How the mammalian endoplasmic reticulum handles aggregation-prone β-sheet proteins

Vincenz-Donnelly, Lisa (2016): How the mammalian endoplasmic reticulum handles aggregation-prone β-sheet proteins. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Misfolded proteins are prone to engage in aberrant intermolecular interactions that can lead to formation of large aggregate structures. Aggregation causes loss-of-function toxicity because the aggregating protein fails to reach its native fold and function. In addition, Protein aggregates may exert gain-of-toxicity, which is due to the shear presence of Aggregate conformations that sequester important cellular factors and disturb cell morphology. Protein aggregation is associated with a large number of human diseases. The endoplasmic reticulum (ER) is a membrane-bound cellular organelle andthe site of synthesis of one third of the eukaryotic proteome including secretory proteins and proteins destined for the endomembrane system. After co-translational translocation into the ER, nascent proteins are assisted to fold by molecular chaperones and are subject to post-translational modifications. Secretory proteins are retained in the ER lumen until they are correctly folded and are then delivered to the Golgi apparatus for further modifications. If a protein fails to fold properly after repeated folding cycles, it is instead targeted for degradation via the ER-associated degradation pathway (ERAD). The aim of the study presented in this thesis was to determine how the human ER quality control (ERQC) machinery deals with aggregation-prone proteins. This is of great interest because protein aggregates are differentially regulated by distinct cellular environments and many of the proteins that aggregate in diseases are in fact synthesised in the ER. To this end, we utilised de novo designed amyloidogenic β-proteins as generic models for protein aggregation. Due to their lack of evolved biological function, these model proteins allow the exclusive study of gain-of-function toxicity and enable us to dissect the effect of the ER environment on amyloidogenic proteins. We determined that ER-targeted versions of the model β-sheet proteins are significantly less toxic and more soluble than their non-targeted counterparts, which form toxic insoluble aggregates in the cytosol and nucleus. We found that the ER-targeted β-protein ER-β23 is recognised by ERQC machinery and efficiently retained in the ER lumen in a soluble polymeric state. Strikingly, ER-β23 interacted with factors of the ERAD pathway,even though it was not efficiently degraded. Instead, ER-β23 inhibited the degradation of other ERAD substrates by sequestering low-abundant ERAD factors. The presented results demonstrate a marked capacity of the ER to prevent the secretion of potentially toxic aggregation-prone proteins and to limit the formation of insoluble aggregates in the ER lumen. In addition, the data reveal a mechanism by which amyloidogenic proteins may disturb ER proteostasis. Another aim of this study was to analyse the effects of small molecule proteostasis modulators. We found that the anti-dopaminergic drugs fluphenazine and droperidolas well as the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib improved proteostasis in the presence of Protein aggregates. In case of the former, this effect was most likely achieved via induction of the cytosol stress response. In summary, the work presented in this thesis provides novel insights into how aggregation-prone proteins behave in the environment of the ER and also demonstrates the potential of using small molecule modulators to improve cellular proteostasis in a disease context.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Protein aggregation, endoplasmic reticulum, proteostasis, chaperones, ERAD
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 6. Dezember 2016
• 1. Bericht­erstatter:in: Hartl, F. Ulrich