SF-1 Induces Nuclear PIP2.
SF-1 Induces Nuclear PIP2.
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DOI：
10.3390/biom13101509
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发表时间：
2023-10-12
期刊：
影响因子：
5.5
通讯作者：
中科院分区：
文献类型：
--
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后生动物的细胞核含有磷脂酰肌醇PI(4，5)P2(PIP2)的非膜池，但这种疏水性脂类如何存在于水核质中仍不清楚。类固醇生成因子-1(NR5A1，SF-1)是一种在体外与PIP2结合的核受体，其共晶结构表明PIP2的酰基链隐藏在SF-1蛋白的疏水核心中，而PIP2头基则暴露在溶剂中。这种结合模式解释了SF-1如何溶解核PIP2；然而，缺乏细胞证据表明SF-1表达与核PIP2相关。在这里，我们使用针对PIP2头基的抗体，研究了四环素诱导SF-1表达是否与PIP2的核积聚有关。事实上，四环素诱导野生型SF-1在HEK细胞核中诱导了一个信号，该信号与PIP2抗体发生交叉反应，但不与针对低丰度磷脂酰肌醇PI(3，4，5)P3(PIP3)的抗体发生交叉反应。核PIP2信号与标记了旗帜的SF-1共同定位于核隔室。为了确定核PIP2信号是否依赖于SF-1与PIP2结合的能力，我们检测了一个经质谱分析显示磷脂结合缺陷的SF-1“口袋突变体”(A270W，L345F)。四环素在HEK细胞中诱导这种口袋突变体SF-1未能诱导可检测到的PIP2抗体交叉反应信号，尽管Tet在这些细胞中诱导野生型和口袋突变SF-1蛋白的表达水平相似。综上所述，这些数据首次表明SF-1的表达在细胞核内诱导了PIP2抗体的交叉反应信号，这与X射线结晶学和生化证据表明SF-1在人类细胞中与PIP2结合是一致的。
Metazoan cell nuclei contain non-membrane pools of the phosphoinositide lipid PI(4,5)P2 (PIP2), but how this hydrophobic lipid exists within the aqueous nucleoplasm remains unclear. Steroidogenic Factor-1 (NR5A1, SF-1) is a nuclear receptor that binds PIP2 in vitro, and a co-crystal structure of the complex suggests the acyl chains of PIP2 are hidden in the hydrophobic core of the SF-1 protein while the PIP2 headgroup is solvent-exposed. This binding mode explains how SF-1 can solubilize nuclear PIP2; however, cellular evidence that SF-1 expression associates with nuclear PIP2 has been lacking. Here, we examined if tetracycline induction of SF-1 expression would associate with nuclear accumulation of PIP2, using antibodies directed against the PIP2 headgroup. Indeed, tetracycline induction of wild-type SF-1 induced a signal in the nucleus of HEK cells that cross-reacts with PIP2 antibodies, but did not cross-react with antibodies against the lower abundance phosphoinositide PI(3,4,5)P3 (PIP3). The nuclear PIP2 signal co-localized with FLAG-tagged SF-1 in the nuclear compartment. To determine if the nuclear PIP2 signal was dependent on the ability of SF-1 to bind PIP2, we examined a “pocket mutant” of SF-1 (A270W, L345F) shown to be deficient in phospholipid binding by mass spectrometry. Tetracycline induction of this pocket mutant SF-1 in HEK cells failed to induce a detectable PIP2 antibody cross-reactive signal, despite similar Tet-induced expression levels of the wild-type and pocket mutant SF-1 proteins in these cells. Together, these data are the first to suggest that expression of SF-1 induces a PIP2 antibody cross-reactive signal in the nucleus, consistent with X-ray crystallographic and biochemical evidence suggesting SF-1 binds PIP2 in human cells.
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影响因子：
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作者：
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