Redox signalling through Ser/Thr protein kinase networks

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。其中包括活性氧（ROS，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上发生快速、受调节的修饰。这些变化被称为翻译后修饰（PTMs），它们改变了蛋白质的功能，并创造了新的“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，相对罕见的氨基酸半胱氨酸（Cys）上的PTMs和邻近的PTMs如何控制被称为蛋白激酶的酶家族的行为，这对于通过在其靶蛋白上调节磷酸基团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，它们作为调节信息交换的特异性“开关”，导致不同的生物反应，如细胞对压力的适应、细胞生长和生存。在其他情况下，Cys的修饰可能变得不可逆，导致氧化反应的“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转的疾病。我们的初步工作表明，在同一酶家族的大约100个成员中，在完全相同的3D位置上发现了Cys残基，这些含有Cys的酶在整个生命树中都有发现，从酵母到人类。这表明，为了感知和控制氧化还原信号，这种氨基酸在正确的时间处于正确的位置至关重要。我们的建议集中在约100个丝氨酸/苏氨酸蛋白激酶的“氧化还原控制”上，这些激酶在称为“激酶激活段”的蛋白质区域中都含有相同的保守Cys残基。我们想知道这些氨基酸是如何感知活性氧的，以及它们是如何将这些信息传递到细胞中形成信号网络的，从概念上讲，它们就像一系列动态的交通信号灯，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，我们的目标将通过一个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（WPs）来实现。除了广泛的初步努力，我们的研究还将集中在两个特定的信号传导“途径”上：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；目的：内源性和基于生物id的100种含Cys的丝氨酸/苏氨酸蛋白激酶的定位；结果：CAMK和AGC激酶在人类细胞中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cys -残基。目的：定义已发表和预测的约100个全长丝氨酸/苏氨酸蛋白激酶的折叠。结果：在氧化还原敏感的丝氨酸/苏氨酸激酶组WP3.1中建立Cys残基相互作用的模型；在体外对激酶中的化学Cys修饰进行定量分析。目的：酶和基于质谱的方法研究氧化还原修饰，最初集中在“AGC”和“CAMK”激酶家族上。结果：Ser/Thr激酶氧化还原调控的生化和细胞分析；wp3.2：重点分析细胞中基于AGC激酶的氧化还原信号传导机制；目的：评估三种AKT信号传导酶的氧化还原和磷酸化调控；结果：定义AGC激酶亚家族模型中的氧化还原信号传导；wp4：重点分析氧化还原反应中基于brsk1 /2的Nrf2信号传导；目的：分析细胞Nrf2调控的上游机制。结果：确定连接BRSK1/2、mTOR和KEAP1/Nrf2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.