Lykidis A, Jackson P, Jackowski S
Department of Biochemistry, St. Jude Children's Research Hospital, Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Biochemistry. 2001 Jan 16;40(2):494-503. doi: 10.1021/bi002140r.
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 alpha-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 alpha-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 CCTalpha[Delta257-309]; however, this protein was tightly regulated by lipid with activities comparable to the full-length CCTalpha, in both the absence and presence of lipid. Activation of CCTalpha[Delta257-309] was dependent exclusively on anionic lipids, whereas full-length CCTalpha 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 CCTalpha can be regulated by lipids by two independent domains: (i) the three amphipathic alpha-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 CCTalpha participates in lipid-mediated regulation and is sufficient for maximum activation of enzyme activity.
磷酸胆碱胞苷转移酶（CCT）调控磷脂酰胆碱（PtdCho）的生物合成速率，其活性受与膜脂相互作用的调控。剖析其羧基末端以确定脂质反应性所需的最小序列。螺旋结构域被认为是脂质相互作用的位点，发现从第257位残基到290位残基的所有三个串联α-螺旋重复序列对于该结构域调节酶活性是必需的。截断羧基末端以去除一个或多个α-螺旋重复序列会产生催化受损的蛋白质，这些蛋白质对脂质无反应，但在体内过表达时仍保留足够的活性来加速PtdCho的生物合成。通过切除第257位残基到309位残基以产生一种蛋白质，该蛋白质保留了与催化核心融合的57个残基的羧基末端结构域，进一步测试了螺旋区域在脂质激活中的作用。该构建体验证了以下假说：螺旋区域在无脂质时抑制活性而非在有脂质时激活酶。该假说预测CCTα[Δ257 - 309]具有组成型活性；然而，该蛋白质在有无脂质的情况下均受脂质严格调控，其活性与全长CCTα相当。CCTα[Δ257 - 309]的激活仅依赖于阴离子脂质，而全长CCTα对阴离子或中性脂质均有反应。在Triton X - 100微团中递送的磷脂酸是第二个脂质激活结构域的首选激活剂。这些数据表明，CCTα可通过两个独立结构域受脂质调控：（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，以及（ii）与阴离子脂质相互作用的最后57个残基。结果表明，这两个结构域在无脂质时均具有抑制作用，在有脂质时具有激活作用。去除这两个结构域会导致一种无反应、失调且活性降低的酶。数据还首次证明，CCTα中57个残基的羧基末端结构域参与脂质介导的调控，并且足以实现酶活性的最大激活。
