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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=688212
• 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。它们是高度动态的细胞器，构成网状，不断地被融合和裂变事件重塑。此外，线粒体从其细菌祖先遗传了能够放出称为线粒体衍生的小囊泡（MDV）的小囊泡的能力，这些囊泡在各种胁迫条件下释放，从氧化应激，热应激，热应激以及暴露于多种药物（例如脂多二氢）（ lps）和thapsigargin。这些囊泡的功能仍然很少了解。我们已经表明，MDV的形成至少被两种蛋白Pink1和Parkin，一种线粒体蛋白和一种与线粒体瞬时相关的细胞质蛋白。在不存在的情况下，MDV会释放，线粒体成分将其递送到溶酶体中，并在其中处理用于线粒体抗原表现（MITAP）。这是一种新的抗原呈现途径。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与MDV形成有关的蛋白质以及该腔室在先天免疫反应中的作用。******为特征MDV，将用诱导剂处理细胞MDV释放（热应力，LPS）。然后，我们将使用细胞分馏方法净化MDVS结构，并与高通量蛋白质组学方法相关联，以识别其构成蛋白质。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方法敲低所选蛋白的表达，以产生稳定的巨噬细胞系，并确定这些蛋白质是否沿MDVS-MITAP途径起作用。 ******为破译MDV在先天免疫反应中的作用，我们将在对照细胞中LPS刺激或缺乏与MDVS生物发生有关的基因中的LPS刺激后的促炎细胞因子产生水平（例如SNX9，RAB9，RAB9 ，rab7）。我们还将测量含有线粒体DNA的MDV的生产水平，以及它们对先天免疫反应的影响。 ******该项目将允许鉴定与MDV-MITAP途径有关的关键蛋白质和分子机，从而为调节MDV的生物发生和功能特性的分子机制提供了有价值的提示。