Molecular mechanisms regulating cellular proteostasis

Abstract

The endoplasmic reticulum (ER) is the largest organelle of eukaryotic cells. It forms an interconnected network of tubules and sheets and plays important roles in calcium storage, carbohydrate metabolism, lipid biogenesis and, importantly, it is the site of synthesis and maturation of the secretory proteome, which makes up between 30 and 40% of a cell’s proteome. Given its important role in protein synthesis, the ER is a major player in maintenance of proteostasis, i.e. in maintenance of the correct amount and quality of the proteome. As such, it is equipped with a quality control machinery that constantly and strictly surveils the state of folding proteins and decides on their secretion or removal. N-glycosylated proteins are assisted during their folding by ER lectins and folding enzymes. The binding of these molecules is regulated by the modification of the glycan tree by ER residing enzymes and decides on the fate of the folding polypeptide. If a protein is able to reach its native conformation it is exported via COPII vesicles in order to reach its functional destination within or outside the cell. In contrast, if a protein misfolds, it is recognized by components of the ER associated degradation (ERAD) machinery and brought to the dislocons. Dislocons are protein complexes built around membrane embedded E3 ubiquitin ligases, which mediate the retrotranslocation of the misfolded protein to the cytosol and polyubiquitination that eventually leads to the destruction by the 26S proteasome. Misfolded proteins might form aggregates, which become resistant to ERAD. In such cases, autophagy may intervene in ill-defined manner in order to remove portions of the ER that contain such aggregates and bring them to lysosomes for destruction. The maintenance of the equilibrium between synthesis, folding, export and degradation of the secretory proteome is crucial for cell function and survival. This equilibrium can be perturbed by several external (hypoxia, nutrient deprivation, changes in temperature, drugs and attack by pathogens) and cell-intrinsic (fluctuations in proteins synthesis, gene mutations, differentiation events and aging) events, leading to ER stress. To cope with such perturbations cells developed an array of transcriptional and translational responses generally termed unfolded protein response (UPR). Activation of the UPR leads to an increase in ER chaperones, folding enzymes and ERAD factors, a diminished translation rate and expansion of the ER membrane. If the ER stress is too strong and overwhelms the cell’s capacity to respond, the UPR eventually leads to the activation of apoptotic programs. With three different projects, we aimed at understanding how cells react when challenged with situations that perturb ER homeostasis. In the first approach, cells were challenged with a drug–induced ER stress that was then removed in order to permit the recovery of pre-stress homeostasis. Monitoring of the levels and localization of ER chaperones with several techniques, including biochemical analysis, confocal and electron microscopy, nuclear mass resonance and mass spectrometry, led to the characterization of recovery dynamics and the description of SEC62 as an autophagy receptor involved in the degradation of excess/damaged ER specifically during the recovery phase. The function of SEC62 in recovER-phagy was shown to be independent on its role in protein translocation as a component of the SEC61 complex. Rather, the role of SEC62 in recovERphagy was shown to be dependent on a functional LC3 interacting region (LIR) in its cytosolic C-terminus. In the second project, we characterized a degradative pathway involved in the removal of aggregated (polymeric) Z-variant of α1 antitrypsin (ATZ). We described a direct delivery of ATZ-containing ER portions to lysosomes, which we termed ER-to-lysosome associated degradation (ERLAD). This delivery is dependent on the ER-phagy receptor FAM134B and its functional LIR domain, the autophagic LC3 lipidation machinery and the ER SNARE syntaxin 17. Interestingly the described mechanism is not relying on the autophagy machinery involved in autophagosome biogenesis and comprising the autophagy genes ATG9, ATG13, ULK1 and ULK2. For the third project, we took a more unbiased approach for observing how cells respond to the expression of ER retained unfolded proteins. For this, we created a panel of inducible cell lines that express model proteins with different chimico-physical features and performed micro-array-based transcriptomics and shotgun mass spectrometry for proteomics data. The comparison of cellular responses to unfolded proteins between each other and with ER-stress inducing chemicals revealed that 1) misfolded proteins induced a much milder cellular response compared to chemicals, 2) ER retention and BiP binding are required for UPR induction and 3) misfolded proteins are well tolerated by cells and only induce a subset of UPR target genes involved in nascent/unfolded protein binding, which mainly depend on the ATF6 branch of the UPR with no (or only marginally) activation of IRE1 and PERK pathways. All in all, the three projects dealt, from different angles, with how cells cope with perturbations of ER homeostasis and how they are able to maintain or reestablish it. The results show that cells possess a highly interconnected network of transcriptional and translational responses that enable them to cope with a wide range of ER perturbations.