CDK-containing macromolecular assemblies

含CDK的大分子组装体

• 批准号: MR/N009738/1
• 负责人: Jane Endicott
• 金额: $ 233.7万
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FN009738%2F1
• 关键词: CDK; containing; macromolecular; assemblies

The behaviour of a cell is defined by the set of genes it expresses and by its commitment to either a quiescent or a proliferating state. The cyclin-dependent kinases (CDKs) bind to non-catalytic cyclin subunits to form CDK-cyclin complexes that play central roles in regulating both gene-expression and cell-proliferation. Consequently, CDK-cyclins are important in normal cells and in a variety of disease conditions. We are proposing a programme of research to understand how CDK-cyclins work, how they in turn are regulated, and how their inappropriate activity can contribute to the development of disease.Because certain CDK-cyclin complexes can function interchangeably, expression of all but one cell-cycle regulatory CDK can be inhibited without compromising the cells ability to divide. The exception is CDK1, which must possess unique properties that make it indispensable. We have solved the structure of CDK1-containing complexes, and found that it has a small number of unique kinetic and structural properties. We propose to use mutagenesis to engineer these properties into CDK2, the closest homologue of CDK1, and see if the resulting CDK2-variant can functionally complement CDK1. In this way, we will identify what is the property of CDK1 that explains its uniqueness, resolving a key conundrum at the heart of CDK-biology.CDK1 and CDK2 play important roles in meiosis, the process by which sperm and egg cells are generated. These CDKs can be regulated by binding of non-cyclin partners and by covalent modifications other than phosphorylation. We will explore how these regulatory mechanisms work, and how conventional and unconventional regulatory mechanisms operate in meiosis.Different CDK-cyclin complexes are characterised as regulating either transcription (e.g. CDK9-cyclin T) or proliferation (e.g. CDK4-cyclin D). A growing body of evidence, however, suggests that this classification is over simplified. In particular cyclin D appears to work together with nuclear hormone receptors (such as the androgen and oestrogen receptors) to regulate transcription, and we will investigate the mechanisms that underlie this. Because inhibition of androgen receptor signalling is a well validated way to combat prostate cancer, a better understanding of how cyclin D contributes to androgen receptor function may suggest ways to tackle forms of prostate cancer that have become resistant to current therapies.Several transcriptional and cell-cycle regulatory CDKs depend on a chaperone system to shepherd them into the correct shape and/or correct set of complexes to carry out their cellular roles. The chaperoning of CDKs is mediated by HSP90, a promiscuous chaperone, and by Cdc37, a more specific adaptor. We have reconstituted the process of "hand-off" from chaperone to functional CDK-cyclin complexes in cell-free experiments. We now propose to characterise the structural mechanism of hand off, using a combination of biophysical and structural techniques.X-ray crystallography has provided a structural explanation of how cyclins both activate CDKs and, to some extent, direct them towards appropriate substrates. For CDK9-cyclin T, a further level of regulation is provided by interchange between two settings: an inhibitory particle termed the 7SK ribonucleoprotein (7SKsnRNP) and an activated setting, where CDK9-cyclin T is recruited to genes that are being expressed. Recent technical developments make large complexes such as the 7SKsnRNP accessible to high resolution structural study by cryoelectron microscopy, and we will use this technique to image the 7SKsnRNP to understand the mechanism by which it holds CDK9-cyclin T inactive, and how it subsequently switches into the activated setting.

细胞的行为是由它表达的一组基因及其对静止或增殖状态的承诺来定义的。细胞周期蛋白依赖性激酶（cyclin-dependent kinases，CDKs）与非催化性细胞周期蛋白亚基结合形成CDK-cyclin复合物，在调控基因表达和细胞增殖中发挥重要作用。因此，CDK-细胞周期蛋白在正常细胞和各种疾病中是重要的。我们提出了一个研究计划，以了解CDK-细胞周期蛋白如何工作，他们反过来又是如何调节，以及他们的不适当的活动如何有助于疾病的发展，因为某些CDK-细胞周期蛋白复合物可以互换功能，除了一个细胞周期调节CDK的表达可以被抑制，而不损害细胞的分裂能力。唯一的例外是CDK 1，它必须具有独特的属性，使其不可或缺。我们已经解决了含有CDK 1的复合物的结构，并发现它具有少数独特的动力学和结构特性。我们建议使用诱变将这些特性工程化为CDK 2，CDK 1的最接近的同源物，并观察所得的CDK 2变体是否可以在功能上补充CDK 1。通过这种方式，我们将确定CDK 1的特性是什么，解释了它的独特性，解决了CDK生物学核心的一个关键难题。CDK 1和CDK 2在减数分裂中发挥重要作用，精子和卵细胞的产生过程。这些CDK可以通过与非细胞周期蛋白伴侣的结合以及通过除磷酸化以外的共价修饰来调节。我们将探讨这些调控机制如何工作，以及常规和非常规调控机制如何在减数分裂中发挥作用。不同的CDK-cyclin复合物的特点是调节转录（例如CDK 9-cyclin T）或增殖（例如CDK 4-cyclin D）。然而，越来越多的证据表明，这种分类过于简单。特别是细胞周期蛋白D似乎与核激素受体（如雄激素和雌激素受体）一起调节转录，我们将研究其背后的机制。因为抑制雄激素受体信号传导是一种有效的对抗前列腺癌的方法，更好地了解细胞周期蛋白D如何促进雄激素受体功能，可能会为解决对当前治疗产生耐药性的前列腺癌提供方法。或正确的复合物来执行它们的细胞角色。CDK的分子伴侣作用由混杂分子伴侣HSP 90和特异性更高的衔接子Cdc 37介导。我们在无细胞实验中重建了从分子伴侣到功能性CDK-细胞周期蛋白复合物的“交接”过程。现在，我们建议使用生物物理学和结构techniques. X射线晶体学的组合，以解释的结构机制的手关闭，提供了一个结构的解释细胞周期蛋白如何既激活CDK，并在一定程度上，直接向适当的基板。对于CDK 9-细胞周期蛋白T，通过两种设置之间的交换提供了进一步的调节水平：称为7SK核糖核蛋白（7SKsnRNP）的抑制性颗粒和激活设置，其中CDK 9-细胞周期蛋白T被募集到正在表达的基因中。最近的技术发展使大型复合物，如7SKsnRNP可通过冷冻电子显微镜进行高分辨率结构研究，我们将使用这种技术对7SKsnRNP进行成像，以了解它使CDK 9-细胞周期蛋白T失活的机制，以及它随后如何切换到激活设置。

项目成果

Exiting the tunnel of uncertainty: crystal soak to validated hit.

• DOI: 10.1107/s2059798322009986
• 发表时间: 2022-11-01
• 期刊: Acta crystallographica. Section D, Structural biology

Modern Methods of Drug Design and Development

现代药物设计和开发方法

• DOI: 10.1016/bs.mie.2023.06.021
• 发表时间: 2023
• 作者: Martin M

Human ex vivo prostate tissue model system identifies ING3 as an oncoprotein.

• DOI: 10.1038/bjc.2017.447
• 发表时间: 2018-03-06
• 期刊: British journal of cancer
• 影响因子: 8.8
• 作者: McClurg UL;Nabbi A;Ricordel C;Korolchuk S;McCracken S;Heer R;Wilson L;Butler LM;Irving-Hooper BK;Pedeux R;Robson CN;Riabowol KT;Binda O
• 通讯作者: Binda O

Differential Regulation of G1 CDK Complexes by the Hsp90-Cdc37 Chaperone System.

• DOI: 10.1016/j.celrep.2017.10.042
• 发表时间: 2017-10-31
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Hallett ST;Pastok MW;Morgan RML;Wittner A;Blundell KLIM;Felletar I;Wedge SR;Prodromou C;Noble MEM;Pearl LH;Endicott JA
• 通讯作者: Endicott JA

Emerging approaches to CDK inhibitor development, a structural perspective.

• DOI: 10.1039/d2cb00201a
• 发表时间: 2023-02-08
• 期刊: RSC CHEMICAL BIOLOGY
• 影响因子: 4.1
• 作者: Hope, Ian;Endicott, Jane A.;Watt, Jessica E.
• 通讯作者: Watt, Jessica E.