Yumiko Kawai

Acetylation-Deacetylation of the Transcription Factor Nrf2 (Nuclear Factor Erythroid 2-related Factor 2) Regulates Its Transcriptional Activity and Nucleocytoplasmic Localization

Activation of Nrf2 by covalent modifications that release it from its inhibitor protein Keap1 has been extensively documented. In contrast, covalent modifications that may regulate its action after its release from Keap1 have received little attention. Here we show that CREB-binding protein induced acetylation of Nrf2, increased binding of Nrf2 to its cognate response element in a target gene promoter, and increased Nrf2-dependent transcription from target gene promoters. Heterologous sirtuin 1 (SIRT1) decreased acetylation of Nrf2 as well as Nrf2-dependent gene transcription, and its effects were overridden by dominant negative SIRT1 (SIRT1-H355A). The SIRT1-selective inhibitors EX-527 and nicotinamide stimulated Nrf2-dependent gene transcription, whereas resveratrol, a putative activator of SIRT1, was inhibitory, mimicking the effect of SIRT1. Mutating lysine to alanine or to arginine at Lys588 and Lys591 of Nrf2 resulted in decreased Nrf2-dependent gene transcription and abrogated the transcription-activating effect of CREB-binding protein. Furthermore, SIRT1 had no effect on transcription induced by these mutants, indicating that these sites are acetylation sites. Microscope imaging of GFP-Nrf2 in HepG2 cells as well as immunoblotting for Nrf2 showed that acetylation conditions resulted in increased nuclear localization of Nrf2, whereas deacetylation conditions enhanced its cytoplasmic rather than its nuclear localization. We posit that Nrf2 in the nucleus undergoes acetylation, resulting in binding, with basic-region leucine zipper protein(s), to the antioxidant response element and consequently in gene transcription, whereas deacetylation disengages it from the antioxidant response element, thereby resulting in transcriptional termination and subsequently in its nuclear export.

Mucopolysaccharide-degrading enzymes from the liver of the squid, Ommastrephes sloani pacificus. I. Hyaluronidase.

Abstract 1. 1. Liver extracts of the squid, Ommastrephes sloani pacificus , contain mucopolysaccharide-degrading enzymes: hyaluronidase (hyaluronate glycanohydrolase, EC 3.2.1.35), β- N -acetylhexosaminidase (EC 3.2.1.30); β-glucuronidase (EC 3.2.1.31) and chondrosulphatase (EC 3.1.6.4). 2. 2. The squid hyaluronidase which was partially purified by gel chromatography on Sephadex G-150 showed the maximum activity at pH 4.5–5.0 and the temperature of 37°. The activity was completely inhibited by Cu 2+ and Fe 3+ . 3. 3. Hyaluronate was the best substrate of the squid hyaluronidase and several sulphated mucopolysaccharides were also degraded. The digestion products of hyaluronate were identical with those obtained by bovine testicular hyaluronidase digestion, indicating that the squid liver hyaluronidase is an endo- β - N -aacetylhexosaminidase. 4. 4. This seems to be the first example of a testicular type hyaluronidase found in invertebrate tissues.

A modified method for chondrosulfatase assay

The turbidimetric method of Dodgson and Price has been modified for chondrosulfatase assay. The method involves removal of undegraded chondroitin sulfates with cetylpyridinium chloride, modification of the barium chloride-gelatin reagent, and substitution of 0.2N hydrochloric acid for the 3% trichloroacetic acid of the original method. By using this modified method, the presence of chondrosulfatase activity was demonstrated in the liver of the squid, Ommastrephes sloani pacificus.

Multiple Nuclear Localization Signals Function in the Nuclear Import of the Transcription Factor Nrf2

Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42–53) and the other (residues 587–593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494–511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins α5 and β1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin α-β heterodimer nuclear import receptor system plays a critical role in the import process.

Transcriptional Activation of the Human Gαi2 Gene Promoter through Nuclear Factor-κB and Antioxidant Response Elements

Very little is known regarding molecular mechanism(s) underlying transcriptional regulation of any G-protein gene despite the importance of G-protein expression in modulating cellular processes. Here we show that phorbol myristate acetate (PMA) and tert-butylhydroquinone (tBHQ), which induce oxidative stress in cells, up-regulate transcription of Gαi2 in K562 cells. Redox-sensing chemicals abrogated this transcriptional effect. A dominant negative I-κB double mutant (S32A/S36A) suppressed PMA-induced transcription by 54–62%, suggesting involvement of nuclear factor-κB (NF-κB). SN50, a cell-permeable peptide that inhibits nuclear import of stress-responsive transcription factors (such as NF-κB), inhibited PMA- and tBHQ-induced transcription. Deletion of an NF-κB-binding motif that maps at +10/+19 in the promoter resulted in 55–60% suppression of PMA-induced transcription, and 81% suppression of tBHQ-induced transcription. Mutation of an antioxidant response element (ARE) that maps at –84/–76 in the promoter resulted in 51 and 86% decrease in PMA- and tBHQ-induced transcription, respectively. In electrophoretic mobility shift assays, this element formed complexes with the transcription factors NF-E2p45 and Nrf2 that are prototypic for binding to the ARE, as well as with c-Fos, which can also interact with the ARE. Chromatin immunoprecipitation analysis demonstrated recruitment of these transcription factors to the promoter. Exogenously transfected Nrf2 transactivated the Gαi2 gene promoter; the cytoskeleton-associated protein, Keap1, abrogated this effect. Taken together, the present studies reveal that transcription factors that bind NF-κB and/or antioxidant response elements play an activating role in the transcription of the human Gαi2 gene.

Mucopolysaccharides from the connective tissues of the amphioxus, Branchiostoma belcherii

Abstract 1. 1. The mucopolysaccharides of amphioxus skin are more similar to those of vertebrates than to cephalopods. 2. 2. The mucopolysaccharides of the notochord differ from those of the hagfish or the lamprey.