Mechanisms of disulfide bond formation in the endoplasmic reticulum

到达细胞外空间的蛋白质在内质网（ER）折叠并形成二硫键，然后再通过分泌途径。二硫键可以在氧化还原继电器中引入，其中电子从客户蛋白通过蛋白质二硫异构酶和氧化酶传递到O2。然而，许多证据支持存在二硫键形成的替代途径。本研究计划的重点是确定内质网中二硫键形成的新途径。为此，我们采用无偏见的发现方法来鉴定在内质网成熟过程中与典型内质网客户端血管内皮生长因子a （VEGF-A）短暂相互作用的蛋白质。通过近距离标记、分离和质谱分析，我们发现在VEGF-A附近线粒体蛋白富集。这表明VEGF-A在与线粒体密切接触的内质网亚结构域成熟，称为线粒体相关膜（MAM），已知富含折叠因子。这使我们假设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，线粒体膜间空间（IMS）是唯一能够形成二硫键的细胞隔室。该项目的目的1是确定merc对内质网定位蛋白折叠的重要性。我们将从基因上破坏merc，并测量对VEGF-A和其他ER客户成熟的影响，以及整体分泌能力。我们还将监测报告内质网折叠应激的未折叠蛋白反应的激活。目的2是确定有助于内质网定位蛋白折叠的线粒体过程。我们将从遗传或药理学上破坏IMS中二硫键形成的途径，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，如电子传递链和三羧酸循环是否在支持er定位的氧化蛋白折叠中发挥作用。目的3是确定merc在细胞-细胞相互作用中的重要性。我们将使用条件培养基从破坏merc的细胞到其他细胞类型，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，如内皮管形成、极化和葡萄糖摄取。二硫键的形成是许多细胞和有机体表型的重要过程。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。该研究计划旨在确定二硫键形成的新途径，并绘制内质网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。

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.