Ca2+ Regulation of Trypanosoma brucei Phosphoinositide Phospholipase C
Ca2+ Regulation of Trypanosoma brucei Phosphoinositide Phospholipase C
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DOI：
10.1128/ec.00019-15
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发表时间：
2015-05-01
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影响因子：
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通讯作者：
Moreno, Silvia N. J.
中科院分区：
文献类型：
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作者：
King-Keller, Sharon;Moore, Christina A.;Moreno, Silvia N. J.
我们从布鲁氏锥虫的原环形式（PCF）中鉴定了一种磷酸肌肽磷脂酶C （PI-PLC）。该蛋白具有典型pi - plc的结构域组织特征，如X和Y催化结构域、EF-hand钙结合基序和C2结构域，但缺乏pleckstrin同源结构域（PH）。此外，布鲁氏T. PI-PLC （TbPI-PLC）含有仅在锥虫PI-PLC中发现的n端肉豆蔻酰化一致序列。含有该n端结构域的肽与绿色荧光蛋白（GFP）融合，被靶向到质膜上。在低于胞质水平的Ca2+浓度下，TbPI-PLC酶活性受到刺激，表明该酶具有组成性活性。TbPI-PLC可水解磷脂酰肌醇（PI）和磷脂酰肌醇4,5-二磷酸（PIP2），对PIP2具有较高的亲和力。我们发现EF-hand基序中单个氨基酸的修饰极大地影响了蛋白质的Ca2+敏感性和底物偏好，证明了该基序在TbPI-PLC的Ca2+调节中的作用。内源性TbPI-PLC定位于细胞内囊泡，可能利用细胞内PIP2来源。通过RNA干扰（RNAi）敲低TbPI-PLC的表达不会导致生长抑制，但寄生虫体内的酶活性仍然存在，导致PIP2水解，并参与肌醇1,4,5-三磷酸(IP3)/二酰基甘油（DAG）途径。
We characterized a phosphoinositide phospholipase C (PI-PLC) from the procyclic form (PCF) of Trypanosoma brucei. The protein contains a domain organization characteristic of typical PI-PLCs, such as X and Y catalytic domains, an EF-hand calcium-binding motif, and a C2 domain, but it lacks a pleckstrin homology (PH) domain. In addition, the T. brucei PI-PLC (TbPI-PLC) contains an N-terminal myristoylation consensus sequence found only in trypanosomatid PI-PLCs. A peptide containing this N-terminal domain fused to green fluorescent protein (GFP) was targeted to the plasma membrane. TbPI-PLC enzymatic activity was stimulated by Ca2+ concentrations below the cytosolic levels in the parasite, suggesting that the enzyme is constitutively active. TbPI-PLC hydrolyzes both phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2), with a higher affinity for PIP2. We found that modification of a single amino acid in the EF-hand motif greatly affected the protein's Ca2+ sensitivity and substrate preference, demonstrating the role of this motif in Ca2+ regulation of TbPI-PLC. Endogenous TbPI-PLC localizes to intracellular vesicles and might be using an intracellular source of PIP2. Knockdown of TbPI-PLC expression by RNA interference (RNAi) did not result in growth inhibition, although enzymatic activity was still present in parasites, resulting in hydrolysis of PIP2 and a contribution to the inositol 1,4,5-trisphosphate (IP3)/diacylglycerol (DAG) pathway.