Chapter Six - γ-Secretase and the Intramembrane Proteolysis of Notch

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Abstract

γ-secretase is the crucial proteolytic activity that releases the Notch intracellular domain and is therefore a central player in the canonical Notch-signaling transduction pathway. We discuss here briefly the discovery of γ-secretase and what is known on its structure and function. Recent work also indicates that the assembly and activity of γ-secretase might be regulated by novel cell biological mechanisms. Finally we explore the recent insight that there are several γ-secretase complexes in mammalian and discuss possibilities to use γ-secretase as a drug target in Alzheimer’s disease and cancer.

Introduction

The study of the Notch-signaling pathway has contributed fundamentally to the understanding of a novel type of cellular signaling process, called regulated intramembrane proteolysis (Brown et al., 2000). This signaling process releases protein fragments at two sides of the cellular membrane, providing cell autonomous signals to the cell interior and sending instructions to neighboring cells via ligand–receptor interactions. Regulated intramembrane proteolysis is now touching a broad field of cell biological research from unicellular organisms to man, and is involved in a myriad of cellular signaling processes (Freeman, 2008, Rawson, 2003, Urban, 2009). The Notch-signaling pathway is of particular interest in this regard as receptors (Notch1-4) and ligands (Delta, Jagged in mammalian) undergo regulated intramembrane proteolysis (De Strooper et al., 1999, Ikeuchi and Sisodia, 2003, LaVoie and Selkoe, 2003, Saxena et al., 2001, Struhl and Greenwald, 1999). The study of the processing of Notch has been instrumental for our understanding of γ-secretase, the main subject of the current review. γ-Secretase is in fact a generic name, coming from the Alzheimer’s research field (Haass and Selkoe, 1993). Indeed, the amyloid precursor protein (APP), which is the precursor of the Aβ or amyloid peptide causally related to the pathogenesis of Alzheimer’s disease (AD), undergoes a very similar consecutive proteolysis as Notch (Annaert and De Strooper, 1999). γ-Secretase occurs after either α- or β-secretase cleavage of APP (Haass and Selkoe, 1993) and results in the release of the notorious Aβ peptide.

Section snippets

Regulated Intramembrane Proteolysis of Notch

Signaling of the Notch receptor is dependent on three types of proteolytic events. After the first cleavage, known as the S1 cleavage, by furin-like convertase in the secretory pathway (Blaumueller et al., 1997, Logeat et al., 1998), the heterodimeric receptor (Blaumueller et al., 1997, Logeat et al., 1998, Rand et al., 2000) proceeds to the cell surface where it is able to interact with Notch ligands presented on neighboring cells. Binding of Delta, Serrate, or Lag-2 ligands like Delta or

Discovery of γ-Secretase

In the mid-1990s genetic linkage analysis of families with autosomal dominant forms of familial AD gave the first crucial clues to the molecular identification of γ-secretase (Alzheimer’s_Disease_Collaborative_Group, 1995, Levy-Lahad et al., 1995, Rogaev et al., 1995, Sherrington et al., 1995). Missense mutations in two previous unknown genes, Presenilin 1&2 (Psen-1 and Psen-2), were sufficient to cause an aggressive, dominant inherited form of AD. It should be said that it was initially

γ-Secretase Cleaves Many Substrates

More than 60 different substrates are known to be processed by the combination sheddase/γ-secretase (reviewed in (McCarthy et al., 2009, Wakabayashi and De Strooper, 2008)) in a similar way as APP and Notch. The sheddases usually belong to the family of the ADAMs, but other sheddases such as BACE1 may cleave a more restricted panel of substrates (reviewed in (Cole and Vassar, 2008)). The released ectodomain can become a soluble ligand (e.g., secreted APP or APPs) or, in the case of Notch, bind

Presenilin: the catalytic subunit and substrate docking

Psens are multipass transmembrane proteins consisting of nine transmembrane domains (TM), with the N-terminus facing the cytosol and the short C-terminus oriented to the extracellular space (Fig. 6.1A) (Henricson et al., 2005, Laudon et al., 2005, Oh and Turner, 2005, Spasic et al., 2006). The catalytic site of Psen consists of two conserved aspartyl residues, located within TM6 en TM7 (Wolfe et al., 1999). Mutations in either of these residues results in loss of γ-secretase activity (Kimberly

γ-Secretases Are Tetrameric Complexes

Co-expression of all four components (Psen, Nct, Aph-1, and Pen-2) increased γ-secretase activity in both Drosophila and mammalian cells and reconstituted γ-secretase activity in Sf9 insect cells and in the budding yeast Saccharomyces cerevisiae (Edbauer et al., 2003, Hayashi et al., 2004, Kimberly et al., 2003, Takasugi et al., 2003, Zhang et al., 2005). The latter experiment was particularly compelling since yeast does not possess such protease activity and contains no orthologs of these four

Structure and Assembly of the Complex

Cysteine-scanning mutagenesis of Psen-1 shows that TM6 and TM7 that harbor the two catalytic aspartates, delineate a water-containing cavity inside the membrane (Sato et al., 2006, Tolia et al., 2006). Recently, it was demonstrated that TM9 and the hydrophobic domain in the large cytoplasmic loop of Psen (between TM6 and TM7) are dynamic parts of the water-containing cavity; also the conserved PAL motif in TM9 contributes to the catalytic center because it can be cross-linked to the active

Consecutive Cleavage Model for γ-Secretase

Recent biochemical studies have indicated that γ-secretase cleaves its substrates at multiple positions in a stepwise manner within their membrane domain (Qi-Takahara et al., 2005, Zhao et al., 2005) (Fig. 6.2). APP has been best investigated in that regard. The current model suggests that proteolysis of APP occurs first close to the cytoplasmic border of the membrane at the ε-site, releasing the AICD in the cytoplasm and leaving long Aβ (Aβ48,Aβ49) species in the membrane. Further cleavages

Regulation of γ-Secretase Activity

The regulation of γ-secretase activity is indeed potentially a very interesting field of research, both from a practical point of view as insight in the regulation of this protease might yield novel drug targets and from a fundamental scientific point of view as we might expect to identify unusual and unexpected novel regulatory mechanisms. Indeed, the above-discussed differential S3 cleavage of Notch supports the concept of γ-secretase being regulated to a certain extent by specific

γ-Secretase as a Drug Target: AD and Cancer

The central role of γ-secretase in Aβ generation makes it a drug target for AD. The interference with Notch signaling is on the other hand an important concern with regard to potential side effects, including gastrointestinal bleeding (van Es et al., 2005), skin cancer (Demehri et al., 2009, Nicolas et al., 2003), and (auto)-immune problems (Hadland et al., 2001, Tournoy et al., 2004). We will discuss below how the field has developed GSIs with Notch-sparing properties. However, Notch

Conclusion

It is clear that work on γ-secretase is a blooming area of research with both fundamental and clinical importance. Over the next years we will see an increasing understanding of the cell biology of this complex, trying to unravel the assembly and regulation of these fascinating enzymes, and also a progressive better understanding of the role of the different γ-secretases in different physiological functions. On the longer run, one hopes that the crystallization of the different complexes will

Acknowledgments

The research in the authors’ laboratory is supported by the Fund for Scientific Research Flanders, KULeuven, VIB, the Federal Office for Scientific Affairs, Belgium (IUAP P6/43/), a Methusalem grant of the KULeuven and the Flemish Government and Memosad (FZ-2007-200611) of the European union. EJ was supported by IWT and a short-term fellowship from EMBO.

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