Conformational diversity in a yeast prion dictates its seeding specificity

A perplexing feature of prion-based inheritance is that prions composed of the same polypeptide can evoke different phenotypes (such as distribution of brain lesions), even when propagated in genetically identical hosts. The molecular basis of this strain diversity and the relationship between strains and barriers limiting transmission between species remain unclear. We have used the yeast prion phenomenon [PSI+]4 to investigate these issues and examine the role that conformational differences may have in prion strains. We have made a chimaeric fusion between the prion domains of two species (Saccharomyces cerevisae and Candida albicans) of Sup35, the protein responsible for [PSI+]. Here we report that this chimaera forms alternate prion strains in vivo when initiated by transient overexpression of different Sup35 species. Similarly, in vitro the purified chimaera, when seeded with different species of Sup35 fibres, establishes and propagates distinct amyloid conformations. These fibre conformations dictate amyloid seeding specificity: a chimaera seeded by S. cerevisiae fibres efficiently catalyses conversion of S. cerevisiae Sup35 but not of C. albicans Sup35, and vice versa. These and other considerations argue that heritable prion strains result from self-propagating conformational differences within the prion protein itself. Moreover, these conformational differences seem to act in concert with the primary structure to determine a prion's propensity for transmission across a species barrier.     

Generation of prion transmission barriers by mutational control of amyloid conformations

Self-propagating beta-sheet-rich protein aggregates are implicated in a wide range of protein-misfolding phenomena, including amyloid diseases and prion-based inheritance. Two properties have emerged as common features of amyloids. Amyloid formation is ubiquitous: many unrelated proteins form such aggregates and even a single polypeptide can misfold into multiple forms--a process that is thought to underlie prion strain variation. Despite this promiscuity, amyloid propagation can be highly sequence specific: amyloid fibres often fail to catalyse the aggregation of other amyloidogenic proteins. In prions, this specificity leads to barriers that limit transmission between species. Using the yeast prion [PSI+], we show in vitro that point mutations in Sup35p, the protein determinant of [PSI+], alter the range of 'infectious' conformations, which in turn changes amyloid seeding specificity. We generate a new transmission barrier in vivo by using these mutations to specifically disfavour subsets of prion strains. The ability of mutations to alter the conformations of amyloid states without preventing amyloid formation altogether provides a general mechanism for the generation of prion transmission barriers and may help to explain how mutations alter toxicity in conformational diseases.     

The physical basis of how prion conformations determine strain phenotypes

A principle that has emerged from studies of protein aggregation is that proteins typically can misfold into a range of different aggregated forms. Moreover, the phenotypic and pathological consequences of protein aggregation depend critically on the specific misfolded form. A striking example of this is the prion strain phenomenon, in which prion particles composed of the same protein cause distinct heritable states. Accumulating evidence from yeast prions such as [PSI+] and mammalian prions argues that differences in the prion conformation underlie prion strain variants. Nonetheless, it remains poorly understood why changes in the conformation of misfolded proteins alter their physiological effects. Here we present and experimentally validate an analytical model describing how [PSI+] strain phenotypes arise from the dynamic interaction among the effects of prion dilution, competition for a limited pool of soluble protein, and conformation-dependent differences in prion growth and division rates. Analysis of three distinct prion conformations of yeast Sup35 (the [PSI+] protein determinant) and their in vivo phenotypes reveals that the Sup35 amyloid causing the strongest phenotype surprisingly shows the slowest growth. This slow growth, however, is more than compensated for by an increased brittleness that promotes prion division. The propensity of aggregates to undergo breakage, thereby generating new seeds, probably represents a key determinant of their physiological impact for both infectious (prion) and non-infectious amyloids.     

Conformational variations in an infectious protein determine prion strain differences

A remarkable feature of prion biology is the strain phenomenon wherein prion particles apparently composed of the same protein lead to phenotypically distinct transmissible states. To reconcile the existence of strains with the 'protein-only' hypothesis of prion transmission, it has been proposed that a single protein can misfold into multiple distinct infectious forms, one for each different strain. Several studies have found correlations between strain phenotypes and conformations of prion particles; however, whether such differences cause or are simply a secondary manifestation of prion strains remains unclear, largely due to the difficulty of creating infectious material from pure protein. Here we report a high-efficiency protocol for infecting yeast with the [PSI+] prion using amyloids composed of a recombinant Sup35 fragment (Sup-NM). Using thermal stability and electron paramagnetic resonance spectroscopy, we demonstrate that Sup-NM amyloids formed at different temperatures adopt distinct, stably propagating conformations. Infection of yeast with these different amyloid conformations leads to different [PSI+] strains. These results establish that Sup-NM adopts an infectious conformation before entering the cell--fulfilling a key prediction of the prion hypothesis--and directly demonstrate that differences in the conformation of the infectious protein determine prion strain variation.     

Prion recognition elements govern nucleation, strain specificity and species barriers

Prions are proteins that can switch to self-perpetuating, infectious conformations. The abilities of prions to replicate, form structurally distinct strains, and establish and overcome transmission barriers between species are poorly understood. We exploit surface-bound peptides to overcome complexities of investigating such problems in solution. For the yeast prion Sup35, we find that the switch to the prion state is controlled with exquisite specificity by small elements of primary sequence. Strikingly, these same sequence elements govern the formation of distinct self-perpetuating conformations (prion strains) and determine species-specific seeding activities. A Sup35 chimaera that traverses the transmission barrier between two yeast species possesses the critical sequence elements from both. Using this chimaera, we show that the influence of environment and mutations on the formation of species-specific strains is driven by selective recognition of either sequence element. Thus, critical aspects of prion conversion are enciphered by subtle differences between small, highly specific recognition elements.     

Structural insights into a yeast prion illuminate nucleation and strain diversity

Self-perpetuating changes in the conformations of amyloidogenic proteins play vital roles in normal biology and disease. Despite intense research, the architecture and conformational conversion of amyloids remain poorly understood. Amyloid conformers of Sup35 are the molecular embodiment of the yeast prion known as [PSI], which produces heritable changes in phenotype through self-perpetuating changes in protein folding. Here we determine the nature of Sup35's cooperatively folded amyloid core, and use this information to investigate central questions in prion biology. Specific segments of the amyloid core form intermolecular contacts in a 'Head-to-Head', 'Tail-to-Tail' fashion, but the 'Central Core' is sequestered through intramolecular contacts. The Head acquires productive interactions first, and these nucleate assembly. Variations in the length of the amyloid core and the nature of intermolecular interfaces form the structural basis of distinct prion 'strains', which produce variant phenotypes in vivo. These findings resolve several problems in yeast prion biology and have broad implications for other amyloids.     

Oligopeptide-repeat expansions modulate 'protein-only' inheritance in yeast.

The yeast [PSI+] element represents a new type of genetic inheritance, in which changes in phenotype are transmitted by a 'protein only' mechanism reminiscent of the 'protein-only' transmission of mammalian prion diseases. The underlying molecular mechanisms for both are poorly understood and it is not clear how similar they might be. Sup35, the [PSI+] protein determinant, and PrP, the mammalian prion determinant, have different functions, different cellular locations and no sequence similarity; however, each contains five imperfect oligopeptide repeats-PQGGYQQYN in Sup35 and PHGGGWGQ in PrP. Repeat expansions in PrP produce spontaneous prion diseases. Here we show that replacing the wild-type SUP35 gene with a repeat-expansion mutation induces new [PSI+] elements, the first mutation of its type among these newly described elements of inheritance. In vitro, fully denatured repeat-expansion peptides can adopt conformations rich in beta-sheets and form higher-order structures much more rapidly than wild-type peptides. Our results provide insight into the nature of the conformational changes underlying protein-based mechanisms of inheritance and suggest a link between this process and those producing neurodegenerative prion diseases in mammals.     

Prion protein remodelling confers an immediate phenotypic switch

In a variety of systems, proteins have been linked to processes historically limited to nucleic acids, such as infectivity and inheritance. These atypical proteins, termed prions, lack sequence homology but are collectively defined by their capacity to adopt multiple physical and therefore functional states in vivo. Newly synthesized prion protein generally adopts the form already present in the cell, and this in vivo folding bias directs the near faithful transmission of the corresponding phenotypic state. Switches between the prion and non-prion phenotypes can occur in vivo; however, the fate of existing protein during these transitions and its effects on the emergence of new traits remain major unanswered questions. Here, we determine the changes in protein-state that induce phenotypic switching for the yeast prion Sup35/[PSI(+)]. We show that the prion form does not need to be specified by an alternate misfolding pathway initiated during Sup35 synthesis but instead can be accessed by mature protein. This remodelling of protein from one stable form to another is accompanied by the loss of Sup35 activity, evoking a rapid change in cellular phenotype within a single cell cycle.     

Atomic structures of amyloid cross-beta spines reveal varied steric zippers

Amyloid fibrils formed from different proteins, each associated with a particular disease, contain a common cross-beta spine. The atomic architecture of a spine, from the fibril-forming segment GNNQQNY of the yeast prion protein Sup35, was recently revealed by X-ray microcrystallography. It is a pair of beta-sheets, with the facing side chains of the two sheets interdigitated in a dry 'steric zipper'. Here we report some 30 other segments from fibril-forming proteins that form amyloid-like fibrils, microcrystals, or usually both. These include segments from the Alzheimer's amyloid-beta and tau proteins, the PrP prion protein, insulin, islet amyloid polypeptide (IAPP), lysozyme, myoglobin, alpha-synuclein and beta(2)-microglobulin, suggesting that common structural features are shared by amyloid diseases at the molecular level. Structures of 13 of these microcrystals all reveal steric zippers, but with variations that expand the range of atomic architectures for amyloid-like fibrils and offer an atomic-level hypothesis for the basis of prion strains.     

Protein-only transmission of three yeast prion strains

Key questions regarding the molecular nature of prions are how different prion strains can be propagated by the same protein and whether they are only protein. Here we demonstrate the protein-only nature of prion strains in a yeast model, the [PSI] genetic element that enhances the read-through of nonsense mutations in the yeast Saccharomyces cerevisiae. Infectious fibrous aggregates containing a Sup35 prion-determining amino-terminal fragment labelled with green fluorescent protein were purified from yeast harbouring distinctive prion strains. Using the infectious aggregates as 'seeds', elongated fibres were generated in vitro from the bacterially expressed labelled prion protein. De novo generation of strain-specific [PSI] infectivity was demonstrated by introducing sheared fibres into uninfected yeast hosts. The cross-sectional morphology of the elongated fibres generated in vitro was indistinguishable from that of the short yeast seeds, as visualized by electron microscopy. Electron diffraction of the long fibres showed the 4.7 A spacing characteristic of the cross-beta structure of amyloids. The fact that the amyloid fibres nucleated in vitro propagate the strain-specific infectivity of the yeast seeds implies that the heritable information of distinct prion strains must be encoded by different, self-propagating cross-beta folding patterns of the same prion protein.     

Relationships of mRNA-protein secondary structures in the human β-globin gene HBB and four variants

Single nucleotide polymorphism is an interesting problem that can alter gene expression,recode amino acids and affect protein function.Protein structural changes have generally been attributed to amino acid replacements,and only a few research efforts have examined the effects of mRNA structural changes to the conformation of the corresponding protein coded by the mRNA.In the present study,the human β-globin HBB gene and four variants were examined.The mRNA secondary structures were constructed using the dynamic extended folding method and the encoded protein secondary structures were obtained from related databases.Comparisons were performed between these structures before and after mutations were introduced into the mature mRNAs and the proteins.We focused on the structural changes from mRNA to protein and found that regular protein conformations tend to match stable mRNA regions,whereas irregular protein conformations,such as β/γ turns and random coils,often match unstable mRNA regions.Mutations within unstable regions can alter the mRNA secondary structure and leave footprints in the protein structure.Comparison of the mRNA-protein secondary structure relationships represents a potential strategy to explore protein functional changes.     

Monoclonal antibodies inhibit prion replication and delay the development of prion disease

Prion diseases such as Creutzfeldt-Jakob disease (CJD) are fatal, neuro-degenerative disorders with no known therapy. A proportion of the UK population has been exposed to a bovine spongiform encephalopathy-like prion strain and are at risk of developing variant CJD. A hallmark of prion disease is the transformation of normal cellular prion protein (PrP(C)) into an infectious disease-associated isoform, PrP(Sc). Recent in vitro studies indicate that anti-PrP monoclonal antibodies with little or no affinity for PrP(Sc) can prevent the incorporation of PrP(C) into propagating prions. We therefore investigated in a murine scrapie model whether anti-PrP monoclonal antibodies show similar inhibitory effects on prion replication in vivo. We found that peripheral PrP(Sc) levels and prion infectivity were markedly reduced, even when the antibodies were first administered at the point of near maximal accumulation of PrP(Sc) in the spleen. Furthermore, animals in which the treatment was continued remained healthy for over 300 days after equivalent untreated animals had succumbed to the disease. These findings indicate that immunotherapeutic strategies for human prion diseases are worth pursuing.     

RNA molecules stimulate prion protein conversion

Much evidence supports the hypothesis that the infectious agents of prion diseases are devoid of nucleic acid, and instead are composed of a specific infectious protein. This protein, PrP(Sc), seems to be generated by template-induced conformational change of a normally expressed glycoprotein, PrP(C) (ref. 2). Although numerous studies have established the conversion of PrP(C) to PrP(Sc) as the central pathogenic event of prion disease, it is unknown whether cellular factors other than PrP(C) might be required to stimulate efficient PrP(Sc) production. We investigated the biochemical amplification of protease-resistant PrP(Sc)-like protein (PrPres) using a modified version of the protein-misfolding cyclic amplification method. Here we report that stoichiometric transformation of PrP(C) to PrPres in vitro requires specific RNA molecules. Notably, whereas mammalian RNA preparations stimulate in vitro amplification of PrPres, RNA preparations from invertebrate species do not. Our findings suggest that host-encoded stimulatory RNA molecules may have a role in the pathogenesis of prion disease. They also provide a practical approach to improve the sensitivity of diagnostic techniques based on PrPres amplification.     

Prions are a common mechanism for phenotypic inheritance in wild yeasts

The self-templating conformations of yeast prion proteins act as epigenetic elements of inheritance. Yeast prions might provide a mechanism for generating heritable phenotypic diversity that promotes survival in fluctuating environments and the evolution of new traits. However, this hypothesis is highly controversial. Prions that create new traits have not been found in wild strains, leading to the perception that they are rare 'diseases' of laboratory cultivation. Here we biochemically test approximately 700 wild strains of Saccharomyces for [PSI(+)] or [MOT3(+)], and find these prions in many. They conferred diverse phenotypes that were frequently beneficial under selective conditions. Simple meiotic re-assortment of the variation harboured within a strain readily fixed one such trait, making it robust and prion-independent. Finally, we genetically screened for unknown prion elements. Fully one-third of wild strains harboured them. These, too, created diverse, often beneficial phenotypes. Thus, prions broadly govern heritable traits in nature, in a manner that could profoundly expand adaptive opportunities.     

Binding of disease-associated prion protein to plasminogen

Transmissible spongiform encephalopathies are associated with accumulation of PrP(Sc), a conformer of a cellular protein called PrP(C). PrP(Sc) is thought to replicate by imparting its conformation onto PrP(C) (ref. 1), yet conformational discrimination between PrP(C) and PrP(Sc) has remained elusive. Because deposition of PrP(Sc) alone is not enough to cause neuropathology, PrP(Sc) probably damages the brain by interacting with other cellular constituents. Here we find activities in human and mouse blood which bind PrP(Sc) and prion infectivity, but not PrP(C). We identify plasminogen, a pro-protease implicated in neuronal excitotoxicity, as a PrP(Sc)-binding protein. Binding is abolished if the conformation of PrP(Sc) is disrupted by 6M urea or guanidine. The isolated lysine binding site 1 of plasminogen (kringles I-III) retains this binding activity, and binding can be competed for with lysine. Therefore, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes.     

Peptide-induced conformational change of the class I heavy chain

There is evidence that peptide ligands take part in the assembly of class I molecules. In particular, addition of peptides to extracts of the mutant cells RMA-S and .174/T2, in which stable assembly of class I does not occur, results in a conformational change in the class I heavy chain and stable association of the heavy chain with beta 2-microglobulin (beta 2m). Thus specific peptides may stabilize or induce a conformational change in the class I heavy chain that results in a rise in the binding affinity of the heavy chain for beta 2m (Fig. 1a). Here we show that peptides have two cooperative roles in class I assembly. Specific short peptides (9-10 amino acids) can induce folding of the heavy chain in the absence of beta 2m. Both short (nine amino acids) and longer sequences (15 amino acids) can stabilize performed low-affinity complexes of heavy chain and beta 2m. To alter the conformation of free heavy chains, the peptides must be exactly the correct size, and they are found to correspond to the sequences isolated from infected cells. This property may therefore be the basis for selection of epitopes presented in vivo.     

Correlation of structural elements and infectivity of the HET-s prion

Prions are believed to be infectious, self-propagating polymers of otherwise soluble, host-encoded proteins. This concept is now strongly supported by the recent findings that amyloid fibrils of recombinant prion proteins from yeast, Podospora anserina and mammals can induce prion phenotypes in the corresponding hosts. However, the structural basis of prion infectivity remains largely elusive because acquisition of atomic resolution structural properties of amyloid fibrils represents a largely unsolved technical challenge. HET-s, the prion protein of P. anserina, contains a carboxy-terminal prion domain comprising residues 218-289. Amyloid fibrils of HET-s(218-289) are necessary and sufficient for the induction and propagation of prion infectivity. Here, we have used fluorescence studies, quenched hydrogen exchange NMR and solid-state NMR to determine the sequence-specific positions of amyloid fibril secondary structure elements of HET-s(218-289). This approach revealed four beta-strands constituted by two pseudo-repeat sequences, each forming a beta-strand-turn-beta-strand motif. By using a structure-based mutagenesis approach, we show that this conformation is the functional and infectious entity of the HET-s prion. These results correlate distinct structural elements with prion infectivity.     

α-氨基酸-甲醇-水三元体系中分子间的异系焓相互作用

探讨α 氨基酸 -甲醇 -水三元体系中溶质分子间相互作用机理 ,为揭示蛋白质的稳定机理、蛋白质变性的原因和生物大分子与小分子间的相互作用提供溶液热力学依据 .利用 2 2 77热活性检测仪的流动测量系统获得了 2 98.1 5K时甘氨酸、L 丙氨酸、和L 脯氨酸分别与甲醇在水溶液中的混合过程焓变以及各自的稀释焓 ,依据McMillan Mayer理论关联得到异系焓相互作用系数 ,讨论了不同氨基酸与甲醇分子的作用机理 .不同氨基酸与甲醇分子间的焓作用系数的大小主要取决于氨基酸分子结构的差异 ,氨基酸的不同侧基 (非极性或极性 )对焓作用系数有着不同的贡献 ,脯氨酸特殊的五元吡咯环结构对hxy 值有较大的正贡献 .     

A combined algorithm for genome-wide prediction of protein function

The availability of over 20 fully sequenced genomes has driven the development of new methods to find protein function and interactions. Here we group proteins by correlated evolution, correlated messenger RNA expression patterns and patterns of domain fusion to determine functional relationships among the 6,217 proteins of the yeast Saccharomyces cerevisiae. Using these methods, we discover over 93,000 pairwise links between functionally related yeast proteins. Links between characterized and uncharacterized proteins allow a general function to be assigned to more than half of the 2,557 previously uncharacterized yeast proteins. Examples of functional links are given for a protein family of previously unknown function, a protein whose human homologues are implicated in colon cancer and the yeast prion Sup35.     

Relative helix-forming tendencies of nonpolar amino acids

An important issue in understanding the relationship between protein sequence and structure is the degree to which different amino acids favour the formation of particular types of secondary structure. Estimates of the 'helix-forming tendency' of amino acids have been made based on 'host-guest' experiments, in which copolymers are made of the amino acid of interest (the 'guest') and a host residue (typically hydroxypropyl- or hydroxybutyl-L-glutamine). Recently, however, short alanine-based peptides were found to form stable monomeric helices in water, contrary to the result predicted from host-guest experiments. We have now measured the helix-forming tendency of five different nonpolar amino acids (Ala, Ile, Leu, Phe, Val) by substituting each in turn for alanine in a 17-residue alanine-based peptide and determining the extent of alpha-helix formation. Our results differ from those of host-guest experiments both in the degree of variation in helix-forming tendency of different amino acids, and in the rank order of the helix-forming tendency. We conclude that the helix-forming tendency of a particular amino acid depends on the sequence context in which it occurs; and the restriction of side-chain rotamer conformations is important in determining the helix-forming tendency.