The fate of endoplasmic reticulum-targeted proteins in the face of proteasome failure

Secreted and membrane-bound proteins are generated in the cytosol and transported into the endoplasmic reticulum (ER) lumen of cells for further processing. The information that guides ER-targeted proteins to their destination is encoded in the first 5-30 amino acids of their sequence. The ER signal peptidase is a complex of five subunits essential for the conversion of secretory and some membrane-bound proteins to their mature form. The enzyme emoves the hydrophobic, N-terminal signal sequences of ER-targeted proteins, while they are translocated into the ER-lumen. The proteasome is a barrel-shaped protein complex and its main function is the degradation of unneeded or damaged proteins that are labeled with ubiquitin chains. So far, it has been shown for a few overexpressed proteins that inhibition of the proteasome causes stabilization of ER-targeted precursor proteins, but the underlying molecular mechanisms have remained elusive. In order to further investigate this mechanism, we screened for ER-guided, transiently- and endogenously expressed proteins that show detectable precursor stabilization during proteasome impairment. In addition, we established an important tool for our further work: an N-terminally FLAG-tagged and C-terminally HA-tagged prostate stem cell antigen (FLAG-PSCA-HA) overexpression construct. Thereby, we were able to analyze cellular localization andglycosylation patterns of accumulated precursor proteins during proteasome inhibition. We also verified that genetical-, as well as protein aggregate-induced proteasome inhibition is able to stabilize the precursor proteins, which excludes side effects of chemical proteasome inhibitors. In another approach, we tested different intracellular stress conditions, e.g. nitrogen stress, ER stress, oxidative stress and heat shock, and their effect on the stability of ER-targeted precursor proteins. We were able to show that only oxidative stress, which eventually leads to proteasome impairment, induces accumulation of ER-guided precursor proteins. Finally, we examined the regulation of the ER-signal peptidase during proteasome disturbance in detail, to discover the role of this enzyme for the stabilization of ER-targeted precursor proteins during defective proteasomal protein degradation. Exposure of cells to inflammatory signals, like the cytokines IFNγ or TNFα, leads to incorporation of β1i (LMP2), β2i (MECL-1) and β5i (LMP7) subunits into the proteasomes of hematopoietic cells. These so-called immunoproteasomes generate more peptides that exhibit hydrophobic or basic C-terminal residues, which are better suited for MHC class I binding and that expand the pool of antigens. In a second, independent project, we studied the processing of immunoproteasome subunits in an astrocyte cell line and generated antibodies against the three enzymatically-active immunoproteasome subunits.