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

描述(申请人提供)：cGMP依赖的蛋白激酶(PKG)是NO-cGMP信号通路的中心酶，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一直被大量用于治疗勃起功能障碍和心血管和肺部疾病等疾病，但由于缺乏结构信息，开发特定的激活剂和抑制剂一直很困难[1，2]。为了成功地进行NO-cGMP介导的信号反应，PKG必须定位于细胞中的特定位置。PKG的定位是NO-cGMP信号转导的一个重要特征，由G-激酶锚定蛋白(GKAPs)介导[3，4，5，6，7，8]。特别是，N末端的可变亮氨酸拉链结构域通过与G-激酶锚定蛋白以同型特异性的方式相互作用，将PKG靶向特定的亚细胞位置[7，9]。尽管越来越多的证据表明，GKAP通过与拉链结构域的结合来靶向PKG，但这一重要的蛋白质-蛋白质相互作用的分子细节仍不清楚。这项建议的具体目的是利用多肽阵列和X射线结晶学相结合的方法来了解PKG的同型特异性靶向机制。增加另一层复杂性，有三种类型的PKG(I1，I2和II)，每种类型在激活、独特的底物集合和组织特异性表达方面都有不同的cGMP依赖[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。虽然它们是具有独特生理作用的功能非冗余蛋白，但人们对它们的功能作用和特定的亚细胞定位知之甚少。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，并比较每种异构体特有的GKAP对接面的分子特征。同时，我计划设计能够选择性地破坏PKG I的两个同工酶和它们各自的结合伙伴之间的相互作用的多肽。最后，我将把高亲和力的多肽加入到共结晶实验中，以了解PKG/GKAP相互作用的分子细节。我的计划是利用同工酶特异性多肽形成稳定的蛋白质/肽复合体，并追求PKG/GAKP复合体的高分辨率晶体结构。拉链结构域结构的解决，以及特异性干扰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，并最终导致治疗剂的开发。 公共卫生相关性：cGMP依赖的蛋白激酶(PKG)是NO-cGMP信号通路的中心酶，调节血小板聚集、平滑肌张力、光转导和白细胞迁移。尽管PKG一直被大量用于治疗勃起功能障碍、心血管和肺部疾病等疾病，但由于缺乏结构信息，开发特定的激活剂和抑制剂一直是困难的。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地靶向PKG，并开发能够调节PKG活性的药物来治疗与NO-cGMP信号功能障碍相关的疾病。

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.