Characterization of molecular mechanisms regulating the formation of mitochondria-derived vesicles and their roles

调节线粒体衍生囊泡形成的分子机制及其作用的表征

• 批准号: RGPIN-2018-06728
• 负责人: Matheoud, Diana
• 金额: $ 2.99万
• 依托单位: Université de Montréal
• 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=761469
• 关键词: Characterization; molecular; mechanisms; regulating; formation

Mitochondria are more than just the powerhouse of the cell. They are highly dynamic organelles constituting a reticulum constantly remodeled by fusion and fission events. Furthermore, mitochondria have inherited from their bacterial ancestors the ability to shed small vesicles called mitochondria-derived vesicles (MDVs), which are release in various stress conditions ranging from oxidative stress, heat stress, and exposure to a variety of drugs such as lipopolysaccharide (LPS) and thapsigargin. The function of these vesicles is still poorly understood. We have shown that the formation of MDVs is actively repressed by at least two proteins, PINK1 and Parkin, a mitochondrial protein and a cytoplasmic protein that transiently associate with mitochondria. In their absence, MDVs are released and mitochondrial components are delivered to lysosomes where they are processed for mitochondrial antigen presentation (MitAP). This is a new antigen presentation pathway. It is thus of prime importance to understand the molecular mechanisms regulating this process. The aim of our project is to characterize the proteins involved in the formation of MDVs and the role of this compartment in the innate immune response.To characterize MDVs, cells will be treated with inducers of MDVs release (heat stress, LPS). We will then use cell fractionation methods, to purify MDVs structures, linked with a high-throughput proteomics approach to identify their constituting proteins. Bioinformatics analyses will be performed on the data to select proteins of interest. We will knock-down the expression of selected proteins, using a shRNA approach, to generate stable macrophage cell lines and determine whether these proteins play a role along the MDVs-MitAP pathway. To decipher the role of MDVs in the innate immune response, we will measure the level of pro-inflammatory cytokines production after LPS stimulation in control cells or in cells deficient in genes implicated in MDVs biogenesis (e.g. Snx9, Rab9, Rab7). We will also measure the production level of MDVs containing mitochondrial DNA, and their implication in the innate immune response. This project will allow the identification of key proteins and molecular machines involved in the MDV-MitAP pathway, providing valuable hints into the molecular mechanisms regulating the biogenesis and functional properties of MDVs.

线粒体不仅仅是细胞的发电站。它们是高度动态的细胞器，构成了一个不断被融合和裂变事件重塑的网。此外，线粒体已经从其细菌祖先继承了脱落小囊泡的能力，这些小囊泡被称为线粒体衍生的囊泡（MDV），其在各种应激条件下释放，所述应激条件包括氧化应激、热应激和暴露于各种药物如脂多糖（LPS）和毒胡萝卜素。这些囊泡的功能仍然知之甚少。我们已经表明，MDV的形成被至少两种蛋白质，PINK 1和Parkin，一种线粒体蛋白质和一种与线粒体瞬时相关的细胞质蛋白质积极抑制。在它们不存在的情况下，MDV被释放并且线粒体组分被递送到溶酶体，在那里它们被加工用于线粒体抗原呈递（MitAP）。这是一种新的抗原呈递途径。因此，了解调控这一过程的分子机制至关重要。我们的项目的目的是表征参与MDV形成的蛋白质以及该区室在先天免疫应答中的作用。为了表征MDV，将用MDV释放的诱导剂（热应激，LPS）处理细胞。然后，我们将使用细胞分级分离方法，纯化MDV结构，与高通量蛋白质组学方法相结合，以确定其组成蛋白。将对数据进行生物信息学分析，以选择目标蛋白质。我们将使用shRNA方法敲低选定蛋白的表达，以产生稳定的巨噬细胞系，并确定这些蛋白是否在MDVs-MitAP通路中发挥作用。为了解释MDV在先天免疫应答中的作用，我们将测量对照细胞或MDV生物发生中涉及的基因（例如Snx 9、Rab 9、Rab 7）缺陷的细胞中LPS刺激后促炎细胞因子产生的水平。我们还将测量含有线粒体DNA的MDV的生产水平，以及它们在先天免疫应答中的意义。 该项目将允许识别参与MDV-MitAP途径的关键蛋白和分子机器，为调节MDV生物发生和功能特性的分子机制提供有价值的提示。