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动力源。它们是高度动态的细胞器，构成网状结构，通过融合和裂变事件不断重塑。此外，线粒体从其细菌祖先那里继承了脱落称为线粒体衍生囊泡（MDV）的小囊泡的能力，这些小囊泡在各种应激条件下释放，包括氧化应激、热应激和暴露于脂多糖等多种药物。 LPS）和毒胡萝卜素。这些囊泡的功能仍然知之甚少。我们已经证明，MDV 的形成受到至少两种蛋白质 PINK1 和 Parkin 的主动抑制，一种是线粒体蛋白，另一种是与线粒体短暂结合的细胞质蛋白。如果没有它们，MDV 就会被释放，线粒体成分会被输送到溶酶体，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，了解调节这一过程的分子机制至关重要。我们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反应中的作用。为了表征 MDV，将用 MDV 释放诱导剂（热应激，LPS）处理细胞。然后，我们将使用细胞分级分离方法来纯化 MDV 结构，并与高通量蛋白质组学方法相结合来鉴定其构成蛋白质。将对数据进行生物信息学分析以选择感兴趣的蛋白质。我们将使用 shRNA 方法敲低选定蛋白质的表达，以生成稳定的巨噬细胞系，并确定这些蛋白质是否在 MDVs-MitAP 途径中发挥作用。为了破译 MDV 在先天免疫反应中的作用，我们将测量对照细胞或缺乏 MDV 生物发生相关基因（例如 Snx9、Rab9、Rab7）的细胞中 LPS 刺激后促炎细胞因子的产生水平。我们还将测量含有线粒体 DNA 的 MDV 的生产水平及其对先天免疫反应的影响。 该项目将鉴定参与 MDV-MitAP 途径的关键蛋白质和分子机器，为调节 MDV 生物发生和功能特性的分子机制提供有价值的线索。