Cryptococus neoformans SOD1p cellular trafficking

新型隐球菌 SOD1p 细胞运输

• 批准号: 6843023
• 负责人: SUDHA CHATURVEDI
• 金额: $ 8.34万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6843023
• 关键词: Cryptococcus neoformans; Golgi apparatus; SDS polyacrylamide gel electrophoresis; centrifugation; endoplasmic reticulum; enzyme linked immunosorbent assay; fungal proteins; host organism interaction; immunoelectron microscopy; intracellular transport; laboratory mouse; microorganism interaction; monoclonal antibody; oxidative stress; polymerase chain reaction; protein transport; regulatory gene; secretory protein; superoxide dismutase; western blottings

DESCRIPTION (provided by applicant): Cryptococcus neoformans (Cn) secretory proteins are believed to be involved in mediating the interaction between the pathogens and host cells during the infection. Cn secretory pathways and machinery involved in the targeting, processing and transport of proteins are largely unexplored. In general, proteins transport to the cell surface follows classical secretory route, i.e. from endoplasmic reticulum to Golgi to plasma membrane, and finally to the cell surface. Interestingly, several physiologically important eukaryotic proteins have been identified that lack a classical signal sequence for secretion, but are still secreted from the cells. Our preliminary data with Cn copper zinc superoxide dismutase (SOD1p) revealed that (a) SOD1p, a cytosolic, single copy, leaderless protein, is secreted outside of the cell through channels in the cell wall, (b) secreted SOD1p is associated with cell wall and capsular material through as yet unknown mechanism, and (c) trapped SOD1p quenches phagocyte oxidative burst more efficiently, and thereby protects Cn from phagocyte killing. Additionally, Cn genomic database searches (Stanford University and The Institute for the Genomic Research) revealed full length CnSTE60RF containing 5121-bp nucleotide with 1706 amino acids, which showed 44% homology to Saccharomyces cerevisiae STE6. S. cerevisiae STE6 mediates export of a lipopeptide mating pheromone a-factor that lacks a classical hydrophobic signal peptide for secretion. Similarly, Cn database searches also revealed 200-300-bp contigs with 50 to 60% homology to nonclassical export genes NCE1, 2, 3 (now named as NCE101, NCE102, NCE103). These genes are well recognized for leaderless protein secretion in S. cerevisiae. These results indicated that Cn possesses nonclassical secretory pathways for protein export. Thus, both classical and nonclassical protein secretory pathways can be studied in Cn to better understand the invasion, survival, multiplication, and dissemination of this pathogen in the host. A number of inter-disciplinary approaches will be used in this investigation based on the previous research training of the principal investigator in Toxoplasma gondii protein trafficking. In specific Aim 1, the nonclassical SOD1p secretion will be compared with classical secretion of capsular protein CAP10p and phospholipase B (PLB1p) using inhibitors of secretion and localization of monoclonal antibodies. In specific Aim 2, two gene regulators of nonclassical secretion (STE6 and NCE102) will be characterized by a combination of molecular genetics and cell biological approaches to determine their relative influence on SOD1p, CAP10p & PLB1p secretions. The results of this investigation are likely to substantially enhance our understanding of protein trafficking in pathogenic fungi in general, and possibly assist in search for new therapeutic targets.

描述（由申请人提供）：据信，新生群（CN）分泌蛋白参与感染过程中病原体和宿主细胞之间的相互作用。蛋白质的靶向，加工和运输涉及的CN分泌途径和机械在很大程度上没有探索。通常，蛋白质转移到细胞表面遵循经典的分泌途径，即从内质网到高尔基体到质膜，最后到细胞表面。有趣的是，已经确定了几种在生理上重要的真核蛋白，它们缺乏分泌的经典信号序列，但仍被分泌在细胞中。我们使用CN铜锌超氧化物歧化酶（SOD1P）的初步数据表明，（a）SOD1P（一种sod1p，一种胞质，单拷贝，无领导蛋白）通过细胞壁上的通道在细胞外部分泌，（b）分泌的SOD1P与细胞壁和旋转的obsefient and of Norked Oust sodectians（b）分泌的SOD1P相关联有效地，从而保护CN免受吞噬细胞的杀戮。此外，CN基因组数据库搜索（斯坦福大学和基因组研究研究所）揭示了含有1706氨基酸的5121 BP核苷酸的全长CNSTE60RF，显示出44％的酿酒酵母Ste6同源性。酿酒酵母Ste6介导了脂肽交配信息素A因子的出口，该信息缺乏经典的疏水信号肽进行分泌。同样，CN数据库搜索还显示了200-300 bp重叠群，与非经典导出基因NCE1、2、3（现在称为NCE101，NCE102，NCE103）具有50％至60％的同源性。这些基因在酿酒酵母中的无领导蛋白分泌方面得到了广泛认可。这些结果表明，CN具有非经典分泌途径用于蛋白质的出口。因此，可以在CN中研究经典和非经典蛋白质分泌途径，以更好地了解宿主中这种病原体的入侵，存活，繁殖和传播。基于先前对弓形虫弓形虫蛋白运输的主要研究者的研究培训，将在这项研究中使用许多跨学科方法。在特定的目标1中，将使用单克隆抗体的分泌抑制剂和定位抑制剂将非经典SOD1P分泌与囊蛋白CAP10P和磷脂酶B（PLB1P）的经典分泌进行比较。在特定的目标2中，两个非经典分泌的基因调节剂（Ste6和NCE102）的特征是分子遗传学和细胞生物学方法的结合，以确定它们对SOD1P，CAP10P和PLB1P分泌的相对影响。这项研究的结果可能会大大增强我们对致病真菌中蛋白质运输的理解，并可能有助于寻找新的治疗靶标。