beta-Helix is a likely core structure of yeast prion Sup35 amyloid fibers

Helicobacter hepaticus, a causal agent of hepatocarcinoma in mice, exhibits a cytolethal distending toxin activity. The three subunits of this holotoxin, CdtA, CdtB, and CdtC, and three CdtB mutants were produced as recombinant histidine-tagged proteins by using an in vitro cell-free protein expression system. We found that the presence of the three H. hepaticus Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticus CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB mutation. These results suggest that the effect of H. hepaticus CDT probably involves the DNase activity of CdtB.     

Wild type huntingtin toxicity in yeast: Implications for the role of amyloid cross-seeding in polyQ diseases

Proteins with expanded polyglutamine (polyQ) regions are prone to form amyloids, which can cause diseases in humans and toxicity in yeast. Recently, we showed that in yeast non-toxic amyloids of Q-rich proteins can induce aggregation and toxicity of wild type huntingtin (Htt) with a short non-pathogenic polyglutamine tract. Similarly to mutant Htt with an elongated N-terminal polyQ sequence, toxicity of its wild type counterpart was mediated by induced aggregation of the essential Sup35 protein, which contains a Q-rich region. Notably, polymerization of Sup35 was not caused by the initial benign amyloids and, therefore, aggregates of wild type Htt acted as intermediaries in seeding Sup35 polymerization. This exemplifies a protein polymerization cascade which can generate a network of interdependent polymers. Here we discuss cross-seeded protein polymerization as a possible mechanism underlying known interrelations between different polyQ diseases. We hypothesize that similar mechanisms may enable proteins, which possess expanded Q-rich tracts but are not associated with diseases, to promote the development of polyQ diseases.     

Yeast Prions Compared to Functional Prions and Amyloids

Saccharomyces cerevisiae is an occasional host to an array of prions, most based on self-propagating, self-templating amyloid filaments of a normally soluble protein. [URE3] is a prion of Ure2p, a regulator of nitrogen catabolism, while [PSI +] is a prion of Sup35p, a subunit of the translation termination factor Sup35p. In contrast to the functional prions, [Het-s] of Podospora anserina and [BETA] of yeast, the amyloid-based yeast prions are rare in wild strains, arise sporadically, have an array of prion variants for a single prion protein sequence, have a folded in-register parallel β-sheet amyloid architecture, are detrimental to their hosts, arouse a stress response in the host, and are subject to curing by various host anti-prion systems. These characteristics allow a logical basis for distinction between functional amyloids/prions and prion diseases. These infectious yeast amyloidoses are outstanding models for the many common human amyloid-based diseases that are increasingly found to have some infectious characteristics.     

Molecular basis for transmission barrier and interference between closely related prion proteins in yeast

Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi. The transmission of prions between species is usually inhibited, being highly sensitive to small differences in amino acid sequence of the prion-forming proteins. To understand the molecular basis of this prion interspecies barrier, we studied the transmission of the [PSI(+)] prion state from Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with prion-forming domains from four other closely related Saccharomyces species. Whereas all the hybrid Sup35 proteins could adopt a prion form in S. cerevisiae, they could not readily acquire the prion form from the [PSI(+)] prion of S. cerevisiae. Expression of the hybrid Sup35 proteins in S. cerevisiae [PSI(+)] cells often resulted in frequent loss of the native [PSI(+)] prion. Furthermore, all hybrid Sup35 proteins showed different patterns of interaction with the native [PSI(+)] prion in terms of co-polymerization, acquisition of the prion state, and induced prion loss, all of which were also dependent on the [PSI(+)] variant. The observed loss of S. cerevisiae [PSI(+)] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid. We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.     

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.     

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。     

Selective Amplification of Classical and Atypical Prions Using Modified Protein Misfolding Cyclic Amplification

With the development of protein misfolding cyclic amplification (PMCA), the topic of faithful propagation of prion strain-specific structures has been constantly debated. Here we show that by subjecting brain material of a synthetic strain consisting of a mixture of self-replicating states to PMCAb, selective amplification of PrP^Sc could be achieved, and that PMCAb mimicked the evolutionary trend observed during serial transmission in animals. On the other hand, using modified PMCAb conditions that employ partially deglycosylated PrP^C (dgPMCAb), an alternative transmissible state referred to as atypical protease-resistant form of the prion protein (atypical PrPres) was selectively amplified from a mixture. Surprisingly, when hamster-adapted strains (263K and Hyper) were subjected to dgPMCAb, their proteinase K digestion profile underwent a dramatic transformation, suggesting that a mixture of atypical PrPres and PrP^Sc might be present in brain-derived materials. However, detailed analysis revealed that the proteinase K-resistant profile of PrP^Sc changed in response to dgPMCAb. Despite these changes, the 263K strain-specific disease phenotype was preserved after passage through dgPMCAb. This study revealed that the change in PrP^Sc biochemical phenotype does not always represent an irreversible transformation of a strain, but rather demonstrated the existence of a wide range of variation for strain-specific physical features in response to a change in prion replication environment. The current work introduced a new PMCA technique for amplification of atypical PrPres and raised a number of questions about the need for a clever distinction between actual strain mutation and variation of strain-specific features in response to a change in the replication environment.     

Amyloids and prions in plants: Facts and perspectives

Amyloids represent protein fibrils that have highly ordered structure with unique physical and chemical properties. Amyloids have long been considered lethal pathogens that cause dozens of incurable diseases in humans and animals. Recent data show that amyloids may not only possess pathogenic properties but are also implicated in the essential biological processes in a variety of prokaryotes and eukaryotes. Functional amyloids have been identified in archaea, bacteria, fungi, and animals, including humans. Plants are one of the most poorly studied groups of organisms in the field of amyloid biology. Although amyloid properties have not been shown under native conditions for any plant protein, studies demonstrating amyloid properties for a set of plant proteins in vitro or in heterologous systems in vivo have been published in recent years. In this review, we systematize the data on the amyloidogenic proteins of plants and their functions and discuss the perspectives of identifying novel amyloids using bioinformatic and proteomic approaches.     

拉格啤酒酵母菌物种和株系的快速分子鉴定

近年来的基因组学研究结果已证实拉格啤酒酵母Saccharomyces pastorianus是一个由艾尔啤酒酵母S. cerevisiae和真贝氏酿酒酵母S. eubayanus杂交而成的杂交种,并可根据地域传承和染色体倍性分为两个株系,即I型/Saaz系和II型/Frohberg系。前者主要为异源3倍体,后者则主要为异源4倍体。为了探讨中国啤酒酿造酵母菌的物种和菌系归属,我们根据拉格啤酒酵母及其两个菌系的基因组特性,制定了一套基于IntFR片段种特异性扩增和ITS-RFLP分析的精确但简便易行的拉格啤酒酵母菌物种和株系鉴定新方法,并以酿酒酵母属内相关种的模式或权威菌株和部分酒精及面包酵母为参照,对保藏于中国普通微生物菌种保藏中心（CGMCC）的41株啤酒酿造酵母菌进行了重新鉴定和分型。这些菌株除1株原定名为贝氏酿酒酵母S. bayanus外,其余菌株的原定名均为S. cerevisiae。研究结果确认了S. bayanus菌株鉴定的正确性,但在其余的40株啤酒酵母菌株中,21株属于S. cerevisiae,1株属于葡萄汁酿酒酵母S. uvarum,18株属于S. pastorianus。菌系鉴定和流式细胞测定结果显示在确认的S. pastorianus菌株中,1株为I型/Saaz系,3倍体;17株为II型/Frohberg系,其中9株为4倍体,两株为3倍体,5株介于3倍至4倍体之间。啤酒酵母物种和株系的确认对优化发酵工艺和菌种选育及遗传改造等具有重要意义。     

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.     

Saccharomyces genome database: underlying principles and organisation

A scientific database can be a powerful tool for biologists in an era where large-scale genomic analysis, combined with smaller-scale scientific results, provides new insights into the roles of genes and their products in the cell. However, the collection and assimilation of data is, in itself, not enough to make a database useful. The data must be incorporated into the database and presented to the user in an intuitive and biologically significant manner. Most importantly, this presentation must be driven by the user's point of view; that is, from a biological perspective. The success of a scientific database can therefore be measured by the response of its users - statistically, by usage numbers and, in a less quantifiable way, by its relationship with the community it serves and its ability to serve as a model for similar projects. Since its inception ten years ago, the Saccharomyces Genome Database (SGD) has seen a dramatic increase in its usage, has developed and maintained a positive working relationship with the yeast research community, and has served as a template for at least one other database. The success of SGD, as measured by these criteria, is due in large part to philosophies that have guided its mission and organisation since it was established in 1993. This paper aims to detail these philosophies and how they shape the organisation and presentation of the database.     

Non-targeted Identification of Prions and Amyloid-forming Proteins from Yeast and Mammalian Cells

The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin.     

裂殖酵母作为外源基因表达系统

虽然裂殖酵母与酿酒酵母同属于子囊真菌,但比其它的酵母相比,裂殖酵母与更高等的真核细胞有许多相似的性质,使得裂殖酵母在分子生物学研究中成为一种提供信息的、准确的真核实验模型.它在外源基因表达方面同样具有前景.主要介绍了裂殖酵母的优点,其表达载体的性质,以及外源蛋白表达的例子.     

Serum amyloid A and protein AA: Molecular mechanisms of a transmissible amyloidosis

Systemic AA-amyloidosis is a complication of chronic inflammatory diseases and the fibril protein AA derives from the acute phase reactant serum AA. AA-amyloidosis can be induced in mice by an inflammatory challenge. The lag phase before amyloid develops can be dramatically shortened by administration of a small amount of amyloid fibrils. Systemic AA-amyloidosis is transmissible in mice and may be so in humans. Since transmission can cross species barriers it is possible that AA-amyloidosis can be induced by amyloid in food, e.g. foie gras. In mice, development of AA-amyloidosis can also be accelerated by other components with amyloid-like properties. A new possible risk factor may appear with synthetically made fibrils from short peptides, constructed for tissue repair.     

Efficient screening of environmental isolates for Saccharomyces cerevisiae strains that are suitable for brewing

We developed an efficient screening method for Saccharomyces cerevisiae strains from environmental isolates. MultiPlex PCR was performed targeting four brewing S. cerevisiae genes (SSU1, AWA1, BIO6, and FLO1). At least three genes among the four were amplified from all S. cerevisiae strains. The use of this method allowed us to successfully obtain S. cerevisiae strains.     

The nuclear experience of CPEB: Implications for RNA processing and translational control

CPEB is a sequence-specific RNA binding protein that promotes polyadenylation-induced translation in early development, during cell cycle progression and cellular senescence, and following neuronal synapse stimulation. It controls polyadenylation and translation through other interacting molecules, most notably the poly(A) polymerase Gld2, the deadenylating enzyme PARN, and the eIF4E-binding protein Maskin. Here, we report that CPEB shuttles between the nucleus and cytoplasm and that its export occurs via the CRM1-dependent pathway. In the nucleus of Xenopus oocytes, CPEB associates with lampbrush chromosomes and several proteins involved in nuclear RNA processing. CPEB also interacts with Maskin in the nucleus as well as with CPE-containing mRNAs. Although the CPE does not regulate mRNA export, it influences the degree to which mRNAs are translationally repressed in the cytoplasm. Moreover, CPEB directly or indirectly mediates the alternative splicing of at least one pre-mRNA in mouse embryo fibroblasts as well as certain mouse tissues. We propose that CPEB, together with Maskin, binds mRNA in the nucleus to ensure tight translational repression upon export to the cytoplasm. In addition, we propose that nuclear CPEB regulates specific pre-mRNA alternative splicing.     

Chaperone Proteins Select and Maintain [PIN+] Prion Conformations in Saccharomyces cerevisiae

Prions are proteins that can adopt different infectious conformations known as “strains” or “variants,” each with a distinct, epigenetically inheritable phenotype. Mechanisms by which prion variants are determined remain unclear. Here we use the Saccharomyces cerevisiae prion Rnq1p/[PIN⁺] as a model to investigate the effects of chaperone proteins upon prion variant determination. We show that deletion of specific chaperone genes alters [PIN⁺] variant phenotypes, including [PSI⁺] induction efficiency, Rnq1p aggregate morphology/size and variant dominance. Mating assays demonstrate that gene deletion-induced phenotypic changes are stably inherited in a non-Mendelian manner even after restoration of the deleted gene, confirming that they are due to a bona fide change in the [PIN⁺] variant. Together, our results demonstrate a role for chaperones in regulating the prion variant complement of a cell.     

Stress-dependent Proteolytic Processing of the Actin Assembly Protein Lsb1 Modulates a Yeast Prion

Yeast prions are self-propagating amyloid-like aggregates of Q/N-rich protein that confer heritable traits and provide a model of mammalian amyloidoses. [PSI⁺] is a prion isoform of the translation termination factor Sup35. Propagation of [PSI⁺] during cell division under normal conditions and during the recovery from damaging environmental stress depends on cellular chaperones and is influenced by ubiquitin proteolysis and the actin cytoskeleton. The paralogous yeast proteins Lsb1 and Lsb2 bind the actin assembly protein Las17 (a yeast homolog of human Wiskott-Aldrich syndrome protein) and participate in the endocytic pathway. Lsb2 was shown to modulate maintenance of [PSI⁺] during and after heat shock. Here, we demonstrate that Lsb1 also regulates maintenance of the Sup35 prion during and after heat shock. These data point to the involvement of Lsb proteins in the partitioning of protein aggregates in stressed cells. Lsb1 abundance and cycling between actin patches, endoplasmic reticulum, and cytosol is regulated by the Guided Entry of Tail-anchored proteins pathway and Rsp5-dependent ubiquitination. Heat shock-induced proteolytic processing of Lsb1 is crucial for prion maintenance during stress. Our findings identify Lsb1 as another component of a tightly regulated pathway controlling protein aggregation in changing environments.