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SYNTHESIS AND BIOCHEMICAL STUDIES OF ATP ANALOG PROBES FOR POST-TRANSLATIONAL MODIFICATIONS

thesis
posted on 2024-04-25, 22:33 authored by Wanzhang PanWanzhang Pan

Post-translational modification (PTM) is an important biological process by which cells regulate their signaling pathways. De-regulation of these signaling pathways often leads to many diseases. Protein AMPylation is a recently discovered PTM that caught a great amount of attention for its involvement in neurodevelopment and neurodegeneration. However, the mechanisms by which protein AMPylation modulates these biological processes remain mostly unknown. FIC domain protein adenylyltransferase (FICD) is one of the only two known AMPylators in eukaryotes, and its physiological role remains largely unexplored. By using a chemical approach, we identified two direct substrates of FICD: Peroxiredoxin 1 (PRX1) and Peroxiredoxin 2 (PRX2). These are antioxidant enzymes responsible for protecting cells from oxidative stress, which has been implicated in many neurodegenerative diseases. In addition, we found that FICD-mediated AMPylation increased PRX1 and PRX2 enzymatic activity in vitro and their protein levels in cells. These findings established a link between FICD-mediated AMPylation and oxidative stress, suggesting a potential neuroprotective role of FICD in neurodegenerative diseases.

Protein phosphorylation is another PTM that has been under extensive study due to its widespread role in cell signaling in many biological processes such as growth, division, metabolism, membrane transport, etc... Deregulation of protein kinases, which catalyze phosphorylation reaction, is often implicated in many diseases, including cancer. To elucidate disease mechanisms and explore alternative therapeutic targets, identifying direct protein substrates of a given disease-relevant kinase is crucial but remains a major challenge. Conventional methods to study phosphorylation involved the use of radiolabeled ATP, which poses health hazards and lacks reliability due to rapid decay of radioactive isotope. In this research, we developed an alternative method with a series of novel γ-modified ATP analog probes bearing a phospho-alkyne reporter handle, and their effectiveness and efficiency for in vitro phosphorylation of recombinant proteins and proteomic substrate labeling in cell lysate were examined.

History

Degree Type

  • Doctor of Philosophy

Department

  • Chemistry

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Scott McLuckey

Additional Committee Member 2

Abram Axelrod

Additional Committee Member 3

Shiliang Tian

Additional Committee Member 4

Chengde Mao

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