Targeting compartmentalized phosphodiesterases to regulate CFTR function

靶向区室化磷酸二酯酶调节 CFTR 功能

• 批准号: 8243487
• 负责人: Wito H Richter
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8243487
• 关键词: Adenylate Cyclase; Advanced Development; Adverse effects; Anions; Biological Models; Biosensor; Cell Line; Cell membrane; Cells; Chimera organism; Chronic Obstructive Airway Disease; Clinical; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Disease; Drug Delivery Systems; Electrolytes; Enzymes; Epithelial Cells; Epithelium; Event; Failure; Family; Feedback; Fluorescence Resonance Energy Transfer; Gastrointestinal tract structure; Genes; Health; Homeostasis; Human; Human Genome; Individual; Ion Transport; Ions; Liquid substance; Lung; Measurement; Measures; Modeling; Mutation; Pharmaceutical Preparations; Phosphodiesterase Inhibitors; Phosphorylation; Physiological; Protein Isoforms; Proteins; Regulation; Regulator Genes; Research Proposals; Role; Shapes; Signal Transduction; Signaling Protein; Smoke; Testing; Therapeutic; Time; Tissues; Variant; Water; apical membrane; base; clinical application; cystic fibrosis patients; inhibitor/antagonist; insight; meetings; member; mutant; novel; novel strategies; phosphodiesterase IV; phosphoric diester hydrolase; public health relevance; research study; response; success

DESCRIPTION (provided by applicant): With this proposal, we will test the overall hypothesis that regulation of cAMP levels in microdomains of signaling, rather than on the global scale, determines the function of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in health and disease. The CFTR is a cAMP-stimulated anion channel that is critical for ion- and water homeostasis across many epithelia including in the airways and the gastrointestinal tract. CFTR hypofunction, as a result of mutations in the CFTR gene in Cystic Fibrosis (CF), or smoke exposure in Chronic Obstructive Pulmonary Disease (COPD), results in reduced levels of this channel in the apical membrane of epithelia which causes abnormal fluid and electrolyte transport. It is well established that CFTR function is stimulated by PKA phosphorylation. However, stimulating CFTR-dependent ion transport in CF model systems by increasing global cellular cAMP to supra-physiological levels with a combination of adenylyl cyclase activators and broad-spectrum PDE inhibitors has been met with mixed results and successes in the past. With this research proposal, we wish to test two novel approaches to overcome this limitation. Specific Aim 1 of this proposal will test the hypothesis that specific isoforms of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade and inactivate cAMP, are physically tethered to signaling complexes involving the CFTR and control the activity of this channel in compartmentalized microdomains of cAMP signaling. We wish to test the idea that inactivation of this localized pool of PDE activity or its displacement from CFTR signaling complexes provides a safer and more effective approach to stimulate CFTR function compared to increasing global cAMP levels, as the latter approach induces significant cellular feedback responses and undesirable side effects that ultimately limit its efficacy. The human genome encodes for 21 PDE genes, which are likely expressed as more than 100 protein variants. Inhibitors of type 4 PDEs (PDE4s) have been shown to stimulate CFTR in immortalized epithelial cell lines. We have now generated preliminary data showing that PDE4 isoforms are the major regulators of CFTR activity in primary lung epithelial cells and are physically tethered to this channel suggesting that these are the PDEs that control cAMP in CFTR microdomains. In Specific Aim 1 of this proposal, we will characterize the interaction of CFTR with individual PDE4 isoforms and test the effect of selective inactivation of these PDE4s on CFTR function in non-CF and CF cells and tissues. In Specific Aim 2 of this proposal, we will generate novel FRET-based biosensors that can measure cAMP levels and PKA activity in the immediate vicinity of the CFTR. This will allow us to trace the pools of cAMP that determine CFTR function, identify the key regulators in this compartment, and explore possible differences in cAMP signaling events between CF and non-CF epithelia. PUBLIC HEALTH RELEVANCE: Abnormal expression and function of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is central to several diseases with high public health relevance including cystic fibrosis (CF). The identification of specific cyclic nucleotide phosphodiesterase 4 (PDE4) isoforms as regulators of CFTR activity will open new opportunities to target PDE4s as a therapeutic approach in CF. This proposal aims to validate the idea that targeting microdomains of cAMP signaling, rather than global cAMP levels, is an effective therapeutic approach, which will advance the development of more selective and, thus, safer drugs.

描述（由申请人提供）：通过该提议，我们将测试总体假设，即在信号传导的微域中而不是在全球范围内调节cAMP水平决定了囊性纤维化跨膜传导调节因子（CFTR）在健康和疾病中的功能。CFTR是cAMP刺激的阴离子通道，其对于许多上皮细胞（包括气道和胃肠道中的上皮细胞）的离子和水稳态至关重要。由于囊性纤维化（CF）中CFTR基因的突变或慢性阻塞性肺病（COPD）中的烟雾暴露，CFTR功能减退导致上皮细胞顶膜中该通道的水平降低，这导致异常的流体和电解质转运。已经确定CFTR功能受PKA磷酸化刺激。然而，在CF模型系统中通过用腺苷酸环化酶激活剂和广谱PDE抑制剂的组合将全局细胞cAMP增加到超生理水平来刺激CFTR依赖性离子转运，在过去已经遇到了混合的结果和成功。通过这项研究计划，我们希望测试两种新的方法来克服这一限制。本提案的具体目标1将检验以下假设：环核苷酸磷酸二酯酶（PDE）的特定亚型（降解和抑制cAMP的酶）与涉及CFTR的信号传导复合物物理连接，并控制cAMP信号传导区室化微域中该通道的活性。我们希望测试这样的想法，即与增加整体cAMP水平相比，PDE活性的这种局部池的失活或其从CFTR信号传导复合物的置换提供了刺激CFTR功能的更安全和更有效的方法，因为后一种方法诱导显著的细胞反馈反应和最终限制其功效的不期望的副作用。人类基因组编码21个PDE基因，这些基因可能表达为100多种蛋白质变体。已显示4型PDE（PDE 4）的抑制剂刺激永生化上皮细胞系中的CFTR。我们现在已经产生了初步的数据，显示PDE4亚型是原代肺上皮细胞中CFTR活性的主要调节剂，并且物理地束缚于该通道，这表明这些是控制CFTR微结构域中cAMP的PDE。在本提案的具体目标1中，我们将表征CFTR与单个PDE 4亚型的相互作用，并检测这些PDE 4的选择性失活对非CF和CF细胞和组织中CFTR功能的影响。在该提案的具体目标2中，我们将产生新的基于FRET的生物传感器，可以测量CFTR附近的cAMP水平和PKA活性。这将使我们能够追踪决定CFTR功能的cAMP池，识别该隔室中的关键调节因子，并探索CF和非CF上皮之间cAMP信号传导事件的可能差异。 公共卫生相关性：囊性纤维化跨膜传导调节因子（CFTR）的异常表达和功能是包括囊性纤维化（CF）在内的几种具有高度公共卫生相关性的疾病的核心。特异性环核苷酸磷酸二酯酶4（PDE4）亚型作为CFTR活性调节剂的鉴定将为靶向PDE4作为CF治疗方法开辟新的机会。该提案旨在验证靶向cAMP信号传导的微域而不是整体cAMP水平是一种有效的治疗方法的想法，这将促进更具选择性，因此更安全的药物的开发。