Mechanisms of Diacylglycerol Signaling Through C1 Domain Proteins

通过 C1 结构域蛋白的二酰甘油信号传导机制

• 批准号: 7734081
• 负责人: James Hurley
• 金额: $ 34.46万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734081
• 关键词: 1,2-diacylglycerol; Activation Analysis; Affinity; Agonist; Amino Acids; Binding; Binding Sites; Class; Complex; Coupled; Crystallization; Diabetes Mellitus; Diglycerides; Distal; Event; Family; Goals; Growth Factor; Helix (Snails); Hormones; Hydrocarbons; Hydrogen Bonding; Immunosuppression; Investigation; Ions; Isoenzymes; Kinetics; Laboratory Study; Length; Lipids; Malignant Neoplasms; Mediator of activation protein; Membrane; Micelles; Molecular; Molecular Conformation; Phorbol Esters; Phospholipase; Phospholipids; Protein Isoforms; Protein Kinase C; Proteins; Receptor Protein-Tyrosine Kinases; Retinal Diseases; Second Messenger Systems; Side; Signal Transduction; Site; Stimulus; Structure; Surface; Tertiary Protein Structure; Therapeutic immunosuppression; Work; Zinc Fingers; arginyllysine; beta-chimaerin; ear helix; extracellular; novel; protein kinase C-delta; protein kinase D; receptor; receptor binding; response; second messenger; simulation; synergism; therapeutic target

Mechanisms of Diacylglycerol Signaling Through C1 Domain Proteins Diacylglycerol (DAG) is a paradigmatic lipid second messenger in metazoan cell signaling, the first to be discovered. The protein kinase C family is the best known target for DAG signals, but other proteins, such as the chimaerins, have also come to the fore as important receptors. PKCs and other DAG receptors are therapeutic targets for cancer, diabetes, and immunosuppression, among others. PKCs and other DAG receptors bind DAG through their C1 domains. This binding event triggers both membrane translocation and allosteric activation through conformational changes. This project aims to characterize these activation mechanisms at atomic-level detail. In addition to the structure of the phorbol ester:PKC delta C1B domain complex , past work in this project has yielded the first and only structure of a full-length C1 domain protein, that of beta2 chimaerin. The approaches used are 1) the crystallization of full-length C1-domain containing proteins; 2) analysis of activation dynamics using molecular simulations; and 3) spectroscopic analysis of activation kinetics and the conformation of active, membrane-associated states inaccessible to crystallization. Diacylglycerol is a central mediator of downstream signaling by a host of hormones coupled through Gq and phospholipase Cbeta, growth factors coupled to tyrosine-kinase linked receptors and phospholipase Cgamma, and many other extra- and intracellular stimuli. The protein kinase C (PKC) isozyme family has historically been the most intensively studied class of targets for diacylglycerol signaling. PKC isoforms are under active investigation as therapeutic targets for cancer and retinopathy in diabetes, and as targets for immunosuppression. In response to diacylglycerol, conventional and novel PKC isozymes, and the protein kinase D isozymes, translocate to membranes, where they phosphorylate Ser and Thr residues in diverse proteins. The diacylglycerol-responsive PKC isozymes are also activated by phorbol esters, which acts as a potent agonist by binding to the same site as diacylglycerol. These PKC isozymes are activated by diacylglycerol and phorbol esters by virtue of their direct binding to motifs known as protein kinase C homology-1 (C1) domains. C1 domains are compact zinc fingers of 50-51 amino acids. Their structure comprises two small b sheets and a single helix folded around two Zn 2+ ions. The two strands in the smaller b sheet are pulled apart, due to a break in their hydrogen bonding induced by a conserved Pro residue. Phorbol ester binds stereospecifically in the groove formed where the two strands pull away from each other, first described by structural studies from this laboratory. The stereospecific phorbol ester binding site is surrounded on three side by bulky hydrophobic side chains, which form a hydrophobic wall around the phorbol ester binding site. Distal to the phorbol ester-binding site, Arg, Lys, and His residues form a basic belt on the surface of the C1 domain. The hydrophobic wall inserts in the hydrocarbon core of phospholipid bilayers upon binding, while the basic belt interacts with acidic phospholipid headgroup. The high affinity of C1 domain/phorbol ester interactions in the presence of bilayers or micelles is due to the synergism between the stereospecific phorbol ester binding and nonspecific binding of acidic phospholipids to the basic and hydrophobic exterior surface of the C1 domain. Translocation and activation of C1 domain proteins requires the interplay of specific and nonspecific activators: both diacylglycerol or phorbol ester and bulk phospholipid. Efforts in FY2008 concentrated on the crystallization of full-length PKC isozymes. We continue to make incremental progress towards this challenging long-term goal.

通过 C1 结构域蛋白的二酰甘油信号传导机制 二酰基甘油 (DAG) 是后生动物细胞信号传导中的典型脂质第二信使，也是第一个被发现的。蛋白激酶 C 家族是 DAG 信号最知名的靶标，但嵌合蛋白等其他蛋白也作为重要受体脱颖而出。 PKC 和其他 DAG 受体是癌症、糖尿病和免疫抑制等的治疗靶点。 PKC 和其他 DAG 受体通过其 C1 结构域结合 DAG。这种结合事件通过构象变化触发膜易位和变构激活。该项目旨在在原子级细节上表征这些激活机制。除了佛波酯：PKC delta C1B 结构域复合物的结构外，该项目过去的工作还产生了第一个也是唯一一个全长 C1 结构域蛋白的结构，即 beta2 嵌合蛋白。使用的方法是 1) 含有全长 C1 结构域的蛋白质的结晶； 2）利用分子模拟分析活化动力学； 3) 活化动力学和无法结晶的活性、膜相关状态的构象的光谱分析。 二酰基甘油是一系列通过 Gq 和磷脂酶 Cbeta 偶联的激素、与酪氨酸激酶连接受体和磷脂酶 Cgamma 偶联的生长因子以及许多其他细胞外和细胞内刺激物偶联的下游信号传导的中心介质。蛋白激酶 C (PKC) 同工酶家族历来是二酰甘油信号传导研究最深入的一类靶标。 PKC 同工型作为癌症和糖尿病视网膜病变的治疗靶点以及免疫抑制的靶点正在积极研究中。响应二酰甘油，传统和新型 PKC 同工酶以及蛋白激酶 D 同工酶易位到膜上，在膜上磷酸化不同蛋白质中的 Ser 和 Thr 残基。二酰甘油反应性 PKC 同工酶也被佛波酯激活，佛波酯通过与二酰甘油相同的位点结合而充当有效的激动剂。这些 PKC 同工酶通过二酰基甘油酯和佛波醇酯直接与称为蛋白激酶 C 同源性 1 (C1) 结构域的基序结合而被激活。 C1 结构域是由 50-51 个氨基酸组成的紧凑锌指。它们的结构包括两个小的 b 片层和围绕两个 Zn 2+ 离子折叠的单个螺旋。由于保守的 Pro 残基导致氢键断裂，较小的 b 片中的两条链被拉开。佛波酯立体定向地结合在两条链相互拉开形成的凹槽中，该实验室的结构研究首次描述了这一点。立体特异性佛波酯结合位点在三侧被庞大的疏水侧链包围，其在佛波酯结合位点周围形成疏水壁。在佛波醇酯结合位点的远端，Arg、Lys 和 His 残基在 C1 结构域的表面形成碱性带。疏水壁在结合后插入磷脂双层的烃核心，而碱性带与酸性磷脂头基相互作用。在存在双层或胶束的情况下，C1 结构域/佛波酯相互作用的高亲和力是由于酸性磷脂与 C1 结构域的碱性和疏水性外表面的立体特异性佛波酯结合和非特异性结合之间的协同作用。 C1 结构域蛋白的易位和激活需要特异性和非特异性激活剂的相互作用：二酰基甘油或佛波醇酯以及大量磷脂。 2008 财年的工作重点是全长 PKC 同工酶的结晶。我们将继续朝着这一具有挑战性的长期目标不断取得进展。