Mechanisms of disulfide bond formation in the endoplasmic reticulum

预定进入细胞外空间折叠的蛋白质在进一步穿越分泌途径之前在内质网(ER)中形成二硫键。二硫键可以在氧化还原继电器中引入，在氧化还原继电器中，电子通过蛋白质二硫键异构酶和氧化物酶传递到O2。然而，许多证据支持二硫键形成的替代途径的存在。本研究计划的重点是确定在内质网中形成二硫键的新途径。为此，我们采取了一种无偏见的发现方法来识别在内质网中成熟过程中与典型的内质网客户-血管内皮生长因子A(VEGF-A)瞬时相互作用的蛋白质。利用邻近标记、分离和质谱仪，我们发现在血管内皮生长因子-A附近的线粒体蛋白有很强的富集性。这表明，VEGF-A在与线粒体密切接触的内质网亚结构域中成熟，称为线粒体相关膜(MAM)，已知该膜含有丰富的折叠因子。这导致我们假设内质网-线粒体接触位点(MERS)支持内质网中二硫键的形成。有趣的是，线粒体膜间间隙(IMS)是唯一能够形成二硫键的另一个细胞间隔。本项目的目标1是确定MERS在内质网定位蛋白折叠中的重要性。我们将从基因上扰乱Mercs，并衡量其对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压力的未折叠蛋白反应的激活。目标2是确定有助于内质网定位的蛋白质折叠的线粒体过程。我们将从基因或药物上破坏IMS中二硫键形成的途径，并监测其对蛋白质成熟和UPR的影响。我们还将确定其他线粒体过程，如电子传输链和三羧酸循环是否在支持内质网局部氧化蛋白质折叠方面发挥作用。目标3是确定MERS在细胞-细胞相互作用中的重要性。我们将把破坏了MERS的细胞的条件培养液应用于其他类型的细胞，包括内皮细胞、巨噬细胞和脂肪细胞。我们将测量分泌体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生物表型的重要过程。然而，我们对这一进程中涉及的机制的了解存在很大差距。这项研究计划旨在确定形成二硫键的新途径，并绘制内质网功能与线粒体之间的独特关系，线粒体对分泌介导的表型至关重要。

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.