Identification of Nuclear Phosphatidylinositol 4,5-Bisphosphate-Interacting Proteins by Neomycin Extraction
Identification of Nuclear Phosphatidylinositol 4,5-Bisphosphate-Interacting Proteins by Neomycin Extraction
复制标题
DOI：
10.1074/mcp.m110.003376
复制
发表时间：
2011-02-01
影响因子：
7
通讯作者：
D'Santos, Clive S.
中科院分区：
文献类型：
--
作者：
Lewis, Aurelia E.;Sommer, Lilly;D'Santos, Clive S.
通过鉴定肌醇磷脂效应蛋白，人们对肌醇磷脂调节的细胞质功能有了很大的了解。然而，磷脂酰肌醇调节的核功能很少，也不太清楚。为了解决这一问题，我们建立了一种基于新霉素提取完整细胞核的蛋白质组学方法，以丰富核磷脂酰肌醇效应蛋白。我们鉴定了168个含有肌醇磷脂结合区的蛋白质。虽然其中绝大多数含有富含赖氨酸/精氨酸的斑块，基序如下：K/R(X-n=(3-7))-K-X-K/R-K/R，但我们也发现了一小部分已知的含有Pleckstrin同源或植物同源结构域模块的磷脂酰肌醇结合蛋白。已鉴定的蛋白质以前没有磷脂酰肌醇相互作用的历史，其中一些蛋白质在RNA剪接和加工以及染色质组装中具有功能作用。其余的蛋白可能代表了其他新的核磷脂酰肌醇效应蛋白，因此加强了我们对磷脂酰肌醇调节的核功能的理解。DNA拓扑是其中的范例：生化分析验证了我们的蛋白质组学数据，支持磷脂酰肌醇4，5-二磷酸和DNA拓扑异构酶IIα之间的直接相互作用。此外，通过定量脂类下拉实验进一步验证了新霉素提取蛋白的一部分为4，5-二磷酸磷脂相互作用蛋白。总而言之，这样的数据集可以作为磷脂酰肌醇调控的核功能的全球视角的资源。分子与细胞蛋白质组学10：10.1074/mcp.M110.003376，1-15,2011年。
Considerable insight into phosphoinositide-regulated cytoplasmic functions has been gained by identifying phosphoinositide-effector proteins. Phosphoinositide-regulated nuclear functions however are fewer and less clear. To address this, we established a proteomic method based on neomycin extraction of intact nuclei to enrich for nuclear phosphoinositide-effector proteins. We identified 168 proteins harboring phosphoinositide-binding domains. Although the vast majority of these contained lysine/arginine-rich patches with the following motif, K/R(X-n = (3-7))-K-X-K/R-K/R, we also identified a smaller subset of known phosphoinositide-binding proteins containing pleckstrin homology or plant homeodomain modules. Proteins with no prior history of phosphoinositide interaction were identified, some of which have functional roles in RNA splicing and processing and chromatin assembly. The remaining proteins represent potentially other novel nuclear phosphoinositide-effector proteins and as such strengthen our appreciation of phosphoinositide-regulated nuclear functions. DNA topology was exemplar among these: Biochemical assays validated our proteomic data supporting a direct interaction between phosphatidylinositol 4,5-bisphosphate and DNA Topoisomerase II alpha. In addition, a subset of neomycin extracted proteins were further validated as phosphatidyl 4,5-bisphosphate-interacting proteins by quantitative lipid pull downs. In summary, data sets such as this serve as a resource for a global view of phosphoinositide-regulated nuclear functions. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.003376, 1-15, 2011.