Investigating the role of lipids and substrate recognition motifs in the function of aspartyl intramembrane proteases

Feilen, Lukas Peter (2023): Investigating the role of lipids and substrate recognition motifs in the function of aspartyl intramembrane proteases. Dissertation, LMU München: Medizinische Fakultät

Abstract

Intramembrane proteases (IMPs) are interesting proteolytic enzymes since they are able to perform the water-requiring hydrolysis reaction in the water-excluding lipid membrane. IMPs are involved in various crucial cellular processes like development or cell signalling but also play a role in the onset and progression of diseases such as Alzheimer’s disease (AD) or certain cancers. The human aspartyl IMP γ-secretase is directly involved in the onset of AD, since it cleaves the C-terminal fragment β (CTFβ) of the β-amyloid precursor protein (APP) releasing amyloid β (Aβ) peptides of which the longer forms like Aβ 42 aggregate into oligomers and plaques which is thought to be causative for AD. γ-Secretase is a multiprotein complex composed of four subunits. The catalytically active subunit is presenilin (PS) which exists in two homologs (PS1 and PS2). PS also has homologs in other organisms like the presenilin/SPP homolog (PSH) found in an archaeabacterium. Like PS, PSH is able to cleave APP-based substrates resulting in the release of an APP intracellular domain (AICD) and Aβ species of various lengths. The Aβ species are released from the substrate in a stepwise cleavage mechanism (processive cleavage). In contrast to PS, the activity of PSH is not dependent on the formation of a complex with accessory proteins and therefore allows to study PS function independent of complex formation. The activity of IMPs is strongly dependent on their environment and can be modulated by several factors like the surrounding pH, the composition of the lipid bilayer or the thickness of the membrane. But little is known about the mechanism underling this modulatory effect of the lipid membrane on IMPs and how the lipid membrane influences the structural dynamics of IMPs. Furthermore, it is also unknown whether – at least – aspartyl IMPs share structural motifs for substrate recognition/binding and what role these motifs might play for their activity. The first part of this study focusses on how the lipid environment modulates the activity of PSH and tries to unravel the underlying mechanism. It was shown that PSH produces longer Aβ peptides when its activity was assessed in a DDM micelle environment. This attenuated processive cleavage was enhanced when PSH was reconstituted into a POPC bilayer and shorter Aβ species were generated. Homology model building could reveal that PSH shares substrate recognition/binding motifs with γ-secretase, namely a small additional helix called transmembrane domain (TMD)6a C-terminal of TMD6 and a cytosolic hybrid β-sheet formed by β-strands from PSH and the APP-based C83 substrate. Mutational analysis could confirm the existence of these elements and their importance for the PSH activity. MD simulations based on this model suggest that the active site geometry is destabilized in the detergent environment compared to the lipid environment and this was validated by performing inhibitor affinity precipitation experiments. Taken together, these results show that PSH share key structural elements with γ-secretase and that the lipid environment is crucial for the correct formation of the active site geometry and promotes the processive cleavage. The second part of this study focused on the hybrid β-sheet of the γ-secretase–C83 complex. To this end, mutational analysis of one of the PS1 β-strands (β2) and of the C83 β-strand (β3) were performed. These mutations were designed to disrupt the β-sheet and all mutations studied here strongly reduced the total activity of γ-secretase. Furthermore, most of the mutations also decreased the processive cleavage resulting in the release of longer Aβ peptides and they also shifted the site of the first γ-secretase cleavage, determining which Aβ product line is used. In addition, the β2-strand mutations also reduced the endoproteolysis of PS1 into an N-terminal fragment (NTF) and a CTF. Taken together, the hybrid β-strand is an important structural element of the γ-secretase–substrate complex which influences the total activity as well as the processive cleavage. The results of this study help to explain how the lipid bilayer can modulate the activity of IMPs by influencing the structural dynamics of the enzymes, especially of important structural elements. One very important structural element of aspartyl IMPs is the hybrid β-sheet formed in the enzyme–substrate complex which influences the total activity as well as the processive cleavage since it is needed to correctly position the scissile bond between the two catalytic aspartates and to drain out excess water molecules from the active site.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 29. August 2023
• 1. Bericht­erstatter:in: Steiner, Harald