Tae Yamamoto

Physiological Significance of Reactive Cysteine Residues of Keap1 in Determining Nrf2 Activity

ABSTRACT Keap1 and Cul3 constitute a unique ubiquitin E3 ligase that degrades Nrf2, a key activator of cytoprotective genes. Upon exposure to oxidants/electrophiles, the enzymatic activity of this ligase complex is inhibited and the complex fails to degrade Nrf2, resulting in the transcriptional activation of Nrf2 target genes. Keap1 possesses several reactive cysteine residues that covalently bond with electrophiles in vitro. To clarify the functional significance of each Keap1 cysteine residue under physiological conditions, we established a transgenic complementation rescue model. The transgenic expression of mutant Keap1(C273A) and/or Keap1(C288A) protein in Keap1 null mice failed to reverse constitutive Nrf2 activation, indicating that cysteine residues at positions 273 and 288 are essential for Keap1 to repress Nrf2 activity in vivo. In contrast, Keap1(C151S) retained repressor activity and mice expressing this molecule were viable. Mouse embryonic fibroblasts from Keap1(C151S) transgenic mice displayed decreased expression of Nrf2 target genes both before and after an electrophilic challenge, suggesting that Cys151 is important in facilitating Nrf2 activation. These results demonstrate critical roles of the cysteine residues in vivo in maintaining Keap1 function, such that Nrf2 is repressed under quiescent conditions and active in response to oxidants/electrophiles.

Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury

We recently used positional cloning to identify the transcription factor Nrf2 (NF‐E2 related factor 2) as a susceptibility gene in a murine model of oxidant‐induced acute lung injury (ALI). NRF2 binds to antioxidant response elements (ARE) and up‐regulates protective detoxifying enzymes in response to oxidative stress. This led us to investigate NRF2 as a candidate susceptibility gene for risk of development of ALI in humans. We identified multiple single nucleotide polymorphisms (SNPs) by resequencing NRF2 in ethnically diverse subjects, and one (—617 C/A) significantly (P< 0.001) diminished luciferase activity of promoter constructs containing the SNP and significantly decreased the binding affinity (P<0.001) relative to the wild type at this locus (—617 CC). In a nested case‐control study, patients with the —617 A SNP had a significantly higher risk for developing ALI after major trauma (OR 6.44; 95% CI 1.34, 30.8;P=0.021) relative to patients with the wild type (—617 CC). This translational investigation provides novel insight into the molecular mechanisms of susceptibility to ALI and may help to identify patients who are predisposed to develop ALI under at risk conditions, such as trauma and sepsis. Furthermore, these findings may have important implications in other oxidative stress related illnesses.–Marzec J. M., Christie, J. D., Reddy, S. P., Jedlicka, A. E., Vuong, H., Lanken, P. N., Aplenc, R., Yamamoto, T., Yamamoto, M., Cho, H.‐Y., Klee‐berger S. R. Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury. FASEB J. 21, 2237–2246 (2007)

Predictive base substitution rules that determine the binding and transcriptional specificity of Maf recognition elements.

Small Maf transcription factors possess a basic region‐leucine zipper motif through which they form homodimers or heterodimers with CNC and Bach proteins. Different combinations of small Maf and CNC/Bach protein dimers bind to cis‐acting DNA elements, collectively referred to as Maf‐recognition elements (MAREs), to either activate or repress transcription. As MAREs defined by function are often divergent from the consensus sequence, we speculated that sequence variations in the MAREs form the basis for selective Maf:Maf or Maf:CNC dimer binding. To test this hypothesis, we analyzed the binding of Maf‐containing dimers to variant sequences of the MARE using bacterially expressed MafG and Nrf2 proteins and a surface plasmon resonance‐microarray imaging technique. We found that base substitutions in the MAREs actually determined their binding preference for different dimers. In fact, we were able to categorize MAREs into five groups: MafG homodimer‐orientd MAREs (Groups I and II), ambivalent MAREs (Group III), MafG:Nrf2 heterodimer‐orientd MAREs (Group IV), and silent MAREs (Group V). This study thus manifests that a clear set of rules pertaining to the cis‐acting element determine whether a given MARE preferentially associates with MafG homodimer or with MafG:Nrf2 heterodimer.

Evaluation of MafG interaction with Maf recognition element arrays by surface plasmon resonance imaging technique

Specific interactions between transcription factors and cis‐acting DNA sequence motifs are primary events for the transcriptional regulation. Many regulatory elements appear to diverge from the most optimal recognition sequences. To evaluate affinities of a transcription factor to various suboptimal sequences, we have developed a new detection method based on the surface plasmon resonance (SPR) imaging technique. Transcription factor MafG and its recognition sequence MARE (Maf recognition elements) were adopted to evaluate the new method. We modified DNA immobilization procedure on to the gold chip, so that a double‐stranded DNA array was successfully fabricated. We further found that a hydrophilic flexible spacer composed of the poly (ethylene glycol) moiety between DNA and alkanethiol self‐assembled monolayers on the surface is effective for preventing nonspecific adsorption and facilitating specific binding of MafG. Multiple interaction profiles between MafG and six of MARE‐related sequences were observed by the SPR imaging technique. The kinetic values obtained by SPR imaging showed very good correlation with those obtained from electrophoretic gel mobility shift assays, although absolute values were deviated from each other. These results demonstrate that the double‐stranded DNA array fabricated with the modified multistep procedure can be applied for the comprehensive analysis of the transcription factor‐DNA interaction.

Molecular Basis Distinguishing the DNA Binding Profile of Nrf2-Maf Heterodimer from That of Maf Homodimer

Abstract Nrf2-small Maf heterodimer activates the transcription of many cytoprotective genes through the antioxidant response element and serves as a key factor in xenobiotic and oxidative stress responses. Our surface plasmon resonance-microarray binding analysis revealed that both Nrf2-MafG heterodimer and MafG homodimer bind to the consensus Maf recognition element with high affinity but bind differentially to the suboptimal binding sequences degenerated from the consensus. We examined the molecular basis distinguishing the binding profile of Nrf2-MafG heterodimer from that of MafG homodimer and found that the Ala-502 residue in the basic region of Nrf2 is a critical determinant of its binding specificity. In Maf proteins, a tyrosine resides in the position corresponding to Ala-502 in Nrf2. We prepared a mutant Nrf2 molecule in which Ala-502 was replaced with tyrosine. In surface plasmon resonance-microarray analysis, heterodimer of Nrf2(A502Y) and MafG displayed a binding specificity similar to that of MafG homodimer. The target genes activated by mutant Nrf2(A502Y)-small Maf heterodimer were largely different, albeit with some overlap, from those activated by wild-type Nrf2-small Maf, indicating that the array of target genes regulated by Nrf2-small Maf heterodimer differs substantially from that regulated by Maf homodimer in vivo. These results suggest that the distinct DNA binding profile of Nrf2-Maf heterodimer is biologically significant for Nrf2 to function as a key regulator of cytoprotective genes. Our contention is supported that the differential DNA binding specificity between Maf homodimers and Nrf2-Maf heterodimers establishes the differential gene regulation by these dimer-forming transcription factors.