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: 7593546
• 负责人: James Hurley
• 金额: $ 29.63万
• 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/7593546
• 关键词: 1,2-diacylglycerol; Activation Analysis; Affinity; Agonist; Amino Acids; Binding; Binding Sites; C elegans Unc-13 protein; Calcium; Class; Collaborations; Complex; Coupled; Crystallization; Diabetes Mellitus; Diglycerides; Distal; Engineering; Esters; Event; Exocytosis; Family; Fluorescence; GTPase-Activating Proteins; 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; Pennsylvania; Phorbol Esters; Phospholipase; Phospholipids; Physiological; Predisposition; Protein Isoforms; Protein Kinase C; Proteins; Proteolysis; Receptor Protein-Tyrosine Kinases; Retinal Diseases; Scaffolding Protein; Second Messenger Systems; Side; Signal Transduction; Site; Stimulus; Structure; Surface; Techniques; Tertiary Protein Structure; Therapeutic immunosuppression; Thinking; Work; Zinc; Zinc Fingers; arginyllysine; base; beta pleated sheet; beta-chimaerin; ear helix; extracellular; mutant; novel; pressure; protein kinase C-delta; protein kinase D; ras Guanine Nucleotide Exchange Factors; receptor; receptor binding; response; second messenger; simulation; synergism; therapeutic target

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 beta sheets and a single helix folded around two zinc ions. The two strands in the smaller beta 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 domainphorbol 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. In recent years, it has become clear that there are several major classes of C1 domain-containing diacylglycerol receptors in addition to PKC. Munc-13 proteins are phorbol ester-binding scaffolding proteins involved in calcium-stimulated exocytosis. RasGRPs are diacylglycerol-activated guanine-nucleotide exchange factors (GEFs) for Ras and Rap1. The alpha- and beta-chimaerins are a family of phorbol ester- and diacylglycerol-responsive GTPase activating proteins. This project takes a holistic approach towards the family of diacylglycerol-binding C1 domain proteins and so includes PKCs and chimaerins. In collaboration with Marcelo Kazanietz (Univ. of Pennsylvania) we characterized the strucure of beta2-chimaerin. We used this information to elucidate the activation mechanism, and engineered a mutant that is supersensitive to DAG activation. This turbo-chimaerin mutant was then used to elucidate the physiological regulatory action of beta2-chimaerin in the Kazanietz lab. Structural and functional studies of C1 domains have established the outlines of a mechanism for diacylglycerol and phorbol ester-induced translocation to membranes. Concomitant with membrane translocation, PKCs, chimaerins, and other C1 domain proteins become allosterically activated. The PKC activation mechanism is thought to require a large conformational change, on the basis of changes in susceptibility to limited proteolysis, translocation kinetics, fluorescence, surface pressure analysis, and other indirect structural techniques.

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β板和一个单螺旋，围绕两个锌离子折叠。由于保守的Pro残留物引起的氢键断裂，因此将较小β板中的两条线拉开。 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结构域的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。 近年来，很明显，除PKC外，还有几类主要含C1域的二酰基甘油受体。 MUNC-13蛋白是参与钙刺激的胞吐作用的佛波酯结合脚手架蛋白。 RASGRP是RAS和RAP1的二酰基甘油激活的鸟嘌呤核苷酸交换因子（GEFS）。 α-和β-Chimaerins是佛波尔酯和二酰基甘油反应性GTPase激活蛋白的家族。该项目对二酰基甘油结合的C1结构域蛋白质的家庭采用了整体方法，因此包括PKCS和Chimaerins。与Marcelo Kazanietz（宾夕法尼亚大学）合作，我们描述了Beta2-Chimaerin的结构。我们使用这些信息来阐明激活机制，并设计了一种超敏感的突变体，该突变体对DAG激活具有超敏感性。然后，使用这种涡轮 - 奇马素突变体来阐明β2-奇马素在Kazanietz实验室中的生理调节作用。 C1结构域的结构和功能研究已经建立了二酰基甘油和佛波酯诱导的易位向膜的机制的轮廓。与膜易位，PKC，嵌合蛋白和其他C1结构域蛋白伴随着变构激活。根据对有限的蛋白水解，易位动力学，荧光，表面压力分析和其他间接结构技术的敏感性的变化，PKC激活机制被认为需要发生巨大的构象变化。