HAP蛋白疏水区的聚合功能及其与细胞凋亡的关系

asy和hap是一对新的细胞凋亡诱发基因 .二者单独转染细胞均能诱发细胞凋亡 ,能在酵母和哺乳动物细胞中形成同源二聚体 ,二者共转染酵母和哺乳动物细胞能形成异源二聚体 .同源二聚体诱发细胞凋亡 ,异源二聚体降低同源二聚体诱发细胞凋亡的活性 .氨基酸序列表明 ,HAP(homologousofASYprotein)含有内质网挽回模体 (KKKAE)、第一疏水区和第二疏水区 .对 3个功能区的缺失突变体研究显示 ,缺失内质网定位信号的HAPΔERS蛋白保留着同源聚合和与ASY异源聚合的能力 ,而分别缺失第一、第二疏水区的突变体HAPΔ4 8 139、HAPΔ15 7 2 18则丧失以上功能 ;通过流式计数法计算 3个缺失突变体诱发细胞凋亡比率 ,用生物统计学方法说明不同的比率与HAP诱发细胞凋亡的比率相比都有显著差异 .说明内质网定位信号、疏水区在HAP蛋白的诱发细胞凋亡过程中起重要作用 ,进一步揭示了HAP诱发细胞凋亡的机制 .     

丝状真菌细胞凋亡研究进展

细胞凋亡是真核生物中保守而重要的细胞死亡机制,与癌症、艾滋病等多种疾病密切相关.与酵母菌这一细胞凋亡模式生物相比,丝状真菌凋亡研究起步较晚但具有其独特的优势.近年来丝状真菌细胞凋亡的内外源诱因、细胞凋亡的特征以及信号传导通路等方面的研究进展迅速.丝状真菌,尤其是构巢曲霉和烟曲霉有望成为细胞凋亡研究新的模式物种.此外,研究丝状真菌细胞凋亡现象在农业和医疗领域也具有重要的应用价值,可为生物防治和人类真菌病的治疗提供新的思路.工业丝状真菌细胞凋亡研究有助于构建性状更加优良的工程菌株.     

植物病原丝状真菌G蛋白偶联受体的研究进展

通过对丝状真菌G蛋白偶联受体(GPCR)的结构、分类以及功能方面进行综述,以期明确丝状真菌与其他模式生物GPCR之间的关系。基于已报道的模式生物及丝状真菌等不同生物中的GPCR,通过SMART保守结构域分析,以及利用Clustal X、MEGA等软件对上述GPCR进行遗传关系分析。明确丝状真菌典型GPCR具有七跨膜结构域,新型GPCR则含有PIPK、RGS等保守结构域,明确不同学者对于GPCR的分类情况,以及新型GPCR所具有的特殊功能,明确模式生物GPCR、丝状真菌GPCR分别各自聚类。丝状真菌中GPCR的数量较模式生物少,不同分类单元中真菌之间GPCR的数量也不尽相同,同时,丝状真菌GPCR除具有典型的七跨膜结构域外,还含有一些其他保守的结构域,上述研究为进一步开展其功能研究提供重要的理论基础。     

丝状真菌糖生物学

蛋白质的糖基化修饰是一种保守的真核生物蛋白质翻译后修饰,存在于从酵母到人的所有真核生物中,赋予了蛋白质功能的多样性。目前对蛋白质糖基化修饰的了解主要来源于对酵母和哺乳动物细胞的研究,但单细胞真核生物或动物细胞水平的研究,很难反映糖基化修饰在多细胞真核生物的发育分化过程中的复杂功能。由于丝状真菌是多细胞真核生物,有相对简单的发育分化过程,因而是研究多细胞真核生物糖基化功能的理想模型之一,在过去10年中,丝状真菌的糖生物学研究开始受到重视,目前的研究结果表明糖基化修饰与丝状真菌的生长、发育密切相关。     

LaeA在丝状真菌次级代谢、生长发育和其他重要生物过程中的作用

LaeA是在构巢曲霉中首次鉴定的一种全局调控因子,其同源蛋白在丝状真菌中广泛存在,具有高度的保守性。LaeA及其同源蛋白序列存在S-腺苷甲硫氨酸结合基序,是一种甲基转移酶,可能影响组蛋白修饰,导致染色体结构的改变,进而调控一系列基因的表达。大量研究表明,LaeA及其同源蛋白参与调控丝状真菌多种次级代谢产物的生物合成,影响丝状真菌的生长发育和形态分化,甚至在丝状真菌生产有机酸和一些工业酶的过程中也发挥着重要作用。本文综述了LaeA及其同源蛋白的作用机制,以及该蛋白在丝状真菌次级代谢、生长发育和其他重要生物过程中的作用,并对存在的问题及应用前景进行讨论与展望。     

两种新的凋亡诱发蛋白的聚合作用及其对细胞凋亡的调控

Asy (apoptosis /saibousi Yutsudo)是日本Yutsudo小组于1999年发现的一个新的人类细胞凋亡诱发基因. 2000年齐兵等人通过酵母双杂交系统从人肺细胞系(W1-38) cDNA 文库中克隆出一个能与ASY蛋白相互作用的蛋白的新基因hap (homologue of asy protein). 已经证明ASY能在酵母细胞和哺乳动物细胞中形成同源二聚体, ASY与HAP能在酵母细胞中形成异源二聚体, ASY和HAP均能诱发肿瘤细胞Saos2和CGL4凋亡. 通过酵母双杂交系统证明HAP能在酵母细胞中形成同源二聚体; 通过交叉免疫沉淀证明HAP与ASY能在人类细胞中形成异源二聚体. 通过AnnexinV, TUNEL, DNA Ladder和流式细胞计数等检测凋亡的技术, 对asy, hap单独转染或共转染的人类正常细胞或肿瘤细胞的凋亡进行定性或定量检测, 证明ASY和HAP不仅诱发人类肿瘤细胞凋亡, 而且能诱发人类正常细胞凋亡, 并且证明由ASY与HAP形成的异源二聚体可降低由ASY和HAP各自形成的同源二聚体诱发细胞凋亡的活性, 揭示ASY与HAP形成同源或异源二聚体是调控人类细胞凋亡的重要机制.     

卵泡颗粒细胞凋亡相关蛋白的研究进展

哺乳动物的卵巢中存在大量卵泡。大多数卵泡在发育过程中发生闭锁而消失,只有少数可以发育到成熟而排卵。卵泡是由卵母细胞与其周围的颗粒细胞构成的。卵泡颗粒细胞的凋亡是卵泡闭锁的主要原因。颗粒细胞凋亡相关蛋白通过参与凋亡通路及凋亡信号转导调节凋亡。本文就哺乳动物卵泡颗粒细胞凋亡相关蛋白的研究进展进行综述。     

酵母Bir1p同源物Survivin对毕赤酵母的分子重组与生长影响

Bir1p是酵母凋亡途径中抑制细胞凋亡的重要蛋白,Survivin是Bir1p的人源同源物,Survivin第34位氨基酸T向A的突变能使其功能逆转.将Survivin及其突变体Survivin(T34A)的基因分别构建到组成型穿梭表达质粒pGAPZA中,整合入毕赤酵母的基因组,分析对酵母细胞凋亡的影响.酵母生长曲线、MTT及流式细胞仪测定数据显示,Survivin和其突变体基因在酵母中的表达分别抑制和促进了酵母凋亡.多样的工艺对工业用酵母的凋亡提出了不同的要求,本研究为工程酵母的理性改造提供了理论依据.     

鱼类和哺乳类Bcl-2家族蛋白的研究进展

细胞凋亡, 即细胞程序性死亡, 在多细胞生物的发育和稳态调控过程中发挥关键作用。Bcl-2家族蛋白是凋亡过程中的主要调控因子, 关于Bcl-2家族蛋白在凋亡过程中的功能及其作用机制一直是研究的热点。已有研究显示Bcl-2家族蛋白不仅作用于线粒体引发凋亡, 并且参与了包括对细胞内质网Ca2+的调控、DNA损伤的修复及与自噬的相互作用等多种反应, 从多方面对细胞的生存状态进行调控。Bcl-2家族蛋白保守存在于脊椎动物和无脊椎动物中, 其功能在进化中存在异同。文章以高等脊椎动物(哺乳动物)和低等脊椎动物(硬骨鱼类)为代表, 总结了近年来Bcl-2家族蛋白在调控宿主凋亡与自噬、DNA损伤及新陈代谢等方面取得的最新进展。该研究为深入了解鱼类和哺乳类Bcl-2家族蛋白的功能和作用机制提供了重要参考。     

凋亡蛋白和Nmi的相互作用及作用位点的筛选鉴定

为研究来源于鸡贫血病毒的小分子蛋白质———凋亡蛋白 (apoptin)诱导肿瘤细胞凋亡的分子机制 ,利用酵母双杂交系统从人白细胞cDNA文库筛选凋亡蛋白相互作用蛋白质 ,核苷酸序列分析及同源性检索表明 ,其中一个约为 1.2kb的克隆与Nmi(N Mycinteractionprotein)高度同源。细胞免疫共沉淀实验结果显示 ,在哺乳动物细胞水平仍能够检测到凋亡蛋白与全长Nmi的特异相互作用。利用构建好的分别缺失C端 11个氨基酸、中间 33～46位氨基酸和二者均缺失的 3个凋亡蛋白突变体进行相互作用位点研究 ,结果表明凋亡蛋白的 33～ 46位氨基酸(核外运信号 )对于凋亡蛋白与Nmi的相互作用是必需的 ,而C端核定位信号 /DNA结合序列对于凋亡蛋白与Nmi的相互作用不是充分必要的     

Apoptosis pathways in fungal growth, development and ageing

Apoptosis is one type of programmed cell death with great importance for development and homeostasis of multicellular organisms. Unexpectedly, during the past decade, evidence has been obtained for the existence of a basal apoptosis machinery in yeast, as unicellular fungus, and in some filamentous fungi, a group of microorganisms that are neither true unicellular nor true multicellular biological systems but something in between. Here, we review evidence for a role of apoptotic processes in fungal pathogenicity, competitiveness, propagation, ageing and lifespan control.     

Apoptosis in yeast

Apoptosis is a highly regulated cellular suicide program crucial for metazoan development. However, dysfunction of apoptosis also leads to several diseases. Yeast undergoes apoptosis after application of acetic acid, sugar- or salt-stress, plant antifungal peptides, or hydrogen peroxide. Oxygen radicals seem to be key elements of apoptotic execution, conserved during evolution. Furthermore, several yeast orthologues of central metazoan apoptotic regulators have been identified, such as a caspase and a caspase-regulating serine protease. In addition, physiological occurrence of cell death has been detected during aging and mating in yeast. The finding of apoptosis in yeast, other fungi and parasites is not only of great medical relevance but will also help to understand some of the still unknown molecular mechanisms at the core of apoptotic execution.     

Autophagy in filamentous fungi

Autophagy is a ubiquitous, non-selective degradation process in eukaryotic cells that is conserved from yeast to man. Autophagy research has increased significantly in the last ten years, as autophagy has been connected with cancer, neurodegenerative disease and various human developmental processes. Autophagy also appears to play an important role in filamentous fungi, impacting growth, morphology and development. In this review, an autophagy model developed for the yeast Saccharomyces cerevisiae is used as an intellectual framework to discuss autophagy in filamentous fungi. Studies imply that, similar to yeast, fungal autophagy is characterized by the presence of autophagosomes and controlled by Tor kinase. In addition, fungal autophagy is apparently involved in protection against cell death and has significant effects on cellular growth and development. However, the only putative autophagy proteins characterized in filamentous fungi are Atg1 and Atg8. We discuss various strategies used to study and monitor fungal autophagy as well as the possible relationship between autophagy, physiology, and morphological development.     

Caspase-independent apoptosis in yeast

Apoptosis is a highly regulated cellular suicide program crucial for metazoan development. Yeast counterparts of central metazoan apoptotic regulators, such as metacaspase Yca1p, have been identified. In spite of the importance of Yca1p in yeast apoptotic process, many other factors such as Aif1p, orthologs of EndoG, AMID and cyclophilin D play important roles in caspase-independent apoptotic pathways. This review summarized recent progress about studies of various intrinsic and extrinsic apoptotic stimuli that may induce yeast cell death via caspase-independent apoptosis.     

Multifunction of autophagy-related genes in filamentous fungi

Autophagy (macroautophagy), a highly conserved eukaryotic mechanism, is a non-selective degradation process, helping to maintain a balance between the synthesis, degradation and subsequent recycling of macromolecules to overcome various stress conditions. The term autophagy denotes any cellular process which involves the delivery of cytoplasmic material to the lysosome for degradation. Autophagy, in filamentous fungi plays a critical role during cellular development and pathogenicity. Autophagy, like the mitogen-activated protein (MAP) kinase cascade and nutrient-sensing cyclic AMP (cAMP) pathway, is also an important process for appressorium turgor accumulation in order to penetrate the leaf surface of host plant and destroy the plant defense. Yeast, an autophagy model, has been used to compare the multi-valued functions of ATG (autophagy-related genes) in different filamentous fungi. The autophagy machinery in both yeast and filamentous fungi is controlled by Tor kinase and both contain two distinct phosphatidylinositol 3-kinase complexes. In this review, we focus on the functions of ATG genes during pathogenic development in filamentous fungi.     

Functional analysis of pathogenicity genes in a genomics world

Genome-wide mutational and expression analyses have been performed in yeast and provide a model for large-scale analysis of gene function in filamentous fungi. The recent completion of the Neurospora crassa genome offers a resource for comparative analysis with plant pathogenic filamentous fungi. These advances have important implications for molecular genetic studies of pathogenicity genes.     

Metabolite profiling of fungi and yeast: from phenotype to metabolome by MS and informatics

Filamentous fungi and yeast from the genera Saccharomyces, Penicillium, Aspergillus, and Fusarium are well known for their impact on our life as pathogens, involved in food spoilage by degradation or toxin contamination, and also for their wide use in biotechnology for the production of beverages, chemicals, pharmaceuticals, and enzymes. The genomes of these eukaryotic micro-organisms range from about 6000 genes in yeasts (S. cerevisiae) to more than 10,000 genes in filamentous fungi (Aspergillus sp.). Yeast and filamentous fungi are expected to share much of their primary metabolism; therefore much understanding of the central metabolism and regulation in less-studied filamentous fungi can be learned from comparative metabolite profiling and metabolomics of yeast and filamentous fungi. Filamentous fungi also have a very active and diverse secondary metabolism in which many of the additional genes present in fungi, compared with yeast, are likely to be involved. Although the 'blueprint' of a given organism is represented by the genome, its behaviour is expressed as its phenotype, i.e. growth characteristics, cell differentiation, response to the environment, the production of secondary metabolites and enzymes. Therefore the profile of (secondary) metabolites--fungal chemodiversity--is important for functional genomics and in the search for new compounds that may serve as biotechnology products. Fungal chemodiversity is, however, equally efficient for identification and classification of fungi, and hence a powerful tool in fungal taxonomy. In this paper, the use of metabolite profiling is discussed for the identification and classification of yeasts and filamentous fungi, functional analysis or discovery by integration of high performance analytical methodology, efficient data handling techniques and core concepts of species, and intelligent screening. One very efficient approach is direct infusion Mass Spectrometry (diMS) integrated with automated data handling, but a full metabolic picture requires the combination of several different analytical techniques.     

L-amino acid oxidase-induced apoptosis in filamentous Botrytis cinerea

As opposed to single-cell yeast and mammalian cell lines, apoptosis has not been greatly investigated in filamentous fungi because antibodies to the relevant fungal apoptosis-related proteins are not available commercially and because multicellular organisms cannot be studied using flow cytometry. Here we demonstrate how antibodies from a nonfungal source could be used to investigate this pathway. We show that apoptosis in the filamentous fungus Botrytis cinerea is triggered by the mitochondria-mediated caspase pathway, with release of the apoptotic factors cytochrome c, caspase 3, and caspase 9, on treatment with Trichoderma harzianum-derived L-amino acid oxidase.     

Factors affecting population development within seawater-displaced fuel tanks

Abstract Populations within model seawater-displaced fuel tanks developed as a succession of eukaryotic species. Growth of filamentous fungi was preceded by the growth of several yeast species. More rapid initiation of growth by filamentous fungi was associated with extracellular metabolites produced by yeasts: both the pH flux towards more acidic conditions and the spore germination induction effect influenced population development. The relative binding affinity of each species for the insoluble hydrocarbon fuel is proposed as a major characteristic determining which species proliferated at the fuel-water interface.