Novel phospholipases C in trypanosomatids

一种新的肌醇特异性磷脂酶C（PI-PLC）已在T.克鲁齐病因学 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识 该序列先前在来自真核细胞的任何其他PI-PLC中没有描述。已经 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的表达水平与锥鞭毛体分化成 无鞭毛体TcPI-PLC在质膜上的过表达刺激了分化， TcPI-PLC表达的减少抑制了该过程。此外，初步证据显示， 这种酶可能参与Ssp-4的脱落，Ssp-4是一种GPI锚定蛋白， 肌醇磷酸神经酰胺在其脂质锚中。这是由TcPI的同时定位提出的- PLC和Ssp-4在细胞的外表面，TcPI-PLC水解的能力 肌醇磷酸神经酰胺在体外，没有其脂质锚的Ssp-4的脱落（如其脂质锚所证明的）。 交叉反应决定簇（CRD）反应性），并增加细胞神经酰胺时，最大的表面 TcPI-PLC的表达发生。神经酰胺也是一种重要的第二信使， 分化基于所有这些发现，我们的假设是TcPI-PLC可能负责多种 当它行进到细胞的外表面时起以下作用：（1）在细胞中水解PIP 2并产生IPS。 寄生虫，这一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，这导致蛋白质脱落到培养基中;以及（3） 宿主细胞中PIP 2的水解导致其细胞骨架的变化并产生IPS，这可能 参与宿主的细胞信号传导。根据这些调查结果，该提案的具体目标是： (1)研究脂肪酸修饰在TcPI-PLC定位和调控膜的作用 （2）为了研究TcPI-PLC是否参与了寄生虫的应激反应， 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在体外的转运机制 细胞

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.