Shiratori Y, Houweling M, Zha X, Tabas I
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
J Biol Chem. 1995 Dec 15;270(50):29894-903. doi: 10.1074/jbc.270.50.29894.
Free cholesterol-loaded macrophages in atheromata synthesize excess phosphatidylcholine (PC), which may be an important adaptive response to the excess free cholesterol (FC) load. We have recently shown that FC loading of macrophages leads to 2-4-fold increases in PC mass and biosynthesis and to the post-translational activation of the membrane-bound form of CTP:phosphocholine cytidylyltransferase (CT), a key enzyme in PC biosynthesis. Herein, we explore further the mechanism of CT activation in FC-loaded macrophages. First, enrichment of membranes from control macrophages with FC in vitro did not increase CT activity, and PC biosynthesis in vivo is up-regulated by FC loading even when CT and FC appear to be mostly in different intracellular sites. These data imply that FC activates membrane-bound CT by a signaling mechanism. That the proposed signaling mechanism involves structural changes in the CT protein was suggested by data showing that two different antibodies against synthetic CT peptides showed increased recognition of membrane-bound CT from FC-loaded cells despite no increase in CT protein. Since CT is phosphorylated, two-dimensional maps of peptides from 32P-labeled control and FC-loaded macrophages were compared: six peptide spots from membrane-bound CT, but none from soluble CT, were dephosphorylated in the FC-loaded cells. Furthermore, incubation of FC-loaded macrophages with the phosphatase inhibitor, calyculin A, blocked increases in both PC biosynthesis and antipeptide-antibody recognition of CT. Last, treatment of membranes from control macrophages with lambda phage protein phosphatase in vitro increased both CT activity (2-fold) and antipeptide-antibody recognition of CT; soluble CT activity and antibody recognition were not substantially affected by phosphatase treatment. In summary, FC loading of macrophages leads to the partial dephosphorylation of membrane-bound CT, and possibly other cellular proteins, which appears to be important in CT activation. This novel regulatory action of FC may allow macrophages to adapt to FC loading in atheromata.
动脉粥样斑块中富含游离胆固醇的巨噬细胞会合成过量的磷脂酰胆碱（PC），这可能是对过量游离胆固醇（FC）负荷的一种重要适应性反应。我们最近发现，巨噬细胞加载FC会导致PC质量和生物合成增加2至4倍，并导致CTP：磷酸胆碱胞苷转移酶（CT）膜结合形式的翻译后激活，CT是PC生物合成中的关键酶。在此，我们进一步探讨FC加载的巨噬细胞中CT激活的机制。首先，体外将对照巨噬细胞膜用FC富集并不会增加CT活性，并且即使CT和FC似乎大多位于不同的细胞内位点，体内PC生物合成仍会因FC加载而上调。这些数据表明FC通过信号传导机制激活膜结合的CT。有数据显示，尽管CT蛋白没有增加，但针对合成CT肽的两种不同抗体对来自FC加载细胞的膜结合CT的识别增加，这表明所提出的信号传导机制涉及CT蛋白的结构变化。由于CT会被磷酸化，因此比较了来自32P标记的对照和FC加载巨噬细胞的肽段二维图谱：在FC加载的细胞中，膜结合CT的六个肽点去磷酸化，而可溶性CT的肽点没有去磷酸化。此外，用磷酸酶抑制剂花萼海绵诱癌素A孵育FC加载的巨噬细胞，会阻断PC生物合成的增加以及CT的抗肽抗体识别。最后，体外使用λ噬菌体蛋白磷酸酶处理对照巨噬细胞膜会使CT活性（增加2倍）和CT的抗肽抗体识别均增加；可溶性CT活性和抗体识别不受磷酸酶处理的显著影响。总之，巨噬细胞加载FC会导致膜结合CT以及可能的其他细胞蛋白部分去磷酸化，这似乎对CT激活很重要。FC的这种新型调节作用可能使巨噬细胞适应动脉粥样斑块中的FC负荷。
