Targeting mechanisms of cGMP dependent protein kinases by peptide arrays and crys

肽阵列和crys的cGMP依赖性蛋白激酶靶向机制

• 批准号: 8102997
• 负责人: Choel Woong Kim
• 金额: $ 30.21万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8102997
• 关键词: Affinity; Affinity Chromatography; Binding; Blood Platelets; Cardiovascular Diseases; Cell physiology; Cells; Collaborations; Complex; Crystallization; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dependence; Development; Dimerization; Disease; Docking; Drops; Engineering; Environment; Enzymes; Erectile dysfunction; Face; Functional disorder; Goals; Homodimerization; Individual; Inositol; Isoenzymes; Laboratories; Lead; Learning; Leucine Zippers; Leukocytes; Lung diseases; MRVI1 gene; Mediating; Molecular; Molecular Target; Muscle Tonus; Muscle relaxation phase; N-terminal; Peptides; Phosphotransferases; Phototransduction; Physiological; Platelet aggregation; Protein Isoforms; Proteins; RNA Splicing; Resolution; Robotics; Role; Sarcoplasmic Reticulum; Signal Pathway; Signal Transduction; Site; Smooth Muscle; Smooth Muscle Myocytes; Solutions; Specificity; Structure; Surface; System; Testing; Therapeutic Agents; Time; Tissues; Variant; X-Ray Crystallography; cGMP-dependent protein kinase Ibeta; design; inhibitor/antagonist; migration; milligram; molecular domain; nanolitre; novel; protein complex; protein protein interaction; public health relevance; receptor; response

DESCRIPTION (provided by applicant): cGMP-dependent protein kinase (PKG) is the central enzyme of NO-cGMP signaling pathway that regulates platelet aggregation, smooth muscle tone, phototransduction, and leukocyte migration [1,2]. Although PKG has been heavily targeted for treating diseases such as, erectile dysfunction and cardiovascular and pulmonary diseases, developing specific activators and inhibitors has been difficult because there is no structural information available[1,2]. For the successful NO-cGMP mediated signaling responses to occur, PKG has to be localized to the specific site in the cell. Localization of PKG is an essential feature of the NO-cGMP signaling and mediated by G Kinase Anchoring Proteins (GKAPs)[3,4,5,6,7,8]. In particular, the variable leucine zipper domain at the extreme N-terminus targets PKG to specific subcellular sites via its interaction with G-kinase anchoring proteins in an isotype specific manner [7,9]. Despite mounting evidence that GKAP target PKGs via its association with the zipper domain, the molecular details of this important protein- protein interaction remain unknown. The specific aims of this proposal are to understand the isotype specific targeting mechanism of PKGs using peptide arrays in combination with X-ray crystallography. Adding another layer of complexity, there are three types of PKG (I1, I2, and II) that each have different cGMP dependence in activation, unique sets of substrates, and tissue specific expression [2,10,11]. My primary focus is the type I isozymes (1 and 2) that have been implicated in erectile dysfunction and many cardiovascular diseases. Although they represent functionally non- redundant proteins with unique physiological roles, their functional roles and specific subcellular localization are poorly understood. In order to elucidate isozyme specific functions and localization, I plan to solve crystal structures of the PKG I1 and 2 zipper domains and compare molecular features of GKAP docking surfaces that are unique to each isoform. In parallel, I plan to engineer peptides that can selectively disrupt interactions between both isozymes of PKG I and their individual binding partners. Lastly, I will incorporate high affinity peptides into the co-crystallization trials in order to understand molecular details of the PKG/GKAP interaction. My plan is to form stable protein/peptide complexes using isozyme specific peptides and pursue high-resolution crystal structures of PKG/GAKP complexes. Solution of the zipper domain structures, and development of small peptides that specifically disrupt isozyme specific targeting, will pave the way to elucidation of the specific functions of PKGs and eventually lead to the development of therapeutic agents. PUBLIC HEALTH RELEVANCE: cGMP-dependent protein kinase (PKG) is the central enzyme of NO-cGMP signaling pathway that regulates platelet aggregation, smooth muscle tone, phototransduction, and leukocyte migration. Although PKG has been heavily targeted for treating diseases such as, erectile dysfunction and cardiovascular and pulmonary diseases, developing specific activators and inhibitors has been difficult because there is no structural information available. My ultimate goal is to rationally target the kinase by obtaining high-resolution crystal structures of PKG and its isozymes and develop pharmacological agents that can modulate the activity of the kinase to treat diseases related to NO-cGMP signaling dysfunction.

描述（申请人提供）：cGMP依赖性蛋白激酶（PKG）是NO-cGMP信号通路的核心酶，调节血小板聚集、平滑肌张力、光转导和白细胞迁移[1,2]。尽管 PKG 已主要用于治疗勃起功能障碍以及心血管和肺部疾病等疾病，但由于没有可用的结构信息，开发特定的激活剂和抑制剂一直很困难[1,2]。 为了成功发生 NO-cGMP 介导的信号反应，PKG 必须定位于细胞中的特定位点。 PKG 的定位是 NO-cGMP 信号传导的一个基本特征，由 G 激酶锚定蛋白 (GKAP) 介导[3,4,5,6,7,8]。特别是，最末端 N 末端的可变亮氨酸拉链结构域通过其与 G 激酶锚定蛋白的相互作用以同种型特异性方式将 PKG 靶向特定的亚细胞位点 [7,9]。尽管越来越多的证据表明 GKAP 通过与拉链结构域的关联来靶向 PKG，但这种重要的蛋白质-蛋白质相互作用的分子细节仍然未知。该提案的具体目的是使用肽阵列结合 X 射线晶体学来了解 PKG 的同种型特异性靶向机制。 PKG 分为三种类型（I1、I2 和 II），这又增加了一层复杂性，每种类型在激活方面都具有不同的 cGMP 依赖性、独特的底物组和组织特异性表达 [2,10,11]。我的主要关注点是 I 型同工酶（1 和 2），它们与勃起功能障碍和许多心血管疾病有关。尽管它们代表了具有独特生理作用的功能上非冗余蛋白质，但它们的功能作用和特定的亚细胞定位却知之甚少。为了阐明同工酶的特定功能和定位，我计划解析 PKG I1 和 2 拉链结构域的晶体结构，并比较每个同工型独特的 GKAP 对接表面的分子特征。与此同时，我计划设计能够选择性破坏 PKG I 两种同工酶及其各自结合配偶体之间相互作用的肽。最后，我将把高亲和力肽纳入共结晶试验中，以了解 PKG/GKAP 相互作用的分子细节。我的计划是使用同工酶特异性肽形成稳定的蛋白质/肽复合物，并追求 PKG/GAKP 复合物的高分辨率晶体结构。拉链结构域结构的解决方案以及特异性破坏同工酶特异性靶向的小肽的开发将为阐明 PKG 的特定功能铺平道路，并最终导致治疗药物的开发。 公共健康相关性：cGMP 依赖性蛋白激酶 (PKG) 是 NO-cGMP 信号通路的核心酶，可调节血小板聚集、平滑肌张力、光转导和白细胞迁移。尽管 PKG 已被广泛用于治疗勃起功能障碍以及心血管和肺部疾病等疾病，但由于没有可用的结构信息，开发特定的激活剂和抑制剂一直很困难。我的最终目标是通过获得 PKG 及其同工酶的高分辨率晶体结构来合理靶向激酶，并开发可以调节激酶活性的药物制剂，以治疗与 NO-cGMP 信号传导功能障碍相关的疾病。