Date of Award

2010

Document Type

Thesis - ECU Access Only

Publisher

Edith Cowan University

Degree Name

Doctor of Philosophy

School

School of Exercise, Biomedical and Health Sciences

Faculty

Faculty of Computing, Health and Science

First Supervisor

Professor Ralph Martins

Second Supervisor

Dr Giuseppe Verdile

Third Supervisor

Associate Professor David Groth

Abstract

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.

LCSH Subject Headings

Edith Cowan University. Faculty of Computing, Health and Science -- Dissertations

Baculoviruses -- Genetics

Alzheimer's disease.";"Multienzyme complexes

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