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Regulation of Normal and Asthmatic Lung Function by G-Protein-Coupled Receptors

G 蛋白偶联受体对正常和哮喘肺功能的调节

基本信息

批准号：
10927794
负责人：
Kirk m Druey
金额：
$ 63.4万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10927794
关键词：
Actins Adenosine Adrenal Cortex Hormones Adrenergic beta-Agonists Affect Affinity Agonist Albuterol Allergens Antifungal Agents Area Aspergillus fumigatus Asthma Autopsy Binding Bradykinin Bronchoconstriction Bronchodilator Agents Calcium Cells Cessation of life Collaborations Coupled Cyclic AMP Disease susceptibility Drug Targeting Endothelin-1 Extracellular Matrix Extrinsic asthma G Protein-Coupled Receptor Signaling G alpha q Protein G-Protein-Coupled Receptors GTP-Binding Protein Regulators GTP-Binding Protein alpha Subunits GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Gene Deletion Goals Grant Guanosine Triphosphate Phosphohydrolases Hepatitis C Hepatitis C Therapy Heterotrimeric GTP-Binding Proteins Histamine Human Hypersensitivity IgE In Vitro Infection Inflammation Mediators Inflammatory Response Inhibitory G-Protein Gi Lead Leukotriene Antagonists Leukotriene D4 Life Ligands Link Lung Mediating Modeling Molds Monoclonal Antibodies Mus Muscle Contraction Muscle Fibers Muscle function Muscle relaxation phase Myosin ATPase Natural Substance PAR-2 Receptor Pathologic Peptide Hydrolases Persons Physiological Proliferating Pulmonary Inflammation RGS Domain RGS Proteins Regulation Relaxation Role Serine Protease Signal Induction Signal Pathway Signal Transduction Slice Smooth Muscle Smooth Muscle Myocytes Specimen Therapeutic Therapeutic Uses Thrombin Tissue Banks Tissues aggressive therapy airway hyperresponsiveness airway obstruction allergic airway inflammation asthmatic asthmatic airway cysteinyl-leukotriene efficacy study eosinophil eosinophilic inflammation experience fungus granule cell inhibitor insight interest mast cell mouse model novel novel therapeutics omalizumab pulmonary function receptor respiratory respiratory smooth muscle success

项目摘要

Asthma, a pathological condition of reversible airway obstruction, is comprised of both inflammation of the lung and hyper-contractility of the bronchial smooth muscle. The major naturally occurring substances that induce bronchial smooth muscle contraction are ligands of G-protein-coupled receptors (GPCRs), such as allergen proteases, thrombin, and those contained in allergen-IgE activated mast cell granules (e.g. histamine, cysteinyl leukotrienes (LTD4), endothelin 1, adenosine, and bradykinin). In general, these agonists induce activation of the heterotrimeric G protein G-alpha q, which increases the concentration of intracellular calcium in smooth muscle cells, promoting actin-myosin interactions and muscle fiber shortening. In contrast, ligands acting on G-alpha-s-coupled receptors, such as albuterol, increase intracellular levels of cyclic AMP (cAMP), facilitating ASM relaxation. Although eosinophilic inflammation typifies allergic asthma, it is not a prerequisite for airway hyper-responsiveness (AHR), suggesting that underlying abnormalities in structural cells including ASM contribute to the asthmatic diathesis. Dysregulation of procontractile, GPCR signaling in ASM could mediate enhanced contractility. 10-15% of people with asthma experience severe, life threatening attacks and even death despite aggressive treatment with bronchodilators and corticosteroids. Nearly half of these (10-20 million) are sensitized (i.e. have IgE-mediated allergy) to filamentous fungi (e.g. Aspergillus fumigatus, Af), which has been designated "severe asthma with fungal sensitization" (SAFS). Current therapies for SAFS including antifungals or omalizumab monoclonal antibody (mAb) targeting IgE have not achieved uniform success. Utilizing a model of allergic airway inflammation in mice induced by respiratory Af exposure, we have 2 overarching aims for this project: 1) identify derangements in ASM contraction signaling downstream of inflammatory mediators; 2) examine the functions of allergen protease activity in AHR, particularly in relation to allergen-ASM interactions. Protease activity is a common and important feature of allergens capable of inducing asthma, most notably from ubiquitous fungi such as Af. Whether any allergens affect ASM contraction directly has never been explored. Previously we found a causal link between fungi and asthma occurring independently of allergenicity; in other words, host inflammatory response to the allergen. The secreted Af protease Alkaline protease 1 (Alp1) was detected in the airways of asthmatic subjects but not controls. In mouse and in vitro studies, we found that Alp1 directly promoted AHR by degrading extracellular matrix (ECM) components, leading to dysregulation of ASM contraction. Subsequently, we demonstrated that Alp1 induces AHR in mice devoid of eosinophils or the protease activated receptor 2 (PAR2) and that Alp1 directly increased contractile force of human ASM cells in vitro. In Fiscal Year (FY) 23, identified an inhibitor of Alp1 protease activity in a compound screen done in collaboration with Dr. Krishnan through an R21 grant. A critical therapeutic gap is to identify moieties with specific affinity for fungal Alp1 but without activity on endogenous serine proteases present in the human airway. The lead compound, which is used therapeutically for hepatitis C (HCV) infections, inhibits the effect of Alp on ASM contraction in vitro and in human airways of murine and human precision-cut lung slices (PCLS) treated with Alp1. We are currently studying the efficacy of the compound in mouse models of fungal asthma. We expect these studies to identify a novel therapeutic application for an HCV inhibitor for fungal asthma and provide novel insight into both ASM-intrinsic and allergen-protease-dependent mechanisms of bronchoconstriction. The second area of emphasis for this project is the study of Regulators of G protein signaling (RGS) proteins in the lung. RGSs bind to the G protein alpha subunits Gi and Gq (but not Gs) through a conserved RGS domain and inactivates them by catalyzing their intrinsic GTPase activity and by blocking downstream effector interactions. Although they are generally considered to act as negative regulators of GPCR signaling pathways, the physiological function of RGS proteins in the lung is mostly unknown. We identified expression of several RGS proteins (RGS4, RGS5) in bronchial smooth muscle of humans and mice.

哮喘是一种可逆性气道阻塞的病理状况，由肺部炎症和支气管平滑肌过度收缩组成。诱导支气管平滑肌收缩的主要天然物质是 G 蛋白偶联受体 (GPCR) 的配体，例如过敏原蛋白酶、凝血酶以及过敏原 IgE 激活的肥大细胞颗粒中所含的物质（例如组胺、半胱氨酰白三烯 (LTD4)、内皮素 1、腺苷和缓激肽）。一般来说，这些激动剂会诱导异源三聚体 G 蛋白 G-α q 的激活，从而增加平滑肌细胞内钙的浓度，促进肌动蛋白-肌球蛋白相互作用和肌纤维缩短。相反，作用于 G-α-s 偶联受体的配体（例如沙丁胺醇）会增加细胞内环 AMP (cAMP) 的水平，从而促进 ASM 松弛。尽管嗜酸性粒细胞炎症是过敏性哮喘的典型表现，但它并不是气道高反应性 (AHR) 的先决条件，这表明包括 ASM 在内的结构细胞的潜在异常会导致哮喘素质。 ASM 中的前收缩 GPCR 信号传导失调可能会介导收缩性增强。尽管使用支气管扩张剂和皮质类固醇进行积极治疗，但 10-15% 的哮喘患者仍会出现严重的、危及生命的发作，甚至死亡。其中近一半（10-2000 万）对丝状真菌（例如烟曲霉，Af）过敏（即 IgE 介导的过敏），这被称为“真菌致敏性严重哮喘”(SAFS)。目前的 SAFS 疗法，包括抗真菌药物或针对 IgE 的奥马珠单抗单克隆抗体 (mAb)，尚未取得一致的成功。利用呼吸道AF暴露引起的小鼠过敏性气道炎症模型，我们这个项目有两个总体目标：1）识别炎症介质下游ASM收缩信号传导的紊乱； 2）检查AHR中过敏原蛋白酶活性的功能，特别是与过敏原-ASM相互作用相关的功能。蛋白酶活性是能够诱发哮喘的过敏原的一个常见且重要的特征，尤其是来自无处不在的真菌，例如 Af。是否有任何过敏原直接影响 ASM 收缩尚未被探索过。此前我们发现真菌与哮喘之间存在独立于过敏性的因果关系。换句话说，宿主对过敏原的炎症反应。在哮喘受试者的气道中检测到分泌的 Af 蛋白酶碱性蛋白酶 1 (Alp1)，但在对照组中未检测到。在小鼠和体外研究中，我们发现Alp1通过降解细胞外基质（ECM）成分直接促进AHR，导致ASM收缩失调。随后，我们证明 Alp1 在缺乏嗜酸性粒细胞或蛋白酶激活受体 2 (PAR2) 的小鼠中诱导 AHR，并且 Alp1 在体外直接增加人 ASM 细胞的收缩力。在第 23 财年 (FY)，通过 R21 拨款与 Krishnan 博士合作进行的化合物筛选中鉴定出了 Alp1 蛋白酶活性的抑制剂。一个关键的治疗空白是鉴定对真菌 Alp1 具有特异性亲和力但对人类气道中存在的内源丝氨酸蛋白酶没有活性的部分。该先导化合物用于治疗丙型肝炎 (HCV) 感染，在体外和经 Alp1 处理的小鼠和人类精密切割肺切片 (PCLS) 的人体气道中，可抑制 Alp 对 ASM 收缩的影响。我们目前正在研究该化合物在真菌性哮喘小鼠模型中的功效。我们期望这些研究能够确定 HCV 抑制剂治疗真菌性哮喘的新治疗应用，并为支气管收缩的 ASM 内在机制和过敏原蛋白酶依赖性机制提供新的见解。该项目的第二个重点领域是肺部 G 蛋白信号传导 (RGS) 蛋白调节因子的研究。 RGS 通过保守的 RGS 结构域与 G 蛋白 α 亚基 Gi 和 Gq（但不是 Gs）结合，并通过催化其内在 GTP 酶活性和阻断下游效应子相互作用来灭活它们。尽管它们通常被认为是 GPCR 信号通路的负调节因子，但 RGS 蛋白在肺部的生理功能大多未知。我们鉴定了人和小鼠支气管平滑肌中几种 RGS 蛋白（RGS4、RGS5）的表达。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Regulation of G-protein-coupled signaling pathways in allergic inflammation.

DOI：
10.1007/s12026-008-8050-0
发表时间：
2009
期刊：
IMMUNOLOGIC RESEARCH
影响因子：
4.4
作者：
Druey, Kirk M.
通讯作者：
Druey, Kirk M.

Alternaria alternata Accelerates Loss of Alveolar Macrophages and Promotes Lethal Influenza A Infection.

DOI：
10.3390/v12090946
发表时间：
2020-08-27
期刊：
Viruses
影响因子：
0
作者：
Percopo CM;Ma M;Mai E;Redes JL;Kraemer LS;Minai M;Moore IN;Druey KM;Rosenberg HF
通讯作者：
Rosenberg HF

Persistent Airway Hyperresponsiveness Following Recovery from Infection with Pneumonia Virus of Mice.

DOI：
10.3390/v13050728
发表时间：
2021-04-22
期刊：
Viruses
影响因子：
0
作者：
Limkar AR;Percopo CM;Redes JL;Druey KM;Rosenberg HF
通讯作者：
Rosenberg HF

Alternaria alternata challenge at the nasal mucosa results in eosinophilic inflammation and increased susceptibility to influenza virus infection.

DOI：
10.1111/cea.13123
发表时间：
2018-06
期刊：
Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology
影响因子：
0
作者：
Ma M;Redes JL;Percopo CM;Druey KM;Rosenberg HF
通讯作者：
Rosenberg HF

Differential Expression of Mitosis and Cell Cycle Regulatory Genes during Recovery from an Acute Respiratory Virus Infection.