Mechanisms of disulfide bond formation in the endoplasmic reticulum

内质网二硫键形成机制

• 批准号: RGPIN-2021-03422
• 负责人: Koritzinsky, Marianne
• 金额: $ 2.62万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=742534
• 关键词: Mechanisms; disulfide; bond; formation; endoplasmic

Proteins destined for the extracellular space fold and form disulfide bonds in the endoplasmic reticulum (ER) prior to further traversing the secretory pathway. Disulfide bonds can be introduced in a redox relay where electrons are passed from the client protein through a protein disulfide isomerase and an oxidase to O2. However, many lines of evidence support the existence of alternative pathways for disulfide bond formation. The focus of this research program is to identify novel pathways for disulfide bond formation in the ER. To this end we have taken an unbiased discovery approach to identify proteins that interact transiently with a canonical ER client, vascular endothelial growth factor A (VEGF-A), during its maturation in the ER. Using proximity labeling, isolation and mass spectrometry, we discovered a strong enrichment for mitochondrial proteins in the vicinity of VEGF-A. This suggests that VEGF-A matures in an ER sub-domain in close contact to the mitochondria called the mitochondria associated membrane (MAM), known to be rich in folding factors. This has led us to hypothesize that ER-mitochondrial contact sites (MERCs) support disulfide bond formation in the ER. Interestingly, the mitochondrial intermembrane space (IMS) is the only other cellular compartment that is capable of disulfide bond formation. Objective 1 of this project is to determine the importance of MERCs for ER localized protein folding. We will genetically disrupt the MERCs and measure the impact on maturation of VEGF-A and other ER clients, as well as overall secretion capacity. We will also monitor activation of the Unfolded Protein Response which reports on ER folding stress. Objective 2 is to identify mitochondrial processes that contribute to ER-localized protein folding. We will genetically or pharmacologically disrupt the pathway involved in disulfide bond formation in the IMS and monitor the impact on protein maturation and UPR as above. We will also determine if other mitochondrial processes such as the electron transport chain and the tricarboxylic acid cycle play roles in supporting ER-localized oxidative protein folding. Objective 3 is to determine the importance of MERCs for cell-cell interactions. We will apply conditioned media from cells with disrupted MERCs to other cell types including endothelial cells, macrophages and adipocytes. We will measure the ability of the secretome to stimulate cell physiological processes such as endothelial tube formation, polarization and glucose uptake. Disulfide bond formation is an essential process important for numerous cellular and organismal phenotypes. However, there are large gaps in our knowledge about the mechanisms involved in this process. This research program aims to identify novel pathways for disulfide bond formation and map a unique relationship between the function of the ER and mitochondria important for secretion-mediated phenotypes.

前往细胞外间隙的蛋白质在进一步穿过分泌途径之前在内质网 (ER) 中折叠并形成二硫键。可以在氧化还原继电器中引入二硫键，其中电子从客户蛋白质通过蛋白质二硫键异构酶和氧化酶传递到 O2。然而，许多证据支持二硫键形成的替代途径的存在。该研究项目的重点是确定内质网中二硫键形成的新途径。为此，我们采取了一种公正的发现方法来识别在 ER 成熟过程中与典型 ER 客户血管内皮生长因子 A (VEGF-A) 短暂相互作用的蛋白质。通过邻近标记、分离和质谱分析，我们发现 VEGF-A 附近的线粒体蛋白大量富集。这表明 VEGF-A 在与线粒体紧密接触的 ER 亚结构域中成熟，称为线粒体相关膜 (MAM)，已知富含折叠因子。这使我们推测内质网线粒体接触位点（MERC）支持内质网中二硫键的形成。有趣的是，线粒体膜间隙（IMS）是唯一能够形成二硫键的细胞区室。 该项目的目标 1 是确定 MERC 对 ER 局部蛋白质折叠的重要性。我们将对 MERC 进行基因破坏，并测量对 VEGF-A 和其他 ER 客户成熟以及整体分泌能力的影响。我们还将监测未折叠蛋白质反应的激活，报告内质网折叠应力。目标 2 是确定有助于内质网局部蛋白质折叠的线粒体过程。我们将从遗传或药理学角度破坏 IMS 中二硫键形成所涉及的途径，并如上所述监测对蛋白质成熟和 UPR 的影响。我们还将确定其他线粒体过程（例如电子传递链和三羧酸循环）是否在支持内质网局部氧化蛋白折叠中发挥作用。 目标 3 是确定 MERC 对细胞间相互作用的重要性。我们将把 MERC 被破坏的细胞的条件培养基应用于其他细胞类型，包括内皮细胞、巨噬细胞和脂肪细胞。我们将测量分泌组刺激细胞生理过程（如内皮管形成、极化和葡萄糖摄取）的能力。二硫键的形成是对许多细胞和生物体表型很重要的重要过程。然而，我们对这一过程所涉及的机制的了解还存在很大差距。该研究项目旨在确定二硫键形成的新途径，并绘制内质网功能和线粒体功能之间的独特关系，这对分泌介导的表型很重要。