Functional characterization of the N-terminal glycine of the GxGD aspartyl protease active site motif in presenilin 1

g-Secretase catalyses the final processing step of the b-amyloid precursor protein (APP) and thereby generates the AD-causing amyloid b-peptide (Ab) as well as the APP intracellular domain (AICD). Apart from APP, g-secretase has more than 20 known substrates. These are type I membrane proteins that undergo ectodomain shedding to become a g-secretase substrate. g-Secretase is a multimeric complex formed of four essential subunits, presenilin (PS), nicastrin (NCT), anterior pharynx defective 1 (APH-1) and presenilin enhancer 2 (PEN-2). PS1 and PS2 are the catalytic subunit of g-secretase and provide the catalytically active aspartates located in transmembrane domains (TMD) 6 and 7. NCT has been shown to act as a g-secretase substrate receptor by recognizing the free amino terminus of the shedded substrate. Following NCT binding, the substrate is believed to translocate to the postulated docking site that has been shown in PS1 to be within three amino acid distance to the critical aspartate in PS1 TMD7. PS is an unusual aspartyl protease, with a novel GxGD active site motif, which is also found in other aspartyl proteases such as SPP and TFPP. The functional characterization of the GxGD motif has only been partially addressed so far. Mutational analysis of the glycine neighbouring the critical aspartate and the amino acid in position x in PS1 have shown that this motif is important for the activity of the enzyme as well as for substrate (APP/Notch) selectivity. To further characterize this conserved motif an extensive mutational analysis of the N-terminal glycine was performed in PS1.

The following results were obtained: 1) PS1 G382 is not critical for g-secretase complex formation. All the PS1 G382 mutants investigated support normal g-secretase complex formation as judged from the normal maturation of NCT that is observed for all PS1 G382 mutants. Surprisingly, none of the PS1 G382 mutants could support endoproteolysis of PS1 except the PS1 G382A mutant. This shows that PS1 G382 is important for endoproteolysis of PS and that PS endoproteolysis requires a small side chain amino acid in this region of the enzyme.

2) PS1 G382 mutants do not support APP cleavage with the exception of the PS1 G382A mutant, which is partially active and is capable of generating Ab and AICD. Furthermore, PS1 G382A increases the generation of Ab43 and is expected to be a FAD mutation if it occurrs in humans. Interestingly, however, also Ab38 generation is increased. This suggests that PS1 G382A changes the PS conformation altering its cleavage specificity.

3) PS1 G382A has an altered response to NSAID treatment (non-steroidal antiinflammatory drugs), which have been shown to modulate g-secretase activity by lowering Ab42 species and increasing Ab38 species. For some g-secretase modulators (GSMs) PS1 G382A responds similar to PS1 wt regarding Ab38 and Ab42 modulation (sulindac sulfide and fenofibrate) whereas for other GSMs (flurbiprofen and naproxen), PS1 G382A has an altered response regarding Ab38 and Ab42 modulation. Furthermore, Ab40 levels are increased by PS1 G382A when treated with flurbiprofen and naproxen. This implies that modulation of Ab38 and Ab42 generation by GSMs is not coupled as it was initially thought.

4) PS1 G382 mutations have a strong impact on the processing of a number of g- secretase substrates. Notch1, Notch2 and Notch3 were partially processed by the PS1 G382A mutant. Surprisingly, PS1 G382A did not support the processing of CD44 and Notch4 implicating that changes in this region are accepted for the processing of some but not for all the g-secretase substrates.

Taken together, the findings in this thesis demonstrate the importance of the GxGD motif in PS1. The conservation of the glycines in the motif in different intramembrane proteases might fulfil the role to restrain the steric hindrance close to the active site in order to allow catalysis of the different substrates.