Dissecting γ-secretase function
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VIB Center for the Biology of Disease, VIB; KULeuven, Center for Human Genetics, Leuven, Belgium Read the full article ‘Important functional role of residue x of the presenilin GxGD protease active site motif for APP substrate cleavage specificity and substrate selectivity of c-secretase’ on page 144. The amyoid-b peptide (Ab) plays a central role in Alzhei- mer’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-secre- tase 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 gener- ation 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 com- posed 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 pro- cessing) 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 patholog- ical 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 character- ization of the c-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 c-secretase endo- and carboxypeptidase-like activities. Whereas the first one (PSEN-Gly382) is critically Received October 24, 2012; accepted October 29, 2012. Address correspondence and reprint requests to Lucía Ch avez- Guti errez, VIB Center for the Biology of Disease, VIB; KULeuven, Center for Human Genetics, Leuven, Belgium. E-mail: [email protected] © 2013 International Society for Neurochemistry, J. Neurochem. (2013) 125, 1--3 1 JOURNAL OF NEUROCHEMISTRY | 2013 | 125 | 1–3 doi: 10.1111/jnc.12077
Transcript of Dissecting γ-secretase function
VIB Center for the Biology of Disease, VIB; KULeuven, Center for Human Genetics, Leuven, Belgium
Read the full article ‘Important functional role of residue x of the presenilin GxGD protease active site motif for APP substrate cleavage specificityand substrate selectivity of c-secretase’ on page 144.
The amyoid-b peptide (Ab) plays a central role in Alzhei-mer’s disease (AD) (Hardy et al. 1998; Hardy and Selkoe2002). Over the past decade, multiple pharmacologicalefforts have been carried out with the aim of lowering Abin AD, either by decreasing its production or by preventingits accumulation in the brain (Karran et al. 2011). Thec-secretase complex cleaves APP into Ab peptides and istherefore an appealing target for reducing brain Ab levels.However, when such putative ‘disease-modifying’ c-secre-tase targeting drugs were tested in Phase II and III clinicaltrials (Imbimbo et al. 2011), severe side-effects wereobserved likely because c-secretase processes many differentsubstrates with key functions in the body (De Strooper et al.2011). Based on these unsuccessful trials, the lesson remainsclear, pharmacological targeting of c-secretase requires thedevelopment of strategies that selectively lower the gener-ation of toxic Ab peptides without altering the processing ofthe other substrates. Therefore, a greater understanding of themolecular mechanisms underlying substrate specificity andcatalysis in the c-secretase complex is essential.c-Secretase is an aspartyl intra-membrane protease com-
posed of four membrane proteins, with presenilin (PSEN) asthe catalytic subunit of the complex (De Strooper et al.2011). Over one hundred mutations in presenilin are knownto cause AD (http://www.molgen.ua.ac.be/ADMutations),which has greatly contributed toward the interest of thepharmaceutical industry in the c-secretase as a drug target.From a mechanistic point of view, the c-secretase complex isa unique enzyme: it hydrolyses substrates in the membrane,and interestingly, proteolysis occurs in a sequential mannerin which cleavages of distinct nature take place (Fig. 1). Thefirst cut (termed e) is in fact an endopeptidase cleavage thatreleases the c-terminal (intracellular) domain of substratesinto the cytosol and generates the actual substrate for the nextseries of cleavages. This ‘enzyme-bound’ N-terminal part isthen processed by a carboxypeptidase-like activity, whichsuccessively remove c-terminal short peptides from itshydrophobic region (transmembrane domain). The decreasein hydrophobicity eventually promotes release of N-terminal(luminal/extracellular) fragments. The carboxypeptidase-likefunctionality, defined as the number of turnovers that
the substrate undergoes after the e-cleavage, is probablyregulated by the catalytic and product release rates at eachproteolytic 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 highresolution, an in-depth understanding of the function of thec-secretase complex requires complementary approaches tofully decipher the complexity of its molecular mechanisms(Fig. 1) and to delineate the corresponding key structuraldeterminants. In this regard, dissection of the mechanismcontributing to the different activities of the enzyme(endopeptidase vs. carboxypeptidase-like proteolytic pro-cessing) is imperative. Indeed, the endopeptidase cleavagehas high physiological relevance as it regulates key cellsignalling pathways (De Strooper and Annaert 2010). On theother hand, the carboxypeptidase-like activity has patholog-ical significance since dysregulation of APP processing atthis level (altered carboxypeptidase-like functionality) maylead to an increased production of longer and moreaggregation-prone Ab products, such as those observed inthe AD brain. Actually, it is proposed that c-secretasecomplex containing FAD linked PSEN mutant generateslonger and more aggregation-prone Ab peptides (relative towt enzyme) because of ‘premature’ release of (intermediary)products (Chavez-Gutierrez et al. 2012).Efforts directed towards the structure-function character-
ization of the c-secretase complex have identified a Glycine-X-Glycine-Aspartic acid motif (GXGD, with X as any aminoacid residue) in presenilin as a key player involved incatalysis (Steiner et al. 2000; Perez-Revuelta et al. 2010).The aspartate in this motif is one of the two aspartate residuespresent in the catalytic site of the enzyme (Wolfe et al.1999). Interestingly, the glycines in this motif play distinctroles in the c-secretase endo- and carboxypeptidase-likeactivities. Whereas the first one (PSEN-Gly382) is critically
Received October 24, 2012; accepted October 29, 2012.Address correspondence and reprint requests to Lucía Ch�avez-
Guti�errez, VIB Center for the Biology of Disease, VIB; KULeuven,Center for Human Genetics, Leuven, Belgium.E-mail: [email protected]
© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 125, 1--3 1
JOURNAL OF NEUROCHEMISTRY | 2013 | 125 | 1–3 doi: 10.1111/jnc.12077
involved in the auto-activation of c-secretase and thee-cleavage (both endopeptidase activities), PSEN-Gly384has a less critical role in the endopeptidase cleavages butremarkably regulates the carboxypeptidase-like activity. Infact, substitution of the PSEN-Gly384 with Ala (G384A)results in an aggressive PSEN FAD linked mutation (Crutset al. 1995). In addition to their crucial roles in the catalyticmechanism, both the PSEN-Gly382 and PSEN-Gly384residues have been implicated in substrate selectivity(Yamasaki et al. 2006). Clearly, mutagenesis studies on thehighly conserved residues of the GXGD presenilin motifhave provided significant mechanistical insights into thefunction of the c-secretase. However, the strict functionalrequirements at these positions have limited the scope ofsuch studies.In this issue, Kretner et al. extend the analysis of the
GXGD motif to the less-conserved residue X (PSEN-Leu383in human c-secretase). In contrast to the glycines andaspartate residues in this motif, extensive mutagenesis atposition 383 in PSEN results in moderate effects onenzymatic activity and provides substantial information thatcorroborates the involvement of the GXGD motif in substrateselectivity and catalysis. Interestingly, this study reveals howdifferent chemical functionalities in the molecular structureof the active site, and perhaps in the substrate binding site(Kornilova et al. 2005), influence substrate recognition/binding, endopeptidase and carboxypeptidase-like activitiesof the c-secretase complex. With regard to APP processing,all of the PSEN-L383 mutants reduce generation of the
shorter Ab38 peptide independently of their effect on theendopeptidase cleavage, with the exception of the PSEN-L383N. The fact that almost all of the mutations impair thecarboxypeptidase-like activity of c-secretase indicates thatstrict steric requirements at this position determine this typeof cleavage. In agreement with these observations, substitu-tion 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-likeactivity. In addition, Asn, but not Asp or Gln substitutions,increases the production of short Ab peptides, indicating thatthe presence of a polar and uncharged residue with similarsize to Leu in position PSEN-383, favours the processing ofAb peptides towards the shorter forms, which could implyincreased carboxypeptidase-like functionality. Furthermore,the study by Kretner et al. shows that mutations whichimpair the endoprotease activity also reduce the functionalityof the carboxypeptidase-like activity and display a limitedresponse to c-secretase modulators (GSM). The occurrenceof such a variety of effects is probably explained by changesin the structure of the enzyme, such as the conformationalchanges 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 thec-secretase or specifically influence the carboxypeptidase-like activity. Interestingly, the other position able to disso-ciate endo- and carboxypeptidase-like activities is position139 in PSEN. The PSEN-M139V mutation (causal of
Fig. 1 Current model for c-secretase function. Substrate recognition/docking into the initial substrate binding site (SBS) precedestranslocation and docking into the active site. Initial endoproteoly-
sis (e-cleavage) releases intracellular domains from substrates andgenerates the actual substrate for the next cleavage. This newsubstrate, which is already bound to the active site, undergoes
carboxypeptidase-like successive cuts that remove short peptides
from its transmembrane domain. The decrease in hydrophobicityeventually results in product release. Whereas the efficiency ofthe endopeptidase cleavage has high biological relevance, mainly
due to the processing of the Notch receptor into NICD, the functionalityof the carboxypeptidase-like activity acquires pathological significancein the context of APP processing, since it determines Ab product
profiles.
© 2013 International Society for Neurochemistry, J. Neurochem. (2013) 125, 1--3
2 Review
familial AD) dramatically impairs Ab processing withoutaffecting the efficiency of the e-cleavage (Chavez-Gutierrezet al. 2012). Therefore, positions 139 and 383 in PSEN seemto be key structural determinants for the carboxypeptidase-like activity of the c-secretase complex and perhaps, thefunctional analogy also implies close spatial proximitybetween these residues.The mechanisms underlying substrate recognition and
binding are far less understood. For instance, the contributionof the initial substrate binding site (SBS) (Fig. 1) to substratespecificity of the endopeptidase activity (e- cleavage) iscompletely unexplored and in fact, little is known about thesubstrate specificity of the active site. Kretner et al. alsoshow that PSEN L383 mutations regulate substrate selectiv-ity. Mutations displaying little effect on APP endopeptidaseprocessing are considerably less functional towards othersubstrates; however, the mechanism involved remains elu-sive 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 motifin substrate recognition and the catalytic mechanism of thec-secretase complex. This work will gain further importancewhen the atomic structure of the active site of the c-secretasebecomes available, that is, the structural–functional relation-ships revealed in this study can be projected and assimilatedinto a full view of the atomic structure of the c-secretasecomplex.
Acknowledgements
The author has no conflict of interest to declare.
References
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Review 3
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