SEARCH

SEARCH BY CITATION

The amyoid-β peptide (Aβ) 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 Aβ in AD, either by decreasing its production or by preventing its accumulation in the brain (Karran et al. 2011). The γ-secretase complex cleaves APP into Aβ peptides and is therefore an appealing target for reducing brain Aβ levels. However, when such putative ‘disease-modifying’ γ-secretase targeting drugs were tested in Phase II and III clinical trials (Imbimbo et al. 2011), severe side-effects were observed likely because γ-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 γ-secretase requires the development of strategies that selectively lower the generation of toxic Aβ peptides without altering the processing of the other substrates. Therefore, a greater understanding of the molecular mechanisms underlying substrate specificity and catalysis in the γ-secretase complex is essential.

γ-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 γ-secretase as a drug target. From a mechanistic point of view, the γ-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 ε) 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 ε-cleavage, is probably regulated by the catalytic and product release rates at each proteolytic step and determines the length of the products.

image

Figure 1. Current model for γ-secretase function. Substrate recognition/docking into the initial substrate binding site (SBS) precedes translocation and docking into the active site. Initial endoproteolysis (ε-cleavage) releases intracellular domains from substrates and generates the actual substrate for the next cleavage. This new substrate, which is already bound to the active site, undergoes carboxypeptidase-like successive cuts that remove short peptides from its transmembrane domain. The decrease in hydrophobicity eventually results in product release. Whereas the efficiency of the endopeptidase cleavage has high biological relevance, mainly due to the processing of the Notch receptor into NICD, the functionality of the carboxypeptidase-like activity acquires pathological significance in the context of APP processing, since it determines Aβ product profiles.

Download figure to PowerPoint

Although a comprehensive picture of the γ-secretase complex will only follow elucidation of its structure at high resolution, an in-depth understanding of the function of the γ-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 Aβ products, such as those observed in the AD brain. Actually, it is proposed that γ-secretase complex containing FAD linked PSEN mutant generates longer and more aggregation-prone Aβ 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 γ-secretase complex have identified a Glycine-X-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 γ-secretase endo- and carboxypeptidase-like activities. Whereas the first one (PSEN-Gly382) is critically involved in the auto-activation of γ-secretase and the ε-cleavage (both endopeptidase activities), PSEN-Gly384 has a less critical role in the endopeptidase cleavages but remarkably regulates the carboxypeptidase-like activity. In fact, substitution of the PSEN-Gly384 with Ala (G384A) results in an aggressive PSEN FAD linked mutation (Cruts et al. 1995). In addition to their crucial roles in the catalytic mechanism, both the PSEN-Gly382 and PSEN-Gly384 residues have been implicated in substrate selectivity (Yamasaki et al. 2006). Clearly, mutagenesis studies on the highly conserved residues of the GXGD presenilin motif have provided significant mechanistical insights into the function of the γ-secretase. However, the strict functional requirements at these positions have limited the scope of such studies.

In this issue, Kretner et al. extend the analysis of the GXGD motif to the less-conserved residue X (PSEN-Leu383 in human γ-secretase). In contrast to the glycines and aspartate residues in this motif, extensive mutagenesis at position 383 in PSEN results in moderate effects on enzymatic activity and provides substantial information that corroborates the involvement of the GXGD motif in substrate selectivity and catalysis. Interestingly, this study reveals how different chemical functionalities in the molecular structure of the active site, and perhaps in the substrate binding site (Kornilova et al. 2005), influence substrate recognition/binding, endopeptidase and carboxypeptidase-like activities of the γ-secretase complex. With regard to APP processing, all of the PSEN-L383 mutants reduce generation of the shorter Aβ38 peptide independently of their effect on the endopeptidase cleavage, with the exception of the PSEN-L383N. The fact that almost all of the mutations impair the carboxypeptidase-like activity of γ-secretase indicates that strict steric requirements at this position determine this type of cleavage. In agreement with these observations, substitution with Arg and Lys, which are bulky residues or Pro, which changes and restricts backbone dihedral angles, display the more severe effect on the carboxypeptidase-like activity. In addition, Asn, but not Asp or Gln substitutions, increases the production of short Aβ peptides, indicating that the presence of a polar and uncharged residue with similar size to Leu in position PSEN-383, favours the processing of Aβ peptides towards the shorter forms, which could imply increased carboxypeptidase-like functionality. Furthermore, the study by Kretner et al. shows that mutations which impair the endoprotease activity also reduce the functionality of the carboxypeptidase-like activity and display a limited response to γ-secretase modulators (GSM). The occurrence of such a variety of effects is probably explained by changes in the structure of the enzyme, such as the conformational changes observed in PSEN mutations causing familial AD (Berezovska et al. 2005).

Collectively, the data suggest that substitutions in position 383 of PSEN may affect the overall conformation of the γ-secretase or specifically influence the carboxypeptidase-like activity. Interestingly, the other position able to dissociate endo- and carboxypeptidase-like activities is position 139 in PSEN. The PSEN-M139V mutation (causal of familial AD) dramatically impairs Aβ processing without affecting the efficiency of the ε-cleavage (Chavez-Gutierrez et al. 2012). Therefore, positions 139 and 383 in PSEN seem to be key structural determinants for the carboxypeptidase-like activity of the γ-secretase complex and perhaps, the functional analogy also implies close spatial proximity between these residues.

The mechanisms underlying substrate recognition and binding are far less understood. For instance, the contribution of the initial substrate binding site (SBS) (Fig. 1) to substrate specificity of the endopeptidase activity (ε- cleavage) is completely unexplored and in fact, little is known about the substrate specificity of the active site. Kretner et al. also show that PSEN L383 mutations regulate substrate selectivity. Mutations displaying little effect on APP endopeptidase processing are considerably less functional towards other substrates; however, the mechanism involved remains elusive until more detailed structural information is available.

In conclusion, the work of Kretner et al. (2012) provides an interesting analysis of the role of the PSEN GXGD motif in substrate recognition and the catalytic mechanism of the γ-secretase complex. This work will gain further importance when the atomic structure of the active site of the γ-secretase becomes available, that is, the structural–functional relationships revealed in this study can be projected and assimilated into a full view of the atomic structure of the γ-secretase complex.

Acknowledgements

  1. Top of page
  2. Acknowledgements
  3. References

The author has no conflict of interest to declare.

References

  1. Top of page
  2. Acknowledgements
  3. References
  • Berezovska O., Lleo A., Herl L. D., Frosch M. P., Stern E. A., Bacskai B. J. and Hyman B. T. (2005) Familial Alzheimer's disease presenilin 1 mutations cause alterations in the conformation of presenilin and interactions with amyloid precursor protein. J. Neurosci. 25, 30093017.
  • Chavez-Gutierrez L., Bammens L., Benilova I. et al. (2012) The mechanism of gamma-secretase dysfunction in familial Alzheimer disease. EMBO J. 31, 22612274.
  • Cruts M., Backhovens H., Wang S. Y. et al. (1995) Molecular genetic analysis of familial early-onset Alzheimer's disease linked to chromosome 14q24.3. Hum. Mol. Genet. 4, 23632371.
  • De Strooper B. and Annaert W. (2010) Novel research horizons for presenilins and gamma-secretases in cell biology and disease. Annu. Rev. Cell Dev. Biol. 26, 235260.
  • De Strooper B., Iwatsubo T. and Wolfe M. S. (2011) Presenilins and gamma-Secretase: Structure, Function, and Role in Alzheimer Disease. Cold Spring Harb. Perspect. Med. 2, a006304.
  • Hardy J. and Selkoe D. J. (2002) The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353356.
  • Hardy J., Duff K., Hardy K. G., Perez-Tur J. and Hutton M. (1998) Genetic dissection of Alzheimer's disease and related dementias: amyloid and its relationship to tau. Nat. Neurosci. 1, 355358.
  • Imbimbo B. P., Panza F., Frisardi V., Solfrizzi V., D'Onofrio G., Logroscino G., Seripa D. and Pilotto A. (2011) Therapeutic intervention for Alzheimer's disease with gamma-secretase inhibitors: still a viable option? Expert Opin. Investig. Drugs 20, 325341.
  • Karran E., Mercken M. and De Strooper B. (2011) The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics. Nat. Rev. Drug Discov. 10, 698712.
  • Kretner B, Fukumori A., Kuhn P.-H., Pérez-Revuelta B. I., Lichtenthaler S. F., Haass C. and Steiner H. (2012) Important functional role of residue x of the presenilin GxGD protease active site motif for APP substrate cleavage specificity and substrate selectivity of γ-secretase. J. Neurochem. 125, 144156.
  • Kornilova A. Y., Bihel F., Das C. and Wolfe M. S. (2005) The initial substrate-binding site of gamma-secretase is located on presenilin near the active site. Proc. Natl Acad. Sci. USA 102, 32303235.
  • Perez-Revuelta B. I., Fukumori A., Lammich S., Yamasaki A., Haass C. and Steiner H. (2010) Requirement for small side chain residues within the GxGD-motif of presenilin for gamma-secretase substrate cleavage. J. Neurochem. 112, 940950.
  • Steiner H., Kostka M., Romig H. et al. (2000) Glycine 384 is required for presenilin-1 function and is conserved in bacterial polytopic aspartyl proteases. Nat. Cell Biol. 2, 848851.
  • Wolfe M. S., Xia W., Ostaszewski B. L., Diehl T. S., Kimberly W. T. and Selkoe D. J. (1999) Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398, 513517.
  • Yamasaki A., Eimer S., Okochi M., Smialowska A., Kaether C., Baumeister R., Haass C. and Steiner H. (2006) The GxGD motif of presenilin contributes to catalytic function and substrate identification of gamma-secretase. J. Neurosci. 26, 38213828.