The thromboxane A2 receptor and peroxisomes

血栓素 A2 受体和过氧化物酶体

• 批准号: RGPIN-2020-04809
• 负责人: Parent, JeanLuc
• 金额: $ 3.64万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=760597
• 关键词: thromboxane; A2; receptor; peroxisomes

G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological functions, such as smell, taste, vision, secretion, neurotransmission, metabolism, inflammatory and immune response. GPCR signaling is thought to stem from the cell surface where receptor interactions with external stimuli can be transduced into cellular responses. Accumulating evidence indicates that many GPCRs also signal from inside the cell. Peroxisomes participate in central pathways of cellular metabolism such as ß-oxydation of fatty acids and detoxification of reactive oxygen species. Peroxisomes have complex biogenesis and degradation pathways, are actively distributed intracellularly and possess their own targeting and translocation machineries. They are dynamic structures whose morphology, abundance, content and function can be rapidly regulated in response to diverse signals. Peroxisomes have been overlooked so far in GPCR biology. Through our research program on the proteins that interact with and regulate TPß (thromboxane A2 receptor), we identified several peroxisomal proteins involved in: A) targeting and translocation of proteins to peroxisomes, B) motility/distribution of peroxisomes, and C) various peroxisomal metabolic pathways. Of particular interest in the latter group is ACOX1, the enzyme involved in the first step of fatty acid ß-oxydation, producing H2O2. TPß co-localization with peroxisomal markers was detected by immunofluorescence microscopy. Interaction between the receptor and peroxisomal proteins were confirmed by co-immunoprecipitation and western blot experiments. To the best of our knowledge, this is the first evidence of the presence of GPCRs in peroxisomes. We will concentrate on the 3 following short-term objectives for the next 5 years: 1)TPß localization to peroxisomes will be studied by electron microscopy at the endogenous level in HeLa cells. Radioligand binding experiments will also be carried out on purified peroxisomes from HeLa cells to further study the presence of TPß. The orientation of TPß in the peroxisomal membrane will be determined. The molecular mechanisms involved in targeting TPß to peroxisomes will be studied. 2)The effects of the stimulation, the blockade and the presence of TPß in peroxisomes on the number, motility, and distribution of this organelle will be characterized. The molecular mechanisms involved in these processes will be explored.  3)The interaction of TPß and peroxisomal enzymes will be studied. We will first focus on the interaction with ACOX1. We will determine whether ACOX1 regulates the functional responses of TPß. We will address whether TPß modulates the activity of ACOX1. To the best of our knowledge, GPCRs have never been described in peroxisomes and their function in this organelle is still unknown. Furthermore, our program will provide significant advancement in our understanding of the molecular regulation of peroxisome biology in response to cellular signals.

G蛋白偶联受体（GPCR）调节多种生理功能，如嗅觉、味觉、视觉、分泌、神经传递、代谢、炎症和免疫反应。GPCR信号传导被认为源于细胞表面，其中受体与外部刺激的相互作用可以被转导成细胞反应。越来越多的证据表明，许多GPCR也从细胞内部发出信号。 过氧化物酶体参与细胞代谢的中心途径，例如脂肪酸的β-氧化和活性氧的解毒。过氧化物酶体具有复杂的生物合成和降解途径，活跃地分布在细胞内，并具有其自身的靶向和易位机制。它们是动态结构，其形态、丰度、含量和功能可以响应于不同的信号而被快速调节。过氧化物酶体在气相化学还原生物学中一直被忽视。通过我们对与TP（血栓烷A2受体）相互作用并调节TP（血栓烷A2受体）的蛋白质的研究计划，我们鉴定了涉及以下的几种过氧化物酶体蛋白质：A）蛋白质靶向和转运至过氧化物酶体，B）过氧化物酶体的运动/分布，以及C）各种过氧化物酶体代谢途径。在后一组中特别感兴趣的是ACOX 1，该酶参与脂肪酸氧化的第一步，产生H2 O2。免疫荧光显微镜检测TP 3与过氧化物酶体标记物的共定位。通过免疫共沉淀和western blot实验证实了GPCR受体与过氧化物酶体蛋白的相互作用，这是首次证明GPCR在过氧化物酶体中的存在。我们将在未来5年内集中精力于以下3个短期目标：1）在HeLa细胞中，通过电子显微镜在内源性水平上研究TP 3在过氧化物酶体中的定位。放射性配体结合实验也将进行纯化的过氧化物酶体从HeLa细胞，以进一步研究的存在下，TP 3。将确定TP 3在过氧化物酶体膜中的取向。将研究靶向TPPs至过氧化物酶体的分子机制。2)刺激的影响，封锁和存在的TPPs在过氧化物酶体的数量，运动性和分布的这个细胞器的特点。3）研究TP酶与过氧化物酶体酶的相互作用。我们将首先关注与ACOX 1的交互。我们将确定ACOX 1是否调节TP 3的功能反应。我们将讨论TP 3是否调节ACOX 1的活性。 据我们所知，GPCR从未在过氧化物酶体中被描述过，它们在这个细胞器中的功能仍然未知。此外，我们的计划将提供我们的过氧化物酶体生物学响应细胞信号的分子调控的理解显着的进步。