Mechanistics of Prothymosin alpha and Nrf2 in the Keap1-Nrf2 mediated Oxidative Stress Response

In an effort to dissect the mechanism of interaction of IDPs, in this thesis we focus on Prothymosin a (ProTa) and nuclear factor erythroid 2-related factor 2 (Nrf2), intrinsically disordered proteins, in the Nrf2 mediated oxidative stress response. Kelch-like ECH-associated protein 1 (Keap1) is an inhibitor of Nrf2, a key transcription factor of cytoprotective genes. Under unstressed conditions, Keap1 interacts with Nrf2 in the cytoplasm via its Kelch domain and suppresses Nrf2 activity. During oxidative stress, Nrf2 is released from Keap1 and is shuttled to the nucleus, where it initiates pro cell survival gene transcription. ProTa also interacts with the Kelch domain and mediates the import of Keap1 into the nucleus to inhibit Nrf2 activity. To gain a molecular basis understanding of the oxidative stress response mechanism, the interaction between ProTa and the Kelch domain of Keap1 has been delineated using nuclear magnetic resonance spectroscopy (NMR), isothermal titration calorimetry (ITC), peptide array analysis, and site-directed mutagenesis. The results revealed that ProTa retains a high level of flexibility, even in the Kelch-bound state. Mutational analysis pinpointed that the region 38NANEENGE45 of ProTa is crucial for the interaction with the Kelch domain, while the flanking residues play relatively minor roles in the affinity of binding. A high yield purification protocol with complete backbone NMR resonance assignment lays the foundation for structural and biophysical studies of the full length-Neh2 domain of the human Nrf2. In this work the full-length Neh2 domain was used to investigate binding to Kelch in the presence of cancer causing somatic mutations. To understand the mechanistic links between Keap1 mutations and cancer pathogenesis, the molecular effects of a series of mutations (G333C, G364C, G379D, G350S, R413L, R415G, A427V, G430C, and G476R on the structural and target recognition properties of Keap1 are investigated. These mutations are found to exert differential effects on the protein stability and target binding. Together with the proposed Hinge-and-Latch mechanism of Nrf2/Keap1 binding, these results provide important insight into the molecular impact of different somatic mutations on Keap1’s function as an Nrf2 repressor