Nrf2 regulates the cellular oxidative stress response, whereas Keap1 represses Nrf2 through its molecular interaction. To elucidate the molecular mechanism of the Keap1 and Nrf2 interaction, we resolved the six-bladed beta propeller crystal structure of the Kelch/DGR and CTR domains of mouse Keap1 and revealed that extensive inter- and intrablade hydrogen bonds maintain the structural integrity and proper association of Keap1 with Nrf2. A peptide containing the ETGE motif of Nrf2 binds the beta propeller of Keap1 at the entrance of the central cavity on the bottom side via electrostatic interactions with conserved arginine residues. We found a somatic mutation and a gene variation in human lung cancer cells that change glycine to cysteine in the DGR domain, introducing local conformational changes that reduce Keap1's affinity for Nrf2. These results provide a structural basis for the loss of Keap1 function and gain of Nrf2 function.

Nrf2 is the regulator of the oxidative/electrophilic stress response. Its turnover is maintained by Keap1-mediated proteasomal degradation via a two-site substrate recognition mechanism in which two Nrf2-Keap1 binding sites form a hinge and latch. The E3 ligase adaptor Keap1 recognizes Nrf2 through its conserved ETGE and DLG motifs. In this study, we examined how the ETGE and DLG motifs bind to Keap1 in a very similar fashion but with different binding affinities by comparing the crystal complex of a Keap1-DC domain-DLG peptide with that of a Keap1-DC domain-ETGE peptide. We found that these two motifs interact with the same basic surface of either Keap1-DC domain of the Keap1 homodimer. The DLG motif works to correctly position the lysines within the Nrf2 Neh2 domain for efficient ubiquitination. Together with the results from calorimetric and functional studies, we conclude that different electrostatic potentials primarily define the ETGE and DLG motifs as a hinge and latch that senses the oxidative/electrophilic stress.

The overproduction of reactive oxygen species (ROS) generates oxidative stress in cells. Oxidative stress results in various pathophysiological conditions, especially cancers and neurodegenerative diseases (NDD). The Keap1-Nrf2 [Kelch-like ECH-associated protein 1-nuclear factor (erythroid-derived 2)-like 2] regulatory pathway plays a central role in protecting cells against oxidative and xenobiotic stresses. The Nrf2 transcription factor activates the transcription of several cytoprotective genes that have been implicated in protection from cancer and NDD. The Keap1-Nrf2 system acts as a double-edged sword: Nrf2 activity protects cells and makes the cell resistant to oxidative and electrophilic stresses, whereas elevated Nrf2 activity helps in cancer cell survival and proliferation. Several groups in the recent past, from both academics and industry, have reported the potential role of Nrf2-mediated transcription to protect from cancer and NDD, resulting from mechanisms involving xenobiotic and oxidative stress. It suggests that the Keap1-Nrf2 system is a potential therapeutic target to combat cancer and NDD by designing and developing modulators (inhibitors/activators) for Nrf2 activation. Herein, we review and discuss the recent advancement in the regulation of the Keap1-Nrf2 system, its role under physiological and pathophysiological conditions including cancer and NDD, and modulators design strategies for Nrf2 activation.

Recognition of acetylated chromatin by the bromodomains and extra-terminal domain (BET) family proteins is a hallmark for transcriptional activation and anchoring viral genomes to mitotic chromosomes of the host. One of the BET family proteins BRD2 interacts with acetylated chromatin during mitosis and leads to transcriptional activation in culture cells. Here, we report the crystal structures of the N-terminal bromodomain of human BRD2 (BRD2-BD1; residues 74 -194) in complex with each of three different Lys-12-acetylated H4 peptides. The BRD2-BD1 recognizes the H4 tail acetylated at Lys-12 (H4K12ac), whereas the side chain of hypoacetylated Lys-8 of H4 binds at the cavity of the dimer interface of BRD2-BD1. From binding studies, we identified the BRD2-BD1 residues that are responsible for recognition of the Lys-12-acetylated H4 tail. In addition, mutation to Lys-8 in the Lys-12-acetylated H4 tail decreased the binding to BRD2-BD1, implicating the critical role of Lys-8 in the Lys-12-acetylated H4 tail for the recognition by BRD2-BD1. Our findings provide a structural basis for deciphering the histone code by the BET bromodomain through the binding with a long segment of the histone H4 tail, which presumably prevents erasure of the histone code during the cell cycle.In eukaryotes, genomic DNA is complexed with core histones, consisting of two H2A-H2B dimers and one H3-H4 tetramer, to form a nucleoprotein architecture called the nucleosome (1, 2). These four core histones include a central core domain, and N-and C-terminal tail regions. The N-terminal histone tails, especially that of histone H4, are rich in lysine residues, onto which several different acetyltransferases and methyltransferases covalently add post-translational modifications (3, 4). The histone codes defined by combinations of such a histone tail modification are considered as a key regulatory mechanism for DNA metabolisms that trigger alteration of the chromatin structure and/or association of several different trans-acting factors (5-7). Among these post-translational modifications, acetylation of the histone tails and its recognition are typical hallmarks for activation of chromatin DNA (4, 7, 8, 10). The acetylated N-terminal tails of the histones are selectively recognized by the bromodomain, an ϳ110-amino acid-long domain found in several chromatin-associated factors, including nuclear histone acetyltransferases, ATP-dependent chromatin-remodeling factors, and the bromodomain and extra-terminal domain (BET) 6 family of nuclear proteins (11, 12). The human BET family, including BRD2, BRD3, BRD4, and BRDT, has a unique architecture with two tandem bromodomains and a conserved extra-terminal domain (13-15). In the intact nuclei, the BET protein BRD2 associates through its bromodomains mainly with the acetylated lysine 12 (K12ac) of H4, one of the active marks of chromatin, and activates transcription (16). Another study indicates that BRD2 binds chromatin containing Lys-12-or Lys-5-acetylated histone H4, although it scarcely bound to those con...

PDB Reference: peptide complex, 2z32.The Nrf2 transcription factor, which plays important roles in oxidative and xenobiotic stress, is negatively regulated by the cytoplasmic repressor Keap1. The -propeller/Kelch domain of Keap1, which is formed by the double-glycine repeat and C-terminal region domains (Keap1-DC), interacts directly with the Neh2 domain of Nrf2. The nuclear oncoprotein prothymosin (ProT ) also interacts directly with Keap1 and may play a role in the dissociation of the Keap1-Nrf2 complex. The structure of Keap1-DC complexed with a ProT peptide (amino acids 39-54) has been determined at 1.9 Å resolution. The Keap1-bound ProT peptide possesses a hairpin conformation and binds to the Keap1 protein at the bottom region of the -propeller domain. Complex formation occurs as a consequence of their complementary electrostatic interactions. A comparison of the present structure with recently reported Keap1-DC complex structures revealed that the DLG and ETGE motifs of the Neh2 domain of Nrf2 and the ProT peptide bind to Keap1 in a similar manner but with different binding potencies.

The BET (bromodomains and extra terminal domain) family proteins recognize acetylated chromatin through their bromodomain and act as transcriptional activators. One of the BET proteins, BRD2, associates with the transcription factor E2F, the mediator components CDK8 and TRAP220, and RNA polymerase II, as well as with acetylated chromatin during mitosis. BRD2 contains two bromodomains (BD1 and BD2), which are considered to be responsible for binding to acetylated chromatin. The BRD2 protein specifically recognizes the histone H4 tail acetylated at Lys12. Here, we report the crystal structure of the N-terminal bromodomain (BD1, residues 74-194) of human BRD2. Strikingly, the BRD2 BD1 protein forms an intact dimer in the crystal. This is the first observation of a homodimer among the known bromodomain structures, through the buried hydrophobic core region at the interface. Biochemical studies also demonstrated BRD2 BD1 dimer formation in solution. The two acetyllysine-binding pockets and a negatively charged secondary binding pocket, produced at the dimer interface in BRD2 BD1, may be the unique features that allow BRD2 BD1 to selectively bind to the acetylated H4 tail.

Histone acetylation constitutes an epigenetic mark for transcriptional regulation. Here we developed a fluorescent probe to visualize acetylation of histone H4 Lys12 (H4K12) in living cells using fluorescence resonance energy transfer (FRET) and the binding of the BRD2 bromodomain to acetylated H4K12. Using this probe designated as Histac-K12, we demonstrated that histone H4K12 acetylation is retained in mitosis and that some histone deacetylase (HDAC) inhibitors continue to inhibit cellular HDAC activity even after their removal from the culture. In addition, a small molecule that interferes with ability of the bromodomain to bind to acetylated H4K12 could be assessed using Histac-K12 in cells. Thus, Histac-K12 will serve as a powerful tool not only to understand the dynamics of H4K12-specific acetylation but also to characterize small molecules that modulate the acetylation or interaction status of histones.

Gankyrin is an oncoprotein commonly overexpressed in most hepatocellular carcinomas. Gankyrin interacts with S6 ATPase of the 19S regulatory particle of the 26S proteasome and enhances the degradation of the tumor suppressors pRb and p53. Here, we report the structure of gankyrin in complex with the Cterminal domain of S6 ATPase. Almost all of the seven ankyrin repeats of gankyrin interact, through its concave region, with the C-terminal domain of S6 ATPase. The intermolecular interactions occur through the complementary charged residues between gankyrin and S6 ATPase. Biochemical studies based on the structure of the complex revealed that gankyrin interacts with pRb in both the presence and absence of S6 ATPase; however, the E182 residue in gankyrin is essential for the pRb interaction. These results provide a structural basis for the involvement of gankyrin in the pRb degradation pathway, through its association with S6 ATPase of the 26S proteasome.