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取决于不同细胞类型感知、处理和响应大量化学信号的能力。其中包括活性氧（ROS，例如过氧化氢），它在各种亚细胞环境中不断产生。实现“氧化还原传感”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、可调节的修饰。这些变化被称为翻译后修饰 (PTM)，它们会改变蛋白质功能并创建新的“信号网络”，从而控制信息在细胞内的传递方式。我们的研究特别感兴趣的是相对罕见的氨基酸半胱氨酸 (Cys) 上及其附近的 PTM 如何控制称为蛋白激酶的酶家族的行为，这对于通过调节磷酸基团向其目标蛋白的添加来控制信号传导信息流非常重要。由于细胞中有限数量的蛋白质可以检测到 ROS，因此它们充当调节信息交换的特异性“开关”，从而导致不同的生物反应，例如细胞对压力的适应、细胞生长和存活。在其他情况下，Cys 修饰可能变得不可逆，导致氧化反应“开启”，从而促进衰老等正常过程中的生存，或导致与神经变性相关的不可逆疾病。我们的初步工作表明，在同一酶家族的约 100 个成员中，在完全相同的 3D 位置中发现了 Cys 残基，并且这些含有 Cys 的酶在从酵母到人类的整个生命树中都有发现。这表明这种氨基酸在正确的时间出现在正确的位置对于感知和控制氧化还原信号传导至关重要。我们的建议重点关注约 100 个 Ser/Thr 蛋白激酶的“氧化还原控制”，这些蛋白激酶在称为“激酶激活片段”的蛋白质区域中均含有相同的保守 Cys 残基。我们想知道这些氨基酸如何感知 ROS，以及它们如何沿着通路传递这些信息以在细胞中形成信号网络，其作用在概念上就像一系列动态红绿灯，调节道路网络中的交通流量。为了快速推进我们对蛋白激酶氧化还原控制的理解，我们的目标将通过由经验丰富的科学家团队承担的 4 个不同但高度互补的工作包 (WP) 来实现。除了广泛的初步努力外，我们的研究还将详细关注两个特定的信号传导“途径”：WP1：细胞氧化还原信号传导：Cys 修饰、蛋白质复合物和相互作用组目标：100 个含 Cys 的 Ser/Thr 蛋白激酶的内源和基于 Bio-ID 的图谱结果：人类细胞中 CAMK 和 AGC 激酶的动态氧化还原图谱。WP2：结构分析，AlphaFold2 (AF2) 使用计算分子动力学进行数据库挖掘和 Cys 残基。目标：定义已发表和预测的约 100 个全长 Ser/Thr 蛋白激酶的折叠。结果：氧化还原敏感 Ser/Thr 激酶组内 Cys 残基相互作用的建模 WP3.1：体外激酶中化学 Cys 修饰的定量分析目标：研究氧化还原的酶促和基于 MS 的方法 修饰，最初关注“AGC”和“CAMK”激酶家族。结果：Ser/Thr 激酶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传导机制的重点分析目标：评估三种 AKT 信号酶的氧化还原和磷酸调节结果：定义 AGC 激酶模型中的氧化还原信号传导 sub-familyWP4：氧化还原反应过程中基于 BRSK1/2 的 Nrf2 信号传导的重点分析目标：分析有助于细胞 Nrf2 调节的上游机制。结果：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