Regulation and Function of γ-Secretase

After we demonstrated g-secretase activity is catalyzed by the presenilin (PS)-containing macromolecular complex using our in vitro g-secretase assay, searching for other components of the g-secretase complex has revealed three additional proteins: nicastrin (NCT), APH-1, and PEN-2 (4) (5) (6), which are required for cellular γ-secretase activity. We have shown that presenilin-1 (PS1) or presenilin-2 (PS2) is the catalytic subunit of γ-secretase (7) (8). Other three subunits play roles in the assembly, stabilization, trafficking, and activation of PS (9). Recently, γ-secretase activating protein (GSAP), which interacts with the γ-secretase complex, is responsible for the selectivity of imatinib (10). γ-Secretase cleaves an array of substrates including APP, Notch, ErbB4, CD44, E-cadherin and other type I membrane proteins (11), reflecting the multitude of physiological functions of this protease. The mechanism that controls the specificity of γ-secretase for various substrates is elusive. Autosomal dominant inheritance of mutations in the PS1 gene is the most common cause of early-onset familial Alzheimer’s disease (FAD) (12) (13). Mutations of APP cause FAD as well (14). Cellular studies suggest both PS1 and APP mutations alter the specificity of γ-secretase, which offers an exceptional system to study the mechanism of γ-secretase specificity and the pathogenic mechanism of diseases. Mechanistic studies of γ-secretase have been hampered due to the complexity of this protease and technical challenges. To overcome these barriers, we have developed various activity-based probes and assays for kinetic analysis, characterization of the active γ-secretase complex and examination of the active site (15) (16) (17) (18) (19). We have demonstrated that the equilibrium of active complexes plays a role in determination of γ-secretase specificity and can be altered by overexpression of PEN2 or PS1 mutants (15). We have found that PS1 FAD mutations directly alter the active site of γ-secretase that affects its specificity for APP and Notch1 cleavage.

Mechanism of cell-type or tissue-specific γ-secretase regulation

γ-Secretase is widely expressed in many cell types and tissues. However, whether distinct γ-secretase complexes exist in different types of cells or tissues, contributing to substrate specificity, remains to be investigated. Notch signaling plays an essential role in determining cell fate specification and homeostasis of multiple organs (20). We have demonstrate that γ-secretase derived from hematopoietic origins is distinct from epithelial derived γ-secretase (21). Furthermore, we have found that γ-secretase specificity and complex formation alter with age and gender in mouse brain (22). Currently, we are elucidating the mechanism of how distinctive γ-secretase complexes in different cell origins and tissues control their activity and specificity, as well as selectivity to inhibitors. To define the structural and molecular basis of γ-secretase specificity, we determine the conformation of the active site from these cell lines and tissues using activity-based inhibitor probes, and determine the subunit composition of active γ-secretase complexes. These studies in examining the structure of the active site and component ratios within the active complex, combined with kinetic analyses, will provide critical insight into the structural and molecular basis of γ-secretase specificity and possibly lead to the development of more-effective γ-secretase-based treatments. These studies will address whether the γ-secretase complex and substrate specificity is associated with cell types, and possibly cell lineage and tissue development. Furthermore, we are exploring whether cell type- or tissue-specific regulatory activating and/or inhibitory molecules that modulate γ-secretase activity and specificity. Our preliminary studies indicate that such molecules present in different tissues.

Substrate feedback mechanism of γ-secretase

γ-Secretase is a macromolecular complex that contains multiple sites capable of interacting with substrates and also with inhibitors/modulators (18). Studies of APP mutations led us to identify a substrate inhibitory domain (ASID) that regulates γ-secretase activity (18) (23). This domain, which is distant to the scissile bond, interacts with an allosteric site, rather than the active site of γ-secretase (18). This work presents a model wherein γ-secretase exists in an active form capable of cleaving multiple substrates but also wherein the substrates themselves serve as key regulators to modulate their own processing by γ-secretase. Autoinhibitory domains within enzymes have widely been utilized as a regulatory mechanism to maintain enzymes in an inactive state (24), such as protease zymogen, protein kinases and phosphatases. This work showing that the APP substrate contains ASID reveals a novel and complementary strategy for regulating enzymatic activity and possibly specificity. We are investigating the action mechanism of the APP ASID and its relationship with the substrate docking site and the active site in the regulation of γ-secretase activity. We are also determining if an ASID within Notch1 substrate plays a similar role in the regulation of γ-secretase activity. Furthermore, we are examining whether this negative regulation mode serves as a novel mechanism to control γ-secretase activity and specificity for proteolysis of multiple substrates and whether these ASID binding sites can be targeted for the development of specific inhibitors.

Reconstitution of the γ-secretase complex

Reconstitution of complex systems from component molecules has been a powerful approach for investigating biological processes of macromolecules. Recently, we have developed an in vitro system (8) that allows for incorporation of highly purified bacteria expressed recombinant γ-secretase subunits into liposomes for studying γ-secretase and presenilinase (PSase) activity that is responsible for endoproteolysis of PS, a critical step for formation of active γ-secretase complexes (9). We have demonstrated that PEN2 alone is both necessary and sufficient for PS1 activation, offering a novel system to study PSase and γ-secretase. We are using a minimal set of proteins (PS1 and PEN2) to reconstitute PSase and γ-secretase activity and characterize their molecular interaction. This bottom up approach addresses the basic process of PS1 activation and provides a foundation for fully reconstituting the γ-secretase complex and elucidating the molecular mechanism of γ-secretase associated with PS1 FAD mutations. Finally, we plan to purify each of the subunits and reconstitute the entire complex using this proteoliposome platform and examine the contributions of each component either individually, or in combination with PS1 in mediating PSase and γ-secretase activity. These studies complement our current understanding of γ-secretase from cellular investigations and provide mechanical insights into γ-secretase activation and catalysis, which opens a new way to investigate γ-secretase at both molecular and atomic levels. Thus, it could facilitate the development of effective therapies for human disorders related to γ-secretase.

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