Novel phospholipases C in trypanosomatids

描述(申请人提供)：一种新的磷脂酰肌醇特异性磷脂酶C(PI-PLC)已经在查加斯病的病原体T.ruzi中被描述。该酶具有N-肉豆蔻酰化和棕榈酰化的共同序列，这是以前在任何其他来自真核细胞的PI-PLC中未曾描述过的。已证实该酶是肉豆蔻酰化和棕榈酰化的。最近，我们证明了TCPI-PLC的表达水平与类鞭毛体向无鞭毛体的分化之间存在相关性。TCPI-PLC在细胞膜上的过表达促进了细胞的分化，而TCPI-PLC的表达则抑制了这一过程。此外，初步证据表明，该酶可能参与SSP-4的脱落，SSP-4是一种GPI锚定的蛋白，其脂质锚中含有肌醇磷酸肌酰胺。这是由于TCPI-PLC和SSP-4同时定位于细胞外表面，TCPI-PLC在体外对肌醇磷酰胺的降解能力，SSP-4在没有脂锚的情况下脱落(由其交叉反应决定簇(CRD)反应所证明)，以及当TCPI-PLC的表面表达达到最大值时细胞神经酰胺的增加。神经酰胺也是参与细胞分化的重要第二信使。根据所有这些发现，我们的假设是，TCPI-PLC在到达细胞外表面时可能负责多种功能：(1)PIP2的水解和寄生虫中IPS的生成，这一效应对寄生虫的分化非常重要；(2)寄生虫糖蛋白的GPI-锚点的糖肌醇磷脂的水解，导致蛋白质脱落到介质中；以及(3)宿主细胞中的PIP2的水解导致其细胞骨架的变化和IPS的生成，这可能参与宿主细胞的细胞信号转导。根据这些发现，该建议的具体目的是：(1)研究脂肪酸修饰在TCPI-PLC膜结合的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，参与寄生虫和哺乳动物磷脂的水解，及其对细胞分化和宿主-寄生虫相互作用的重要性；(3)研究TCPI-PLC向细胞外表面的转运机制。

DESCRIPTION (provided by applicant): 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 IPS that 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.