Colorimetric RhoB GTPase Activity Assay
Bibliographic record
Abstract
The Ras homologous protein (Rho) GTPase subfamily, including RhoA, RhoB, and RhoC are small molecules (~21 kDa) that act as molecular switches in a wide range of signaling pathways to orchestrate biological processes associated with both physiological and tumorigenic cellular states. The Rho GTPases are crucial regulators of actin cytoskeleton rearrangements and FA dynamics and are required for effective cell migration and invasion, as well as cell cycle progression and apoptosis. The Rho GTPases activity is regulated by conformational switching between GTP-bound (active) and GDP-bound (inactive) states. This GTP/GDP cycling is tightly controlled by the guanine nucleotide exchange factors (GEFs), which function as activators by catalyzing the exchange of GDP for GTP and by the GTPase-activating proteins (GAPs), which enable hydrolysis of GTP leading to the Rho GTPase inactivation. Here, we describe a detailed protocol to perform a RhoB G-LISA activation assay to detect the level of GTP-loaded RhoB in vitro. This is the first colorimetric assay designed to specifically measure RhoB activation. This method was developed by adapting the RhoA G-LISA Activation Assay Kit (Cytoskeleton, Inc.) and allow the precise measurement of RhoB activity in less than 3 hours. This rapid methodology can be broadly used to assess the level of GTP-loaded RhoB in any kind of cellular models, to appreciate either the role RhoB activation in physiological processes, diseases, oncogenic transformation or for drug discovery in high throughput screens., [摘要] 包括RhoA，RhoB和RhoC在内的Ras同源蛋白（Rho）GTPase亚家族是小分子（〜21 kDa ），在广泛的信号传导途径中充当分子开关，以协调与生理和致瘤细胞状态相关的生物学过程。该Rho GTP酶是至关重要的调节肌动蛋白细胞骨架重排和FA动态和所需要的有效细胞迁移和侵袭，以及细胞周期进程和凋亡，其Rho GTP酶的活动是监管下的构象之间切换GTP结合（有源）和GDP -- GTP / GDP循环受鸟嘌呤核苷酸交换因子（GEF）严格控制，鸟嘌呤核苷酸交换因子（GEF）通过催化GTP的GDP交换和GTPase激活蛋白（GAP）来激活，从而实现水解作用。导致Rho GTPase失活的GTP 的详细信息。在此，我们描述了执行RhoB G-LISA活化测定以检测体外GTP负载的RhoB的水平的详细协议。这是第一个专门用于测量RhoB活化的比色测定。该方法是通过改编RhoA G-LISA激活检测试剂盒（Cytoskeleton，Inc.）而开发的，可在不到3 小时的时间内精确测量RhoB活性，这种快速方法可广泛用于评估GTP负载的RhoB 的水平在任何种类的细胞模型中，要了解RhoB激活在生理过程，疾病，致癌转化中的作用或在高通量筛选中发现药物的作用。[背景 ] 尽管Rho GTP酶的RhoA，RhoB的，和RhoC占有率超过85 Pasento的氨基酸序列相同，他们中发挥不同作用在肿瘤发生中通过交互使用不同的信号通路。而贡献小号中的RhoA和RhoC在肿瘤发展已详述许多研究表明，RhoB在癌症进展中的作用尚不清楚.RhoB 控制着几个基本过程，例如细胞形态，运动性，粘附，细胞内转运以及细胞增殖和有丝分裂（Spiering和Hodgson ，2011; Ridley 等，2003 ; 鞠而吉尔克斯，2018; 扎维等，2019; Svensmark而Brakebusch，。2019）中RhoB的在肿瘤发生中的作用似乎是复杂的，因为它很可能是RhoB的功能于一身的前后背景的方式，应对具体的信号在肿瘤微环境（Mazieres 等，2004）。此外，RhoB的具有几个特性是未发现其它Rho GTP酶，而小卢GTP酶一般都是定位于细胞色素 RhoB不仅定位在质膜和细胞质中，而且在对刺激的反应中也定位在质膜和细胞质上，还定位在内体和多囊泡体上（Wheeler 和Ridley ，2004; Vega和Ridley ，2018; Zaoui 等人， 2019） 。此外，RhoB的具有“GCI” （甘氨酸，半胱氨酸，异亮氨酸）三肽，定位于高变C-末端区域（残基188-190）（Wang和Sebti ，2005）。中棕榈酰化半胱氨酸这三肽调控对RhoB的稳定性而RhoB的亚细胞定位（佩雷斯·萨拉等，2009） 。这些独特的功能有助于多样性，并可能完全相反的功能RhoB的在致癌上下文。体外，GTP酶测定是广泛用于研究一般Rho GTP酶性质在活跃状态和非活跃状态之间循环，以及蛋白质表达和稳定性。我们的研究旨在了解RhoB GTPase活性在癌细胞信号调节细胞骨架重排，细胞过程中的作用。迁移和侵袭。该色度RhoB的GTP酶活性测定本文描述的呈现方法来研究激活RhoB的GTP酶在这些多步流程，至关重要的癌症细胞播散和转移的殖民化。这种方法可能有重大价值的监测水平GTP-可以在各种情况下加载RhoB，例如细胞运动的基础研究以及治疗性化合物的开发。
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".