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.

二酰基甘油通过C1域蛋白的机制 二酰基甘油（DAG）是内唑细胞信号传导中的范式脂质第二信使，是第一个被发现的。蛋白激酶C家族是最著名的DAG信号靶标，但是其他蛋白质（例如嵌合蛋白）也已成为重要受体。 PKC和其他DAG受体是癌症，糖尿病和免疫抑制等的治疗靶标。 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。该项目旨在将这些激活机制表征为原子级细节。除了Phorbol酯的结构：PKC Delta C1B结构域复合物外，该项目的过去工作还产生了Beta2 Chimaerin的全长C1域蛋白的第一个也是唯一的结构。所使用的方法是1）全长C1结构域含有蛋白质的结晶； 2）使用分子模拟分析激活动力学； 3）激活动力学的光谱分析以及与结晶无法接近的活性，膜相关状态的构象。 二酰基甘油是通过GQ和磷脂酶CBETA耦合的许多激素的下游信号传导的中心介体，生长因子与酪氨酸激酶连接受体以及磷脂酶Cgamma以及许多其他外部和细胞内刺激。从历史上看，蛋白激酶C（PKC）同工酶家族一直是二酰基甘油信号传导一类研究的靶标。 PKC同工型正在积极研究糖尿病的癌症和视网膜病的治疗靶点，并作为免疫抑制的靶标。为了响应二酰基甘油，常规和新型的PKC同工酶，以及蛋白激酶D同工酶，转移到膜上，在那里它们磷酸化的Ser和THR残基在多种蛋白质中。二酰基甘油反应性PKC同工酶也被佛波酯激活，佛波酯通过与二酰基甘油的位点结合来充当有效的激动剂。这些PKC同工酶通过二酰基甘油和佛波酯的直接结合而被称为蛋白激酶C同源1（C1）结构域激活。 C1结构域是50-51个氨基酸的紧凑锌手指。它们的结构包括两个小B板和一个单个螺旋，在两个Zn 2+离子周围折叠。较小的B纸中的两条线被拉开，这是由于其由保守的Pro残留物引起的氢键断裂。 Phorbol Ester在形成的凹槽中结合了立体特异性的结合，在该凹槽中，两条线彼此脱离，首先是由该实验室的结构研究描述的。立体特异性的佛波酯结合位点被笨重的疏水侧链包围在三侧，该链链在凤凰酯结合位点周围形成疏水壁。佛经酯结合位点的远端，arg，lys及其残留物在C1域的表面形成基本皮带。疏水壁在结合后插入磷脂双层的烃核，而基本皮带与酸性磷脂头组相互作用。在双层或胶束存在下，C1结构域/佛波酯相互作用的高亲和力是由于立体特异性的phorbol酯结合与酸性磷脂与基本和疏水性外部表面C1结构域之间的协同作用。 C1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：二酰基甘油或凤凰酯和大量的磷脂。 2008财年的努力集中在全长PKC同工酶的结晶上。我们继续朝着这个充满挑战的长期目标取得渐进的进步。