Presentación de la Escuela Universitaria de Ciencias de la Salud

The different proteolytic cleavages of APP occur in distinct subcellular locations and are carried out by specific secretases (see figures 1.2 and 1.4).

1.6.1

α-cleavage

There are several candidate proteases for α-secretases, all of which are members of the ADAMs (A Disintegrin And Metalloprotease) family of proteases: ADAM9, 10 and 17. Increasing expression of each of ADAM9, 10 and 17 increases sAPPα production, whereas knocking down expression decreases sAPPα production. ADAM9, 10 and 17 are all expressed in the brain (Asai et al., 2003; Allinson et al., 2004). It appears that all of these cleave APP in vivo, and they may be able to functionally substitute for each other. For example, knockout of ADAM10 causes a reduction in sAPPα, though not complete inhibition (Hartmann et al., 2002; Vardy et al., 2005). Although ADAM17 is capable of cleaving APP, its inhibitor profile and kinetics do not match with physiological observations for α-secretase (Parvathy et

al., 1998), so it is most likely not the main enzyme responsible for α-cleavage of APP (Allinson et al., 2003). ADAM17 cleavage of APP is stimulated by protein kinase C activation, this occurs at the cell surface and intracellular compartments (such as the late Golgi/ TGN), so it maybe involved in stimulated rather than constitutive α-cleavage of APP (Jolly-Tornetta and Wolf, 2000).

1.6.2

β-cleavage

β-secretase has been identified as BACE1 (β-site APP cleaving enzyme). BACE1 was identified in a screen of a cDNA library from HEK (human embryonic kidney) cells, when expression of one of the cDNA clones increased generation of Aβ (Vassar et al., 1999). Purification of this clone demonstrated that BACE1 had the substrate specificity, optimum pH and inhibitor profile for β-secretase. BACE1 is an aspartyl protease, with a transmembrane domain and an active site made from two separate motifs. It is expressed at low levels by most tissues of the body, including the brain. In the brain BACE1 expression is highest in the hippocampus, the cortex and cerebellum, whereas expression in glial cells is very low/non-existent (however BACE1 expression has been detected in reactive astrocytes surrounding amyloid plaques in mouse brain (Rossner et al., 2001)). Expression of HA-tagged BACE in HEK cells showed it is localised to the Golgi, and endosomes (where it is at its optimum pH), with small amounts in the ER and lysosomes (Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999). BACE1 in the endoplasmic reticulum is in its immature form, it is cleaved by a furin in the Golgi to form mature BACE1 (Benjannet et al., 2001). BACE1 has a transmembrane domain that is required for its activity (Yan et al., 2001). Most BACE1 cleavage of APP occurs in the late TGN and endosomes (an acid environment is required for activity), however there is some

in the ER (Schrader-Fischer and Paganetti, 1996; Benjannet et al., 2001; Yan et al., 2001). BACE1 as β-secretase is reviewed in (Cole and Vassar, 2007).

1.6.3

γ-cleavage

γ-secretase is a complex composed of several sub units: Aph1a, Pen-2, nicastrin and either presenilin 1 or 2 (De Strooper, 2003; Vardy et al., 2005) (but it can function in the absence of nicastrin) (Zhao et al., 2010). γ-secretase is an aspartyl protease, and the presenilin subunit (either PS1 or PS2 can be present) provides the catalytic aspartic acid residues. Pen-2 is required for an activating cleavage of the presenilin. The other subunits may only be required for stability and maturation of the complex (De Strooper, 2003). Mutations in γ-secretase account for more than half the FAD cases demonstrating its importance in the generation of Aβ in AD (Vardy et al., 2005). γ-secretase cleavage of APP has been reported in a number of subcellular locations such as the nuclear envelope, the ER, the trans- Golgi network (TGN), the cell surface and late endosomes. The majority of γ- cleavage however, occurs after APP has been transported to the cell surface, then endocytosed (Kaether et al., 2006; Hare, 2010). It has also been reported that Aβ40 and Aβ42 generation occurs in separate subcellular locations (Hartmann et al., 1997; Xu et al., 1997).

There is some variability in the cleavage sites of the secretases on APP. α- secretase cleaves APP at L17 of Aβ, but other α-cleavage sites have been identified around this area (at E11 and Q15). These are called α’, and are thought to be due to several different enzymes being responsible for α-cleavage (Simons et al., 1996). There is also an additional β-cleavage site, called β’ (the β-cleavage site is D1 of Aβ, whereas the β’ site is at E11). Both β and β’ cleavage of APP are carried out by BACE1 (Vassar et al., 1999). The γ-secretase cleavage site is not very sequence

specific which means a range of different sized Aβ peptides are produced: it can vary from 39 to 43 amino acids long (Vardy et al., 2005). It has been hypothesised that this is because γ-secretase initially cleaves C99 at the ε-site genereating Aβ48 or Aβ49, then continutes “nibbling” at the C-terminus of Aβ sequentially removing two or three amino acids at a time resulting in the generation of a range of Aβ species (Takami et al., 2009).

Figure 1.5 Amyloid precursor protein structure and cleavage sites. (A) The cleavage points of APP by α- β- and γ-secretase. α and α’ cleavages are carried out by α-secretase, β and β’ cleavages are carried out by BACE1, and γ and ε cleavages are carried out by the γ-secretase complex. Cleavage at the β- and either of the γ-sites generates Aβ (shown in red). Numbering of the residues corresponds to the 695 isoform of APP. The yellow box represents the membrane. (B) Scale diagram of APP695 isoform. The signal sequence is shown in blue, Aβ in red, and motifs important for endocytosis and trafficking in yellow. The pale yellow box represents the membrane. “N-linked” represents the N-linked glycosylation on APP. “O-linked” represents the complex O-linked glycosylation. The location of the KPI and Ox2 domain splice site is shown by the insert.

TNIKTEEISEVKMMDAEFRHDSGYEVHHQKLVFFAEDVGS NKGAIIGLMVGGVVIATV IVITLVMLK

β β`α` α`α γγ ε 649 596 638 N -l in k e d N -l in k e d O -l in k e d L18 P299 V298 N467 N467 S581D596 A638 G681YENPTY Y653TSI KPI+Ox2 A B ε γ-secretase cleavage generating Aβ42 γ-secretase cleavage generating Aβ40