Structural mechanisms of regulation and assembly in the nephronophthisis INVS-NPHP3-NEK8-ANKS6 module

肾结核INVS-NPHP3-NEK8-ANKS6模块调节和组装的结构机制

• 批准号: MR/L017032/1
• 负责人: Richard Bayliss
• 金额: $ 48.3万
• 依托单位: University of Leicester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FL017032%2F1
• 关键词: Structural; mechanisms; regulation; assembly; nephronophthisis

Cystic kidney diseases represent a major health burden for which there are few effective treatments. Yet we are far from understanding the cell biology behind these diseases. Here, we propose to study the regulation and function of proteins that are mutated in nephronophthisis, an inherited childhood cystic kidney disease. These proteins coordinate signals received by developing kidney cells at antenna-like structures on the cell surface, called primary cilia. Defective signalling leads to loss of cell division control and cyst formation. Our aim is to provide new insights into cystic kidney disease mechanisms that will ultimately lead to new therapies.We will concentrate our efforts principally on two proteins, NEK8 and INVS. NEK8 belongs to a family of enzymes called protein kinases. These modify other proteins to modulate their functions, and are often found in signalling pathways that pass information from the cell surface to the cell nucleus so that cells may respond to their environment. The activity of protein kinases, that is to say the efficiency with which they modify other proteins, is strictly controlled. Protein kinases and other proteins that control them are very commonly mutated in diseases. The mutations result in uncontrolled kinase activity so that signalling pathways malfunction: they might pass signals to the nucleus when they should not, or they might block signals that ought to be passed on. We discovered that INVS controls the activity of NEK8, and we mapped the part of INVS responsible down to a region of the protein that is usually disrupted in patients that have INVS mutations. In many patients, it seems that disease is caused by missing just this region of INVS. In the first part of this project, we will investigate how INVS controls the activity of NEK8. To simplify matters, we will study just the two proteins in isolation. Crystallography will be used to produce atomic-resolution models of NEK8, both alone and associated with INVS, to show how its activity is controlled in the maximum possible detail. Crystallography produces detailed but static models. So we will apply advanced biochemical methods that enable us to track the activity of NEK8 over time under a number of different experimental conditions. This will provide us with a detailed understanding of what effects INVS has on NEK8, and what is therefore lacking in patients that have mutations in INVS.In the second part of this project, we will broaden our study to encompass two further proteins that are associated with NEK8 and INVS and that are also mutated in cystic kidney disease. We will investigate whether and how these proteins associate and how these interactions relate to the activity of NEK8. Finally, we will examine mutations in these proteins that were found in cystic kidney disease patients, to discover their effects on the interactions between proteins. Although many of the genes involved in cystic kidney disease are known, we have very little insight into how mutations in these genes cause disease. In this project, we will explore the idea that these mutations disrupt the interactions between a group of proteins, leading to loss of control of NEK8 activity. These insights will influence the way we think about signalling in the cilia, and how this goes awry in a range of associated diseases. We will produce a detailed model to explain how NEK8 activity is controlled by INVS and generate the first chemical compounds that block NEK8 activity. Our work will provide the basis for strategies to rescue ciliary signalling in cells with mutations in NEK8, INVS and interacting proteins. If successful, this might eventually lead to new therapies for patients.

囊性肾病是一种主要的健康负担，几乎没有有效的治疗方法。然而，我们还远未了解这些疾病背后的细胞生物学。在这里，我们建议研究的调节和功能的蛋白质突变的肾单位，遗传性儿童囊性肾病。这些蛋白质协调发育中的肾细胞在细胞表面的天线状结构（称为初级纤毛）上接收的信号。信号传导缺陷导致细胞分裂控制丧失和囊肿形成。我们的目标是为囊性肾病的发病机制提供新的见解，最终导致新的治疗方法。我们将主要集中精力研究两种蛋白质，NEK8和INVS。NEK8属于蛋白激酶家族。这些修饰其他蛋白质以调节它们的功能，并且通常在将信息从细胞表面传递到细胞核的信号通路中发现，以便细胞可以对其环境做出反应。蛋白激酶的活性，即它们修饰其他蛋白质的效率，受到严格控制。蛋白激酶和控制它们的其他蛋白质在疾病中非常常见地突变。这些突变导致激酶活性不受控制，从而导致信号传导通路发生故障：它们可能在不应该传递信号时将信号传递到细胞核，或者它们可能阻止应该传递的信号。我们发现INVS控制NEK8的活性，我们将INVS负责的部分定位到蛋白质的一个区域，该区域通常在INVS突变患者中被破坏。在许多患者中，疾病似乎是由于缺少INVS的这一区域引起的。在本项目的第一部分，我们将研究INVS如何控制NEK8的活动。为了简化问题，我们将只研究两种蛋白质。晶体学将用于产生NEK 8的原子分辨率模型，无论是单独的还是与INVS相关的，以显示其活动如何在最大可能的细节中被控制。晶体学产生详细但静态的模型。因此，我们将应用先进的生物化学方法，使我们能够在许多不同的实验条件下跟踪NEK 8随时间的活动。这将使我们详细了解INVS对NEK8的影响，以及INVS突变的患者因此缺乏什么。在本项目的第二部分，我们将扩大我们的研究范围，包括与NEK8和INVS相关的另外两种蛋白质，它们也在囊性肾病中突变。我们将研究这些蛋白质是否以及如何关联，以及这些相互作用如何与NEK8的活性相关。最后，我们将检查在囊性肾病患者中发现的这些蛋白质中的突变，以发现它们对蛋白质之间相互作用的影响。虽然许多参与囊性肾病的基因是已知的，但我们对这些基因的突变如何导致疾病的了解很少。在这个项目中，我们将探索这些突变破坏一组蛋白质之间的相互作用，导致NEK8活性失控的想法。这些见解将影响我们对纤毛信号的看法，以及这种信号在一系列相关疾病中如何出错。我们将产生一个详细的模型来解释NEK8活性是如何被INVS控制的，并产生第一个阻断NEK8活性的化合物。我们的工作将为在NEK8、INVS和相互作用蛋白突变的细胞中拯救纤毛信号传导的策略提供基础。如果成功，这可能最终为患者带来新的治疗方法。

项目成果

Mechanistic basis of Nek7 activation through Nek9 binding and induced dimerization.

• DOI: 10.1038/ncomms9771
• 发表时间: 2015-11-02
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Haq T;Richards MW;Burgess SG;Gallego P;Yeoh S;O'Regan L;Reverter D;Roig J;Fry AM;Bayliss R
• 通讯作者: Bayliss R

Mitotic Regulation by NEK Kinase Networks.

• DOI: 10.3389/fcell.2017.00102
• 发表时间: 2017
• 期刊: Frontiers in cell and developmental biology
• 影响因子: 5.5
• 作者: Fry AM;Bayliss R;Roig J
• 通讯作者: Roig J

The Ys and wherefores of protein kinase autoinhibition

• DOI: 10.1016/j.bbapap.2015.04.025
• 发表时间: 2015-10-01
• 期刊: BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
• 影响因子: 3.2
• 作者: Bayliss, Richard;Haq, Tamanna;Yeoh, Sharon
• 通讯作者: Yeoh, Sharon

Nek7 conformational flexibility and inhibitor binding probed through protein engineering of the R-spine

• DOI: 10.1042/bcj20200128
• 发表时间: 2020-04-01
• 期刊: BIOCHEMICAL JOURNAL
• 影响因子: 4.1
• 作者: Byrne, Matthew J.;Nasir, Nazia;Bayliss, Richard
• 通讯作者: Bayliss, Richard