摘要/Abstract
摘要： 目的·研究核受体结合因子2（nuclear receptor-binding factor 2，NRBF2）的不同磷酸化水平对Ⅲ型磷脂酰肌醇-3-激酶复合物Ⅰ（class Ⅲ phosphatidylinositol 3-kinase complex Ⅰ，PI3KC3-C1）的活性影响，寻找一种活性最高的PI3KC3-C1作为自噬特异性小分子抑制剂筛选的靶标。方法·首先通过建立体外蛋白纯化体系，分别获得四元不含NRBF2的PI3KC3-C1和五元包含不同磷酸化水平NRBF2的PI3KC3-C1，通过凝胶过滤色谱检测复合物的完整性，然后体外测定纯化的不同PI3KC3-C1的激酶活性差异，观察NRBF2的磷酸化对PI3KC3-C1活性的影响。结果·纯化的PI3KC3-C1具有良好的纯度和产量。包含NRBF2的五元PI3KC3-C1表现出比四元的蛋白复合物更高的激酶活性，其中包含持续去磷酸化NBRF2的PI3KC3-C1具有最高的酶活性。结论·NRBF2确实作为PI3KC3-C1的组分之一稳定存在，NRBF2的存在可提升PI3KC3-C1的激酶活性；包含持续去磷酸化NRBF2的PI3KC3-C1可以作为一种新的自噬调控靶标，用来筛选自噬特异性小分子抑制剂。
关键词: 核受体结合因子2, Ⅲ型磷脂酰肌醇-3-激酶复合物Ⅰ, 激酶活性检测, 自噬调控
Abstract:
Objective · To study the effects of different phosphorylation levels of nuclear receptor-binding factor 2 (NRBF2) on the activity of class Ⅲ phosphatidylinositol 3-kinase complex Ⅰ (PI3KC3-C1), and find a type of PI3KC3-C1 with the highest kinase activity as a target for the screening of autophagy-specific inhibitors. Methods · First, in vitro protein purification system was established to obtain quaternary NRBF2-free PI3KC3-C1 and quinary PI3KC3-C1 containing different phosphorylation levels of NRBF2. The integrity of the complex was determinedgel filtration chromatography. The different kinase activity of purified PI3KC3-C1 was detected in vitro, and thus the effect of phosphorylation of NRBF2 on PI3KC3-C1 activity was observed. Results · The purified PI3KC3-C1 had good purity and yield. The five-membered PI3KC3-C1 containing NRBF2 showed higher kinase activity than the quaternary protein complex, and PI3KC3-C1 containing the continuous dephosphorylated NBRF2 had the highest kinase activity. Conclusion · NRBF2 does exist as one of the components of PI3KC3-C1. The presence of NRBF2 promotes the kinase activity of PI3KC3-C1, and PI3KC3-C1 containing continuous dephosphorylation of NRBF2 may serve as a new regulatory target for the screening of autophagy-specific inhibitor.
Key words: nuclear receptor-binding factor 2 (NRBF2), class Ⅲ phosphatidylinositol 3-kinase complex Ⅰ (PI3KC3-C1), kinase activity detection, autophagy regulation
PDF全文下载地址:
点我下载PDF
