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β- Secretase BACE, a key enzyme in the Aβ plaque formation cascade -A structural biology perspective. - Sayali Kukday. Background. Beta-site APP Cleaving Enzyme1 (BACE1) was discovered in 1999, by a team of scientists at a biotech company, Amgen at Thousand Oaks, California.
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β-Secretase BACE, a key enzyme in the Aβ plaque formation cascade-A structural biology perspective -SayaliKukday
Background • Beta-site APP Cleaving Enzyme1 (BACE1) was discovered in 1999, by a team of scientists at a biotech company, Amgen at Thousand Oaks, California. • Martin Citron, Robert Vassar and their colleagues at Amgen carried out a series of cloning experiments before discovering the gene responsible for increase in production of β-amyloid. • BACE1 is an aspartic protease responsible for the production and accumulation of Aβ peptides in brain tissue to form amyloid plaques, a characteristic of Alzheimer’s disease (AD). • Clinical features of AD include brain tissue deterioration and memory loss. • BACE1 is mostly expressed in pancreas and brain. • Present in neurons but almost in negligible amount in glial cells of the brain.
Steps involved in APP cleavage by BACE APP Amyloid Precursor Protein (APP) cleavage by BACE and γ-secretase to produce Aβ peptides (Aβ40 or Aβ42) and AICD (APP Intracellular Domain) Citron M, (2002) Journal of Neuroscience Research. 70, 373–379 β-secretase Cytoplasm β-APPs γ-secretase A β AICD
Structure of Beta-site APP cleaving enzyme (BACE1) • Monomer containing 501 amino acids. • 2 aspartic protease motifs that possess the sequence DTGS (residues 93-96) and DSGT (289-292) are present in the active site, mutation of either aspartic acid abolishes the catalytic activity of the enzyme. • Single C-terminal transmembrane domain (461-477). • Active site extends into the lumen; correct topological orientation required for APP cleavage. • 6 Cys residues are present in the catalytic domain to form 3 intramolecular disulfide bonds. Active site cleft Tyr71 (Flap) Structure of BACE1 (PDB ID 2ohl) Murray CW et al (2007) J. Med. Chem. 50,1116-1123. Modified using Rasmol
Active Site • Active site is covered by flaps. A single flap of 14 residues in length forms an α-hairpin structure that is perpendicular to cleft which houses the active site and covers its central part. • During the catalytic cycle, the flaps must open to allow entrance of substrate (APP) into the catalytic cleft and also to release hydrolytic products. • Structural difference between bound and unbound BACE1- a large flap movement of about 4.5 Å at the tip. The flap in the unbound enzyme is in an open position allowing access to the active-site cleft. • Binding of flap tightly covers inhibitor and this would restrict its movement preventing APP entry into the active site making BACE1 a primary target in the development of inhibitor drugs for treating Alzheimer’s disease. Active site pocket Structure of BACE showing 2-aminoquinoline sitting in the active site pocket (PDB ID 2ohl) Murray CW et al (2007) J. Med. Chem. 50,1116-1123 Modified using RASMOL and Powerpoint
Catalytic Mechanism • The catalytic aspartates (Asp32 and Asp228) are involved in general acid-base catalysis which leads to the proteolytic cleavage of the substrate (APP). • Amine group of the 2-aminoquinoline (inhibitor which is positively charged at the normal operating pH of BACE1-around 4.5), places itself between the side chains of the Asp32 and Asp228 forming H-bonds. An additional H-bond is formed by the protonated pyridine group with the side chain of Asp32. • The compound binds to an open flap formed by the Tyr71 residue of the enzyme. The flap then tightly covers the inhibitor with flap residue Tyr 71 binding the side chains of 2-aminoquinoline. Tyr71 2-aminoquinoline Asp32 Asp228 Active site of BACE showing interaction of catalytic residues Tyr71, Asp32 and Asp 228 with the inhibitor 2-Aminoquinoline (PDB ID 2ohl) Murray CW et al (2007) J. Med. Chem. 50,1116-1123 Modified using RASMOL and Powerpoint
Conclusions • The initial and rate-limiting step in Aβ production is catalyzed by BACE1. • A β aggregation is the cause for the pathological characteristics observed in AD. • Therefore, BACE1 seems to be a promising therapeutic target for the prevention and treatment of AD. • Targeting BACE1 can prove beneficial as inhibitors for other similar aspartic proteases like renin, HIV proteases have been developed and are being used as treatment. • Another possible theraupetic strategy would be to divert APP into the α-secretase pathway, decreasing plaque formation since α-secretase cleavage does not result in Aβ production.
