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

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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.