NBCE1

引言 作为肥胖症与II型糖尿病的药物治疗靶点，胰高血糖素样肽-1受体（GLP-1R）在多个组织中广泛分布且具有细胞类型特异的生理功能。例如，GLP-1R在胰腺胰岛中主要调节胰岛素分泌和细胞增殖，在大脑中则具有抑制食欲、神经保护、抗炎等功能。考虑到受体的成熟、定位、激活、内吞等过程都受到蛋白质相互作用的影响，探索受体与细胞表面其他膜蛋白的互作能够为GLP-1R的调控机制研究提供重要线索。然而，由于大部分膜受体表达量低、互作瞬时且微弱，对内源性GLP-1R开展细胞膜蛋白的互作组研究，首先需要发展新的研究手段。 2024年9月3日，上海科技大学iHuman研究所、生命科学与技术学院水雯箐课题组联合庄敏课题组，在Nature Chemical Biology上发表了题为Endogenous cell membrane interactome mapping for the GLP-1 receptor in different cell types的研究论文。该工作发展了基于配体的邻近标记技术，实现对内源表达的GLP-1R的互作蛋白质组的特异标记、组学鉴定与动态调控分析。 研究者首先设计了GLP-1配体与邻近标记酶偶联的两种探针，预测该探针在结合并激活受体后能够标记与受体发生互作的邻近细胞膜蛋白（图1）。在过表达受体的293T细胞以及内源表达受体的两种细胞（INS-1E胰岛β细胞和SK-N-SH类神经细胞）中，研究者通过一系列验证实验，证明了偶联抗坏血酸过氧化物酶（APEX2）的探针具有与天然GLP-1相当的结合受体、激活下游信号及诱导内吞的活性，且能实现高特异性的细胞表面标记。 图1  基于配体的邻近标记的实验设计（Credit: Nature Chemical Biology） 而后，研究者将邻近标记与定量蛋白质组学技术结合，在INS-1E胰岛β细胞与SK-N-SH类神经细胞中分别解析了内源GLP-1R的细胞膜蛋白互作组。有趣的是，从这两种细胞得到的互作组在分子组成上完全独立，并且与数据库中记录的过表达GLP-1R的潜在互作蛋白的重合度也很低，这反映出内源性受体在不同细胞类型中的互作蛋白质组的独特性。 对于两种细胞中新发现的若干GLP-1R潜在互作蛋白，研究者首先利用生化实验验证了11个候选蛋白与GLP-1R的相互作用。其次，在INS-1E β细胞中，证明5个互作蛋白的敲低会显著提高高糖条件下GLP-1刺激的胰岛素释放效应。进一步的机制研究暗示，这些细胞膜互作蛋白主要通过抑制GLP-1R介导的cAMP信号通路来削弱GLP-1刺激的胰岛素释放。综合功能实验结果，本工作最终发现了3个未知的位于细胞膜上的GLP-1R调控因子（SLC4A4，ATP1B3，GPC4）。最后，研究者在INS-1E β细胞中完成了时间分辨的邻近标记组学实验，在多时间点上追踪受体激活与内吞过程中的膜蛋白互作组变化，构建了内源GLP-1R的动态细胞膜蛋白互作组图谱（图2）。 图2  INS-1E细胞中内源GLP-1R的细胞膜蛋白互作组图谱（Credit: Nature Chemical Biology） 综上所述，本工作不仅为内源GLP-1R的细胞膜蛋白互作组解析提供了一种新型的化学生物学技术，也为深入理解其细胞类型特异的功能调控机制奠定了基础。GLP-1R属于G蛋白偶联受体家族，这种基于配体的邻近标记策略有望推广至该家族中其他重要的药物靶标，为研究组织特异性的药物作用机制提供新的理论依据。 参考文献 https://www.nature.com/articles/s41589-024-01714-1 责编|探索君 排版|探索君 文章来源|“BioArt” End 往期精选 围观 一文读透细胞死亡(Cell Death) | 24年Cell重磅综述（长文收藏版） 热文 Cell | 是什么决定了细胞的大小？ 热文 Nature | 2024年值得关注的七项技术 热文 Nature | 自身免疫性疾病能被治愈吗？科学家们终于看到了希望 热文 CRISPR技术进化史 | 24年Cell综述

A recent study has found that an ion transporter protein that regulates the pH of specific brain cells can repair the blood-brain barrier and restore normal brain function after ischemic stroke. The study has revealed novel and specific therapeutic targets for ischemic stroke and related brain conditions for which no targeted treatments exist currently. Ischemic stroke is a leading cause of death and disability, affecting about 15 million worldwide each year. Among the various factors contributing to the pathogenesis of this condition is the loss of the blood-brain barrier, a highly selective protective cellular barrier that prevents harmful chemicals from entering the brain through the blood. Understanding the mechanisms that regulate the integrity of the blood-brain barrier, developing strategies to repair it, and reversing brain damage will have far-reaching benefits for patients suffering from stroke and related neurological conditions. A recent study from the laboratory of Dr. Hyun Kyoung Lee, associate professor at Baylor College of Medicine and an investigator at the Jan and Dan Duncan Neurological Research Institute (Duncan NRI) at Texas Children's Hospital, has found that an ion transporter protein that regulates the pH of specific brain cells can repair the blood-brain barrier and restore normal brain function after ischemic stroke. The study, published in Cell Reports, is the first to reveal novel and specific therapeutic targets for ischemic stroke and related brain conditions for which no targeted treatments exist currently. The blood-brain barrier and pH regulation are disrupted in stroke and other neurological conditions "Disruption of blood-brain barrier integrity is a pathophysiological hallmark of stroke and several devastating neurological disorders," Dr. Lee, a Cynthia and Anthony G. Petrello Endowed Scholar in Neurological Research, said. "A damaged blood-brain barrier often leads to severe consequences such as brain edema, neuronal damage, and eventually, motor and cognitive deficits. Very little was known about how stroke damages the blood-brain barrier prior to this study. While endothelial cells form the core of the blood-brain barrier, emerging evidence suggests astrocytes, which are the most abundant and diverse type of support cells in the central nervous system, play a critical role in maintaining the integrity of this structure. In addition to this barrier, a strict balance of pH (a measure of acidity or alkalinity of a solution) within the brain cells and their surrounding environment is crucial for optimal brain function. Dysregulation of pH homeostasis in the brain is often implicated in a range of neurological conditions. Ischemic stroke injury is associated with a drastic reduction in pH. However, until this study, the precise reason for this alteration and the role of astrocytes in this process was not known. Astrocytic Slc4a4 plays a critical role in the maintenance of the blood-brain barrier Previous studies have demonstrated that a sodium-carbonate cotransporter 1 (Slc4a4), which is enriched in the astrocytes, is responsible for shuttling acid-base ions across the cell membrane in a bidirectional manner to regulate both intra- and extracellular pH in response to internal and external stimuli. Moreover, patients with Slc4a4 variants have been associated with many brain disorders, including ischemic stroke. This is the first study to investigate whether and how Slc4a4 governs astrocyte-endothelial cell interaction in blood-brain barrier maintenance and repair after stroke. To test the biological role of Slc4a4, the Duncan NRI team generated a conditional mouse model of Slc4a4 using which they could specifically delete the expression of this gene in the brain astrocytes during development and in adult mice. Using this mouse model, they found that deleting Slc4a4 from astrocytes altered their structure and function. "We found that the brain vasculature was altered in the absence of astrocytic Slc4a4 -- there was >40% increase in the diameter of the blood vessels in the brain, a three-fold increase in the number of small molecules that were able to infiltrate the brain along with the loss of junctional markers -- which together provided compelling evidence that the blood-brain barrier was disrupted in the absence of astrocytic Slc4a4 in these mice," Dr. Qi Ye, lead author and postdoctoral fellow in the Lee lab said. Slc4a4 is critical for remodeling the blood-brain barrier after ischemic stroke To test if astrocytic Slc4a4 was important for remodeling the blood-brain barrier after ischemic stroke, they used a cortical photothrombotic stroke model in Slc4a4 animal models. This stroke injury model in animals closely mimics the size, location, as well as reactive gliosis, a scar-like inflammatory response to ischemic stroke injury in human brains. In this model using multi-omics analyses, they found that loss of astrocytic Slc4a4 increased the secretion of a CCL2, a pro-inflammatory molecule, which activated the CCR2 receptor present on the nearby endothelial cells to increase permeability, damaging the blood-brain barrier and increasing leakage between the two cell types. Finally, they found this breakdown of the blood-brain barrier was mediated by increased levels of specific metabolites (arginine and nitrous oxide) caused by changes in astrocytic pH levels. "By revealing the precise mechanism and key players involved in the breakdown of the blood-brain barrier after stroke, this study has opened potential new avenues to treat ischemic stroke and several associated brain pathologies," Dr. Lee said. "It is promising that key players identified in this pathway -- the Slc4a4 transporter protein, the CCL2-CCR2 axis as well the metabolites -- are 'druggable' and can be developed into potential therapeutic targets."