γ-secretase, a four subunit protease complex, works as an integral membrane protein that cleaves single-pass transmembrane proteins at residues within the transmembrane domain. γ-secretase cleaves over 100 type-1 membrane proteins. Among all substrates that γ-secretase cleaved, amyloid precursor protein (APP) and Notch receptor are mostly studied due to their essential role in Alzheimer's disease (AD) pathogenesis and cancer development, respectively. Though, γ-secretase is also very important in other signal transduction pathway, such as receptor tyrosine kinases (RTK) pathway. γ-secretase maturation and its’ hardcore function in amyloidogenic pathway and notch pathway will be discussed below. Also, some inhibitors of γ-secretase, which appeal to the scientists, are expected to be potential drugs of AD and cancers induced by γ-secretase mutations.
γ -Secretase maturation
The γ-secretase complex consists of four individual proteins: Presenilins (PS), Nicastrin (Nct), PEN-2 and APH-1. Maturation of γ-secretase follows a stepwise assembly. PS provides the active core of the protease. The PS (~50 kDa) span the cellular membrane for several times. PS is synthesized as a full length (FL) protein but PS-FL is unstable and is quickly either endoproteolysed or degraded. So, it should be stabilized before its function. The stabilization of PS is accompanied by a proteolytic “maturation” cleavage performed by an unknown “presenilinase”. This cleavage leads to the two segements named amino-terminal fragment (PS-NTF, ~30 KDa) and carboxy-terminal fragment (PS-CTF, ~20 KDa), each contributes separately one aspartyl residue to the catalytic site. This two aspartate residues (Asp257 and Asp385) located in transmembrane domains (TMD) 6 and 7, respectively, are essential for the catalytic activity of the protease. A second member of the complex is Nct, which is the first to be discovered through coimmunoprecipitation with PS-directed antibody. Nct is a glycosylated 130 kDa integral membrane protein that binds relatively well to both the PS-NTF and the PS-CTF. Four hydrophilic residues in the proximal one third of the N-terminal portion of the Nct TMD are critical for interaction between Nct and the rest of the γ-secretase complex. Nct interacts initially with Aph-1, followed by the interaction with PS and Pen-2. Pen-2, a ∼10 kDa protein with two TMDs, was discovered as a gene product that can interfere with PS activity in a genetic study involving C. elegans. elegans. Pen-2 is required for endoproteolysis of PS-FL into PS-NTF/CTF and for γ-secretase activity. Another subunit is Aph-1, a ∼29 kDa protein with seven TMDs, was discovered in the same genetic screen as Pen-2. The GXXXG motif of Aph-1’s TMD4 is essential for assembly into the γ-secretase complex as it plays a major role in intramembrane helix-helix interactions. Enormous investigations of the γ-secretase revealed that each of the four essential γ-secretase subunits is tightly and independently controlled. More researches about the interaction of these subunits and the co-factors need be established in the future.
Accumulation of β-amyloid plaque is the hallmark of AD in the patients’ brain. γ-secretase is thought to contribute to the development of Alzheimer’s disease by generating β-amyloid peptides (Aβ peptides), particularly those that are prone to aggregation such as Aβ42. Aβ peptides are generated through the processing of amyloid precursor protein (APP) by β-secretase and γ-secretase in a stepwise fashion. After the β-secretase enzyme (BACE1, aspartyl protease) cleaves the APP extracellular domain, γ-secretase cleaves the remaining part to release the Aβ peptide and the AICD (App intracellular domain).The AICD will regulate the calcium signal in the nucleus. And The Aβ peptides can aggregate into small neurotoxic oligomeric structures (App oligomers) and eventually form the typical plaques seen in Alzheimer’s disease. γ-secretase cleaves APP at different positions, possibly in a consecutive way, resulting in the release of Aβ peptides that ranged from 37 to 49 amino-acid residues. Aβ40 is the most abundant product cleaved and is found in the artificial cerebrospinal fluid and the plasma. Aβ42 occurs less usually and only account for 10 % of the Aβ40 in the organism. But Aβ42 is considered pathogenic, it aggregates faster than Aβ40 and is more toxic to the cells. The mutations of subunit PS in the γ-secretase always cause the relative amount of Aβ42 and Aβ40, whose ratio was usually regarded as an indicator of pathogenic mutations. So γ-secretase, especially the PS domain is considered as the drug target in screening of drugs for AD therapy.
Ligands (like Jagged-1,-2 and Delta-like-1,-3 and -4) located on adjacent signal-presenting cells would activate notch receptors in the signal receiving cells’ membrane. Notch receptor will undergo a two-step cleavage with different enzymes. The activated notch receptor will have a conformational change and expose the cleavage site 2 (S2) for ADAM family metalloproteases (also named TACE) that cleave notch at an extracellular, membrane-proximal region. The membrane-bound Notch segment that results from this cleavage, known as Notch Intracellular Truncation domain (NEXT), is a γ-secretase substrate. γ-secretase performs the subsequent cleavage at cleavage site 3 (S3), releasing Notch intracellular domain (NICD) from the membrane and allowing for signal transduction through binding with the CBL-1, Su(H), Lag-1 family of DNA binding proteins.
Figure. 1 Proteolytic processing of APP and Notch
Several inhibitors have been developed clinically due to its’ profound decrease of brain Aβ concentrations in a mouse model of Alzheimer's disease. And also, it increased the survival rate of mouse models of leukaemia by inhibition of the notch signaling. However, the first clinical trial of γ-secretase inhibitors raised many important questions about therapeutic potential. These inhibitors will lead to serious gastrointestinal side-effects due to the inhibition of notch.
So we can conclude that, the γ-secretase have two main function. One is the APP cleavage to produce Aβ, whose disorder will induce AD. Another is the constitutive notch function, whose inhibition would lead to gastrointestinal side effect (so called notch-related toxicity).The balance of that two aspects is the essential point for curing AD, but it’s hard to establish the certain level of inhibition which can give attention to both of that two aspects. Semagacestat (also named LY450139) is in a phase 3 clinical trial for Alzheimer’s disease. And in the phase 2 clinical trial of semagacestat, the notch-related toxicity is rather mild maybe due to the γ-secretase incompletely inhibited. It has shown a 50% decrease of Aβ40 in the phase 2 clinical trial. But the Aβ40 response is biphasic and it will recovery in the later phase. Begacestat, another γ-secretase inhibitor, has a limited Notch-related toxicity while inhibiting APP cleavage. This type of drug is thought promising in clinical trials for the future AD therpy.
To be brief, γ-secretase is a very special point for several other signal transduction, since its’ role in cleaving the intermembrane sites, transmitting the upstream signal into the intercellular part and eventually modifying the gene transcription as well as mRNA translation. The intracellular signaling is also important for γ-secretase, since this protein has such complex roles in different signaling pathways and cleave many important proteins at the same time. It is worth to consider to regulate the downstream signaling in the different pathway, which maybe is a direction to avoid the side-effect caused by directly inhibition of γ-secretase itself.Some researches show that γ-secretase is also localized on the mitochondria, which is proposed to be a part in the cell apoptosis. This observation interprets that some AD patients have the multitude mitochondria defect as well.
|Research Areas||Related Targets||Featured Products|
|Cardiovascular and blood system||CaMK-II||NGP-555|
|Endocrinology and Metabolic Disease||AChE||Amyloid β-Peptide (12-28)|