Redox signalling through Ser/Thr protein kinase networks

生物体的生长和生存取决于不同类型细胞感知、处理和响应大量化学信号的能力。这些包括活性氧物质（ROS，如过氧化氢），其在各种亚细胞环境中连续产生。可以实现“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中发生的快速，受调节的修饰。这些变化被称为翻译后修饰（PTM），改变蛋白质功能并创建新的“信号网络”，控制信息如何在细胞内传递。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）上和附近的PTM如何控制称为蛋白激酶的酶家族的行为，这对于通过向其靶蛋白调节磷酸基团来控制信号传导信息的流动非常重要。由于ROS可以通过细胞中有限数量的蛋白质检测到，因此它们充当调节信息交换的特异性“开关”，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，导致在正常过程（例如衰老）期间促进存活的氧化反应的“开启”，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，Cys残基在相同酶家族的约100个成员中完全相同的3D位置，并且这些含Cys的酶在整个生命树中发现，从酵母到人类。这指出了这种氨基酸在正确的时间处于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'氧化还原控制'的100丝氨酸/苏氨酸蛋白激酶，都含有相同的保守的半胱氨酸残基的蛋白质的区域称为'激酶激活段'。我们想知道这些氨基酸是如何感知ROS的，以及它们如何沿着沿着通路传递这些信息以形成细胞中的信号网络，在概念上就像一系列动态交通灯一样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4个不同但高度互补的工作包（WP）来实现，由经验丰富的科学家团队进行。除了广泛的初步努力，我们的研究还将详细关注两个特定的信号传导“途径”：WP 1：细胞氧化还原信号传导：Cys修饰，蛋白质复合物和相互作用体目标：内源性和基于Bio-ID的100个含Cys的Ser/Thr蛋白激酶图谱结果：人类细胞中CAMK和AGC激酶的动态氧化还原图谱。WP 2：使用计算分子动力学进行结构分析、AlphaFold 2（AF 2）数据库挖掘和Cys残基。目标：定义约100种全长Ser/Thr蛋白激酶的已发表和预测折叠。结果：氧化还原敏感的Ser/Thr激酶组中Cys残基相互作用的建模WP3.1：体外激酶中化学Cys修饰的定量分析目的：研究氧化还原修饰的酶和基于MS的方法，最初集中在“AGC”和“CAMK”激酶家族。结果：Ser/Thr激酶中氧化还原调节的生物化学和细胞分析WP 3.2：重点分析细胞中基于AGC激酶的氧化还原信号传导机制目标：评估三种AKT信号传导酶的氧化还原和磷酸调节结果：定义模型AGC激酶亚家族中的氧化还原信号传导WP 4：重点分析氧化还原反应期间基于BRSK 1/2的Nrf 2信号传导目标：对细胞Nrf 2调节的上游机制进行分析。结果：定义连接BRSK 1/2、mTOR和KEAP 1/Nrf 2的氧化还原信号通路。

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.