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在纳米盘上与14-3-3和KRAS4B复合的BRAF的复合和表征

Ningdi F Liu^1,2, Masahiro Enomoto¹, Christopher B Marshall¹

¹Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Protein science : a publication of the Protein Society

|May 15, 2024

概括

通过RAF激酶激活RAS-MAPK通路对于细胞生长和癌症至关重要. 这项研究揭示了RAS GTPase和膜相互作用如何促进RAF二分化和活性,为癌症信号提供了新的见解.

科学领域:

生物化学生物化学
细胞信号传递细胞信号传递
分子生物学分子生物学

背景情况:

RAF类激酶是RAS-RAS-MAPK通路的核心,调节细胞生长,并与癌症有关.
RAF激活涉及RAS招募到膜,促进从一个不活跃的单体到一个活跃的二元体的结构变化.
精确的RAF激活机制,特别是RAS和膜的作用,仍然不完全理解.

研究的目的:

通过重建一个功能信号复合体来阐明RAF激活的机械细节.
研究RAS GTPase和膜性质对RAF形状变化和激酶活性的影响.
提供RAF-RAS-MAPK信号调节的生物物理视角.

主要方法:

在纳米盘双层系统上重建BRAF-14-3-3-KRAS4B复合物.
复杂组件的GTP依赖性验证.
生物层干涉测量 (BLI) 来评估结合亲和度以及脂质组成和KRAS4B密度的影响.
使用复制的系统剖析酶活性.

主要成果:

一个GTP依赖的二极体BRAF,14-3-3二极体和KRAS4B的活性复合物被成功重组.
膜脂组合 (DOPS) 和更高的KRAS4B密度增强了BRAF:14-3-3与RAS-nanodiscs的结合.
KRAS4B和纳米盘单独没有刺激BRAF活动,但RAS-纳米盘复合体激活了单体和二元BRAF状态.

结论:

重建的无细胞系统提供了一个整体的生物物理方法来研究RAF-RAS-MAPK信号复杂调节.
RAS GTPase和膜相互作用对于调解RAF形状重组和激酶激活至关重要.
这项工作加深了对细胞膜RAF激活机制的理解,这与癌症研究有关.

关键词:

克拉斯 (Kras) 是一个国家.在MAPK信号传输中.在RAF中使用RAF基因酶.生物层干涉度测试是生物层干涉度测试.在体外溶解中的溶解.监管机制的调节机制.纳米光盘的使用方法

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