Novel phospholipases C in trypanosomatids

描述（由申请人提供）：一种新的磷酸肌醇特异性磷脂酶C （PI-PLC）在恰加斯病的病原克氏锥虫中被描述。该酶具有n -肉豆蔻酰化和棕榈酰化的一致序列，这在以前的任何其他真核细胞的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，我们证明了TcPI-PLC的表达水平与密交体向无尾体的分化之间存在相关性。质膜中TcPI-PLC的过表达促进了分化，而TcPI-PLC表达的降低抑制了分化。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锚定蛋白，其脂质锚定中含有肌醇磷酸神经酰胺。这可以通过TcPI-PLC和Ssp-4在细胞外表面的同时定位、TcPI-PLC在体外水解肌磷酸神经酰胺的能力、无脂锚的Ssp-4的脱落（通过其交叉反应性决定因素（CRD）反应性证明）以及当TcPI-PLC在细胞外表面最大表达时细胞神经酰胺的增加来证明。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可能负责多种功能，因为它移动到细胞的外表面：(1)PIP2的水解和IPS在寄生虫中产生，这种作用对它们的分化很重要；(2)寄生虫糖蛋白gpi锚点的糖肌醇磷脂水解，导致蛋白质脱落到培养基中；(3)宿主细胞中PIP2的水解导致其细胞骨架发生变化，产生可能参与宿主细胞信号传导的IPS。根据这些发现，本研究的具体目的是：(1)研究脂肪酸修饰在TcPI-PLC定位和膜结合调控中的作用；(2)探讨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.