Lipid activation of CTP: phosphocholine cytidylyltransferase alpha: characterization and identification of a second activation domain.
Lipid activation of CTP: phosphocholine cytidylyltransferase alpha: characterization and identification of a second activation domain.
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CTP 的脂质激活：磷酸胆碱胞苷酰转移酶 α：第二个激活域的表征和鉴定。
DOI：
10.1021/bi002140r
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Jackowski,S
中科院分区：
文献类型：
--
作者：
Lykidis,A;Jackson,P;Jackowski,S
磷脂酰转移酶（CCT）控制磷脂酰胆碱（PtdCho）生物合成的速率，其活性受与膜脂相互作用的控制。羧基端被解剖以描绘脂质反应所需的最小序列。螺旋结构域被认为是脂质相互作用的一个位点，从残基257到290的三个串联α-螺旋重复序列被发现是该结构域调节酶活性所必需的。截断羧基末端以去除一个或多个α-螺旋重复序列产生催化受损的蛋白质，这些蛋白质对脂质没有反应，但在体内过度表达时保留足够的活性以加速PtdCho的生物合成。螺旋区域在脂质活化中的作用通过切除残基257到309得到一个保留了57个残基的羧基末端结构域融合到催化核心的蛋白质进一步测试。这个结构验证了螺旋区域在没有脂质的情况下抑制活性而不是在有脂质的情况下激活酶的假设。这一假设预测了CCTα的本构活性[Δ257−309]；然而，无论是否存在脂质，这种蛋白都受到脂质的严格调控，其活性与全长CCTα相当。CCTα的激活[Δ257−309]完全依赖于阴离子脂质，而全长CCTα对阴离子或中性脂质均有反应。在Triton X-100胶束中传递的磷脂酸是第二脂质激活域的首选激活剂。这些数据表明，CCTα可以通过两个独立的结构域被脂质调节：(i)与中性和阴离子脂质混合物相互作用的三个两亲性α-螺旋重复序列和（ii）与阴离子脂质相互作用的最后57个残基。结果表明，这两个结构域在没有脂质的情况下是抑制的，在有脂质的情况下是激活的。去除这两个结构域会导致酶活性降低，无反应，失调。这些数据还首次证明了CCTα中57个残基羧基末端结构域参与脂质介导的调节，并足以最大限度地激活酶活性。
The CTP:phosphocholine cytidylyltransferase (CCT) governs the rate of phosphatidylcholine (PtdCho) biosynthesis, and its activity is governed by interaction with membrane lipids. The carboxy-terminus was dissected to delineate the minimum sequences required for lipid responsiveness. The helical domain is recognized as a site of lipid interaction, and all three tandem α-helical repeats from residues 257 through 290 were found to be required for regulation of enzymatic activity by this domain. Truncation of the carboxy-terminus to remove one or more of the α-helical repeats yielded catalytically compromised proteins that were not responsive to lipids but retained sufficient activity to accelerate PtdCho biosynthesis when overexpressed in vivo. The role of the helical region in lipid-activation was tested further by excising residues 257 through 309 to yield a protein that retained a 57-residue carboxy terminal domain fused to the catalytic core. This construct tested the hypothesis that the helical region inhibits activity in the absence of lipid rather than activates the enzyme in the presence of lipid. This hypothesis predicts constitutive activity for CCTα[Δ257−309]; however, this protein was tightly regulated by lipid with activities comparable to the full-length CCTα, in both the absence and presence of lipid. Activation of CCTα[Δ257−309] was dependent exclusively on anionic lipids, whereas full-length CCTα responded to either anionic or neutral lipids. Phosphatidic acid delivered in Triton X-100 micelles was the preferred activator of the second lipid-activation domain. These data demonstrate that CCTα can be regulated by lipids by two independent domains: (i) the three amphipathic α-helical repeats that interact with both neutral and anionic lipid mixtures and (ii) the last 57 residues that interact with anionic lipids. The results show that both domains are inhibitory in the absence of lipid and activating in the presence of lipid. Removal of both domains results in a nonresponsive, dysregulated enzyme with reduced activity. The data also demonstrate for the first time that the 57-residue carboxy-terminal domain in CCTα participates in lipid-mediated regulation and is sufficient for maximum activation of enzyme activity.