Project Summary/Abstract
Inflammation is a tightly regulated and necessary biological process that protects humans from invading
pathogens and repairs tissue following injury. Failure to resolve inflammation is associated with the pathology
of many chronic diseases including cancers, cardiovascular diseases, diabetes, and neurodegeneration.
Complex cellular signaling, mediated by the enzymatic oxidation of polyunsaturated fatty acids (PUFA) by
cyclooxygenases and lipoxygenases, induces and then resolves inflammation, restoring the human body to
homeostasis. The structure of PUFAs also make them susceptible to autoxidation or nonenzymatic oxidation.
The resulting autoxidation products include lipid electrophiles (LE) that react with nucleophilic amino acids to
form protein post-translational modifications (PTM). These PTMs are deleterious to protein function and thus
can be toxic to the cell. Accumulation of LE PTMs is associated with the chronic diseases mentioned above
and clinical trials attempting to treat these diseases have used antioxidants and LE scavengers to prevent the
formation and accumulation of toxic PTMs. We discovered that LEs are generated in the mitochondria of cells
through an autoxidation process and exert their toxic effects by targeting mitochondrial proteins. Mapping each
LE back to the PUFA source and the pathway of formation is difficult in mammalian cells because of the
presence of multiple PUFAs and PUFA metabolizing enzymes. Therefore, we propose using Staphylococcus
aureus as a simplified model system to study PUFA autoxidation in cells. We discovered that S. aureus
autoxidizes PUFAs to LEs that deleteriously adduct cellular proteins, killing the bacterium. S. aureus is also
genetically tractable, metabolically similar to mitochondria, and does not have PUFA metabolizing enzymes.
These characteristics will allow us to use S. aureus to unambiguously define the pathways that contribute to LE
generation. S. aureus does not synthesize PUFAs, facilitating our ability to trace LEs back to their specific
PUFA source. Alkynyl PUFAs, tools that I developed and validated during my Ph.D., will enable the
identification of the protein targets of LEs. The autoxidation chemistry of alkynyl PUFAs is indistinguishable
from their native PUFA counter parts and S. aureus will metabolize alkynyl PUFAs to alkynyl LEs. The resulting
protein PTMs will be enriched by attaching an affinity tag to the alkynyl LE modified proteins through click
chemistry. Following affinity purification, quantitative proteomics will identify the protein targets of LEs for
comparison to other treatment conditions and for downstream validation of each pathway for its role in the
cellular toxicity of LEs. In sum, my research program will identify LEs formed through autoxidation, determine
their PUFA and cellular pathway source of formation, and elucidate the cellular pathways that are modified by
LEs. The findings of this research will define the molecular mechanisms of LE formation in cells and inform
future design of LE scavengers, antioxidants, and other small molecules that can modulate LE protein PTMs to
treat chronic human diseases.
