The identification of myriocin-binding proteins
The identification of myriocin-binding proteins
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DOI：
10.1016/s1074-5521(99)80038-6
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发表时间：
1999-04-01
影响因子：
--
通讯作者：
Schreiber, SL
中科院分区：
文献类型：
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作者：
Chen, JK;Lane, WS;Schreiber, SL
背景：多球壳菌素是一种天然产物，可有效诱导小鼠细胞毒性 T 淋巴细胞系 (CTLL-2) 凋亡，并抑制丝氨酸棕榈酰转移酶 (SPT) 活性，已在细胞提取物中检测到该活性，并被认为可启动鞘脂生物合成。因为 SPT 从未经过生化纯化，也未对其进行全面评估。 尚未进行多球菌素结合蛋白的研究，我们使用基于多球菌素的亲和层析分离了特定靶标。结果：合成了多球菌素衍生物，并使用 CTLL-2 增殖和 SPT 活性测定进行了评估。在这些结果的指导下，制备了亲和层析基质，并从 CTLL-2 裂解物中分离出两种特异性多球菌素结合蛋白，并对这些蛋白进行了分析 结论：含多壳菌素的基质结合具有 SPT 活性的因子的能力以及作为多壳菌素结合蛋白的 LCB1 和 LCB2 的独特分离表明，这两种 蛋白质直接负责 SPT 活性，并且多球菌素直接作用于这些多肽。
Background: Myriocin is a natural product that potently induces apoptosis of a murine cytotoxic T lymphocyte cell line (CTLL-2 and inhibits a serine palmitoyltransferase (SPT) activity that has been detected in cell extracts and is thought to initiate sphingolipid biosynthesis. Because SPT has never been biochemically purified and a comprehensive appraisal of myriocin-binding proteins has not been conducted, we isolated specific targets using myriocin-based affinity chromatography.Results: Myriocin derivatives were synthesized and evaluated using CTLL-2 proliferation and SPT activity assays. Guided by these results, affinity chromatography matrices were prepared and two specific myriocin-binding proteins were isolated from CTLL-2 lysates, Analyses of these polypeptides establish conclusively that they are murine LCB1 and LCB2, mammalian homologs of two yeast proteins that have been genetically linked to sphingolipid biosynthesis.Conclusions: The ability of myriocin-containing matrices to bind factors that have SPT activity and the exclusive isolation of LCB1 and LCB2 as myriocin-binding proteins demonstrates that the two proteins are directly responsible for SPT activity and that myriocin acts directly upon these polypeptides.