Beta-secretase
Created by Amber Fauber
Beta-secretase(2XFK), also known as BACE-1, from Homo sapiens is an enzyme discovered in 1999. BACE-1 is an aspartyl protease, and its functional role is specified cleavage of protein chains during maturation (4). It is usually found in the endoplasmic reticulum and Golgi, removing essential proteins in the neural process (4). Examples of such proteins include neuregulin, a protein regulating formation of myelin sheaths near nerve axons and voltage-gated sodium channels, which are vital for nerve signal transmission (4). BACE-1 contains a deep active site cleft to clench protein chains and a pair of Asp amino acids to cleave proteins (4). Its structure also possesses a long tail, which binds the enzyme to the membrane surface (4). The tail restricts the enzyme to this surface so it does not roam freely throughout the cell. The molecular weight of BACE-1 is 43722.54 Da, and its isoelectric point(pI) is 5.04 (8).
Beta-secretase is a type-I integral membrane glycoprotein (5). It consists of a 21 residue cleavable signal sequence, a large ectodomain of 434 amino acids, a single transmembrane domain of 22 amino acids and a short cytoplasmic tail (12). Its compact globular structure is formed by two domains: residues 47-146 and residues 146-385 (5). Because BACE-1 is an aspartyl protease, it possesses two lobes with an active site between the lobes (12). The active site consists of residues 93-96 and 289-292 (12). Each lobe yields one aspartate residue of the catalytic dyad (12). Present on the active site are 2 conserved aspartic acid residues, Asp-32 and Asp-228 (12). The two aspartyl residues are in close proximity and are generally found in eukaryotic aspartic proteinase (8). The active site residues sit in the center of the cleft along with their surrounding hydrogen bond network (9). BACE-1 contains one subunit, which comprises the voltage-gated sodium channels used for signaling impulses in nerve cells (5). Any problems with the sodium channel will inhibit the passage of neuronal signals through the cell, which can ultimately lead to seizures in humans (5). Furthermore, sodium channel metabolism is significantly distorted in the brains of Alzheimer patients compared to normal counterparts of comparable age (5).
The secondary structure of the human beta-secretase monomer contains two domains with a characteristic aspartic protease fold (6). The secondary structure also consists of alpha helices, beta sheets, 3/10 helices and random coils. More specifically, BACE-1 is 14% helical (12 helices) and 40% beta sheet (30 strands). Shortening the amyloid precursor protein in the area between the BACE-1 cleavage site and the membrane interrupts processing, signifying the secondary structure influences enzyme-substrate interaction (6). The secondary structure serves as a binding site for the ligand, N-((1S,2R)-3-(((1S)-2-(Cyclohexylamino)-1—Methyl-2-oxoethyl)Amino)-2-hydroxy-1(phenylmethyl)propyl)-3-(ethylamino)-5-((methylsulfonyl)(phenyl)amino)benzamide(6). This ligand functions as an inhibitor to BACE-1 by way of edge to face interaction with Arg-296 (2).
In typical aspartyl proteases, a lysine side-chain in the pro-segment creates a salt bridge to two catalytic aspartates (9). In the tertiary structure of BACE-1, there is no salt bridge and proline, the corresponding residue, does not interact with catalytic residues (9). This distinction in BACE-1 reveals that it does not suppress activity, and it facilitates correct folding in the active protease domain (9). Furthermore, the tertiary structure consists of 3 disulfide bonds. All ectodomain cysteines are needed for complete BACE-1 activity and serve a functional role in disulfide bonding (9). The association of disulfide bonds facilitates enzyme folding. The three conserved cysteines on the ectodomain are Cys-278, Cys-330 and Cys-380(9). Adjacent to Cys-216, Cys-330 and Cys-380 are added sequences with no homology to pepsin family members, which can be visualized as loops within the structure (9). These loops extend from and encircle the end of the active site cleft (9). This specific side of the active site cleft binds the N-terminal end of the substrate, indicating the presence of an extended substrate binding pocket (9).
PSI-BLAST is an algorithm used to find areas of similarity between primary structures (10). The program compares nucleotide or protein sequences to calculate the statistical importance of matches (10). This program can also be utilized to reveal functional and evolutionary connections between sequences as well as gene family constituents. Significant matches have values below 0.05 (10). The DALI Server finds proteins with similar tertiary structures by comparing structural alignment (7). Structural similarity is measured using Z scores; Z scores above 2.0 are significant and denote comparable protein folding (7). Cathepsin-E(1TZS) was identified by both servers as a comparable protein. The results of PSI-BLAST (E= 3e-134) and DALI (Z=33.1) indicate that cathepsin-E has primary and tertiary structural similarities to BACE-1(7, 10).
Cathepsin-E and BACE-1 are both aspartic proteases. Aspartic proteases exhibit varying substrate specificities, but are typically active in the cleavage of peptide bonds between two hydrophobic amino acid residues (13). There are five hydrogen bonds between the two active site Asp residues and the surrounding residues for BACE-1, but only four hydrogen bonds for cathepsin- E (13). The common feature between the proteins is the single active site Asp residue, which forms two hydrogen bonds with Leu/Met residues (13); the other Asp forms one to two hydrogen bonds with the Thr residue (13). Protein folding is inhibited by interaction between the disulfide bonds; cathepsin-E and BACE-1 have two matching Cys positions (13).
BACE-1 is typically found in the brain (13). Cathepsin-E is generally found in immune system cells, and its deficiency is linked to the development of atopic dermatitis, an inflammatory skin disease (13). This has impeded the development of therapeutic drugs for Alzheimer’s disease because it interacts with the same type of compounds as BACE-1 (13). When finding drugs to combat Alzheimer’s disease, the drugs should only inhibit BACE-1 because inhibition of cathepsin-E will induce atopic dermatitis (13). The two structures differ in their respective microenvironments surrounding the active site. Asp-96 in cathepsin-E forms an additional hydrogen bond with Ser-99, and Asp-281 does not form hydrogen bond with Leu/Met residue (13). This seemingly insignificant difference could be used to create a highly selective inhibitor.
BACE-1 is of interest to researchers because of its link to Alzheimer’s disease. Alzheimer’s is a type of dementia that creates problems with memory and cognitive skills (1). Signs and symptoms gradually emerge and develop, worsening over time. This disease accounts for 50-80% of dementia cases and is the sixth leading cause of death in the United States (1). Currently, there is no cure, but treatments are available to slow the symptoms. Inhibition of BACE-1 can potentially render therapeutic benefits against Alzheimer’s disease. BACE-1 reacts by cutting off a protein projecting from cells in the brain, isolating the cell membrane-bound fragment Cys-99 (3). These two sequential cleavages of the amyloid precursor protein are speculated to be one of the primary steps for amyloid accumulation, toxic debris to brain cells (3). The neurons progressively die, which is reflected through memory loss and mental capacity in humans (3). Hydroxyethylamine inhibitors interact with the non-prime enzyme side by way of edge to face interaction with Arg-296 (2). Preliminary studies conducted in transgenic mice led to the discovery of the first BACE-1 inhibitor, GSK188909 (2). This inhibitor was administered orally to the mice and successfully lowered brain amyloid levels (2).