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.     

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.     

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.     

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.     

The structural basis of yeast prion strain variants

Among the many surprises to arise from studies of prion biology, perhaps the most unexpected is the strain phenomenon whereby a single protein can misfold into structurally distinct, infectious states that cause distinguishable phenotypes. Similarly, proteins can adopt a spectrum of conformations in non-infectious diseases of protein folding; some are toxic and others are well tolerated. However, our understanding of the structural differences underlying prion strains and how these differences alter their physiological impact remains limited. Here we use a combination of solution NMR, amide hydrogen/deuterium (H/D) exchange and mutagenesis to study the structural differences between two strain conformations, termed Sc4 and Sc37 (ref. 5), of the yeast Sup35 prion. We find that these two strains have an overlapping amyloid core spanning most of the Gln/Asn-rich first 40 amino acids that is highly protected from H/D exchange and very sensitive to mutation. These features indicate that the cores are composed of tightly packed beta-sheets possibly resembling 'steric zipper' structures revealed by X-ray crystallography of Sup35-derived peptides. The stable structure is greatly expanded in the Sc37 conformation to encompass the first 70 amino acids, revealing why this strain shows increased fibre stability and decreased ability to undergo chaperone-mediated replication. Our findings establish that prion strains involve large-scale conformational differences and provide a structural basis for understanding a broad range of functional studies, including how conformational changes alter the physiological impact of prion strains.     

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.     

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.     

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.     

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.     

Structure of the cross-beta spine of amyloid-like fibrils

Numerous soluble proteins convert to insoluble amyloid-like fibrils that have common properties. Amyloid fibrils are associated with fatal diseases such as Alzheimer's, and amyloid-like fibrils can be formed in vitro. For the yeast protein Sup35, conversion to amyloid-like fibrils is associated with a transmissible infection akin to that caused by mammalian prions. A seven-residue peptide segment from Sup35 forms amyloid-like fibrils and closely related microcrystals, from which we have determined the atomic structure of the cross-beta spine. It is a double beta-sheet, with each sheet formed from parallel segments stacked in register. Side chains protruding from the two sheets form a dry, tightly self-complementing steric zipper, bonding the sheets. Within each sheet, every segment is bound to its two neighbouring segments through stacks of both backbone and side-chain hydrogen bonds. The structure illuminates the stability of amyloid fibrils, their self-seeding characteristic and their tendency to form polymorphic structures.     

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.     

A yeast prion provides a mechanism for genetic variation and phenotypic diversity

A major enigma in evolutionary biology is that new forms or functions often require the concerted effects of several independent genetic changes. It is unclear how such changes might accumulate when they are likely to be deleterious individually and be lost by selective pressure. The Saccharomyces cerevisiae prion [PSI+] is an epigenetic modifier of the fidelity of translation termination, but its impact on yeast biology has been unclear. Here we show that [PSI+] provides the means to uncover hidden genetic variation and produce new heritable phenotypes. Moreover, in each of the seven genetic backgrounds tested, the constellation of phenotypes produced was unique. We propose that the epigenetic and metastable nature of [PSI+] inheritance allows yeast cells to exploit pre-existing genetic variation to thrive in fluctuating environments. Further, the capacity of [PSI+] to convert previously neutral genetic variation to a non-neutral state may facilitate the evolution of new traits.     

Conformational change of glutathione-S-transferase by its co-expression with prion domain of yeast Ure2p

Ure2 protein from Saccharomyces cerevisisae has a changeable structure similar to that ofrnammalian prion protein. Its N-terminal is the prion domain (PrD) consisting of 65 amino acids which plays a critical role in yeast prion development. In this study, PrD gene was recombinated with glutathione-S-transferase(GST) gene, and a soluble GST-PrD(sGST-PrD) fusion protein was expressed in E. coli. sGST-PrD could spontaneously polymerize into amyloid fibrils in vitro, displaying typical β-sheet-type structure; it had increased resistance to proteinase K and exhibited amvloid-like optical properties. Moreover, the aggregated GST-PrD(aGST-PrD) could induce sGST-PrD to aggregate into fibrils. These results indicate that PrD could change the conformation of GST moiety in a recombinant protein with PrD to form a prion-like chimeric protein, which proves that PrD has the ability to mediate a prion-like conversion of other proteins fused with it.     

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.     

Review： Protein folding pathology in domestic animals

Fibrillar proteins form structural elements of cells and the extracellular matrix. Pathological lesions of fibrillar microanatomical structures, or secondary fibrillar changes in globular proteins are well known. A special group concerns histologically amorphous deposits, amyloid. The major characteristics of amyloid are: apple green birefringence after Congo red staining of histological sections, and non-branching 7-10nm thick fibrils on electron microscopy revealing a high content of cross beta pleated sheets. About 25 different types of amyloid have been characterised. In animals, AA-amyloid is the most frequent type. Other types of amyloid in animals represent: AIAPP (in cats), AApoAⅠ, AApoAⅡ, localised AL-amyloid, amyloid in odontogenic or mammary tumors and amyloid in the brain. In old dogs Aβ and in sheep APrP^sc-amyloid can be encountered. AA-amyloidosis is a systemic disorder with a precursor in blood, acute phase serum amyloid A (SAA). In chronic inflammatory processes AA-amyloid can be deposited. A rapid crystallization of SAA to amyloid fibrils on small beta-sheeted fragments, the ‘amyloid enhancing factor‘ (AEF), is known and the AEF has been shown to penetrate the enteric barrier. Amyloid fibrils can aggregate from various precursor proteins in vitro in particular at acidic pH and when proteolytic fragments are formed. Molecular chaperones influence this process. Tissue data point to amyloid fibrillogenesis in lysosomes and near cell surfaces. A comparison can be made of the fibrillogenesis in prion diseases and in enhanced AA-amyloidosis. In the reactive form, acute phase SAA is the supply of the precursor protein, whereas in the prion diseases, cell membrane proteins form a structural source. AI3-amyloid in brain tissue of aged dogs showing signs of dementia forms a canine counterpart of senile dementia of the Alzheimer type (ccSDAT) in man. Misfolded proteins remain potential food hazards. Developments concerning prevention of amyloidogenesis and therapy of amyloid deposits are shortly commented.     

Epigenetic regulation of translation reveals hidden genetic variation to produce complex traits

Phenotypic plasticity and the exposure of hidden genetic variation both affect the survival and evolution of new traits, but their contributing molecular mechanisms are largely unknown. A single factor, the yeast prion [PSI(+)], may exert a profound effect on both. [PSI(+)] is a conserved, protein-based genetic element that is formed by a change in the conformation and function of the translation termination factor Sup35p, and is transmitted from mother to progeny. Curing cells of [PSI(+)] alters their survival in different growth conditions and produces a spectrum of phenotypes in different genetic backgrounds. Here we show, by examining three plausible explanations for this phenotypic diversity, that all traits tested involved [PSI(+)]-mediated read-through of nonsense codons. Notably, the phenotypes analysed were genetically complex, and genetic re-assortment frequently converted [PSI(+)]-dependent phenotypes to stable traits that persisted in the absence of [PSI(+)]. Thus, [PSI(+)] provides a temporary survival advantage under diverse conditions, increasing the likelihood that new traits will become fixed by subsequent genetic change. As an epigenetic mechanism that globally affects the relationship between genotype and phenotype, [PSI(+)] expands the conceptual framework for phenotypic plasticity, provides a one-step mechanism for the acquisition of complex traits and affords a route to the genetic assimilation of initially transient epigenetic traits.     

Misfolded proteins partition between two distinct quality control compartments

The accumulation of misfolded proteins in intracellular amyloid inclusions, typical of many neurodegenerative disorders including Huntington's and prion disease, is thought to occur after failure of the cellular protein quality control mechanisms. Here we examine the formation of misfolded protein inclusions in the eukaryotic cytosol of yeast and mammalian cell culture models. We identify two intracellular compartments for the sequestration of misfolded cytosolic proteins. Partition of quality control substrates to either compartment seems to depend on their ubiquitination status and aggregation state. Soluble ubiquitinated misfolded proteins accumulate in a juxtanuclear compartment where proteasomes are concentrated. In contrast, terminally aggregated proteins are sequestered in a perivacuolar inclusion. Notably, disease-associated Huntingtin and prion proteins are preferentially directed to the perivacuolar compartment. Enhancing ubiquitination of a prion protein suffices to promote its delivery to the juxtanuclear inclusion. Our findings provide a framework for understanding the preferential accumulation of amyloidogenic proteins in inclusions linked to human disease.     

常见蔬菜及淀粉类食物抗氧化活性初探

本研究采用红细胞溶血,脑匀浆脂质过氧化的生物测定法,测定了多种常见绿叶蔬菜、瓜类、豆类、非绿叶蔬菜及淀粉类食物的抗氧化活性,初步确定了许多日常食物都具有一定的抗氧化活性,尤其是绿叶菜中的通菜、芫荽及淀粉类食物中的莲藕显示出极高的抗氧化能力.     

人和鸡溶菌酶结构保守性的对比研究

人体内的溶菌酶在第56位、第67位残基发生自发突变的情况下可以通过分子间的聚集形成淀粉样病变,产生一系列的临床症状甚至导致死亡.本研究基于STAMP结构比对算法,使用VMD软件主要从结构角度比较了人、鸡、狗、鼠四种脊椎动物溶菌酶的相似程度及亲缘关系,结果发现鸡的溶菌酶与人的溶菌酶结构最相似,亲缘关系最接近,并且人和鸡的溶菌酶的第56位和第67位两个残基位点在结构上高度保守.该发现为现在流行的以鸡溶菌酶为模型研究人溶菌酶引起的淀粉样病变的致病机制及治愈方法的可行性提供了必要的理论依据.