Tweety proteins: their roles in pericytes and macrophages

Tweety 蛋白：它们在周细胞和巨噬细胞中的作用

• 批准号: 10665494
• 负责人: William A Coetzee
• 金额: $ 16.95万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10665494
• 关键词: Action Potentials; Adult; Animal Model; Anions; Area; Biochemistry; Blood; Blood Vessels; Blood capillaries; Blood flow; Bone Marrow; Brain; Cell secretion; Cells; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Cryoelectron Microscopy; DNA; Data; Dimethylxanthenone Acetic Acid; Drug Targeting; Dyes; Electrophysiology (science); Endocrine; FDA approved; Family; Gene Expression Profile; Hormone secretion; Human; Hypoxia; IRF3 gene; Immune; Immune System Diseases; Immunology; Inflammation; Inflammatory; Innate Immune System; Interferon Type II; Interferon-beta; Interferons; Interleukin-1 beta; Interleukin-6; Ion Channel; Joints; Lung; Macrophage; Measures; Mediating; Membrane Potentials; Molecular Biology; Mus; Muscle; Muscle Cells; Natural Immunity; Neurons; Pathway interactions; Pericytes; Phosphorylation; Physiological; Pilot Projects; Positioning Attribute; Potassium Channel; Production; Proteins; Publications; Pump; RNA; Role; STING agonists; Sentinel; Signal Pathway; Small Interfering RNA; Stimulator of Interferon Genes; Surface; Swelling; TNF gene; Testing; Therapeutic; Transcript; Viral; cell type; chemokine; cytokine; design; environmental change; experimental study; first responder; follow-up; knock-down; member; mouse model; patch clamp; pathogen; public database; response; transcriptome sequencing; uptake

Ion channels, transporters, exchangers, and pumps in neurons or muscle myocytes regulate the formation of action potentials and contractile activity. In endocrine cells, they regulate hormone secretion. Ion channels have key roles even in non-excitable cells, for example, by setting the membrane potential and regulating the influx of Ca2+ into cells. There are hundreds of channels, and most are relatively well characterized. A number, however, remains understudied, which is the focus of RFA-RM-22-024. One focus area of our lab is to investigate the roles of pericytes, specialized cells on the abluminal surface of capillary blood vessels. Pericytes have multiple functions, including forming new blood vessels and regulating blood flow. Pericytes are also sentinels of the innate immune system and directly interact with several types of immune cells by secreting chemokines and cytokines, including IFN-γ, TNF-α, IL-1β, and IL-6. We have developed a mouse model in which mCherry is explicitly expressed in pericytes, which allows isolation of pericytes and enrichment to high purity. We have performed global RNA-seq and focused on the transcriptional profiles of >650 ion channels, exchangers, and pumps expressed in brain pericytes. Transcripts of several Cl- and K+ channels were present. Of note, members of the tweety family (TTYH1, TTYH2, and TTYH3) were amongst the top expressing channels in brain pericytes. This finding was corroborated by public databases, showing that Tthy2 is specifically expressed in pericytes of adult mouse lung vascular and perivascular cells. Tthy2 was initially characterized as swelling-dependent volume-regulated anion channels, but later cryo-EM studies could not identify structural features that are consistent with known characteristics of an anion conduction pore. We hypothesize that Tthy2 might be a component or regulator of a volume-regulated anion channel or that it may have non-channel functions. In Aim 1, we will investigate whether tweety proteins act as components of anion channels in microvascular pericytes. We will record volume-regulated anion currents (VRAC) from primary human brain vascular pericytes and compare data with or without CRISPR knockdown of TTYH2. Experiments are also designed to investigate other types of currents. In Aim 2, we will follow up on preliminary findings suggesting that TTYH2 participates as an immune sentinel. Specifically, we found that TTYH2 may be a negative regulator of the cGAS-STING pathway, which controls the production of IFN-β and IL-6 in response to foreign (e.g., viral) DNA. We will test the physiological function of TTYH2 by examining IRF3 phosphorylation and IFN-β type I IFN and IL-6 production after stimulating the cGAS-STING pathway by treating cells with cGAMP or the STING agonist DMXAA. This multi-PI R03 proposal by Drs. William Coetzee and Stefan Feske bring together their unique expertise to better understand the roles of channels, particularly TTYH2, in vascular function and innate immunity. There are currently no FDA- approved drugs that target ion channels for immunological disorders, and the completion of the proposed studies takes us an essential step in the direction of this missed therapeutic opportunity.

神经元或肌肉心肌细胞中的离子通道，转运蛋白，交换器和泵调节形成 行动潜力和收缩活动。在内分泌细胞中，它们调节骑马分泌物。离子通道具有 例如，即使在不可观的细胞中，关键角色也是通过设置膜电位并控制的影响 Ca2+进入细胞。有数百个渠道，大多数渠道的特征相对较好。但是，一个数字 仍然是理解的，这是RFA-RM-22-024的重点。我们实验室的一个重点领域是调查角色 时期，毛细血管血管的放血表面上的专门细胞。周细胞有多个 功能，包括形成新的血管和控制血流。周细胞也是哨兵 先天免疫系统，并通过分泌趋化因子和几种类型的免疫细胞相互作用 细胞因子，包括IFN-γ，TNF-α，IL-1β和IL-6。我们已经开发了一个鼠标模型 在周细胞中明确表达，这使得周细胞和富集的纯度很高。我们有 执行了全局RNA-seq，并专注于> 650个离子通道，交换和 在脑周细胞中表达的泵。存在几个Cl-和K+通道的转录本。值得注意的是，会员 在Tyety家族（TTYH1，TTYH2和TTYH3）中，大脑周细胞中的最高表达渠道之一。 公共数据库证实了这一发现 成年小鼠肺血管和血管周围细胞。 tthy2最初被描述为肿胀依赖性 体积调节的阴离子通道，但后来的冷冻EM研究无法识别出的结构特征 与阴离子传导孔的已知特征一致。我们假设tthy2可能是 在AIM 1中， 我们将调查Tweety蛋白是否充当微血管周细胞中阴离子通道的组成部分。我们 将记录体积调节的阴离子电流（VRAC） 带有或不带CRISPR敲低的数据。实验还旨在调查其他类型的 在AIM 2中，我们将跟进初步发现，表明TTYH2是作为免疫力参与的 Sentinel。特别是，我们发现TTYH2可能是CGAS sting途径的负调节器， 控制IFN-β和IL-6的产生，以响应外国（例如病毒）DNA。我们将测试生理 TTYH2通过检查IRF3磷酸化和IFN-β型IFN和IL-6产生后的功能 通过用CGAMP或STING激动剂DMXAA处理细胞，CGAS刺激途径。这个多PI R03 博士的提案。 William Coetzee和Stefan Feske汇集了他们独特的专业知识，以更好地了解 通道，尤其是TTYH2的作用，在血管功能和先天免疫中。目前没有FDA- 旨在以免疫疾病的离子通道的批准药物，并完成了拟议的研究 我们迈出了这个错过的治疗机会的方向的重要一步。