Gamma secretase

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Background

γ-secretase belongs to the family of intramembrane-cleaving proteases (i-CLiPs), which includes the presenilin family of aspartyl proteases, the zinc metalloprotease, site-2 protease family, and the rhomboid family of serine proteases. All i-CLiPs enzymatically cleave their substrates within the plane of the lipid bilayer in a process termed regulated intramembrane proteolysis. γ-secretase is mainly involved in intramembranous proteolysis of type I membrane proteins. It cleaves numerous functionally important proteins, such as Notch, E-cadherin, ErbB4, CD44, tyrosinase, TREM2 and Alcadein, suggesting the participation of γ-secretase in a vast range of biological activities. The best-studied γ-secretase substrates are APP for its roles in Alzheimer’s Disease, and Notch for its importance in development and cell fate determination.^[4]

Structure of Gamma Secretase Complex

γ-secretase has been identified as an aspartyl protease accountable for cleaving over 90 integral membrane proteins after they have undergone ectodomain shedding. GS has been characterized as a high molecular weight complex that consists of four essential subunits in a 1:1:1:1 heterodimer^[5]: , , , and .^[6] PSs play a very significant role in AD and is considered a vital catalytic subunit in γ-secretase. PS are multi-transmembrane proteins with nine transmembrane helixes; it is assumed the amino-terminus is located in the cytosol while the carboxyl-terminus is exposed to the luminal/extracellular space. Functional PS requires endoproteolytic cleavage between TM6 and TM7 which generates a 27–28 kDa amino-terminal fragment (NTF) and a 16–17 kDa carboxyl-terminal fragment (CTF). ^[4] The two aspartyl residues in PS1 and PS2 (D257 in TM 6 and at D385 in TM 7) play crucial roles in intramembranous cleavage and AD plaque formation; substitutions of these residues reduces cleavage of APP and Notch1 proteins.^[7] PS, NTF, and CTF bind to form stable and active PS heterodimers at a 1:1 stoichiometry. ^[4] The remaining three subunits (NCT, APH-1, PEN-2) help with stabilizing GS by forming a mature enzyme. NCT contains a large extracellular (or ectodomain) domain, transmembrane helix, and smaller cytoplasmic domain.^[5]The ectodomain of NCT recognizes and binds to the amino-terminal stubs of previously cleaved transmembrane proteins. APH-1 aids the formation of a pre-complex, which interacts with PS1 or PS2^[7]; it contains two different isoforms from two paralogous genes on chromosomes 1 (APH-1A) and 15 (APH-1B). While PEN-2 works in enzyme maturation^[5]; it enters the formed complex to initiate the cleavage of PS1 or PS2 to form an N-terminal 28-kDa fragment and a C-terminal 18-kDa fragment, both APH-1 and PEN-2 are critical to the γ-secretase complex.^[7] The γ-secretase complex has a molecular weight of approximately 170 kDa, with an additional 30–70 kDa derived from NCT glycosylation, reaching a total size of about 230 kDa with 19 TMs.^[4]

Function

Once all of the subunits are present, the complex must be correctly assembled for γ-secretase to function properly. The complex is first assembled in the endoplasmic reticulum. The events leading to the formation of a mature γ-secretase complex start from the formation of an initial scaffolding complex composed of APH-1 and NCT. Once the scaffold is created, the full-length PS can attach itself.^[1]The proximal C-terminus of the PS holoprotein binds to the APH-1-NCT subcomplex by interacting with the TM domain of NCT. Following PS binding, PEN-2 is incorporated into the complex by interacting with TM4 of PS. At the final step, the loop domain between TM6 and TM7 of PS is cleaved by endoproteolysis. Alternatively, the APH-1-NCT subcomplex may bind directly to a cognate PS1-PEN-2 structure to generate the mature γ-secretase complex.^[4]The active complex is then shuttled to the Golgi where it is glycosylated. Only after the assembly of all and the glycosylation will GS become active.^[5]

Alzheimer's Pathway

On the cell surface, amyloid precursor protein (APP) can be proteolyzed directly by α-secretase followed by γ-secretase, a process that does not generate Aβ, or APP can be reinternalized in clathrin-coated pits into another endosomal compartment containing the proteases BACE1 and γ-secretase resulting in the production of Aβ. FRET analysis indicates that γ-secretase activity is present on the cell surface, where it complements α-secretase activity, and in endosomal compartments, where it complements BACE1 activity.^[7] The cleavage of various substrates appears to be dependent on the subcellular compartment; APP is mainly cleaved in the TGN and early endosomal domains thus, a disturbance in the localization of the γ-secretase complex may play some role in abnormal Aβ generation and AD pathogenesis.^[6] The initial cleavage of APP by α- or β-secretase, results in membrane-bound C-terminal fragments of APP (APP αCTF and βCTF). αCTF and βCTF are further cleaved by γ-secretase to generate p83 or Aβ, respectively. The p83 fragment is rapidly degraded and widely believed to have a negligible function, whereas is neurotoxic.^[4] γ-secretase-mediated cleavage is unique in that the cleavage takes place within the transmembrane domain, though the exact site can vary. γ-cleavage can yield both Aβ40, the majority species, and Aβ42, the more amyloidogenic species, as well as release the intracellular domain of APP (AICD). Recent data has shown that PS/γ-secretase also mediates ζ-site cleavage (Aβ46) and ε-site cleavage (Aβ49)^[6]; the existence of different Aβ species, including the shorter Aβ38 fragments suggests that γ-secretase cleaves APP in a sequential manner, first at the ε-site, followed by the ζ-site, and the γ-site.2 Upon Aβ formation, Aβ is then dumped into the extracellular space following vesicle recycling or degraded in lysosomes.^[7]

Alzheimer's Disease

Although the majority of Aβ is secreted out of the cell, Aβ can be generated in several subcellular compartments within the cell, such as the ER, Golgi/TGN, and endosome/lysosome. In addition, extracellular Aβ can be internalized by the cell for degradation. The intracellular existence of Aβ implies that Aβ may accumulate within neurons and contribute to disease pathogenesis. Confirming this, intraneuronal Aβ immunoreactivity has been found in the hippocampal and entorhinal cortical regions which are prone to early AD pathology in patients with mild cognitive impairment. In Down Syndrome (DS) patients, the accumulation of intracellular Aβ precedes extracellular plaque formation and the level of intraneuronal Aβ decreases as the extracellular Aβ plaques accumulate. Intraneuronal Aβ can also impair amygdala-dependent emotional responses by affecting the ERK/MAPK signaling pathway. Inhibition of dynamin-mediated but not clathrin-mediated Aβ internalization was also found to reduce Aβ-induced neurotoxicity. One recent study suggests that internalized Aβ can aggregate within the cell and disrupt the vesicular membrane, thus contributing to its pathological effect. There are two main toxic species, Aβ40 and Aβ42, with Aβ42 more hydrophobic and more prone to fibril formation while only making up about 10% of the Aβ peptide produced. Studies done on familial AD (FAD) mutations consistently show increases in the ratio of Aβ42/40, suggesting that elevated levels of Aβ42 relative to Aβ40 is critical for AD pathogenesis, probably by providing the core for Aβ assembly into oligomers, fibrils, and amyloidogenic plaques.^[6] In addition to generating Aβ, γ-secretase cleavage of APP also generates an APP intracellular domain (AICD) within the cell. AICD has been found to possess transcriptional transactivation activity and can regulate the transcription of multiple genes including APP, GSK-3b, KAI1, neprilysin, BACE1, p53, EGFR, and LRP1. In addition, free AICD can induce apoptosis and may play a role in sensitizing neurons to toxic stimuli. ^[4] However, as the intracellular domain of APP, one important function of AICD is to facilitate the interaction of APP with various cytosolic factors that regulate APP's intracellular trafficking and/or signal transduction function. Interestingly, it seems that AICD-mediated APP interaction with different factors is controlled by the phosphorylation state of AICD.^[6]

Relevance

There are 32 APP, 179 PSEN1 (presenilin 1 gene locus), and 14 PSEN2 gene mutations that result in early-onset, autosomal dominant, fully penetrant AD. In APP, mutations cluster around the γ-secretase cleavage site, although the most famous APP mutation (APP-swe) causes a change in amino acids adjacent to the BACE1 cleavage site.^[7] AD-related loci are found on chromosome 1 and chromosome 14; two homologous genes, PSEN1(encoding PS1) on chromosome 14 and PSEN2 (encoding PS2) on chromosome 1.2 PSEN gene mutations (which gives rise to proteins presenilin, PS1 and PS2) predominantly alter the amino acids in their nine transmembrane domains. The common thread to all these mutations is that they increase production of the less soluble and more toxic Aβ42 relative to Aβ40.5 Mutations in the PSEN1 gene, encoding presenilin-1 (PS1), are the most common cause of familial Alzheimer’s disease (FAD). ^[8] These familial mutations lead to the heritable form of Alzheimer’s disease.^[5]

Structural highlights

The structural information of the γ-secretase complex has been primarily obtained by electron microscopy analysis with a maximum resolution of 12 Å, revealing a globular structure with several extracellular domains, three water-accessible cavities, and a potential substrate-binding surface groove in the TM region. Recently a three-dimensional structure of the intact human γ-secretase complex was determined by cryo-electron microscopy with a resolution of 4.5 Å. The overall structural model comprises a horseshoe-shaped structure with 19 TMs and a bilobed ectodomain representing Nicastrin. The extracellular domain of Nicastrin contains a large lobe and a small lobe. The large lobe of Nicastrin thought to be responsible for substrate recognition, associates with the small lobe through a hydrophobic pivot at the center. The current speculative model suggests that PS1 and PEN-2 are located to the “thick” end of the horseshoe shape, whereas APH-1 and Nicastrin are located toward the “thin” end. PEN-2 spans the membrane twice, with N- and C-terminal domains facing the lumen of the ER. Analysis of the APH-1 sequence shows that it contains seven potential TM domains, with the N-terminal domain facing the extracellular space and the C-terminal domain facing the cytosol. Further work is required to elucidate structural details of other γ-secretase components at the atomic level.2 However, strong evidence suggests that the γ-secretase complex resides primarily in the ER, Golgi/TGN, endocytic and intermediate compartments, most of which (except the TGN) are not major subcellular localizations for APP.^[6]

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