Efficient production of a mature and functional gamma secretase protease

Baculoviral protein expression in insect cells has been previously used to generate large quantities of a protein of interest for subsequent use in biochemical and structural analyses. The MultiBac baculovirus protein expression system has enabled, the use of a single baculovirus to reconstitute a protein complex of interest, resulting in a larger protein yield. Using this system, we aimed to reconstruct the gamma (γ)-secretase complex, a multiprotein enzyme complex essential for the production of amyloid-β (Aβ) protein. A MultiBac vector containing all components of the γ-secretase complex was generated and expression was observed for all components. The complex was active in processing APP and Notch derived γ-secretase substrates and proteolysis could be inhibited with γ-secretase inhibitors, confirming specificity of the recombinant γ-secretase enzyme. Finally, affinity purification was used to purify an active recombinant γ-secretase complex. In this study we demonstrated that the MultiBac protein expression system can be used to generate an active γ-secretase complex and provides a new tool to study γ-secretase enzyme and its variants

Efficient production of a mature and functional gamma secretase protease

Baculoviral protein expression in insect cells has been previously used to generate large quantities of a protein of interest for subsequent use in biochemical and structural analyses. The MultiBac baculovirus protein expression system has enabled, the use of a single baculovirus to reconstitute a protein complex of interest, resulting in a larger protein yield. Using this system, we aimed to reconstruct the gamma (γ)-secretase complex, a multiprotein enzyme complex essential for the production of amyloid-β (Aβ) protein. A MultiBac vector containing all components of the γ-secretase complex was generated and expression was observed for all components. The complex was active in processing APP and Notch derived γ-secretase substrates and proteolysis could be inhibited with γ-secretase inhibitors, confirming specificity of the recombinant γ-secretase enzyme. Finally, affinity purification was used to purify an active recombinant γ-secretase complex. In this study we demonstrated that the MultiBac protein expression system can be used to generate an active γ-secretase complex and provides a new tool to study γ-secretase enzyme and its variants

The structure and function of Alzheimer's gamma secretase enzyme complex

The production and accumulation of the beta amyloid protein (Aβ) is a key event in the cascade of oxidative and inflammatory processes that characterizes Alzheimer’s disease (AD). A multi-subunit enzyme complex, referred to as gamma (γ) secretase, plays a pivotal role in the generation of Aβ from its parent molecule, the amyloid precursor protein (APP). Four core components (presenilin, nicastrin, aph-1, and pen-2) interact in a high-molecular-weight complex to perform intramembrane proteolysis on a number of membrane-bound proteins, including APP and Notch. Inhibitors and modulators of this enzyme have been assessed for their therapeutic benefit in AD. However, although these agents reduce Aβ levels, the majority have been shown to have severe side effects in pre-clinical animal studies, most likely due to the enzymes role in processing other proteins involved in normal cellular function. Current research is directed at understanding this enzyme and, in particular, at elucidating the roles that each of the core proteins plays in its function. In addition, a number of interacting proteins that are not components of γ-secretase also appear to play important roles in modulating enzyme activity. This review will discuss the structural and functional complexity of the γ-secretase enzyme and the effects of inhibiting its activity

Reconstructing the gamma-secretase complex using the MultiBac baculovirus system for expression of heterologous multi-protein complexes

A small 4 kDa peptide, referred to as β-amyloid (Aβ), which characteristically accumulates and is deposited in the Alzheimer‟s disease (AD) brain, is known to play a central role in the disease pathogenesis. The Aβ peptide is generated by proteolytic processing of its larger parent molecule, the amyloid precursor protein (APP). The final stage in this process, which releases the Aβ peptide, is the cleavage of the APP-Cterminal fragment (APP-C99/C100) by a multi-subunit enzyme complex is referred to as γ-secretase. The enzyme‟s ability to cleave a number of substrates has hampered the development of suitable inhibitors that target γ-secretase with the aim of specifically reducing Aβ production. A clearer understanding of the enzyme‟s structure and function is required in order to develop more specific therapeutic agents that will attenuate/modulate γ-secretase activity. Four core proteins, namely Presenilin (PS), Nicastrin (NCT), Anterior-pharynx defective homolog-1 (aph1aL/ aph1aS/ aph1b) and Presenilin enhancer-2 (pen2), interact to form the active γ-secretase complex. The assembly and function of the enzyme components are slowly becoming apparent. However, there are many unknown/ understudied aspects of the γ-secretase enzyme complex. Reconstructing the γ-secretase complex using appropriate protein expression models is required to obtain further insight into the structure and function of this unusual enzyme. One such in vitro system is the baculoviral expression system. The major objective of this thesis was to use a unique baculoviral expression system (referred to as MultiBac) to express four protein components of the γ-secretase complex and to assess the ability of the reconstituted enzyme to cleave the direct precursor to Aβ: APP-C100. This MultiBac is a novel highly versatile system that has been devised to generate recombinant baculovirus DNA that can be used to express multi-subunit protein complexes. The basis of the system is the use of transfer vectors to facilitate the assembly of polycistronic expression cassettes in the pFBDM vector, ultimately generating one baculovirus expressing multiple proteins, negating the need of multiple viruses for the expression of multi-subunit complexes. This, results in improved protein production and also in equivalent amounts, which is essential for any type of structural analysis, particularly for multi-subunit membrane bound complexes such as γ-secretase. In addition, the versatility of the pFBDM vector facilitates the incorporation of variations within the multi-protein complex such as mutations or different isoforms of complex components, thus providing more models which can be used to to gain further insight into critical active domains with the protein complex. Here, I detail the use of the MultiBac protein expression system to reconstruct the γ- secretase complex. Difficulties in generating some of the inserts required and expressing all of the protein components were experienced in initial attempts to use this system for reconstituting γ-secretase activity. However, a new cloning strategy was developed and implemented resulting in the successful expression of all four components in Sf21 insect cells. Preliminary activity experiments showed that complex to be active by generating a ~4 kDa „Aβ-like‟ species from the cleavage of APP-C100. In addition, incorporating octa-his and CBP tags also provided the opportunity to generate purified protein in future studies. The successful reconstruction of the γ-secretase enzyme using the MultiBac baculovirus system now provides a new model that can be utilised to gain further insight into γ-secretase function and structure. In addition, with the addition of other substrates, in particularly Notch, the system can be adapted to screen and characterise agents that selectively attenuate/modulate APP-C100 cleavage. This in turn will lead to the development of AD drugs that more specifically modify the γ-secretase enzyme‟s Aβ42 producing activity, thus producing therapeutic agents with reduced side-effects