05-565 Sigma-AldrichAnti-Nucleolin Antibody, clone 3G4B2

The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation.

The interactions between proteins and nucleic acids have a fundamental function in many biological processes, including gene transcription, RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins that bind individual mRNAs in mammalian cells has been greatly augmented by recent surveys, no systematic study on the non-sequence-specific engagement of native human proteins with various types of nucleic acids has been reported.We designed an experimental approach to achieve broad coverage of the non-sequence-specific RNA and DNA binding space, including methylated cytosine, and tested for interaction potential with the human proteome. We used 25 rationally designed nucleic acid probes in an affinity purification mass spectrometry and bioinformatics workflow to identify proteins from whole cell extracts of three different human cell lines. The proteins were profiled for their binding preferences to the different general types of nucleic acids. The study identified 746 high-confidence direct binders, 139 of which were novel and 237 devoid of previous experimental evidence. We could assign specific affinities for sub-types of nucleic acid probes to 219 distinct proteins and individual domains. The evolutionarily conserved protein YB-1, previously associated with cancer and drug resistance, was shown to bind methylated cytosine preferentially, potentially conferring upon YB-1 an epigenetics-related function.The dataset described here represents a rich resource of experimentally determined nucleic acid-binding proteins, and our methodology has great potential for further exploration of the interface between the protein and nucleic acid realms.

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex-binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex-binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.

Non-malignant cells can be transformed via the activation of kinases that control degradation of neural-restrictive silencer factor (REST). Here, we identify a mechanism that contributes to the activation of genes, expression of which is controlled by responsive elements containing overlapping binding sites for REST and nucleolin. We demonstrate that both phosphorylated and non-phosphorylated nucleolin-bound DNA; however, only phosphorylated nucleolin successfully competed with either full-length REST or a REST-derived DNA-binding peptide, REST68, for binding to the overlapping binding sites. We show that this interplay between the two transcription factors regulates the activation of cell survival and immunomodulatory genes in tumors and non-malignant cells with activated protein kinase C, which is accompanied with alterations in cell proliferation and apoptosis. We propose a model for the regulation of these genes, which brings a new insight into the molecular mechanisms that control cellular transformation driven by activation of protein kinases.

Transcription factors, POU5F1/OCT4 and NANOG, whose expression is restricted to the inner cell mass (ICM) in mouse and human blastocysts, are used to characterize undifferentiated embryonic stem cells (ESC) in vitro. However, POU5F1 may not be a useful marker in domestic animals due to its expression in both ICM and trophectoderm (TE), while NANOG mRNA and protein expression have only been described fully in mice. In an effort to identify ESC markers for domestic animals, expression patterns of NANOG, POU5F1, and the cell surface markers (SSEA1, SSEA4, TRA-1-60, TRA-1-81) were examined in preimplantation goat embryos, a species that has proven to be a superior choice for the production of transgenic proteins in milk (biopharming). Our results indicate that while goat embryos express POU5F1, SSEA1, and SSEA4 proteins, their expression is not strictly restricted to the ICM. In a unique staining pattern, NANOG protein was localized to the nucleoplasm and nucleoli in ICM cells, but was localized strictly to nucleoli in TE. This pattern may reflect down-regulation of protein by sequestration/degradation utilizing a nucleolar mechanism known to operate in stem cells. Furthermore, NANOG mRNA in TE was also significantly down-regulated as compared with that in ICM. Taken together, this novel expression pattern of NANOG in goat preimplantation embryos suggests that NANOG could serve as marker of pluripotency in goats and may be useful in derivation and characterization of caprine ESC. This study is the first to characterize both NANOG mRNA and protein expression in any species other than the mouse.