Nucleus Accumbens-Associated Protein 1 Binds DNA Directly through the BEN Domain in a Sequence-Specific Manner.

Nucleus accumbens-associated protein 1 (NAC1) is a nuclear protein that harbors an amino-terminal BTB domain and a carboxyl-terminal BEN domain. NAC1 appears to play significant and diverse functions in cancer and stem cell biology. Here we demonstrated that the BEN domain of NAC1 is a sequence-specific DNA-binding domain. We selected the palindromic 6 bp motif ACATGT as a target sequence by using a PCR-assisted random oligonucleotide selection approach. The interaction between NAC1 and target DNA was characterized by gel shift assays, pull-down assays, isothermal titration calorimetry (ITC), chromatin-immunoprecipitation assays, and NMR chemical shifts perturbation (CSP). The solution NMR structure revealed that the BEN domain of human NAC-1 is composed of five conserved α helices and two short ß sheets, with an additional hitherto unknown N-terminal α helix. In particular, ITC clarified that there are two sequential events in the titration of the BEN domain of NAC1 into the target DNA. The ITC results were further supported by CSP data and structure analyses. Furthermore, live cell photobleaching analyses revealed that the BEN domain of NAC1 alone was unable to interact with chromatin/other proteins in cells.

Pregnancy by Assisted Reproductive Technology Is Associated with Shorter Telomere Length in Neonates.

Telomere length (TL) influences the development of lifestyle-related diseases, and neonatal TL may influence their prevalence. Various factors have been reported to affect neonatal TL. Although the fetus is exposed to multiple conditions in utero, the main factors affecting the shortening of neonatal TL are still not known. In this study, we sought to identify factors that influence fetal TL. A total of 578 mother-newborn pairs were included for TL analysis. TL was measured in genomic DNA extracted from cord blood samples using quantitative PCR. The clinical factors examined at enrollment included the following intrauterine environmental factors: maternal age, assisted reproductive technology (ART) used, body mass index (BMI), gestational diabetes mellitus (GDM), maternal stress, smoking, alcohol consumption, preterm delivery, small-for-gestational-age, neonatal sex, and placental weight. Univariate and multivariate regression analyses were used to verify the relationship between neonatal TL and these clinical factors. The median neonatal TL to single-copy gene ratio was 1.0. Pregnancy with ART was among the 11 factors associated with shorter neonatal TL. From multiple regression analysis, we determined that neonatal TL was significantly shorter for pregnancies in the ART group than in the other groups. We conclude that pregnancy with ART is associated with shorter neonatal TL.

Elucidation of the aberrant 3' splice site selection by cancer-associated mutations on the U2AF1.

The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.

Generation and characterization of antagonistic anti-human interleukin (IL)-18 monoclonal antibodies with high affinity: Two types of monoclonal antibodies against full-length IL-18 and the neoepitope of inflammatory caspase-cleaved active IL-18.

Interleukin-18 (IL-18) is a pro-inflammatory cytokine that evokes both innate and acquired immune responses. IL-18 is initially synthesized as an inactive precursor and the cleavage for processing into a mature, active molecule is mediated by pro-inflammatory caspases following the activation of inflammasomes. Two types of monoclonal antibodies were raised: anti-IL-1863-68 antibodies which recognize full-length1-193 and cleaved IL-18; and anti-IL-18 neoepitope antibodies which specifically recognize the new N-terminal 37YFGKLESK44 of IL-18 cleaved by pro-inflammatory caspase-1/4. These mAbs were suitable for Western blotting, capillary Western immunoassay (WES), immunofluorescence, immunoprecipitation, and function-blocking assays. WES analysis of these mAbs allowed visualization of the IL-18 bands and provided a molecular weight corresponding to the pro-inflammatory caspase-1/4 cleaved, active form IL-1837-193, and not to the inactive precursor IL-18, in the serum of patients with adult-onset Still's disease (6/14, 42%) and hemophagocytic activation syndrome (2/6, 33%). These monoclonal antibodies will be very useful in IL-18 and inflammasome biology and for diagnostic and therapeutic strategies for inflammatory diseases.

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.

Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography.

Nucleophosmin (NPM1) is a multifunctional phosphoprotein which plays important roles in diverse biological processes. NPM1 can form homo- or hetero-oligomers through its N-terminal region, and bind DNA and RNA through its C-terminal region. However, the monomer-oligomer distribution of NPM1, and the extent of NPM1 binding and unbinding to RNA in living cells, are not fully understood. In this work, we analysed molecular complexes of NPM1 using size exclusion chromatography. We found that a substantial fraction of NPM1 behaves as an oligomer in HeLa cells. Furthermore, we identified three distinct oligomeric states of NPM1 using molecular characterization techniques such as subcellular localization and RNA binding. Finally, we found that heterozygous expression of a leukemia-associated NPM1 mutant significantly decreases the RNA binding level. Our data demonstrate that size exclusion chromatography provides a powerful tool for analysing NPM1 oligomers.

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.

Protein complex formation and intranuclear dynamics of NAC1 in cancer cells.

Nucleus accumbens-associated protein 1 (NAC1) is a cancer-related transcription regulator protein that is also involved in the pluripotency and differentiation of embryonic stem cells. NAC1 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 and the dynamics of intranuclear NAC1 in cancer cells remain poorly understood. In this study, analysis of HeLa cell lysates by fast protein liquid chromatography (FPLC) 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. Furthermore, live cell photobleaching analyses with green fluorescent protein (GFP)-fused NAC1 proteins revealed the intranuclear dynamics of NAC1. Collectively our results demonstrate that NAC1 forms a protein complex to function as a transcriptional regulator in cancer cells.

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/ß 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.

Abnormal cytoplasmic dyslocalisation and/or reduction of nucleophosmin protein level rarely occurs in myelodysplastic syndromes.

The Nucleophosmin1 (NPM1) gene located in chromosome 5q35 is affected by chromosomal translocation, mutation and deletion in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). NPM1 haploinsufficiency reportedly causes MDS-like disorders in knockout mice. Here, we studied mRNA and protein expression in bone marrow (BM) samples from 36 patients with MDS. The NPM1 expression levels of mRNA and protein were not related to chromosome 5 abnormalities and were almost the same as those in normal BM and AML cells. However, the protein levels in AML cells with NPM1 mutations were slightly lower than in those without mutation. Immunochemical studies showed no difference in the staining intensity and subcellular localisation between MDS and normal BM cells. It was concluded that abnormal cytoplasmic localisation and/or significant reduction of NPM1 protein level rarely occurs in MDS. The increase in the number of nuclear NPM1-positive cells may be related to the progression of MDS.