Ca2+ Regulation of Trypanosoma brucei Phosphoinositide Phospholipase C.

Eukaryotic Cell Pub Date : 2015-05-01 Epub Date: 2015-03-13 DOI:10.1128/EC.00019-15
Sharon King-Keller, Christina A Moore, Roberto Docampo, Silvia N J Moreno
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引用次数: 10

Abstract

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 Ca(2+) 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 Ca(2+) sensitivity and substrate preference, demonstrating the role of this motif in Ca(2+) 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.

Abstract Image

Abstract Image

Abstract Image

布鲁氏锥虫磷酸肌肽磷脂酶C的Ca2+调控。
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Ca(2+)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对Ca(2+)的敏感性和底物偏好,证明了该基序在TbPI-PLC的Ca(2+)调控中的作用。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。
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来源期刊
Eukaryotic Cell
Eukaryotic Cell 生物-微生物学
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审稿时长
1 months
期刊介绍: Eukaryotic Cell (EC) focuses on eukaryotic microbiology and presents reports of basic research on simple eukaryotic microorganisms, such as yeasts, fungi, algae, protozoa, and social amoebae. The journal also covers viruses of these organisms and their organelles and their interactions with other living systems, where the focus is on the eukaryotic cell. Topics include: - Basic biology - Molecular and cellular biology - Mechanisms, and control, of developmental pathways - Structure and form inherent in basic biological processes - Cellular architecture - Metabolic physiology - Comparative genomics, biochemistry, and evolution - Population dynamics - Ecology
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