Novel Isoform-specific Targeting of PKA

PKA 的新型异构体特异性靶向

• 批准号: 8640917
• 负责人: SUSAN S. TAYLOR
• 金额: $ 68.27万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 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/8640917
• 关键词: A kinase anchoring protein; Animal Disease Models; Architecture; Automobile Driving; Binding; Biogenesis; Biological; Cell Respiration; Cell membrane; Cells; Collaborations; Complex; Crista ampullaris; Crystallography; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diet; Disease; Electron Microscopy; Engineering; Fatty acid glycerol esters; Fluorescence Microscopy; Goals; Holoenzymes; Image; Insulin Resistance; Intervention; Knowledge; Length; Life; Liver; Maps; Mass Spectrum Analysis; Membrane; Metabolism; Mitochondria; Modeling; Molecular; Morphology; Motivation; Mus; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organelles; Peptides; Phosphorylation; Play; Protein Isoforms; Proteins; Recruitment Activity; Reporter; Resolution; Role; Signal Transduction; Singlet Oxygen; Site; Specificity; Sphingosine; Structure; Symptoms; Technology; Tissues; cellular imaging; feeding; molecular dynamics; novel; programs; protein expression; response; scaffold; sphingosine kinase; structural biology; tomography

While our past efforts have focused on defining the domains and motifs that constitute the building blocks for AKAP targeting of PKA, our focus now has shifted to understanding how large macromolecular signaling scaffolds are assembled. Our multi-scale approaches are allowing us not only to achieve a mechanistic understanding of such scaffolds at atomic level resolution but also to understand the cellular architecture of PKA scaffolds that are assembled at the mitochondria and how these scaffolds change in response to cAMP and as a function of diet and disease. We are focusing, in particular, on the uniqueness of the PKA isoforms as revealed by newly solved structures of full-length holoenzymes and on a newly discovered and highly conserved mitochondrial PKA substrate, ChChdS, that is localized to the intermembrane space. ChchdS nucleates a large scaffold that bridges the inner and outer membranes and plays a major role in crista biogenesis [1]. Also recruited to this complex is a novel Rl-specific AKAP, Sphingosine Kinase Interacting Protein (SKIP), recently discovered by Scott and Taylor [2, 3]. Depletion of ChchdS leads to loss of crista and loss of oxidative metabolism while ChchdS is enriched in livers of mice fed on a high fat diet (HFD), which is associated with insulin resistance and obesity, hallmark symptoms of Type II diabetes. We will use the expertise of the Analytical Core (Core A) to define the molecular features of these complexes while the Multi-Scale Ceil Imaging Core (Core B) will be used to define the architecture of the mitochondria and how it changes dynamically in response to cAMP and to diet and depletion of ChchdS. In parallel, we are using miniSOG (small Singlet Oxygen Generator) techno-logy, recently discovered by Tsien and Ellisman [4] to finely map localized sites of PKA signaling. MinSOG, a genetically encoded tag that generates singlet oxygen that can then be used for EM contrast, has the potential to do for EM what GFP did for fluorescence microscopy. Moving from atomic level resolution to defining the dynamic molecular architecture of organelles is the driving motivation for Protect I as we try to understand how specificity in PKA signaling is achieved through dynamic and polyvalent signaling scaffolds. Our specific goals are as follows. In Aim I we will crystallize an Rl-specific AKAP bound to full-length Rip, an isoform that is enriched in mitochondria. In Aim II we define the molecular and dynamic features of ChChdS using peptide arrays, structural biology, and mass spectrometry, in Aim III we use miniSOG reporters to define PKA targeting sites and to correlate PKA signaling with mitochondria dynamics. In Aim IV we will collaborate with Olefsky to elucidate tissue-specific changes in PKA signaling and mitochondria morphology in response to a HFD. RELEVANCE: Although PKA plays a major role in regulating metabolism and mitochondrial dynamics, we lack knowledge of how macromolecular signaling scaffolds target specific PKA isoforms to mitochondria and how they are regulated. The multi scale strategy outlined here will define structural and cellular PKA scaffolds at the mitochondria and elucidate how these scaffolds and mitochondrial dynamics change in response to diet induced insulin resistance and obesity, hallmarks of type II diabetes. Novel targets for intervention will likely emerge as well as enhanced molecular understanding of biological signaling by PKA.

尽管我们过去的努力一直集中在定义域和图案上 构成AKAP靶向PKA的构件，我们的重点已转移到了解如何 组装大的大分子信号传导支架。我们的多尺度方法使我们不允许 仅是为了在原子水平分辨率下对此类支架的机械理解，但也 了解在线粒体上组装的PKA脚手架的细胞结构以及如何 脚手架响应营地和饮食和疾病的作用。我们特别关注的是 关于PKA同工型的独特性，如全长全酶的新求解结构所揭示的 在新发现且高度保守的线粒体PKA底物上，该基材本地化为 膜间空间。 CHCHD对大型脚手架有核能，该支架桥接内膜和外膜 并在Crista生物发生中起主要作用[1]。也招募到这个综合体是一种新颖的RL特异性AKAP， Scott和Taylor [2，3]最近发现的鞘氨醇激酶相互作用蛋白（SKIP）。耗尽 CHCHD会导致Crista的丧失和氧化代谢的丧失，而CHCHS则富含小鼠的肝脏富含 在高脂肪饮食（HFD）上，与胰岛素抵抗和肥胖有关，类型的标志症状 II糖尿病。我们将使用分析核心（核心A）的专业知识来定义 这些复合物虽然多尺度的CEIL成像核心（核心B）将用于定义 线粒体及其如何动态变化，以响应营地，饮食和CHCHD的饮食和耗竭。 同时，我们使用的是Minisog（小单重氧生成器）技术，最近发现 Tsien和Ellisman [4]可以精细地绘制PKA信号的局部位点。 Minsog，一种基因编码的标签 生成可用于EM对比的单线氧气，有可能对EM做什么 GFP用于荧光显微镜。从原子水平分辨率转移到定义动态分子 在我们试图了解PKA的特殊性时，细胞器的结构是保护我的驱动动力 信号是通过动态和多价信号支架实现的。我们的具体目标如下。 在目的中，我将结晶一个与全长RIP结合的RL特异性AKAP，这是一种富含的同工型 线粒体。在AIM II中，我们使用肽阵列定义CHCHD的分子和动态特征， 结构生物学和质谱法，在AIM III中，我们使用Minisog记者定义PKA靶向 位点并将PKA信号与线粒体动力学相关联。在AIM IV中，我们将与Olefsky合作 为了阐明响应HFD的PKA信号传导和线粒体形态的组织特异性变化。 相关性：尽管PKA在调节新陈代谢和线粒体动力学中起着重要作用，但我们 缺乏了解大分子信号传导支架如何将特定的PKA同工型靶向线粒体和 它们的监管方式。此处概述的多尺度策略将定义结构和细胞PKA 线粒体的脚手架，并阐明了这些脚手架和线粒体动力学的变化 对饮食诱导的胰岛素抵抗和肥胖症的反应，这是II型糖尿病的标志。新颖的目标 干预可能会出现，并增强PKA对生物信号传导的分子理解。