Redox signalling through Ser/Thr protein kinase networks

通过 Ser/Thr 蛋白激酶网络的氧化还原信号传导

• 批准号: BB/X002780/1
• 负责人: Patrick Eyers
• 金额: $ 136.26万
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX002780%2F1
• 关键词: Redox; signalling; through; Ser; Thr

The growth and survival of organisms depends upon an ability of different cell types to sense, process and respond to a huge number of chemical signals. These include reactive oxygen species (ROS, such as hydrogen peroxide), which is continuously generated in various sub-cellular environments. One way that 'redox-sensing' can be achieved is through rapid, regulated, modifications that take place in chemically-reactive hot-spots found in some proteins. These changes, which are called post-translational modifications (PTMs), alter protein function and create new 'signalling networks', which control how information is relayed within cells. Our study is particularly interested in how PTMs on, and adjacent to, the relatively rare amino acid called Cysteine (Cys) control the behaviour of a family of enzymes termed protein kinases, which are important for controlling the flow of signalling information by the regulated addition of phosphate groups to their target proteins. Because ROS can be detected by a limited number of proteins in cells, they act as specificity 'switches' that modulate information exchange, leading to different sets of biological responses, such as cellular adaption to stress, cell growth and survival. In other circumstances, Cys modifications may become irreversible, leading to the switching 'on' of oxidant responses that promote survival during normal processes such as ageing, or lead to irreversible diseases associated with neurodegeneration. Our preliminary work has shown that Cys residues are found in exactly the same 3D location in ~100 members of the same enzyme family, and these Cys-containing enzymes are found throughout the tree of life, from yeasts to humans. This points to a fundamental importance of this amino acid being in the right place at the right time in order to sense and control redox signalling. Our proposal focusses on the 'redox control' of ~100 Ser/Thr protein kinases that all contain the same conserved Cys residue in a region of the protein called the 'kinase activation segment'. We want to know how these amino acids sense ROS, and how they pass this information along pathways to form signalling networks in cells, acting conceptually like a series of dynamic traffic lights that regulate the flow of traffic in a road network.To rapidly advance our understanding of redox control of protein kinases, our objectives will be achieved through 4 distinct, but highly complementary, work packages (WPs), undertaken by an experienced team of scientists. Alongside broad initial efforts, our studies will also focus on two specific signalling 'pathways' in detail:WP1: Cellular redox signaling: Cys modifications, protein complexes and interactomesObjectives: Endogenous and Bio-ID-based mapping of 100 Cys-containing Ser/Thr protein kinasesOutcomes: Dynamic redox mapping for CAMK and AGC kinases in human cells.WP2: Structural analysis, AlphaFold2 (AF2) database mining and Cys-residue using computational molecular dynamics.Objectives: Define published and predicted folds for ~100 full-length Ser/Thr protein kinases.Outcomes: Modelling of Cys residue interactions within the redox-sensitive Ser/Thr kinome WP3.1: Quantitative analysis of chemical Cys modifications in kinases in vitroObjectives: Enzymatic and MS-based approaches to study redox modifications, focussing initially on 'AGC' and 'CAMK' kinase families.Outcomes: Biochemical and cellular analysis of redox regulation in Ser/Thr kinasesWP3.2: Focused analysis of AGC kinase-based redox signaling mechanisms in cellsObjectives: Evaluating redox and phospho-regulation in three AKT signalling enzymesOutcomes: Define redox signalling in the model AGC kinase sub-familyWP4: Focused analysis of BRSK1/2-based signalling to Nrf2 during the redox responseObjectives: Analysis of upstream mechanisms contributing to cellular Nrf2 regulation.Outcomes: Define redox signalling pathways linking BRSK1/2, mTOR and KEAP1/Nrf2.

生物体的生长和生存依赖于不同类型的细胞感知、处理和响应大量化学信号的能力。其中包括在各种亚细胞环境中持续产生的活性氧物种(ROS，如过氧化氢)。实现“氧化还原感应”的一种方法是通过在某些蛋白质中发现的化学反应热点进行快速、受调控的修饰。这些变化被称为翻译后修饰(PTM)，改变了蛋白质的功能，并创建了新的“信号网络”，控制着信息在细胞内的传递方式。我们的研究特别感兴趣的是，在相对罕见的氨基酸半胱氨酸(Cys)上及其附近的PTM如何控制一系列名为蛋白激酶的酶的行为，这些酶对于通过调节磷酸基团添加到目标蛋白来控制信号信息流是重要的。由于ROS可以被细胞中有限数量的蛋白质检测到，它们扮演着调节信息交换的特异性‘开关’的角色，导致不同的生物反应，如细胞对压力的适应、细胞的生长和生存。在其他情况下，半胱氨酸的修饰可能变得不可逆转，导致氧化剂反应的启动，从而在衰老等正常过程中促进生存，或导致与神经退化相关的不可逆转疾病。我们的初步工作表明，在同一酶家族的约100个成员中，半胱氨酸残基位于完全相同的3D位置，这些含有半胱氨酸的酶存在于从酵母到人类的整个生命树中。这表明了这种氨基酸在正确的时间出现在正确的位置以感知和控制氧化还原信号的根本重要性。我们的建议集中在~100Ser/Thr蛋白激酶的氧化还原控制上，这些蛋白激酶在蛋白质的一个区域都含有相同的保守半胱氨酸残基，称为‘激酶激活片段’。我们想知道这些氨基酸是如何感知ROS的，以及它们如何沿着细胞中的路径传递这些信息，从而在细胞中形成信号网络，在概念上就像一系列动态红绿灯一样，调节道路网络中的交通流量。为了迅速推进我们对蛋白激酶氧化还原控制的理解，我们的目标将通过4个截然不同但高度互补的工作包(WPS)来实现，由一个经验丰富的科学家团队进行。除了广泛的初始努力外，我们的研究还将详细地关注两个特定的信号通路：WP1：细胞氧化还原信号：CyS修饰、蛋白质复合体和相互作用目标：100个含有半胱氨酸的Ser/Thr蛋白激酶的内源性和基于Bio-ID的定位结果：人类细胞中CAMK和AGC激酶的动态氧化还原定位。WP2：结构分析，AlphaFold2(AF2)数据库挖掘和使用计算分子动力学的半胱氨酸残基。目标：定义约100个全长Ser/Thr蛋白激酶的已发表和预测的折叠。结果：氧化还原敏感的Ser/Thr激动组WP3.1中半胱氨酸残基相互作用的模拟：体外对半胱氨酸化学修饰的定量分析目的：酶和基于MS的方法研究氧化还原修饰，结果：对丝氨酸/苏氨酸酶中氧化还原调节的生化和细胞分析WP3.2：细胞中基于AGC激酶的氧化还原信号机制的重点分析目的：评估三种AKT信号酶中的氧化还原和磷酸化调节结果：在模型AGC激酶亚家族中定义氧化还原信号WP4：重点分析氧化还原反应过程中基于BRSK1/2的Nrf2信号。目的：定义连接BRSK1/2、mTOR和Keap1/Nrf2的氧化还原信号通路。

项目成果

Classification of Cushing's syndrome PKAc mutants based upon their ability to bind PKI

• DOI: 10.1042/bcj20230183
• 发表时间: 2023-06-01
• 期刊: BIOCHEMICAL JOURNAL
• 影响因子: 4.1
• 作者: Omar，Mitchell H.;Kihiu，Maryanne;Scott，John D.
• 通讯作者: Scott，John D.

Considerations for defining +80 Da mass shifts in mass spectrometry-based proteomics: phosphorylation and beyond.

• DOI: 10.1039/d3cc02909c
• 发表时间: 2023-09-26
• 期刊: CHEMICAL COMMUNICATIONS
• 影响因子: 4.9
• 作者: Daly, Leonard A.;Clarke, Christopher J.;Po, Allen;Oswald, Sally O.;Eyers, Claire E.
• 通讯作者: Eyers, Claire E.

Mechanistic and evolutionary insights into isoform-specific 'supercharging' in DCLK family kinases

DCLK 家族激酶中异构体特异性“增压”的机制和进化见解

• DOI: 10.7554/elife.87958.2
• 发表时间: 2023
• 作者: Venkat A

Custom Workflow for the Confident Identification of Sulfotyrosine-Containing Peptides and Their Discrimination from Phosphopeptides.

• DOI: 10.1021/acs.jproteome.3c00425
• 发表时间: 2023-12-01
• 期刊: JOURNAL OF PROTEOME RESEARCH
• 影响因子: 4.4
• 作者: Daly, Leonard A.;Byrne, Dominic P.;Perkins, Simon;Brownridge, Philip J.;Mcdonnell, Euan;Jones, Andrew R.;Eyers, Patrick A.;Eyers, Claire E.
• 通讯作者: Eyers, Claire E.

ERK1/2 inhibitors act as monovalent degraders inducing ubiquitylation and proteasome-dependent turnover of ERK2, but not ERK1.

• DOI: 10.1042/bcj20220598
• 发表时间: 2023-05-15
• 期刊: The Biochemical journal