Research Project
9440621
Project Summary Many human diseases are caused when a protein or enzyme is mutated or misfolded. For example, the dysfunction of the HECT (Homologous to E6AP Carboxyl Terminus) E3 ubiquitin ligases has been linked to neurodevelopmental syndromes (i.e. Angelman, Prader-?Willi and Wolfram), Huntington?s, cancer (i.e. breast, lung, prostate, and Wilms? tumors), and viral infections (i.e. HIV, HPV, Ebola, and Influenza A/B). It is paramount that we understand how these enzymes work to aid in the future development of therapies to treat HECT E3 ubiquitin ligase dysfunction and enhance human health. The HECT E3 ubiquitin ligases are enzymes involved in the ubiquitylation-?signaling pathway that coordinate the posttranslational attachment of the 8.5 kDa signaling protein ubiquitin to their specific target proteins in the cell. The objective of this project is to understand the structural and biochemical basis for HECT-?dependent ubiquitylation. We will elucidate the unique mechanisms that the 28 human members of the HECT E3 ubiquitin ligase family use to attach ubiquitin to their intracellular substrates. All 28 HECT E3 ubiquitin ligases encoded in the human genome contain the characteristic HECT domain, consisting of an N-?terminal lobe and a C-?terminal lobe, that is responsible for catalyzing ubiquitin chain attachment to a target protein. Currently the mechanism that each of these 28 unique enzymes uses and the identities of specific residues in and around the active site required for catalysis remain unclear. The long term scientific goal of the PI is to fully investigate the 3D structures and underlying enzymology for the C-?terminal lobes of all 28 human HECT E3 ubiquitin ligases to learn how this region of each enzyme controls polyubiquitin chain assembly and linkage specificity. The major foci of this proposal will be to determine the role of dynamics and conformational flexibility in HECT-? dependent ubiquitylation (Aim 1), and to decipher the catalytic mechanisms and the role of dimerization in regulating HECT E3 ligase activity (Aim 2). Our preliminary studies using NMR spectroscopy and other biochemical approaches suggest that some of the HECT E3 ubiquitin ligases use novel mechanisms found exclusively in their HECT domain C-? terminal lobes that contain the absolutely conserved catalytic cysteine. These inherent differences provide an enticing opportunity to expand our current understanding of HECT-?dependent ubiquitylation. Our findings will offer new insight into the molecular mechanisms used by the HECT E3 ubiquitin ligases and help us learn how and why HECT E3 ubiquitin ligase dysfunction occurs and can possibly be controlled. Undergraduate students will be an essential part of the success of this R15 AREA research project. The contribution from Biochemistry and Molecular Biology (BCMB) undergraduate students at Clark University will be integral to the completion of the proposed work and, as a result of their importance in this research program, they will receive extensive guidance from the PI and will share in manuscript preparation and publication authorship.
