IDENTIFICATION OF CATHEPSIN L AS A CHLOROQUINE TARGET USING A NOVEL AMINO ACID PROBE AND THE ROLE OF TIPARP IN BACTERIAL INFECTIONS
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Mammalian cells have evolved sophisticated mechanisms to respond to either pathogenic or metabolic stress. Such a response can activate key processes such as protein translation, autophagy, pathogen clearance, or cytokine response. These processes must be carefully regulated both during initial stress response but also after the stress resolution. This thesis discusses two independent research directions regarding metabolic or pathogen induced stress. In Chapter 1, the mechanisms by which proteins sense metabolic stress and in turn activate catabolic or anabolic processes via mTORC1 are discussed. In particular, we emphasize proteins that bind free amino acids at the lysosome. Building on this, we review established tools for microscopy studies of free amino acids in cells. There is a need for more high turn-on fluorescent amino acid probes to help visualize pools of free amino acids under various stress conditions. With this in mind, we established NS560 as a novel pan-amino acid, turn-on fluorescent probe. Chapter 2 focuses on both in vitro validation of this probe but also identification of lysosomal pockets of free amino acid storage. Using NS560, we uncover an unexpected impact of chloroquine on free amino acids and propose a mechanism by which chloroquine binds and inhibits Cathepsin L in cells. Protein post-translational modifications (PTMs) are critical for stress response to pathogens. Discussed in Chapter 3, one particular type of PTM, ADP-ribosylation is well characterized to be involved in viral and bacterial infections. It is a large, reversible, and diverse PTM that directly impacts protein structure and function. There is a lack of studies focused on mono-ADP ribosyl transferases enzymes and their role in bacterial infections. In Chapter 4, we identify TiPARP to be highly upregulated during V. cholera or S flexneri infections. We propose this enzyme is playing a role in turning off immune response.
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Cerione, Richard