Insights into the architecture of the Ure2p yeast protein assemblies from helical twisted fibrils

The protein Ure2 from baker's yeast is associated with a heritable and transmissible phenotypic change in the yeast Saccharomyces cerevisiae. Such prion properties are thought to arise from the fact that Ure2p is able to self-assemble into insoluble fibrils. Assemblies of Ure2p are composed of full-length proteins in which the structure of the globular, functional, C-terminal domain is retained. We have carried out structural studies on full-length, wild-type Ure2p fibrils with a regularly twisted morphology. Using electron microscopy and cryo-electron microscopy with image analysis we show high-resolution images of the twisted filaments revealing details within the fibrillar structure. We examine these details in light of recent proposed models and discuss how this new information contributes to an understanding of the architecture of Ure2p yeast prion fibrils.     

毕赤酵母表面展示南极假丝酵母脂肪酶B全细胞催化合成葡萄糖月桂酸酯

利用表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的毕赤酵母细胞为全细胞催化剂,以葡萄糖为酰基受体,月桂酸为酰基供体,在非水相体系中催化合成糖酯。用硅胶柱层析对产物进行初提,再用制备液相色谱进一步分离纯化,并用高效液相色谱-质谱鉴定纯品性质。对该酶法合成糖脂反应体系进行了优化,其中考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、酶量、底物摩尔比、水活度和温度等几个影响酯化反应的因素。结果表明:在5mL反应体系中,以叔戊醇/二甲基亚砜(DMSO30%,V/V)为反应介质,添加初始水活度为0.11的全细胞催化剂0.5g,葡萄糖0.5mmol/L,月桂酸1.0mmol/L,60°C下反应72h后,葡萄糖月桂酸单酯的转化率达到48.7%。     

酵母细胞表面展示技术及其在非水相酶催化合成中的应用

酵母展示技术是将外源蛋白与酵母细胞壁蛋白融合,并将外源蛋白表达在酵母细胞表面。酵母展示技术已广泛应用于各种功能蛋白的表达及筛选。以下重点介绍酵母展示技术在脂肪酶展示体系构建及其在脂肪酸甲酯、短链芳香酯及糖酯生物合成中的应用。     

NLS-RARα蛋白相互作用蛋白的筛选与验证

急性早幼粒细胞白血病(APL)具有特征性染色体易位,产生的PML-RARα融合基因在其发生发展中有重要作用．PML-RARα融合蛋白在细胞内被中性粒细胞弹性蛋白酶(neutrophil elastase,NE)切割为PML突变蛋白(核定位信号NLS缺失)和RARα突变体 (NLS-RARα,包含有PML的核定位信号),这两段蛋白质在APL的发生中可能具有重要作用．为进一步研究NLS-RARα的生物学功能,运用酵母双杂交技术在白血病cDNA文库中筛选与其作用的蛋白质．首先PCR技术扩增NLS-RARα编码序列,克隆至诱饵载体pGBKT7,测序鉴定后将其转化酵母AH109．免疫印迹检测到诱饵蛋白表达后,将含有诱饵载体的AH109与含有白血病cDNA文库的酵母Y187交配,在含有X-α-gal的营养缺陷性培养基上选择和筛选二倍体酵母．经回转实验和测序分析验证得到8个与NLS-RARα相互作用的蛋白质．为进一步验证这些相互作用,克隆其中的JTV-1蛋白,利用间接免疫荧光,GST pull-down和免疫共沉淀技术成功验证了它与NLS-RARα的相互作用．为进一步探讨APL的发生机制提供了新的线索．     

Self-fertilization is the main sexual reproduction mechanism in native wine yeast populations

Saccharomyces cerevisiae is a model eukaryotic organism for classical genetics and genomics, and yet its ecology is still largely unknown. In this work, a population genetic analysis was performed on five yeast populations isolated from wine-making areas with different enological practices using simple sequence repeats and restriction fragment length polymorphism of mitochondrial DNA as molecular markers on 292 strains. In accordance with other studies, genome size estimation suggests that native S. cerevisiae strains are mainly homothallic and diploids. Analysis of mtDNA data showed that yeast populations from nonindustrial areas have 40% higher genetic diversity than populations isolated from industrial areas, demonstrating that industrial enological practices are likely to affect native yeast populations negatively by reducing its biodiversity. On the other hand, genetic differentiation analysis based on their microsatellite showed no correlation between genetic and geographic distance and a nonsignificant value when a Mantel test was applied. Finally, in the five populations studied, positive inbreeding ( F _is) values from 0.4 to 0.75, a low but significant level of linkage disequilibrium and a high number of multilocus genotypes were detected. These results strongly advocate that sexual reproduction is frequent enough to erase clonal signature in natural populations and that self-fertilization is the main mating system.     

Growth, lipid accumulation, and fatty acid composition in obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts

Obligate psychrophilic, facultative psychrophilic, and mesophilic yeasts were cultured in a carbon-rich medium at different temperatures to investigate whether growth parameters, lipid accumulation, and fatty acid (FA) composition were adaptive and/or acclimatory responses. Acclimation of facultative psychrophiles and mesophiles to a lower temperature decreased their specific growth rate, but did not affect their biomass yield (Y_X/S). Obligate and facultative psychrophiles exhibited the highest Y_X/S. Acclimation to lower temperature decreased the lipid yield (Y_L/X) in mesophilic yeasts, but did not affect Y_L/X in facultative psychrophilic ones. Similar Y_L/X were found in both groups of psychrophiles, suggesting that lipid accumulation is not a distinctive characteristic of adaptation to permanently cold environments. The unsaturation of FAs was one major adaptive feature of the yeasts colonizing permanently cold ecosystems. Remarkable amounts of α-linolenic acid were found in obligate psychrophiles at the expense of linoleic acid, whereas it was scarce or absent in all the other strains. Increased unsaturation of FAs was also a general acclimatory response of facultative psychrophiles to a lower temperature. These results improve the knowledge of the responses enabling psychrophilic yeasts to cope with the cold and may be of support for potential biotechnological exploitation of these strains.     

The S‐phase checkpoint: targeting the replication fork

The S‐phase checkpoint is a surveillance mechanism, mediated by the protein kinases Mec1 and Rad53 in the budding yeast Saccharomyces cerevisiae (ATR and Chk2 in human cells, respectively) that responds to DNA damage and replication perturbations by co‐ordinating a global cellular response necessary to maintain genome integrity. A key aspect of this response is the stabilization of DNA replication forks, which is critical for cell survival. A defective checkpoint causes irreversible replication‐fork collapse and leads to genomic instability, a hallmark of cancer cells. Although the precise mechanisms by which Mec1/Rad53 maintain functional replication forks are currently unclear, our knowledge about this checkpoint function has significantly increased during the last years. Focusing mainly on the advances obtained in S. cerevisiae, the present review will summarize our understanding of how the S‐phase checkpoint preserves the integrity of DNA replication forks and discuss the most recent findings on this topic.     

Solution structures and DNA binding properties of the N-terminal SAP domains of SUMO E3 ligases from Saccharomyces cerevisiae and Oryza sativa

SUMO E3 ligase of the Siz/PIAS family that promotes sumoylation of target proteins contains SAP motif in its N-terminal region. The SAP motif with a consensus sequence of 35 residues was first proposed to be as a new DNA binding motif found in diverse nuclear proteins involved in chromosomal organization. We have determined solution structures of the SAP domains of SUMO ligases Siz1 from yeast and rice by NMR spectroscopy, showing that the structure of the SAP domain (residues 2-105) of rice Siz1 is a four-helix bundle with an up-down-extended loop-down-up topology, whereas the SAP domain (residues 1-111) of yeast Siz1 is comprised of five helices where the fifth helix alpha5 causes a significant change in the alignment of the four-helix bundle characteristic to the SAP domains of the Siz/PIAS family. We have also demonstrated that both SAP domains have binding ability to an A/T-rich DNA, but that binding affinity of yeast Siz1 SAP is at least by an order of magnitude higher than that of rice Siz1 SAP. Our NMR titration experiments clearly showed that yeast Siz1 SAP uses alpha2-helix for DNA binding more effectively than rice Siz1 SAP, which would result from the dislocation of this helix due to the existence of the extra helix alpha5. In addition, based on the structures of the SAP domains determined here and registered in Protein Data Bank, general features of structures of the SAP domains are discussed in conjunction with equivocal nature of their DNA binding.     

Physiological characterization of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts from agro-industrial and environmental origins with possible probiotic function

Probiotics are defined as live microorganisms, which when administered in adequate amount confer a health benefit to the host. Most of studied or commercialized probiotics contain bacteria and very few of them present yeast in its composition. In this last case, the microorganisms almost always belong to Saccharomyces genus. In the present study, it was of interest to screen among 103 non-Saccharomyces yeasts a candidate for probiotic by using in vitro and in vivo criteria. In vitro assays included growth at 37°C and production of antagonistic compounds against enteropathogenic indicators, and the in vivo assays evaluated the colonization ability of mouse gastrointestinal tract without pathologic consequences and the protective ability in mice experimentally challenged with Clostridium difficile. In conclusion, Pichia kluyveri strain 898 showed to be a potential candidate for probiotic use, based on the criteria cited above, particularly as demonstrated by its protective effect against experimental infection in mice. Interestingly, an in vivo inhibition against C. difficile observed in the animal models did not correlate with the results obtained with the in vitro assays.