In vitro fluorescence assay to measure GDP/GTP exchange of guanine nucleotide exchange factors of Rho family GTPases.
In vitro fluorescence assay to measure GDP/GTP exchange of guanine nucleotide exchange factors of Rho family GTPases.
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DOI：
10.1093/biomethods/bpab024
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发表时间：
2022
影响因子：
3.6
通讯作者：
Koleske AJ
中科院分区：
文献类型：
--
作者：
Blaise AM;Corcoran EE;Wattenberg ES;Zhang YL;Cottrell JR;Koleske AJ
鸟嘌呤核苷酸交换因子（GEF）是通过GTP负载促进GTP酶活化的酶。全外显子组测序已经确定了与疾病相关的GEF中的罕见变异，表明GEF在人类发育中起着关键作用。然而，这些罕见变异的后果只能通过测量它们对细胞活性的影响来理解。在这里，我们提供了一个详细的，用户友好的协议，用于纯化和基于荧光的分析的两个GEF结构域内的蛋白质，三。该分析提供了一种直接的定量工具来测试GEF结构域对其各自GTP酶的活性，以及利用高通量筛选来鉴定调节剂和抑制剂。该方案可适用于其他Rho家族GEF的表征。这种分析对于全面了解全球环境基金遗传变异在人类发育和疾病中的作用至关重要。
Guanine nucleotide exchange factors (GEFs) are enzymes that promote the activation of GTPases through GTP loading. Whole exome sequencing has identified rare variants in GEFs that are associated with disease, demonstrating that GEFs play critical roles in human development. However, the consequences of these rare variants can only be understood through measuring their effects on cellular activity. Here, we provide a detailed, user-friendly protocol for purification and fluorescence-based analysis of the two GEF domains within the protein, Trio. This analysis offers a straight-forward, quantitative tool to test the activity of GEF domains on their respective GTPases, as well as utilize high-throughput screening to identify regulators and inhibitors. This protocol can be adapted for characterization of other Rho family GEFs. Such analyses are crucial for the complete understanding of the roles of GEF genetic variants in human development and disease.
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影响因子：
--
作者：
Evelyn CR;Ferng T;Rojas RJ;Larsen MJ;Sondek J;Neubig RR
通讯作者：
Neubig RR
肌动蛋白调节波复合物的结构和控制。
影响因子：
64.8
作者：
Chen, Zhucheng;Borek, Dominika;Padrick, Shae B.;Gomez, Timothy S.;Metlagel, Zoltan;Ismail, Ayman M.;Umetani, Junko;Billadeau, Daniel D.;Otwinowski, Zbyszek;Rosen, Michael K.
通讯作者：
Rosen, Michael K.
DOI：
10.1074/jbc.m112.435941
发表时间：
2013-03-22
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Hong L;Kenney SR;Phillips GK;Simpson D;Schroeder CE;Nöth J;Romero E;Swanson S;Waller A;Strouse JJ;Carter M;Chigaev A;Ursu O;Oprea T;Hjelle B;Golden JE;Aubé J;Hudson LG;Buranda T;Sklar LA;Wandinger-Ness A
通讯作者：
Wandinger-Ness A
影响因子：
3.7
作者：
Lee, Donghoon M.;Rodrigues, Francisco F.;Harris, Tony J. C.
通讯作者：
Harris, Tony J. C.
影响因子：
30.8
作者：
Girard, Simon L.;Gauthier, Julie;Rouleau, Guy A.
通讯作者：
Rouleau, Guy A.