Biophysical basis for enzyme mediated deglycation in protein repair

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Project Summary/Abstract Organisms across all domains of life decorate their protein molecules with an incredible diversity of chemical modifications. Modifications on proteins are critical for their function, affecting protein structure, stability, and interaction partners. Many of the proteins and the enzymes that read, write, and erase these modifications are closely tied to human diseases ranging from neurological disorders to cancer to type 2 diabetes. While these proteins and pathways can be targets to treat these diseases, we lack a high-resolution, mechanistic understanding of how the cell installs, recognizes, and leverages certain post-translational modifications, specifically ubiquitination and spontaneous, non-enzymatic modifications. Our lab is working to understand how protein-protein interactions dynamically regulate post-translational modifications to alter proteome landscape and impact human disease. Protein glycation is an understudied post-translational modification that arises when a sugar covalently attaches to a primary amine. This process occurs spontaneously under normal physiological conditions and is a bio-marker in aging and the development, or worsening, of diseases such as diabetes, Alzheimer's disease, osteoarthritis, and atherosclerosis. Early glycation events are reversible and represent one of the few protein repair mechanisms in the cell. Deglycation is mediated by an unusual “hybrid” kinase/deglycase called Fructosamine-3-kinase (FN3K). FN3K facilitates the removal of protein-linked glycans by directly phosphorylating the attached sugar and destabilizing the sugar-protein linkage. FN3K and FN3K homologs are found in all branches of the tree of life. The glycation of intracellular proteins is not well studied, yet the conservation of FN3K and FN3K-related proteins underscores an important biological role for these enzymes. In this project, my lab will use a multidisciplinary approach, including techniques and expertise in structural biology, enzymology, and systems biology, to address sharply focused mechanistic questions regarding FN3K-mediate protein repair. We hypothesize that an improved mechanistic understanding of FN3K will reveal new biological insight into this ancient repair process, and that we can leverage this insight to better diagnose and treat diseases associated with elevated glycation. In order to distinguish our contributions from those of others, we will integrate reductionist and global approaches to develop a deeper and more complete understanding of the regulation and repair of glycated proteins. Over the five-year funding period, the goals of this project are to: (i) determine the structural and biophysical basis for FN3K-mediated protein repair (ii) systematically characterize the binding kinetics and enzymatic activity of FN3K and FN3K-RP on diverse substrates; (iii) identify sites-specific FN3K deglycation sites and their potential cross-talk with other PTMs. The successful completion of this work will establish the molecular mechanisms that govern the protein deglycation repair process and will ultimately provide needed breakthroughs in biomedical research.