Decreasing aggregate size and increasing the number of growing aggregate ends facilitates both aggregate transmission and growth. Our previous work showed that overexpression of 11 proteins with Q/N-rich domains facilitates the de novo aggregation of Sup35 into the [PSI⁺] prion, presumably by a cross-seeding mechanism. We now discuss our recent paper, in which we showed that overexpression of most of these same 11 Q/N-rich proteins, including Pin4C and Cyc8, destabilized pre-existing Q/N rich prions. Overexpression of both Pin4C and Cyc8 caused [PSI⁺] aggregates to enlarge. This is incompatible with a previously proposed “capping” model where the overexpressed Q/N-rich protein poisons, or “caps,” the growing aggregate ends. Rather the data match what is expected of a reduction in prion severing by chaperones. Indeed, while Pin4C overexpression does not alter chaperone levels, Pin4C aggregates sequester chaperones away from the prion aggregates. Cyc8 overexpression cures [PSI⁺] by inducing an increase in Hsp104 levels, as excess Hsp104 binds to [PSI⁺] aggregates in a way that blocks their shearing.