61 research outputs found

    (1)H, (15)N and (13)C backbone resonance assignments of the Kelch domain of mouse Keap1.

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    Kelch-like ECH-associated Protein 1 (Keap1) is a multi-domain protein that functions as an inhibitor of the transcription factor nuclear factor E2-related factor 2 (Nrf2) in the cellular response to oxidative stress. Under normal conditions, Keap1 binds to Nrf2 via its C-terminal Kelch domain and the interaction ultimately leads to the ubiquitin-dependent degradation of Nrf2. It has been proposed that designing molecules to selectively disrupt the Keap1-Nrf2 interaction can be a potential therapeutic approach for enhancing the expression of cytoprotective genes. Here, we reported the (1)H, (13)C, and (15)N backbone chemical shift assignments of the Kelch domain of mouse Keap1. Further, unlabeled Nrf2 peptide containing the Kelch-binding motif was added to the (15)N-labeled Kelch sample. (1)H-(15)N HSQC spectra of the protein in the absence and presence of an equimolar concentration of the Nrf2 peptide were presented. A significant number of resonance signals were shifted upon addition of the peptide, confirming the protein-peptide interaction. The results here will not just facilitate the further studies of the binding between Keap1 and Nrf2, it will also be valuable for probing interactions between the Kelch domain and small molecules, as well as a growing list of protein targets that have been identified recently

    Molecular effects of cancer-associated somatic mutations on the structural and target recognition properties of Keap1.

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    Kelch-like ECH-associated protein 1 (Keap1) plays an important regulatory role in the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent oxidative stress response pathway. It functions as a repressor of Nrf2, a key transcription factor that initiates the expression of cytoprotective enzymes during oxidative stress to protect cells from damage caused by reactive oxygen species. Recent studies show that mutations of Keap1 can lead to aberrant activation of the antioxidant pathway, which is associated with different types of cancers. To gain a mechanistic understanding of the links between Keap1 mutations and cancer pathogenesis, we have investigated the molecular effects of a series of mutations (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C and G476R) on the structural and target recognition properties of Keap1 by using nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) and isothermal titration calorimetry (ITC). Depending on their locations in the protein, 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 in the literature, our results provide important insight into the molecular affect of different somatic mutations on Keap1\u27s function as an Nrf2 repressor

    Easily Established and Multifunctional Synthetic Nanobody Libraries as Research Tools

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    Nanobodies, or VHHs, refer to the antigen-binding domain of heavy-chain antibodies (HCAbs) from camelids. They have been widely used as research tools for protein purification and structure determination due to their small size, high specificity, and high stability, overcoming limitations with conventional antibody fragments. However, animal immunization and subsequent retrieval of antigen-specific nanobodies are expensive and complicated. Construction of synthetic nanobody libraries using DNA oligonucleotides is a cost-effective alternative for immunization libraries and shows great potential in identifying antigen-specific or even conformation-specific nanobodies. This review summarizes and analyses synthetic nanobody libraries in the current literature, including library design and biopanning methods, and further discusses applications of antigen-specific nanobodies obtained from synthetic libraries to research
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