Yuko Nariai

Nitric oxide regulates actin reorganization through cGMP and Ca2+/calmodulin in RAW 264.7 cells

Abstract Nitric oxide (NO) has been reported to be involved in the regulation of pseudopodia formation, phagocytosis and adhesion in macrophages through the reorganization of actin. In the present study, we directly separated the globular (G) and filamentous (F) actin from quiescent or NO-stimulated macrophage-like cell line RAW 264.7 cells in order to investigate the dynamic redistribution of actin pools. We also focused on the regulatory mechanisms of actin assembly, induced by NO and its possible subsequent signaling pathway. We showed that predominant G-actin coexisted with Triton X-100-insoluble filamentous (TIF) and Triton X-100-soluble filamentous actin in resting RAW 264.7 cells. The exogenous NO produced by (±)-( E )-2-[( E )-hydroxyimino]-6-methoxy-4-methyl-5-nitro-3-hexenamide (NOR1), the endogenous NO induced by lipopolysaccharide (LPS) plus interferon-γ (IFNγ), and dibutyryl-cGMP increased the contents of TIF-actin in dose- and time-dependent manners and altered its morphology. The increase in the TIF-actin contents induced by NOR1 or LPS plus IFNγ was efficiently blocked by the radical scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide and the soluble guanylate cyclase inhibitor 1 H -[1,2,4]oxadiazolo[4,3- a ]quinoxalin-1-one or the arginine analogue N G -monomethyl- l -arginine acetate, respectively. Preincubation with the calmodulin antagonist W-7 almost completely blocked the NO-induced TIF-actin increase and morphological change. On the other hand, preincubation with C3 transferase, an inhibitor of Rho protein, efficiently prevented the change in cell morphology, but had no effect on the TIF-actin increase. We postulate that cGMP and subsequent Ca 2+ /calmodulin may be key regulators of actin reorganization in NO-stimulated RAW 264.7 cells.

PAD4 regulates proliferation of multipotent haematopoietic cells by controlling c-myc expression

Peptidylarginine deiminase 4 (PAD4) functions as a transcriptional coregulator by catalyzing the conversion of histone H3 arginine residues to citrulline residues. Although the high level of PAD4 expression in bone marrow cells suggests its involvement in haematopoiesis, its precise contribution remains unclear. Here we show that PAD4, which is highly expressed in lineage− Sca-1+ c-Kit+ (LSK) cells of mouse bone marrow compared with other progenitor cells, controls c-myc expression by catalyzing the citrullination of histone H3 on its promoter. Furthermore, PAD4 is associated with lymphoid enhancer-binding factor 1 and histone deacetylase 1 at the upstream region of the c-myc gene. Supporting these findings, LSK cells, especially multipotent progenitors, in PAD4-deficient mice show increased proliferation in a cell-autonomous fashion compared with those in wild-type mice. Together, our results strongly suggest that PAD4 regulates the proliferation of multipotent progenitors in the bone marrow by controlling c-myc expression.

Nuclear localization signal in a cancer-related transcriptional regulator protein NAC1

Nucleus accumbens-associated protein 1 (NAC1) might have potential oncogenic properties and participate in regulatory networks for pluripotency. Although NAC1 is described as a transcriptional regulator, the nuclear import machinery of NAC1 remains unclear. We found, using a point mutant, that dimer formation was not committed to the nuclear localization of NAC1 and, using deletion mutants, that the amino-terminal half of NAC1 harbored a potential nuclear localization signal (NLS). Wild type, but not mutants of this region, alone was sufficient to drive the importation of green fluorescent protein (GFP) into the nucleus. Bimax1, a synthetic peptide that blocks the importin α/β pathway, impaired nuclear localization of NAC1 in cells. We also used the binding properties of importin to demonstrate that this region is an NLS. Furthermore, the transcriptional regulator function of NAC1 was dependent on its nuclear localization activity in cells. Taken together, these results show that the region with a bipartite motif constitutes a functional nuclear import sequence in NAC1 that is independent of NAC1 dimer formation. The identification of an NAC1 NLS thus clarifies the mechanism through which NAC1 translocates to the nucleus to regulate the transcription of genes involved in oncogenicity and pluripotency.

Monoclonal Nonspecific Suppressor Factor β (MNSFβ) Inhibits the Production of TNF-α by Lipopolysaccharide-Activated Macrophages

Abstract The monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by a murine T cell hybridoma, shows a pleiotropic antigen-nonspecific suppressive function. Most recently, a cDNA encoding a subunit of MNSF (MNSFβ) has been isolated and characterized. Recombinant form of MNSFβ (rMNSFβ) inhibits lymphokine functions, as does native MNSF. In this study, we investigated whether rMNSFβ also affects macrophage function in terms of LPS-induced TNF-α production by a mouse macrophage cell line, J774. rMNSFβ suppressed the TNF-α production in a dose-dependent manner. This suppressive effect was remarkably reduced when rMNSFβ was added after 6 h of LPS stimulation. In addition, enhancement of TNF-α production by IFN-γ was also suppressed by rMNSFβ. The suppressive effect was partly neutralized by the addition of the serine/threonine phosphatase inhibitor, okadaic acid. This finding suggests that serine/threonine protein phosphatases type 1 and/or 2A may be implicated in the mechanism of action of MNSF.

The SUMO-targeted ubiquitin ligase RNF4 localizes to etoposide-exposed mitotic chromosomes: Implication for a novel DNA damage response during mitosis

RNF4, a SUMO-targeted ubiquitin ligase (STUbL), localizes to the nucleus and functions in the DNA damage response during interphase of the cell cycle. RNF4 also exists in cells undergoing mitosis, where its regulation and function remain poorly understood. Here we showed that administration of etoposide, an anticancer DNA topoisomerase II poison, to mitotic human cervical cancer HeLa cells induced SUMO-2/3-dependent localization of RNF4 to chromosomes. The FK2 antibody signals, indicative of poly/multi-ubiquitin assembly, were detected on etoposide-exposed mitotic chromosomes, whereas the signals were negligible in cells depleted for RNF4 by RNA interference. This suggests that RNF4 functions as a STUbL in the etoposide-induced damage response during mitosis. Indeed, RNF4-depletion sensitized mitotic HeLa cells to etoposide and increased cells with micronuclei. These results indicate the importance of the RNF4-mediated STUbL pathway during mitosis for the maintenance of chromosome integrity and further implicate RNF4 as a target for topo II poison-based therapy for cancer patients.

G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity.

The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (Kd = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research.

Plant Aurora kinases interact with and phosphorylate transcription factors

Aurora kinase (AUR) is a well-known mitotic serine/threonine kinase that regulates centromere formation, chromosome segregation, and cytokinesis in eukaryotes. In addition to regulating mitotic events, AUR has been shown to regulate protein dynamics during interphase in animal cells. In contrast, there has been no identification and characterization of substrates and/or interacting proteins during interphase in plants. The Arabidopsis thaliana genome encodes three AUR paralogues, AtAUR1, AtAUR2, and AtAUR3. Among them, AtAUR1 and AtAUR2 are considered to function redundantly. Here, we confirmed that both AtAUR1 and AtAUR3 are localized in the nucleus and cytoplasm during interphase, suggesting that they have functions during interphase. To identify novel interacting proteins, we used AlphaScreen to target 580 transcription factors (TFs) that are mainly functional during interphase, using recombinant A. thaliana TFs and AtAUR1 or AtAUR3. We found 133 and 32 TFs had high potential for interaction with AtAUR1 and AtAUR3, respectively. The highly AtAUR-interacting TFs were involved in various biological processes, suggesting the functions of the AtAURs during interphase. We found that AtAUR1 and AtAUR3 showed similar interaction affinity to almost all TFs. However, in some cases, the interaction affinity differed substantially between the two AtAUR homologues. These results suggest that AtAUR1 and AtAUR3 have both redundant and distinct functions through interactions with TFs. In addition, database analysis revealed that most of the highly AtAUR-interacting TFs contained a detectable phosphopeptide that was consistent with the consensus motifs for human AURs, suggesting that these TFs are substrates of the AtAURs. The AtAURs phosphorylated several highly interacting TFs in the AlphaScreen in vitro. Overall, in line with the regulation of TFs through interaction, our results indicate the possibility of phosphoregulation of several TFs by the AtAURs (280/300).

Cancer-related transcription regulator protein NAC1 forms a protein complex with CARM1 for ovarian cancer progression

NAC1 is a cancer-related transcription regulator protein that is overexpressed in various carcinomas, including ovarian, cervical, breast, and pancreatic carcinomas. NAC1 knock-down was previously shown to result in the apoptosis of ovarian cancer cell lines and to rescue their sensitivity to chemotherapy, suggesting that NAC1 may be a potential therapeutic target, but protein complex formation of intranuclear NAC1 in ovarian cancer cells remain poorly understood. In this study, analysis of ovarian cancer cell lysates by fast protein liquid chromatography on a sizing column showed that the NAC1 peak corresponded to an apparent molecular mass of 300–500 kDa, which is larger than the estimated molecular mass (58 kDa) of the protein. Liquid chromatography-tandem mass spectrometry analysis identified CARM1 as interacting with NAC1 in the protein complex. Furthermore, tissue microarray analysis revealed a significant correlation between CARM1 and NAC1 expression levels. Ovarian cancer patients expressing high levels of NAC1 and CARM1 exhibited poor prognosis after adjuvant chemotherapy. Collectively, our results demonstrate that high expression levels of NAC1 and its novel binding partner CARM1 may serve as an informative prognostic biomarker for predicting resistance to chemotherapy for ovarian cancer.

Structural basis for promotion of duodenal iron absorption by enteric ferric reductase with ascorbate

Dietary iron absorption is regulated by duodenal cytochrome b (Dcytb), an integral membrane protein that catalyzes reduction of nonheme Fe3+ by electron transfer from ascorbate across the membrane. This step is essential to enable iron uptake by the divalent metal transporter. Here we report the crystallographic structures of human Dcytb and its complex with ascorbate and Zn2+. Each monomer of the homodimeric protein possesses cytoplasmic and apical heme groups, as well as cytoplasmic and apical ascorbate-binding sites located adjacent to each heme. Zn2+ coordinates to two hydroxyl groups of the apical ascorbate and to a histidine residue. Biochemical analysis indicates that Fe3+ competes with Zn2+ for this binding site. These results provide a structural basis for the mechanism by which Fe3+ uptake is promoted by reducing agents and should facilitate structure-based development of improved agents for absorption of orally administered iron.Ganasen et al. report the crystallographic structures of human duodenal cytochrome b and its complex with ascorbate and Zn2+. This study provides mechanistic insights into how reducing agents promote the uptake of orally administered iron and may facilitate the development of such interventions.