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: 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=764434
• 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.

在进一步穿越分泌途径之前，蛋白质注定要用于细胞外空间折叠并形成内质网（ER）的二硫键。二硫键可以在氧化还原继电器中引入，在该继电器中，通过蛋白质硫化物异构酶和氧化酶从客户蛋白传递到客户蛋白和O2。但是，许多证据支持二硫键形成的替代途径。该研究计划的重点是确定ER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，以鉴定与经典ER客户量在ER成熟期间与规范性ER客户（VEGF-A）瞬时相互作用的蛋白质。使用接近标记，分离和质谱法，我们发现了VEGF-A附近线粒体蛋白的强富集。这表明VEGF-A在ER子域中成熟，与线粒体相关膜（MAM）密切接触，已知在折叠因子中富含。这导致我们假设ER-线粒体接触位点（MERCS）支持ER中的二硫键形成。有趣的是，线粒体膜间空间（IMS）是唯一能够二硫键形成的其他细胞室。 该项目的目标1是确定MERC对ER局部蛋白质折叠的重要性。我们将在基因上破坏Mercs，并衡量对VEGF-A和其他ER客户的成熟以及整体分泌能力的影响。我们还将监视未折叠的蛋白质反应的激活，该反应报告了ER折叠应力。目标2是确定有助于ER定位蛋白折叠的线粒体过程。我们将在遗传或药理上破坏IMS中二硫键形成的途径，并监测上述蛋白质成熟和UPR的影响。我们还将确定其他线粒体过程（例如电子传输链和三羧酸循环）是否在支持ER定位的氧化蛋白折叠方面起着作用。 目标3是确定MERCS对细胞 - 细胞相互作用的重要性。我们将将来自MERCS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的能力，例如内皮管形成，极化和葡萄糖摄取。二硫键形成是对众多细胞和有机表型重要的重要过程。但是，我们关于此过程中涉及的机制的知识存在很大的差距。该研究计划旨在确定二硫键形成的新途径，并绘制ER和线粒体对分泌介导的表型重要的独特关系。