Dissecting γ‐secretase function

The amyoid-b peptide (Ab) plays a central role in Alzheimer’s disease (AD) (Hardy et al. 1998; Hardy and Selkoe 2002). Over the past decade, multiple pharmacological efforts have been carried out with the aim of lowering Ab in AD, either by decreasing its production or by preventing its accumulation in the brain (Karran et al. 2011). The c-secretase complex cleaves APP into Ab peptides and is therefore an appealing target for reducing brain Ab levels. However, when such putative ‘disease-modifying’ c-secretase targeting drugs were tested in Phase II and III clinical trials (Imbimbo et al. 2011), severe side-effects were observed likely because c-secretase processes many different substrates with key functions in the body (De Strooper et al. 2011). Based on these unsuccessful trials, the lesson remains clear, pharmacological targeting of c-secretase requires the development of strategies that selectively lower the generation of toxic Ab peptides without altering the processing of the other substrates. Therefore, a greater understanding of the molecular mechanisms underlying substrate specificity and catalysis in the c-secretase complex is essential. c-Secretase is an aspartyl intra-membrane protease composed of four membrane proteins, with presenilin (PSEN) as the catalytic subunit of the complex (De Strooper et al. 2011). Over one hundred mutations in presenilin are known to cause AD (http://www.molgen.ua.ac.be/ADMutations), which has greatly contributed toward the interest of the pharmaceutical industry in the c-secretase as a drug target. From a mechanistic point of view, the c-secretase complex is a unique enzyme: it hydrolyses substrates in the membrane, and interestingly, proteolysis occurs in a sequential manner in which cleavages of distinct nature take place (Fig. 1). The first cut (termed e) is in fact an endopeptidase cleavage that releases the c-terminal (intracellular) domain of substrates into the cytosol and generates the actual substrate for the next series of cleavages. This ‘enzyme-bound’ N-terminal part is then processed by a carboxypeptidase-like activity, which successively remove c-terminal short peptides from its hydrophobic region (transmembrane domain). The decrease in hydrophobicity eventually promotes release of N-terminal (luminal/extracellular) fragments. The carboxypeptidase-like functionality, defined as the number of turnovers that the substrate undergoes after the e-cleavage, is probably regulated by the catalytic and product release rates at each proteolytic step and determines the length of the products. Although a comprehensive picture of the c-secretase complex will only follow elucidation of its structure at high resolution, an in-depth understanding of the function of the c-secretase complex requires complementary approaches to fully decipher the complexity of its molecular mechanisms (Fig. 1) and to delineate the corresponding key structural determinants. In this regard, dissection of the mechanism contributing to the different activities of the enzyme (endopeptidase vs. carboxypeptidase-like proteolytic processing) is imperative. Indeed, the endopeptidase cleavage has high physiological relevance as it regulates key cell signalling pathways (De Strooper and Annaert 2010). On the other hand, the carboxypeptidase-like activity has pathological significance since dysregulation of APP processing at this level (altered carboxypeptidase-like functionality) may lead to an increased production of longer and more aggregation-prone Ab products, such as those observed in the AD brain. Actually, it is proposed that c-secretase complex containing FAD linked PSEN mutant generates longer and more aggregation-prone Ab peptides (relative to wt enzyme) because of ‘premature’ release of (intermediary) products (Chavez-Gutierrez et al. 2012). Efforts directed towards the structure-function characterization of the c-secretase complex have identified a GlycineX-Glycine-Aspartic acid motif (GXGD, with X as any amino acid residue) in presenilin as a key player involved in catalysis (Steiner et al. 2000; Perez-Revuelta et al. 2010). The aspartate in this motif is one of the two aspartate residues present in the catalytic site of the enzyme (Wolfe et al. 1999). Interestingly, the glycines in this motif play distinct roles in the c-secretase endoand carboxypeptidase-like activities. Whereas the first one (PSEN-Gly382) is critically