Novel phospholipases C in trypanosomatids

锥虫中的新型磷脂酶 C

• 批准号: 7408626
• 负责人: Silvia N Moreno
• 金额: $ 28.1万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7408626
• 关键词: 1,2-diacylglycerol; Binding; Cell Differentiation process; Cell membrane; Cell physiology; Cell surface; Cells; Ceramides; Chagas Disease; Consensus Sequence; Cytoskeleton; Diglycerides; Enzymes; Eukaryotic Cell; Fatty Acids; Generations; Glycoproteins; Hydrolysis; In Vitro; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Lipids; Membrane; Metabolism; Modification; N-Myristoylation; Parasites; Pathway interactions; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipase C; Phospholipids; Phosphorylation; Physiological; Process; Protein Kinase C; Protein Overexpression; Proteins; Regulation; Research Personnel; Role; Second Messenger Systems; Signal Transduction; Surface; Travel; Trypanosoma cruzi; base; biological adaptation to stress; inositolphosphoceramides; myristoylation; novel; palmitoylation; programs; relating to nervous system; response; second messenger

A novel Phosphoinositide-specific phospholipase C (PI-PLC) has been described in T. cruzi, the etiologic agent of Chagas' disease. This enzyme possesses an N-myristoylation and palmitoylation consensus sequence that had not been described previously in any other PI-PLC from eukaryotic cells. It has been confirmed that the enzyme is myristoylated and palmitoylated. Recently, we demonstrated that there is a correlation between the expression levels of the TcPI-PLC and the differentiation of trypomastigotes into amastigotes. The overexpression of TcPI-PLC in the plasma membrane stimulated differentiation and reduction in the TcPI-PLC expression inhibited the process. In addition, preliminary evidence showed that the enzyme could be involved in shedding of Ssp-4, a GPI-anchored protein containing inositolphosphoceramide in its lipid anchor. This was suggested by the simultaneous localization of TcPI- PLC and Ssp-4 in the external surface of the cells, the ability of the TcPI-PLC to hydrolyze inositolphosphoceramide in vitro, the shedding of Ssp-4 without its lipid anchor (as demonstrated by its cross-reactive determinant (CRD) reactivity), and the increase in cellular ceramide when maximal surface expression of TcPI-PLC takes place. Ceramide is also an important second messenger involved in cellular differentiation. Based on all these findings our hypothesis is that TcPI-PLCs could be responsible for multiple functions as it travels to the outer surface of the cells: (1) hydrolysis of PIP2 and generation of IPS in the parasites, this effect being important for their differentiation; (2) hydrolysis of the glycoinositolphospholipids of GPI-anchors of parasite glycoproteins, which results in shedding of proteins to the medium; and (3) hydrolysis of PIP2 from the host cells leading to changes in its cytoskeleton and generation of IPSthat could be involved in cell signaling in the host. According to these findings, the specific aims of the proposal are: (1) To investigate the role of fatty acid modifications in TcPI-PLC localization and regulation of membrane binding; (2) To investigate whether TcPI-PLC is involved in the stress response of the parasite, in the hydrolysis of parasite and mammalian phospholipids, and its importance for cell differentiation and host- parasite interactions; (3) To investigate the transport mechanism of TcPI-PLC to the outer surface of the cells.

一种新的磷脂酰肌醇特异性磷脂酶C(PI-PLC)已在克氏锥虫的病原学中被描述 恰加斯病的病原体。该酶具有N-肉豆蔻酰化和棕榈酰化的共同点 这是以前在任何其他来自真核细胞的PI-PLC中未曾描述的序列。一直以来 证实该酶是肉豆蔻酰化和棕榈酰化的。最近，我们演示了有一个 TCPI-PLC表达水平与类星形胶质细胞分化的相关性 无鞭毛虫。TCPI-PLC在细胞膜上的过表达促进细胞分化和分化 TCPI-PLC表达的降低抑制了这一过程。此外，初步证据显示， 该酶可能参与SSP-4的脱落，SSP-4是一种GPI锚定蛋白，含有 肌醇磷酰胺在其脂质锚中。这是由TCPI的同时本地化提出的- PLC和SSP-4在细胞外表面，TCPI-PLC的水解能力 肌醇磷酰胺在体外，SSP-4在没有其脂质锚的情况下的脱落(如其 交叉反应决定簇(CRD)反应性)，以及细胞神经酰胺在最大表面积时的增加 TCPI-PLC的表达发生。神经酰胺也是细胞内重要的第二信使。 差异化。基于所有这些发现，我们的假设是TCPI-PLC可能负责多个 当它移动到细胞外表面时起作用：(1)PIP2的水解和在细胞内产生IPS 寄生虫，这种作用对它们的分化很重要；(2)糖肌醇磷脂的水解 寄生虫糖蛋白的GPI-锚定，导致蛋白质脱落到介质中；以及(3) 宿主细胞中PIP2的水解导致其细胞骨架的变化，并产生可以 参与宿主的细胞信号传递。根据这些研究结果，该建议的具体目标是： (1)探讨脂肪酸修饰在TCPI-PLC膜定位和调控中的作用 (2)研究TCPI-PLC是否参与寄生虫的应激反应 寄生虫和哺乳动物磷脂的水解及其对细胞分化和宿主的重要性 (3)研究TCPI-PLC的外表面转运机制。 细胞。