Gamma Secretase

Introduction

Background

Gamma Secretase (GS) is a transmembrane aspartatic protease. It catalyzes peptide bond hydrolysis of type I integral membrane proteins such as Notch, amyloid precursor protein (APP), and various other substrates. It recognizes and catalyzes the reaction with its substrate using 3 residue segments. These 3 residue segments give rise to specific cleavage points for each substrate. APP is the primary substrate of focus, and it is cleaved to form amyloid-β peptides (Aβ). This product is important for various neural processes, and it is well known for its implications with Alzheimer's disease (AD). This has made GS a popular drug target, specifically using gamma secretase inhibitors. However, due to the nature of the enzyme having various neural functions, there are dangerous side effects when it is inhibited.

Overall Structure

Gamma secretase is composed of 20 transmembrane components (TMs) and has 4 subunits: Nicastran (NCT), Presenilin (PS1), Anterior Pharynx-defective 1 (APH-1), and Presenilin Enhancer 2 (PEN-2). These subunits are stabilized by hydrophobic interactions and 4 phosphatidylcholines.These have interfaces between: PS1 and PEN-2, APH-1 and PS1, APH-1 and NCT. has a large extracellular domain and 1 TM. It is important to substrate recognition and binding. serves as the active site of the protease and contains 9 TMs, each varying in length. The site of autocatalytic cleavage is located between in PS1. Major conformational changes take place in this subunit upon substrate binding. serves as a scaffold for anchoring and supporting the flexible conformational changes of PS1. Activation of the active site is dependent on the binding of . PEN-2 is also important in maturation of the enzyme.^[1]

Structural highlights

Substrate Structure

Figure 1. APP fragment conformational change in gamma secretase. APP bound to gamma secretase undergoes a conformational change. The free state consists of 2 helices. The N-terminal helix unfolds into a coil and the C-terminal helix unwinds into a β-strand. This β-strand interacts with PS1 and is the site of cleavage by gamma secretase.

GS has multiple substrates, but the substrate of main concern is APP. APP is composed of an N-terminal loop and a transmembrane helix. It uses as a mechanism of entry into the enzyme via the lid complex, and once in place, the TM helix is anchored by . Upon binding to GS, the C-terminal extracellular helix unwinds. The N-terminal end of the TM helix unwinds into a β-strand (Fig. 1). In order to differentiate between different substrates like APP and notch, the β-strand is often the main point of identification for the enzyme. Substrate binding induces a structural change in GS, creating 2 β-strands that form a β-sheet with the substrate. This β-sheet is in close proximity with the active site, and guides the process of catalysis.^[2]

Lid Complex

The is the first point of entry and recognition for the substrate. This lid is located within the NCT subunit between Asn55 and Asn435. This lobe of NCT is divided into two separate subunits, being the large and small lobes. Phe287 from the large lobe acts as pivot between them. This of the small subunit, and these residues compose a greasy pocket that provides an environment for easy movement. The lid consists of 5 aromatic residues which are highly involved with stabilizing the closed conformation. In particular, this conformation is stabilized by . Once the substrate binds and the lid is opened, a charged, hydrophilic pocket is revealed. This pocket contains , and is further involved with substrate binding and recognition once the lid is removed. However, this lid complex is relatively far away from the catalytic site of the enzyme in PS1. Once the substrate binds, the enzyme undergoes a conformational change to shorten this distance, and the rotation of the large lobe in relation to the small lobe reorients the substrate for cleavage by aligning the pocket in NCT to the active site in PS1. ^[3]

Active Site

The is located between TM6 and TM7 of the PS1 subunit, which is mainly hydrophilic and disordered. Each of these transmembrane helices has an aspartate residue, , which are located approximately 10.6 A˚ apart when inactive.^[3] The PAL sequence of is in close proximity with the catalytic aspartates and is important to substrate recognition. GS becomes active upon substrate binding, when TM2 and TM6 each rotate about 15 degrees to more closely associate. Two β-strands are induced in PS1, creating an with the β-strand of APP.^[2] The β-strand of the substrate interacts via main chain H-bonds with the PAL sequence, stabilizing the active site. Asp257 and Asp385 hydrogen bond to each other and are located 6–7 Å away from the scissile peptide bond of the substrate, allowing catalysis to occur.^[1] The cleavage site is between the helix and the N-terminal β-strand of APP. GS cleaves in 3 residue segments which is driven by the presence of three amino acid binding pockets in the active site. GS can cleave via different pathways, depending on its starting point, but the 2 most commonly used pathways produce Aβ48 and Aβ49.^[4]. Tripeptide cleavage starting between Thr719 and Leu720 results in Aβ48. Cleavage between Leu720 and Val721 yields Aβ49.^[2]

Relevance

Gamma secretase has been determined to be highly involved with diseases such as Alzheimer's disease (AD). In this, beta-amyloid build up leads to amyloidplaques in brain.^[5] These plaques then go on to cause severe neural dysfunction over time. Inhibition of GS could be potential AD treatment, but as stated earlier, this is a hard model to accomplish as the protease is relevant with several different substrates. Complete inhibition would cause other severe implications beyond that of AD, making treatment more difficult than what meets the eye. However, what is known is that there are many different regions that give rise to GS malfunction when they are mutated. Over 200 of these mutations have been linked to causing AD. In particular, these mutations target so called "hot spots" on the enzyme and heavily impact the interface between PS1 and APP, affecting the integrity of catalysis and ultimately creating the plaques that impair neural function. Currently, in order to combat this complex situation, differences in binding between different substrates are being utilized to create drugs that selectively inhibit APP binding with GS, and possibly create a more ideal target for AD treatment^[2].