Date of Award
M. Lane Gilchrist
Proteoliposome, Transmembrane Protein, Lipid Microenvironment, High-throughput, Tethering, Phase Separation
Gamma-Secretase (γ-secretase) is a transmembrane protease of increasing interest, which has been shown to have significant connections to both cancer and Alzheimer’s disease. γ-secretase cleaves both Notch-1, a transmembrane signaling protein, and Amyloid precursor protein (APP), a transmembrane protein whose cleavage may result in the formation of β-amyloid plaques in the brain. Notch-1 and APP are widely studied proteins that have substantial impacts on the development and proliferation of cancer and Alzheimer’s disease, respectively. Notch-1 partakes in the signaling of apoptosis in damaged and mutated cells, thus its cleavage by γ-secretase within the plasma membrane has ramifications on cell growth and proliferation. However, the APP molecule is the key protein in the metabolic pathway that produces small amyloid fragments. These fragments, in undesirable conditions, have the propensity to aggregate and form, as stated above, amyloid plaques, depending on the fragment length. These plaques have been long believed to inhibit neuronal function if they are not degraded or removed from the intracellular space, specifically in the brain.
Due to these widespread mental and physical health impacts, isolation and modulation of the cleavage of such proteins in intact, controlled bilayers in a highly reproducible, and potentially high-throughput, process is a key goal in understanding these and a vast array of intramembrane proteases for the development of pharmaceutical therapies. The work presented looks to the development of one such platform, yielding crucial spatial and temporal information within these complex lipid microenvironments. Synthetic, biomimetic membranes were studied and manipulated to develop biologically relevant systems in which to resuspend isolated proteins. A formulation of sphingomyelin, 1,2-Dioleoyl-sn-phosphatidylcholine (DOPC), and cholesterol was chosen due to its attributes in resembling fundamental lipodomics within a human brain cell. It is shown that this canonical formulation and subsequent formulations with added complex mixtures, yield a lipid system that retains visible phase separation to a quantifiable degree. These lipid formulations, when fused with solid silica support structures such as planar surfaces or silica microbeads, allows for the reconstitution of the three of proteins of interest.
These assay and high throughput platforms are essential to understanding key functions and potential modulations of these protein pathways, however this approach does not fully replicate the biological environment these proteins experience within an active cell. Two approaches are shown in this work to increase the biological relevancy of these platforms. Tethering of the solid support structures with a series of polyethylene glycol (PEG) polymers culminating in a functionalized capping moiety that can yield overall increases to protein mobility, and added functionality of the platform. Additionally, added dopants of more complex lipid components into the basic lipid membrane analogue shows the ability to increase complexity of the formulation and closes the gap between the synthetic membrane and the protein’s true biological lipid environment.
These platforms are highly robust and rugged in nature and lend themselves to be useful in future high-throughput screening and functional assay processes in pharmaceutical research. The coupling of both planar surface support structures and micro bead structures in tandem can be analyzed through confocal, super-resolution, and atomic force microscopy, leading to a fuller understanding of these complex spatial reaction-diffusion systems prevalent within human cells. The systems developed in this research, apart from being tested with the aforementioned proteins, are not protein-specific and thus could yield a viable platform on which to test any number of isolated transmembrane proteins in a highly reproducible manner.
Houlihan, William J., "Modification of Lipid Microenvironments on Solid Support Structures for Use in Transmembrane Protein Assays" (2019). CUNY Academic Works.