Investigating Factors That Affect Cis/Trans Equilibrium And Isomerization Of Xaa-Pro Peptide Bonds Using Pin1 And Its Substrates Associated With Alzheimer!S Disease

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Peptidyl-prolyl isomerase, Pin1, is an extremely important cell regulatory enzyme, participating in cell cycle checkpoints, neuronal growth and development and cellular signaling. As such, Pin1 has been shown to play a role in many diseases, such as asthma, cancer and Alzheimer!s Disease. As the baby boomer population reaches the age of increasing risk of Alzheimer!s disease, the need to understand its progression becomes increasingly important. There are many factors affecting the free energy difference between the cis and trans conformers of Xaa-Pro peptide bonds. The cis isomer of a peptide bond is more rare than the trans isomer and linked to important cell signalling and regulatory events. These rare peptide bonds are prevalent in two substrates of Pin1, the amyloid precursor protein cytoplasmic tail and the Tau protein, at high levels upon phosphorylation, a state associated with the progression of Alzheimer!s disease. In this thesis I have investigated one factor that can affect peptide bonds conformation, proline modification, within peptides corresponding to the Tau protein by Nuclear Magnetic Resonance (NMR) Spectroscopy. The purpose herein was to increase the cis conformer population for laboratory purposes, but the principles can be applied to biological situations. Specifically, modification at C! of the proline to increase the steric block to rotation stabilizes the cis conformer. In addition to the importance of understanding the cis/trans equilibrium, the features affecting the interconversion between the two conformations is also important for biological processes. Via NMR Spectroscopy, I have investigated how the rate of isomerization of the cytoplasmic tail of the amyloid precursor protein affects the levels of two separate but competing proteolytic pathways, amyloidogenic and non-amyloidogenic processing. To this end, I have generated eight mutants in the catalytic domain of Pin1 and assessed the affect of these mutations on the rate of isomerization in vitro. These mutants have a range of activity. Introduction of these mutants into cellular knockdowns of Pin1 by our collaborators reveals that the rate of isomerization regulates the levels of these processing pathways.
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