Protein-disulfide isomerase: the decisive off-switch of a disintegrin and metalloprotease-17

该项目的重点是特定巯基开关的基本原理，该开关导致跨膜蛋白酶A去整合素和金属蛋白酶（ADAM 17）的失活。这种失活是由两个二硫桥异构化引起的胞外域结构变化的结果。这种构象变化的先决条件是催化巯基转换的细胞外蛋白二硫键异构酶（PDI）的还原状态。这一过程可能是一种安全机制，因为ADAM17的过度活性与癌症和慢性炎症等病理情况有关。然而，它们的充分释放必须确保适当的发展，再生和免疫防御。因此，必须严格调节ADAM 17的活性，因此硫醇开关也是如此。相应地，GRP78保护蛋白酶免受巯基开关的影响，并且PDI必须位于细胞表面并且处于还原状态。这些内质网驻留蛋白的移动和PDI的减少最有可能受到调节。由于结构变化导致ADAM17失活，因此随后将发生降解或巯基开关逆转，可能是由于与生物合成类似的折叠事件。我们的目的是进一步表征巯基开关关于适配器分子的识别，主要异构酶，并分析运输到细胞表面和再循环的ADAM 17和PDIs，以及调节细胞外PDIs的氧化还原状态。我们将研究PDIs和GRP78的相反作用，并检查硫醇开关是否可逆。将探讨这一过程的生物学意义。为此，我们将下调蛋白质的表达，生物素化细胞表面蛋白和游离巯基，进行复合体分析，并应用流式细胞术，荧光显微镜，Western印迹，SPR和MST测量以及增殖和迁移测定等方法。

This project focuses on the underlying principles of the specific thiol switch, which leads to the inactivation of the transmembrane protease A Disintegrin and Metalloprotease (ADAM17). This inactivation is the consequence of a structural change in its ectodomain, caused by isomerization of two disulfide bridges. A prerequisite for this conformational change is the reduced state of extracellular protein-disulfide isomerases (PDIs) which are catalyzing the thiol switch. This process might function as a safety mechanism, since the over-activity of ADAM17 is associated with pathological situations, like cancer and chronic inflammation. However, their adequate release must be ensured for proper development, regeneration and immune defense. Hence, the activity of ADAM17 must be tightly regulated and so the thiol switch. In line, GRP78 protects the protease against the thiol switch and PDIs have to be at the cell surface and in a reduced state. Traveling of those endoplasmic reticulum resident proteins and reduction of PDIs are most likely regulated. Since the structural change leads to inactive ADAM17, this will be followed by degradation or by the reverse of the thiol switch, possible due to folding events similar to the biosynthesis. Our aim is to further characterize the thiol switch regarding the identification of adapter molecules, the principal isomerase and to analyse the transport to the cell surface and recycling of ADAM17 and PDIs, as well as the regulation of the redox state of extracellular PDIs. We will study the opposite roles of PDIs and GRP78 and examine whether the thiol switch is reversible. The biological relevance of this process will be explored. For that, we will down-regulate the expression of proteins, biotinylate cell surface proteins and free thiol groups, perform complexome profiling and apply methods like flow cytometry, fluorescence microscopy, Western blot, SPR and MST measurements, as well as proliferation and migration assays.