申克孢子丝菌Pall和Pall样蛋白生物信息学分析

为初步了解申克孢子丝菌Sporothrix schenckii基因组中真菌特有蛋白PalI和PalI样蛋白的生物学特征,利用在线生物信息学分析软件,对两蛋白理化特征、功能域、二级结构、抗原表位等进行分析预测。发现两蛋白理论分子量分别为24.6kDa和75.6kDa,等电点均偏碱性; 属于同一蛋白家族,具有相同的功能域和多个PKC磷酸化位点; 两蛋白在近氨基端区域相似度较高,并与其他模式真菌中同源蛋白有很高相似度; 同时,两蛋白都有跨膜区,氨基端序列三级结构极为相似,并有丰富的抗原表位。通过生物信息学分析对申克孢子丝菌PalI和PalI样蛋白生物学特征有了初步的认识,为后续实验研究提供有力支持。     

Candida albicans Rim13p, a protease required for Rim101p processing at acidic and alkaline pHs

Candida albicans is an important commensal of mucosal surfaces that is also an opportunistic pathogen. This organism colonizes a wide range of host sites that differ in pH; thus, it must respond appropriately to this environmental stress to survive. The ability to respond to neutral-to-alkaline pHs is governed in part by the RIM101 signal transduction pathway. Here we describe the analysis of C. albicans Rim13p, a homolog of the Rim13p/PalB calpain-like protease member of the RIM101/pacC pathway from Saccharomyces cerevisiae and Aspergillus nidulans, respectively. RIM13, like other members of the RIM101 pathway, is required for alkaline pH-induced filamentation and growth under extreme alkaline conditions. Further, our studies suggest that the RIM101 pathway promotes pH-independent responses, including resistance to high concentrations of lithium and to the drug hygromycin B. RIM13 encodes a calpain-like protease, and we found that Rim101p undergoes a Rim13p-dependent C-terminal proteolytic processing event at neutral-to-alkaline pHs, similar to that reported for S. cerevisiae Rim101p and A. nidulans PacC. However, we present evidence that suggests that C. albicans Rim101p undergoes a novel processing event at acidic pHs that has not been reported in either S. cerevisiae or A. nidulans. Thus, our results provide a framework to understand how the C. albicans Rim101p processing pathway promotes alkaline pH-independent processes.     

The Sur7 protein regulates plasma membrane organization and prevents intracellular cell wall growth in Candida albicans

The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Δ mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Δ mutant are similar to the effects of inhibiting β-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains.     

Establishment of the ambient pH signaling complex in Aspergillus nidulans: PalI assists plasma membrane localization of PalH

The Aspergillus nidulans ambient pH signaling pathway involves two transmembrane domain (TMD)-containing proteins, PalH and PalI. We provide in silico and mutational evidence suggesting that PalI is a three TMD (3-TMD) protein with an N-terminal signal peptide, and we show that PalI localizes to the plasma membrane. PalI is not essential for the proteolytic conversion of the PacC translation product into the processed 27-kDa form, but its absence markedly reduces the accumulation of the 53-kDa intermediate after cells are shifted to an alkaline pH. PalI and its homologues contain a predicted luminal, conserved Gly-Cys-containing motif that distantly resembles a Gly-rich dimerization domain. The Gly44Arg and Gly47Asp substitutions within this motif lead to loss of function. The Gly47Asp substitution prevents plasma membrane localization of PalI-green fluorescent protein (GFP) and leads to its missorting into the multivesicular body pathway. Overexpression of the likely ambient alkaline pH receptor, the 7-TMD protein PalH, partially suppresses the null palI32 mutation. Although some PalH-GFP localizes to the plasma membrane, it predominates in internal membranes. However, the coexpression of PalI to stoichiometrically similar levels results in the strong predominance of PalH-GFP in the plasma membrane. Thus, one role for PalI, but possibly not the only role, is to assist with plasma membrane localization of PalH. These data, considered along with previous reports for both Saccharomyces cerevisiae and A. nidulans, strongly support the prevailing model of pH signaling involving two spatially segregated complexes: a plasma membrane complex containing PalH, PalI, and the arrestin-like protein PalF and an endosomal membrane complex containing PalA and PalB, to which PacC is recruited for its proteolytic activation.     

部分模式真菌中Prp5蛋白生物信息学分析

【目的】利用生物信息学方法对7种模式真菌中Prp5蛋白进行分析,为进一步研究Prp5蛋白在RNA剪接中的作用及其他独特的生物学功能提供基础。【方法】利用多种在线网站与软件分析和预测了稻瘟菌、裂殖酵母、酿酒酵母、粗糙脉胞菌、构巢曲霉、新生隐球菌、白色念珠菌Prp5蛋白的基本特性、二级结构、结构域及三级结构,同时对7种Prp5蛋白进行同源比对,并构建了系统进化树。【结果】所有研究蛋白均为不稳定的亲水性蛋白,稻瘟菌、粗糙脉胞菌、构巢曲霉的Prp5蛋白及裂殖酵母Prp11蛋白均呈碱性,而新生隐球菌与白色念珠菌的Prp5蛋白显酸性;进化分析显示稻瘟菌和粗糙脉胞菌亲缘关系最近,且7种模式生物被分为两个类群;二级结构以α螺旋为主;所有蛋白均含有DEAD和Helicase_C功能结构域;三级结构分析显示DEAD结构域较稳定,而Helicase_C结构域在空间结构上存在一定的变化。【结论】Prp5蛋白在不同真菌中具有保守性,因此可能发挥相似的生物学功能。然而酿酒酵母Prp5蛋白结构上略有变化,表明其在功能上可能具有一定独特性。研究结果对指导Prp5蛋白的进一步研究具有一定的参考价值。     

Nitric oxide and nitrosative stress tolerance in yeast

The opportunistic human fungal pathogen Candida albicans encounters diverse environmental stresses when it is in contact with its host. When colonizing and invading human tissues, C. albicans is exposed to ROS (reactive oxygen species) and RNIs (reactive nitrogen intermediates). ROS and RNIs are generated in the first line of host defence by phagocytic cells such as macrophages and neutrophils. In order to escape these host-induced oxidative and nitrosative stresses, C. albicans has developed various detoxification mechanisms. One such mechanism is the detoxification of NO (nitric oxide) to nitrate by the flavohaemoglobin enzyme CaYhb1. Members of the haemoglobin superfamily are highly conserved and are found in archaea, eukaryotes and bacteria. Flavohaemoglobins have a dioxygenase activity [NOD (NO dioxygenase domain)] and contain three domains: a globin domain, an FAD-binding domain and an NAD(P)-binding domain. In the present paper, we examine the nitrosative stress response in three fungal models: the pathogenic yeast C. albicans, the benign budding yeast Saccharomyces cerevisiae and the benign fission yeast Schizosaccharomyces pombe. We compare their enzymatic and non-enzymatic NO and RNI detoxification mechanisms and summarize fungal responses to nitrosative stress.     

Yeast PalA/AIP1/Alix Homolog Rim20p Associates with a PEST-Like Region and Is Required for Its Proteolytic Cleavage

The Saccharomyces cerevisiae zinc finger protein Rim101p is activated by cleavage of its C-terminal region, which resembles PEST regions that confer susceptibility to proteolysis. Here we report that Rim20p, a member of the broadly conserved PalA/AIP1/Alix family, is required for Rim101p cleavage. Two-hybrid and coimmunoprecipitation assays indicate that Rim20p binds to Rim101p, and a two-hybrid assay shows that the Rim101p PEST-like region is sufficient for Rim20p binding. Rim101p-Rim20p interaction is conserved in Candida albicans, supporting the idea that interaction is functionally significant. Analysis of Rim20p mutant proteins indicates that some of its broadly conserved regions are required for processing of Rim101p and for stability of Rim20p itself but are not required for interaction with Rim101p. A recent genome-wide two-hybrid study (T. Ito, T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, and Y. Sakaki, Proc. Natl. Acad. Sci. USA 98:4569-4574, 2000) indicates that Rim20p interacts with Snf7p and that Snf7p interacts with Rim13p, a cysteine protease required for Rim101p proteolysis. We suggest that Rim20p may serve as part of a scaffold that places Rim101p and Rim13p in close proximity.