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Summer Research Fellowship Programme of India's Science Academies

Purification, crystallization and interaction study of human Keap1 protein with Nrf2 and direct inhibitors.

Nayana J

Christ (Deemed to be University), Hosur Main Road, Bhavani Nagar, S.G. Palya, Bengaluru, Karnataka 560029

Dr. Mukesh Kumar

Professor, Homi Bhabha National Institute and Scientific Officer (H), Protein Crystallography Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400085.

Abstract

Keap1 (Kelch-like ECH-associated protein 1) is a redox sensor protein that in humans is encoded by the Keap1 gene. At normal conditions, Keap1 binds Nrf2 through its C-terminal Kelch domain, ubiquitinates Nrf2 in the cytoplasm and targets it for degradation by the 26S proteosome ; Nrf2 level in cytoplasm is thus maintained. Stress conditions lead to suspension of Keap1–Nrf2 interactions due to modification of the important cysteine residues in Keap1 that causes conformational changes leading to release of Nrf2. Hence Nrf2 is available for translocating to the nucleus to allow transcription of cytoprotective genes to combat the cellular oxidative stress. Keap1-Nrf2 protein-protein interaction has thus become an important therapeutic target for cancer and neurodegenerative diseases. Direct inhibitors bind non-covalently to kelch domain of Keap1 and prevent Nrf2’s binding with the protein. The recombinant double mutant (E540A/E542A)] Keap1 was expressed in competent BL21 (DE3) RIL cells. Protein was purified using Ni- NTA affinity chromatography. The purified protein was subjected to crystallization by hanging drop vapour diffusion method. Interaction studies were carried out by biophysical methods such as ITC.

Keywords: Nrf2, Keap1, Direct inhibitors

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