Microcystin induction small Maf protein involve in transcriptional regulation of GST from freshwater mussel Cristaria plicata.

The small Mafs, MafF, MafG and MafK play critical roles in morphogenesis and homeostasis through associating with Cap "n" Collar family of transcription factors. In this study, we tried to identify a small Maf protein in the freshwater mussel Cristaria plicata. The MafK cDNA of C. plicata, designated as CpMafK, was cloned from the hemocytes using degenerate primers by the rapid amplification of cDNA ends PCR. The full length cDNA of CpMafK is 2170 bp, which includes an open reading frame of 570 bp, encoding 189 amino acids. CpMafK possesses four conserved domains and shows a low level (54-63%) of sequence similarity to small Mafs from other species. The results of Real-time quantitative PCR revealed that CpMafK mRNA was constitutively expressed in tissues, and the highest expression level was in hepatopancreas. After microcystin challenge, the expression levels of CpMafK mRNA were up-regulated in hemocytes and hepatopancreas. The cDNA of CpMafK was cloned into the plasmid pET-32, and the recombinant protein was expressed in Escherichia coli BL21(DE3). CpMafK could combine to the promoters of CpGST1 and CpGST2 with high-affinity in vitro. Therefore, CpMafK could regulate the expression of detoxification.

Small Maf deficiency recapitulates the liver phenotypes of Nrf1- and Nrf2-deficient mice.

Nrf1 and Nrf2 (NF-E2-related factors 1 and 2, respectively) are transcription factors that belong to the Cap'n'collar (CNC) family and play critical roles in various tissues, including the liver. Liver-specific Nrf1 knockout mice show hepatic steatosis, accompanied by dysregulation of various metabolic genes. Nrf2 knockout mice show impairment in the induction of antioxidant and xenobiotic-metabolizing enzyme genes. Although it has been shown that small Maf (sMaf) proteins act as obligatory partners of CNC proteins, their precise contributions to the function of CNC proteins remain unclear especially in the context of adult liver functions. To address this issue, we generated mice that conditionally lack expression of all sMaf proteins in the liver. The liver-specific sMaf-deficient mice develop hepatic steatosis and dysregulation of genes involved in lipid and amino acid metabolism and proteasomal subunit expression. Importantly, the gene expression profiles in the sMaf-deficient livers share a strong similarity with those in Nrf1-deficient livers. In addition, the basal expression levels of a number of Nrf2 target genes were diminished in the sMaf-deficient livers. These results provide the first genetic evidence that sMaf proteins are indispensable for liver functions as heterodimeric partners for Nrf1 and Nrf2.

Inhibitor of differentiation proteins protect against oxidative stress by regulating the antioxidant-mitochondrial response in mouse beta cells.

AIMS/HYPOTHESIS: Oxidative stress is implicated in beta cell glucotoxicity in type 2 diabetes. Inhibitor of differentiation (ID) proteins are transcriptional regulators induced by hyperglycaemia in islets, but the mechanisms involved and their role in beta cells are not clear. Here we investigated whether or not oxidative stress regulates ID levels in beta cells and the role of ID proteins in beta cells during oxidative stress. METHODS: MIN6 cells were cultured in H2O2 or ribose to induce oxidative stress. ID1, ID3 and small MAF proteins (MAFF, MAFG and MAFK) were inhibited using small interfering RNA. Isolated islets from Id1(-/-), Id3(-/-) and diabetic db/db mice were used. RESULTS: ID1-4 expression was upregulated in vivo in the islets of diabetic db/db mice and stimulated in vitro by ribose and H2O2. Id1/3 inhibition reduced the expression of multiple antioxidant genes and potentiated oxidative stress-induced apoptosis. This finding was associated with increased levels of intracellular reactive oxygen species, altered mitochondrial morphology and reduced expression of Tfam, which encodes a mitochondrial transcription factor, and respiratory chain components. Id1/3 inhibition also reduced the expression of small MAF transcription factors (MafF, MafG and MafK), interacting partners of nuclear factor, erythroid 2-like 2 (NFE2L2), master regulator of the antioxidant response. Inhibition of small MAFs reduced the expression of antioxidant genes and potentiated oxidative stress-induced apoptosis, thus recapitulating the effects of Id1/3 inhibition. CONCLUSIONS/INTERPRETATION: Our study identifies IDs as a novel family of oxidative stress-responsive proteins in beta cells. IDs are crucial regulators of the adaptive antioxidant-mitochondrial response that promotes beta cell survival during oxidative stress through a novel link to the NFE2L2-small MAF pathway.

Declining signal dependence of Nrf2-MafS-regulated gene expression correlates with aging phenotypes.

Aging is a degenerative process characterized by declining molecular, cell and organ functions, and accompanied by the progressive accumulation of oxidatively damaged macromolecules. This increased oxidative damage may be causally related to an age-associated dysfunction of defense mechanisms, which effectively protect young individuals from oxidative insults. Consistently, older organisms are more sensitive to acute oxidative stress exposures than young ones. In studies on the Drosophila Nrf2 transcription factor CncC, we have investigated possible causes for this loss of stress resistance and its connection to the aging process. Nrf2 is a master regulator of antioxidant and stress defense gene expression with established functions in the control of longevity. Here, we show that the expression of protective Nrf2/CncC target genes in unstressed conditions does not generally decrease in older flies. However, aging flies progressively lose the ability to activate Nrf2 targets in response to acute stress exposure. We propose that the resulting inability to dynamically adjust the expression of Nrf2 target genes to the organism's internal and external conditions contributes to age-related loss of homeostasis and fitness. In support of this hypothesis, we find the Drosophila small Maf protein, MafS, an Nrf2 dimerization partner, to be critical to maintain responsiveness of the Nrf2 system: overexpression of MafS in older flies preserves Nrf2/CncC signaling competence and antagonizes age-associated functional decline. The maintenance of acute stress resistance, motor function, and heart performance in aging flies overexpressing MafS supports a critical role for signal responsiveness of Nrf2 function in promoting youthful phenotypes.

The Keap1-Nrf2 system as an in vivo sensor for electrophiles.

The Keap1-Nrf2 regulatory system plays a central role in cytoprotection from electrophilic and oxidative stress. In unstressed conditions, Nrf2 is constantly ubiquitinated by the Cul3-Keap1 ubiquitin E3 ligase complex and is degraded in the proteasome. Upon the exposure to electrophilic and oxidative stress, reactive cysteine residues in Keap1 are covalently modified, which abrogates the E3 ligase activity of the Cul3-Keap1 complex. Consequently Nrf2 is stabilized and induces the transcription of various cytoprotective genes. Structural analyses have revealed the overall structure of the Keap1 homodimer as well as structural features of the association between Keap1 and Nrf2, which has greatly enhanced our understanding of the molecular mechanisms involved in the regulation of the Keap1-Nrf2 system. Recently nitric oxide signaling has been shown to activate Nrf2, suggesting that Nrf2 is a mediator of the cytoprotective effect of nitric oxide. Analyses of Nrf2-null mice have revealed a critical contribution of Nrf2 to the protection from various diseases caused by electrophilic and oxidative stress. In contrast, constitutive activation of Nrf2 has been found in many cancers, resulting in resistance against chemotherapy and radiotherapy in cancer cells. Thus, Nrf2 is a promising target for drug development. The development of Nrf2 inducers and inhibitors is an important challenge for enhancing therapies for stress-induced diseases and cancers, respectively.

Molecular determinants for small Maf protein control of platelet production.

MafG and p45 possess basic region-leucine zipper (bZip) domains and form a heterodimer called NF-E2, a key regulator of megakaryopoiesis. NF-E2 binds to the Maf recognition element (MARE) and activates transcription of many platelet genes. Since the bZip domain, which mediates DNA binding and heterodimerization, is the only functional domain established for MafG, it has been assumed that MafG is required only for p45 binding to MARE and to facilitate p45-mediated transcriptional activation. Analysis of the C-terminal region of MafG, which is distinct from the bZip domain, revealed that this region contains a nuclear matrix-targeting signal. We used a transgenic complementation rescue assay to delineate the function of the MafG C terminus in vivo. Transgenic mice expressing a mutant MafG protein lacking the C terminus (MafGΔC) were crossed into a MafG-null background. The compound mutant mice displayed severe thrombocytopenia and splenomegaly, which phenocopied p45-null mice. The MafG C terminus is essential for proplatelet formation and platelet gene activation but not for p45 binding to MARE. These results demonstrate that the MafG C terminus is required for NF-E2 function and suggest that efficient targeting of NF-E2 to a specific nuclear scaffold is important to achieve high-level activity.

Heterodimerization with small Maf proteins enhances nuclear retention of Nrf2 via masking the NESzip motif.

Nrf2 is the key transcription factor regulating the antioxidant response. When exposed to oxidative stress, Nrf2 translocates to cell nucleus and forms heterodimer with small Maf proteins (sMaf). Nrf2/sMaf heterodimer binds specifically to a cis-acting enhancer called antioxidant response element and initiates transcription of a battery of antioxidant and detoxification genes. Nrf2 possesses a NESzip motif (nuclear export signal co-localized with the leucine zipper (ZIP) domain). Heterodimerization with MafG via ZIP-ZIP binding enhanced Nrf2 nuclear retention, which could be abrogated by the deletion of the ZIP domain or site-directed mutations targeting at the ZIP domain. In addition, dimerization with MafG precluded Nrf2zip/CRM1 binding, suggesting that Nrf2/MafG heterodimerization may simultaneously mask the NESzip motif. MafG-mediated nuclear retention may enable Nrf2 proteins to evade cytosolic proteasomal degradation and consequently stabilize Nrf2 signaling. For the first time, we show that under the physiological condition, the NESzip motif can be switched-off by heterodimerization.