Suicidal [PSI+] is a lethal yeast prion

[PSI(+)] is a prion of the essential translation termination factor Sup35p. Although mammalian prion infections are uniformly fatal, commonly studied [PSI(+)] variants do not impair growth, leading to suggestions that [PSI(+)] may protect against stress conditions. We report here that over half of [PSI(+)] variants are sick or lethal. These "killer [PSI(+)]s" are compatible with cell growth only when also expressing minimal Sup35C, lacking the N-terminal prion domain. The severe detriment of killer [PSI(+)] results in rapid selection of nonkiller [PSI(+)] variants or loss of the prion. We also report variants of [URE3], a prion of the nitrogen regulation protein Ure2p, that grow much slower than ure2Δ cells. Our findings give a more realistic picture of the impact of the prion change than does focus on "mild" prion variants.     

Yeast prions [URE3] and [PSI+] are diseases

Viruses, plasmids, and prions can spread in nature despite being a burden to their hosts. Because a prion arises de novo in more than one in 10(6) yeast cells and spreads to all offspring in meiosis, its absence in wild strains would imply that it has a net deleterious effect on its host. Among 70 wild Saccharomyces strains, we found the [PIN+] prion in 11 strains, but the [URE3] and [PSI+] prions were uniformly absent. In contrast, the "selfish" 2mu DNA was in 38 wild strains and the selfish RNA replicons L-BC, 20S, and 23S were found in 8, 14, and 1 strains, respectively. The absence of [URE3] and [PSI+] in wild strains indicates that each prion has a net deleterious effect on its host.     

Strain-specific sequences required for yeast [PSI+] prion propagation

Amyloid polymorphism underlies the prion strain phenomenon where a single protein polypeptide adopts different chain-folding patterns to form self-propagating cross-β structures. Three strains of the yeast prion [PSI], namely [VH], [VK], and [VL], have been previously characterized and are amyloid conformers of the yeast translation termination factor Sup35. Here we define specific sequences of the Sup35 protein that are necessary for in vivo propagation of each of these prion strains. By sequential substitution of residues 5–55 of Sup35 by proline and insertion of glycine at alternate sites in this segment, specific mutations have been identified that interfere selectively with the propagation of each of the three prion strains in yeast: the [VH] strain requires amino acid residues 7–21; [VK] requires residues 9–37; and [VL] requires residues 5 to at least 52. Minimal polypeptide segments capable of encoding prion conformations were defined by assembly of recombinant Sup35 fragments on purified prion nuclei to form amyloid fibers in vitro, whose infectivity was assayed in yeast. For the [VK] and [VL] strains, the minimal fragments approximately coincide with the strain-specific sequences defined by mutations of the N-terminal portion of the intact Sup35 (1–685); and for the [VH] strain, a longer Sup (1–53) fragment is required. Polymorphic structures of other amyloids might similarly involve different stretches of polypeptides to form cross-β amyloid cores with distinct molecular recognition surfaces.     

羟基脲抑制盐酸胍对于酵母朊病毒[PSI+]的治愈

摘 要：目的 研究细胞分裂与盐酸胍作用下酵母朊病毒[PSI+]聚集体解聚的关系。方法 本研究借助重组表达Sup35p-GFP的菌株,采用表型分析方法与半变性琼脂糖凝胶电泳（SDD-AGE）结合蛋白质免疫印迹技术,分析了羟基脲抑制细胞分裂情况下对于盐酸胍解聚酵母朊病毒聚集体的影响。结果 羟基脲抑制细胞分裂的情况下,表型分析的数据显示盐酸胍不能治愈酵母朊病毒[PSI+],蛋白水平的实验数据证实了这一结果并发现,羟基脲的存在使得盐酸胍解聚酵母朊病毒聚集体的能力明显下降。结论 这暗示着盐酸胍治愈朊病毒[PSI+]是需要细胞进行分裂的。     

Implications of the Actin Cytoskeleton on the Multi-Step Process of [PSI+] Prion Formation

Yeast prions are self-perpetuating misfolded proteins that are infectious. In yeast, [PSI⁺] is the prion form of the Sup35 protein. While the study of [PSI⁺] has revealed important cellular mechanisms that contribute to prion propagation, the underlying cellular factors that influence prion formation are not well understood. Prion formation has been described as a multi-step process involving both the initial nucleation and growth of aggregates, followed by the subsequent transmission of prion particles to daughter cells. Prior evidence suggests that actin plays a role in this multi-step process, but actin’s precise role is unclear. Here, we investigate how actin influences the cell’s ability to manage newly formed visible aggregates and how actin influences the transmission of newly formed aggregates to future generations. At early steps, using 3D time-lapse microscopy, several actin mutants, and Markov modeling, we find that the movement of newly formed aggregates is random and actin independent. At later steps, our prion induction studies provide evidence that the transmission of newly formed prion particles to daughter cells is limited by the actin cytoskeletal network. We suspect that this limitation is because actin is used to possibly retain prion particles in the mother cell.     

Cell division is essential for elimination of the yeast [PSI+] prion by guanidine hydrochloride

Guanidine hydrochloride (Gdn.HCl) blocks the propagation of yeast prions by inhibiting Hsp104, a molecular chaperone that is absolutely required for yeast prion propagation. We had previously proposed that ongoing cell division is required for Gdn.HCl-induced loss of the [PSI+] prion. Subsequently, Wu et al.[Wu Y, Greene LE, Masison DC, Eisenberg E (2005) Proc Natl Acad Sci USA 102:12789-12794] claimed to show that Gdn.HCl can eliminate the [PSI+] prion from alpha-factor-arrested cells leading them to propose that in Gdn.HCl-treated cells the prion aggregates are degraded by an Hsp104-independent mechanism. Here we demonstrate that the results of Wu et al. can be explained by an unusually high rate of alpha-factor-induced cell death in the [PSI+] strain (780-1D) used in their studies. What appeared to be no growth in their experiments was actually no increase in total cell number in a dividing culture through a counterbalancing level of cell death. Using media-exchange experiments, we provide further support for our original proposal that elimination of the [PSI+] prion by Gdn.HCl requires ongoing cell division and that prions are not destroyed during or after the evident curing phase.     

Curing of yeast [PSI+] prion by guanidine inactivation of Hsp104 does not require cell division

Propagation of the yeast prion [PSI+], a self-replicating aggregated form of Sup35p, requires Hsp104. One model to explain this phenomenon proposes that, in the absence of Hsp104, Sup35p aggregates enlarge but fail to replicate thus becoming diluted out as the yeast divide. To test this model, we used live imaging of Sup35p-GFP to follow the changes that occur in [PSI+] cells after the addition of guanidine to inactivate Hsp104. After guanidine addition there was initially an increase in aggregation of Sup35p-GFP; but then, before the yeast divided, the aggregates began to dissolve, and after approximately 6 h the Sup35-GFP looked identical to the Sup35-GFP in [psi+] cells. Although plating studies showed that the yeast were still [PSI+], this reduction in aggregation suggested that curing of [PSI+] by inactivation of Hsp104 might be independent of cell division. This was tested by measuring the rate of curing of [PSI+] cells in both dividing and nondividing cells. Cell division was inhibited by adding either alpha factor or farnesol. Remarkably, with both of these methods, we found that the rate of curing was not significantly affected by cell division. Thus, cell division is not a determining factor for curing [PSI+] by inactivating Hsp104 with guanidine. Rather, curing apparently occurs because Sup35-GFP polymers slowly depolymerize in the absence of Hsp104 activity. Hsp104 then counteracts this curing possibly by catalyzing formation of new polymers.     

Genetic variation in hepcidin expression and its implications for phenotypic differences in iron metabolism

At the core of iron homeostasis is hepcidin, a small acute phase antimicrobial peptide that now also appears to synchronously orchestrate the response of iron transporter and regulatory genes. In this perspective article, Drs Bayele and Srai discuss cis and trans acting factors that may influence hepcidin variation in humans and their potential role in iron metabolism control. See related papers on page 1293 and 1297.Iron homeostasis, like other physiological processes, relies on precise and timely interactions between key proteins involved in either its uptake or release. At the core of this is hepcidin, a small acute phase antimicrobial peptide that now also appears to synchronously orchestrate the response of iron transporter and regulatory genes to ensure proper balance between how much dietary iron is absorbed by the small intestine or released into the circulation by macrophages.¹ Several studies suggest that there are strong genetic components that underlie hepcidin regulation beyond the usual suspects (i.e. infection, inflammation, erythropoiesis, hypoxia and iron), in a manner that could impinge on phenotypic differences in susceptibility to iron-overload or anemia. Based on variation in hepcidin expression phenotypes, new emerging data suggest that there are heritable regulatory polymorphisms within the promoter that are linked to diseases of iron metabolism. Here we provide a perspective of what factors could determine such variability, giving some insight into how gene-gene, gene-environment, gene-nutrient interactions and even circadian rhythms may contribute to hepcidin expression variation and diseases associated with such variation.     

Genetic and phenotypic variation of West Nile virus in New York, 2000-2003

West Nile virus (WNV) strains circulating during the first five years of WNV transmission in New York were collected, partial nucleotide sequences were determined, and in vitro and in vivo phenotypic analyses of selected strains were undertaken to determine whether observed increases in the intensity of enzootic and epidemic transmission in New York State during 2002 and 2003 were associated with viral genetic changes. Functionally diverse regions of the WNV genome were also compared to determine whether some regions may be more or less variable than others. The complete envelope coding regions of 67 strains and fragments of the nonstructural protein 5 (NS5) and 3' noncoding regions of 39 strains collected during 2002 and 2003 were examined. West Nile virus in New York remains relatively genetically homogeneous. Viral genetic diversity was greater in 2002 and 2003 at both the nucleotide and amino acid levels than in previous years due to the emergence of a new WNV genotype in 2002. This genotype persisted and became dominant in 2003. Envelope and NS5 coding regions were approximately two-fold more likely than the 3' untranslated region to contain nucleotide substitutions, and the envelope region was approximately three-fold more likely to contain amino acid substitutions than the NS5 region. Variation was noted in in vivo mosquito transmission assays, but not in in vitro growth studies. Strains belonging to the epizootiologically dominant clade were transmitted after approximately two fewer days of extrinsic incubation, providing a possible mechanism for the dominance of this clade. The observed increase in the intensity of WNV transmission beginning in 2002 was associated with an increase in viral genetic diversity that was the result of the emergence of an additional phylogenetic clade. This genotype seems to possess an advantage over previously recognized WNV strains in mosquito transmission phenotype.     

Genetic variation in cancer predisposition: mutational decay of a robust genetic control network

A computational model of cancer progression is used to study how mutations in genes that control tumor initiation and progression accumulate in populations. The model assumes that cancer occurs only after a cell lineage has progressed through a series of stages. The greater the number of stages, the more strongly the individual is protected against cancer. It is shown that an extra stage initially improves the survival of individuals by decreasing mortality from cancer. However, the additional buffering by an extra stage reduces the impact of any single hereditary mutation and therefore allows the accumulation of more nonlethal mutations in the population. Extra stages thereby lead to the evolution of partially decreased cancer mortality and significantly increased genetic predisposition to disease in the population as a whole. In general, the model illustrates how all robust control networks allow the accumulation of deleterious mutations. An increase in the number of buffering components leads to significant mutational decay in the protection provided by each buffering component and increased genetic predisposition to disease. An extra buffering component's net contribution to survival and reproduction is often small.     

Influence of varied [Ca2+]0 and [Na+]0 on slow responses of newborn and adult rat hearts

The electrophysiological properties of slow responses of rat hearts were investigated in newborn (two-day-old) and adult (few-month-old) rats under conditions of high [K+]0 (25 mM) with isoproterenol (1 X 10(-7)M) using the standard techniques of intracellular microelectrode recording. The potentials of slow responses were measured in varied [Ca2+]0: in adults the peak potential varied linearly with a slope of about 31 mV for a 10-fold change in [Ca2+]0, while in newborns it varied, but not linearly, and showed a positive peak potential in low [Ca2+]0 (0.5 mM). In a low [Na+]0 solution (55% [Na+]0), with constant [Ca2+]0 (2 mM), the peak potential was reduced in newborn hearts, while it was little affected in adult hearts. These results suggest that the peak potential of the slow response depends mainly on [Ca2+]0 in adult rat hearts, while it depends on both [Ca2+]0 and [Na+]0 in newborns.     

Effects of Q/N-rich, polyQ, and non-polyQ amyloids on the de novo formation of the [PSI+] prion in yeast and aggregation of Sup35 in vitro

Prions are infectious protein conformations that are generally ordered protein aggregates. In the absence of prions, newly synthesized molecules of these same proteins usually maintain a conventional soluble conformation. However, prions occasionally arise even without a homologous prion template. The conformational switch that results in the de novo appearance of yeast prions with glutamine/aspargine (Q/N)-rich prion domains (e.g., [PSI+]), is promoted by heterologous prions with a similar domain (e.g., [RNQ+], also known as [PIN+]), or by overexpression of proteins with prion-like Q-, N-, or Q/N-rich domains. This finding led to the hypothesis that aggregates of heterologous proteins provide an imperfect template on which the new prion is seeded. Indeed, we show that newly forming Sup35 and preexisting Rnq1 aggregates always colocalize when [PSI+] appearance is facilitated by the [RNQ+] prion, and that Rnq1 fibers enhance the in vitro formation of fibers by the prion domain of Sup35 (NM). The proteins do not however form mixed, interdigitated aggregates. We also demonstrate that aggregating variants of the polyQ-containing domain of huntingtin promote the de novo conversion of Sup35 into [PSI+]; whereas nonaggregating variants of huntingtin and aggregates of non-polyQ amyloidogenic proteins, transthyretin, alpha-synuclein, and synphilin do not. Furthermore, transthyretin and alpha-synuclein amyloids do not facilitate NM aggregation in vitro, even though in [PSI+] cells NM and transthyretin aggregates also occasionally colocalize. Our data, especially the in vitro reproduction of the highly specific heterologous seeding effect, provide strong support for the hypothesis of cross-seeding in the spontaneous initiation of prion states.