Complex Modulation in Gamma-Secretase Interactions

The activity of the gamma-secretase protein complex provided a crucial step in limiting Alzheimer’s disease spread, but is tricky to consider as a drug target because this enzyme can be configured from different molecular modules. To understand the logic of how each of these different modules interact, we will methodically take the enzyme apart at the molecular level. Through this process, we can begin to answer which drug is best suited to (or ill-suited to) which combination of modules.

The goal of this research is to create a unique system where all the modular permutations of this enzyme can be appraised against multiple substrates. This simply cannot be done in mammalian cells as one cannot eliminate five genes at the same time with the goal of studying them by replacing them each one-by-one. However this can, and has been done in yeast, where we have created highly active recombinant enzymes from gamma secretase’s four sub-units. This means we can ask which version of the enzyme goes with which substrate.

The “gain” here will be a quick screen to find all the beneficial virtues – and/or detrimental attributes – of any candidate drug or modulator. For the former, the beneficiary might be a drug company and for the general public the benefit could be in knowing which nutrients or nutraceuticals might be worth considering as part of a preventative strategy.

Specific Aim 1: Investigating individual gamma secretase complexes with a reconstituted yeast system

(i) Validation of the system by examining the various complex permutations in our yeast system.
(ii) Definition of the substrate specificity of each complex permutation.
(iii) Test the effect of potential modulators on the substrate specificity and the structural stability of the complexes.

Specific Aim 2: Characterization of brain region specific “preferential” association between PS and Aph1 isoforms in correlation with aging

(i) Defining neuroanatomical and chronological variation of gamma-secretase enzyme complexes from wild type and premature aging mice.
(ii) Alteration of gamma-secretase complexes in mammalian cells with the environmental conditions identified in Aim 1.

Gamma secretase is a multi sub-unit enzyme lying at a common point shared between pathways of Alzheimer’s disease (AD) pathogenesis, cell differentiation and oncogenesis. While the activities of different sub-unit permutations are likely distinct there are absolute limitations in the tools of mammalian genetics to methodically alter these components. To get around this problem we have placed the enzyme in yeast, where the enzyme isoforms can be reconstituted one-by-one, and the biochemistry of enzymatic action and mode of substrate interaction can be studied with precision without the hindrance or distorting effect of multiple selection pressures that would operate in mammalian cells. We have improved a prior yeast cell paradigm by removing non-physiological mutations and by optimization to increase enzymatic output and maturation. Our studies will allow high-throughput techniques to be brought to bear on all versions of an important human enzyme to define composition and substrate selectivity – parallel studies of mammalian tissue will reveal how these complexes with different specificities are modulated during differentiation and aging.

Grantee institution at the time of this grant: University of Toronto