Novel phospholipases C in trypanosomatids

锥虫中的新型磷脂酶 C

• 批准号: 7091733
• 负责人: Silvia N Moreno
• 金额: $ 29.5万
• 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/7091733
• 关键词: cell membrane; ceramides; enzymes; fatty acylation; hydrolysis; lipids; phosphatidylinositols; phospholipase C; proteins; second messengers

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.

描述（由申请人提供）：一种新的肌醇特异性磷脂酶C（PI-PLC）已在T.克氏锥虫病的病原体。该酶具有N-肉豆蔻酰化和棕榈酰化的共有序列，这在以前的真核细胞的任何其他PI-PLC中都没有描述过。已证实该酶是肉豆蔻酰化和棕榈酰化的。最近，我们发现TcPI-PLC的表达水平与锥鞭毛体向无鞭毛体的分化之间存在相关性。TcPI-PLC在质膜上的过表达促进了分化，而TcPI-PLC表达的减少抑制了这一过程。此外，初步证据表明，该酶可能参与Ssp-4的脱落，Ssp-4是一种在其脂质锚中含有肌醇磷酸神经酰胺的GPI锚定蛋白。这通过TcPI-PLC和Ssp-4在细胞外表面的同时定位、TcPI-PLC在体外水解肌醇磷酸神经酰胺的能力、Ssp-4在没有其脂质锚的情况下的脱落（如通过其交叉反应性决定簇（CRD）反应性所证明的）以及当发生TcPI-PLC的最大表面表达时细胞神经酰胺的增加来表明。神经酰胺也是参与细胞分化的重要第二信使。基于所有这些发现，我们的假设是TcPI-PLC在其行进到细胞外表面时可能负责多种功能：（1）在寄生虫中水解PIP 2并产生IPS，该作用对于它们的分化是重要的;（2）水解寄生虫糖蛋白的GPI锚的糖肌醇磷脂，这导致蛋白质脱落到培养基中;和（3）来自宿主细胞的PIP 2的水解导致其细胞骨架的变化和可能参与宿主中的细胞信号传导的IPS的产生。根据这些发现，该计划的具体目标是：（1）研究脂肪酸修饰在TcPI-PLC定位和调节膜结合中的作用;（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以及其在细胞分化和宿主-寄生虫相互作用中的重要性;（3）探讨TcPI-PLC向细胞外表面转运的机制。