Abstract

Every cell consists of carefully orchestrated biomolecules such as lipids, carbohydrates, and proteins. To maintain internal stability (homeostasis), cells maintain the right amount of these molecules at the right time and at the right place. This process is especially true for proteins since they are the foundation functional units within the cell. Proteins form structures and perform chemistry that bestows cells overarching functional roles. Cells maintain protein homeostasis (proteostasis) by modulating synthesis, folding, and degradation processes (turnover) to maintain the abundance levels for all proteins. This is the foundational kinetic model of proteostasis that is covered in this work, and it comprises protein abundance and turnover essential for protein homeostasis. When proteostasis is lost, cells may also fail to perform their internal cellular functions which will impact their external role. The sustained loss of proteostasis leads to disease. In the area of proteomics, we seek out the mechanisms of proteome change that result in the loss of normal proteostasis that are associated with disease states. As biochemists we explore the role of different proteins within biological systems and disease states. Predominantly, these studies involve isolating proteins (generally one at time) to measure abundance levels, function, and structure. In more recent years, technological advances in liquid chromatography and mass spectrometry (LC-MS) ushered in the golden age of proteomics. With LC-MS we can explore thousands of proteins in a single experiment to measure their expression levels. This work covers the fundamentals of this process as well as examples of LC-MS based proteomics for biomarker discovery and individual protein dynamics. In a sense, these experiments are like taking a snapshot of what proteins are found within a biological system at a given moment. However, cells are not static systems, rather they are dynamic systems in which proteins are being created and destroyed to maintain proteostasis. In this regard, LC-MS has recently become a powerful tool to explore protein turnover for thousands of proteins. Combined with protein abundance measurements, protein turnover yields a dynamic image of the internal state of the cell. This work applies the ideas within the kinetic model of proteostasis to explore the changes in protein homeostasis associated with Apolipoprotein E (ApoE) isoforms. ApoE isoforms are a genetic risk factor of ongoing research because of their role in disease and longevity. This work reviews some of the proposed mechanisms associated with ApoE genotype, and the LC-MS experiment we created to measure both proteome wide abundance and turnover changes associated with ApoE genotype. Our findings not only provide evidence that unifies previous ApoE studies, and it provides a benchmark for how to incorporate both quantitative and kinetic proteomics to monitor proteostasis.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

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