Abstract
Intramembrane-cleaving proteases (I-CLiPs) play crucial roles in physiological and pathological processes, such as Alzheimer’s disease and cancer. However, the mechanisms of substrate recognition by I-CLiPs remain poorly understood. The aspartic I-CLiP presenilin is the catalytic subunit of the γ-secretase complex, which releases the amyloid-β peptides (Aβs) through intramembrane proteolysis of the transmembrane domain of the amyloid precursor protein (APPTM). Here we used solution NMR to probe substrate docking of APPTM to the presenilin homologs (PSHs) MCMJR1 and MAMRE50, which cleaved APPTM in the NMR tube. Chemical shift perturbation (CSP) showed juxtamembrane regions of APPTM mediate its docking to MCMJR1. Binding of the substrate to I-CLiP decreased the magnitude of amide proton chemical shifts δH at the C-terminal half of the substrate APPTM, indicating that the docking to the enzyme weakens helical hydrogen bonds and unwinds the substrate transmembrane helix around the initial ε-cleavage site. The APPTM V44M substitution linked to familial AD caused more CSP and helical unwinding around the ε-cleavage site. MAMRE50, which cleaved APPTM at a higher rate, also caused more CSP and helical unwinding in APPTM than MCMJR1. Our data suggest that docking of the substrate transmembrane helix and helical unwinding is coupled in intramembrane proteolysis and FAD mutation modifies enzyme/substrate interaction, providing novel insights into the mechanisms of I-CLiPs and AD drug discovery.
Highlights
(such as V44M in APPTM) can cause familial AD (FAD) characterized by early onset of dementia and increased Aβ42/Aβ40 ratio
We used solution NMR to probe the interaction between APPTM and presenilin homologs (PSHs) in intramembrane proteolysis and show that juxtamembrane residues in APPTM make initial contacts with PSH, and that unwinding of the substrate’s transmembrane helix is coupled with its recognition
MAMRE50 (45% sequence identity) from the archaeon Methanocella arvoryzae MRE50 were used as I-CLiPs in this study, while APPTM was used as substrate[22]
Summary
(such as V44M in APPTM) can cause familial AD (FAD) characterized by early onset of dementia and increased Aβ42/Aβ40 ratio. Over 90 physiological substrates (e.g. Notch) are known for presenilin/γ-secretase, with no apparent consensus recognition motif 8. Understanding substrate/enzyme interaction in I-CLiPs will contribute to our fundamental understanding of I-CLiPs and may provide novel insights for selective inhibition of γ-secretase in AD drug discovery. How I-CLiPs recognize their transmembrane helical substrates remains a central, unresolved question in I-CLiP mechanism with important implications for AD drug discovery. We have solved the NMR structure of the APPTM dimer (Fig. 1A) and characterized the structural effects of FAD mutations such as V44M18. V44M, an FAD mutation initially identified in French population ( the name “French mutation”), causes dementia as early as forty years of age[19]. We used solution NMR to probe the interaction between APPTM and PSHs in intramembrane proteolysis and show that juxtamembrane residues in APPTM make initial contacts with PSH, and that unwinding of the substrate’s transmembrane helix is coupled with its recognition
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