Human myeloid cell nuclear differentiation antigen binds specifically to nucleolin (original) (raw)
Related papers
Blood, 1994
We have previously shown that the human myeloid cell nuclear differentiation antigen (MNDA) is expressed at both the antigen and mRNA levels specifically in human monocytes and granulocytes and earlier stage cells in the myeloid lineage. A 200 amino acid region of the MNDA is strikingly similar to a region in the proteins encoded by a family of interferon-inducible mouse genes, designated Ifi-201, Ifi-202, Ifi-203, etc, that are not regulated in a cell- or tissue-specific fashion. However, a new member of the Ifi-200 gene family, D3, is induced in mouse mononuclear phagocytes but not in fibroblasts by interferon. The same 200 amino acid region, duplicated in the mouse Ifi-200 gene family, is also repeated in the recently characterized human IFI 16 gene that is constitutively expressed specifically in lymphoid cells and is induced in myeloid cells by interferon gamma. The 1.8-kb MNDA mRNA, which contains an interferon-stimulated response element in the 5' untranslated region, was...
Journal of Proteomics, 2012
One of the challenges of the proteomic analysis by 2D-gel is to visualize the low abundance proteins, particularly those localized in the organelles. An additional problem with nuclear proteins lies in their strong interaction with nuclear acids. Several experimental procedures have been tested to increase, in the nuclear extract, the ratio of nuclear proteins compared to contaminant proteins, and also to obtain reproducible conditions compatible with 2D-gel electrophoresis. The NaCl procedure has been chosen. To test the interest of this procedure, the nuclear protein expression profiles of macrophages and dendritic cells have been compared with a proteomic approach by 2D-gel electrophoresis. Delta2D software and mass spectrometry analyses have allowed pointing out some proteins of interest. We have chosen some of them, involved in transcriptional regulation and/or chromatin structure for further validations. The immunoblotting experiments have shown that most of the observed changes are due to post-translational modifications, thereby exemplifying the interest of the 2D gel approach. Finally, this approach allowed us to reach not only high abundance nuclear proteins but also lower abundance proteins, such as the HP1 proteins and reinforces the interest of using 2DE-gel in proteomics because of its ability to visualize intact proteins with their modifications.
Electrophoresis, 1997
Two-dimensional electrophoresis reveals a nuclear matrix-associated nucleolin complex of basic isoelectric point A monoclonal antibody was raised against a salt-extractable fraction of nuclear matrix / intermediate filament scaffolds of polarized MDCK cells. The antibody recognized an-100 kDa protein in total cell lysates and nuclear matrices of various human cells and tissues and stained nucleolar structures in immunofluorescence microscopy. By partial sequencing of five peptides derived from immunoprecipitated protein, the targeted antigen was found to be homologous to human nucleolin. After two-dimensional electrophoresis of total HeLa cell lysates, immunoreactive bands were detected at isoelectric point (PO 5.5-6.1, characteristic for nucleolin, and at p l 8.5-9. Whereas the protein focusing at acidic p l was found in Triton X-100-soluble cellular fractions, the antigen focusing at basic p l was exclusively contained in the residual nuclear fraction and was solubilized upon treatment of nuclear matrices with RNAse. The component solubilized by RNAse treatment was still detected at basic p l in two-dimensional electrophoresis. However, upon immunoprecipitation of the antigen from the RNAse-released fraction in the presence of sodium dodecyl sulfate (SDS), the nuclear matrix-derived antigen was positioned at pf 5-6. The present data indicate that the nuclear matrix-bound nucleolin is associated dissociation under
Journal of Cellular Biochemistry, 2002
The human myeloid nuclear differentiation antigen (MNDA) is a hematopoietic cell specific nuclear protein. MNDA and other related gene products interact with and alter the activity of a large number of proteins involved in regulating specific gene transcription. MNDA and related genes exhibit expression characteristics, which suggest functions unique to specific lineages of cells, in addition to mediating the effects of interferons. Cells of the human K562 myeloid line do not express MNDA and are relatively immature compared to lines that express MNDA (HL-60, U937, and THP1). The hypothesis that MNDA influences the expression of specific genes was tested by creating MNDA expressing K562 cells using stable retroviral mediated gene transfer followed by evaluation of transcription profiles. Two macroarrays containing a total of 2,350 cDNAs of known genes showed a specific up-regulation of Dlk1 expression in MNDA expressing K562 cell clones. Real time quantitative RT-PCR analysis confirmed an average of over 3-and 7-fold upregulation of Dlk1 in two clones of MNDA expressing K562 cells. The effects on Dlk1 were also confirmed by Northern blotting. Dlk1 is essential for normal hematopoiesis and abnormal expression is a proposed marker of myelodysplastic syndrome. Additional screening of transcription profiles after induced erythroid and megakaryoblastic differentiation showed no additional gene transcripts altered by the presence of MNDA. These results indicate that MNDA alters expression of a gene essential for normal hematopoiesis.
Cloning of cDNA encoding a 100 kDa nucleolar protein (nucleoline) of Chinese hamster ovary cells
Nucleic Acids Research
Nucleoline (100 kDa) is the major nucleolar protein in exponentially growing cells that behaves like a nucleolar organizer protein and plays a key role in rDNA transcription and prerRNA processing. We reported the isolation of 5 cDNA clones by probing a cDNA library, constructed in the expression vector A gtll, with a polyclonal serum raised against nucleoline. A new immunoassay, using hybrid proteins (pgal-cDNA encoded protein) was developped to establish that the isolated cDNAs encoded parts of nucleoline. A further confirmation resulted from the sequence comparison between the cDNA encoded peptide and a 42 aa peptide isolated from rat nucleoline (1). The 5 cDNAs overlapped extensively and covered more than 90 X of a full length cDNA. By probing a Northern blot with the 1O0kDa cDNA, a 2650 nucleotide polyA+ RNA was detected that contained just enough information to code for nucleoline.
Nucleolin Binds to the Proliferating Cell Nuclear Antigen and Inhibits Nucleotide Excision Repair
Molecular and cellular pharmacology, 2009
Nucleolin is over-expressed in malignant tumors and is used as a marker for cell proliferation and to reliably predict tumor growth rate. However, it is not known whether nucleolin expression is directly involved in or is a consequence of carcinogenesis. Using GST-pull down assays, we have determined that the recombinant nucleolin interacts with the Proliferating Cell Nuclear Antigen (PCNA). Co-immunoprecipitation assays indicate that the nucleolin-PCNA interaction also occurs in intact cells and this interaction increases after exposure of colon carcinoma RKO cells to UV radiation. Moreover, our data indicate that PCNA and nucleolin co-localize in some areas within the RKO cell nuclei. The functional significance of this interaction is evaluated on Nucleotide Excision Repair (NER) since PCNA is a primary mediator of this cellular function. Our data indicate that overexpression of nucleolin decreases the repair efficiency of UV damaged plasmid DNA in RKO cells. Co-transfection with ...
Cytometry, 2001
Background. Various attributes of nucleoli, including abundance of the nucleolar product (rRNA), correlate with cell-proliferative status and are useful markers for tumor diagnosis and prognosis. However, there is a paucity of methods that can quantitatively probe nucleolus. The aim of the present study was to utilize the morphometric capacity of the laser scanning cytometer (LSC) to analyze nucleoli and measure expression of the nucleolar protein nucleolin (NCL) in individual cells and correlate it with their state of proliferation. Materials and Methods. Human lymphocytes were mitogenically stimulated, and at different time points their nucleoli were detected immunocytochemically using NCL Ab. The frequency of nucleoli per nucleus, their area, and the level of expression of NCL, separately in the nuclear and nucleolar compartments, were estimated in relation to the G 0 to G 1 transition and the cell cycle progression. Results. During the first 24 h of stimulation, when the cells underwent G 0 to G 1 transition, their RNA content was increased nearly 8-fold, the level of NCL per nucleus also increased 8-fold, the NCL per nucleolus increased 12-fold, nucleolear area increased 3-fold, and NCL/nucleolar area increased nearly 4-fold. During the subsequent 24 -48 h of stimulation, when cells were progressing through S, G 2 , and M and reentering the next cycle, the number of nucleoli per nucleus was increased and a massive translocation of NCL from nucleoli to nucleoplasm was observed; its overall level per nucleus, however, still remained high, at 6-fold above of that of G 0 cells. Conclusions. While high expression of NCL in the nucleolar compartment correlates with the rate of rRNA accumulation in the cell and is a sensitive marker of the G 0 to G 1 transition, the cells progressing through the remainder of the cycle are better distinguished from G 0 cells by high overall level of NCL within the nucleus. Such an analysis, when applied to tumors, may be helpful in obtaining the quantitative parameters related to the kinetic status of the tumor-cell population and tumor prognosis. The capability of LSC to measure the protein translocation between nucleolus and nucleoplasm can be used to study the function and regulatory mechanisms of other proteins that reside in these compartments. Cytometry 45: 206 -213, 2001.
Nucleolar proteins with altered expression in leukemic cell lines
2012
The function of the nucleolus is intimately connected to cell proliferation, division and growth. Many cancer cells have enlarged nucleoli, and several nucleolar proteins have been linked to tumorigenesis. In order to find proteins whose expression is altered in the nucleoli of leukemic cells, we carried out twodimensional difference gel electrophoresis (2-D DIGE) analyses. Prohibitin (PHB) and TAR-DNA-binding protein-43 (TDP-43) were strongly expressed in the nucleoli of the pre-B-ALL cell line MHH-CALL3. Our results demonstrate that leukemic cells have differences in their nucleolar protein composition, and suggest that it may be possible to exploit these differences in identification of leukemia subtypes. (K.J. Teittinen).
Characterization of a 48-kDa nucleic-acid-binding fragment of nucleolin
European Journal of Biochemistry, 1989
Nucleolin (C23 or 100 kDa) is an abundant single-stranded-nucleic-acid-binding nucleolar protein proposed to be involved in the early stages of ribosome assembly. A stable 48-kDa fragment of the protein was produced either by proteolytic activity present in nucleolar extracts or by added trypsin. The hydrodynamic and DNAbinding properties of the 48-kDa fragment were compared with the parent molecule. Protein sequencing indicated that the fragment begins at residue 282; amino acid composition of the fragment including 10-12 methylated arginine residues suggested that the fragment contains the entire COOH-terminal two-thirds of the protein. The 48-kDa fragment was more globular than nucleolin, as indicated by a lower frictional coefficient (1.3 vs. 2.0 for nucleolin) and a similar sedimentation coefficient (4.1 -4.3 S) in spite of the reduction in molecular mass. Although the 48-kDa fragment retained single-stranded-DNA-binding activity, the binding capacity and the ability to reassociate DNA were about fivefold and sixfold lower, respectively, than nucleolin. Similarly, tenfold higher concentrations of the 48-kDa fragment were required to form nucleoprotein aggregates. These results suggcst that nucleolin contains a globular COOH-terminal domain for nucleic-acid binding and a NH,-terminal region which is involved in protein-protein interactions and modulating nucleic-acid-binding activity.
Nucleolin, a major conserved multifunctional nucleolar phosphoprotein of proliferating cells
Journal of Applied Biomedicine, 2010
Nucleolin is the major nucleolar protein of animal, plant and yeast proliferating cells. It is enriched in the most soluble nuclear or nucleolar protein extract, containing ribonucleoproteins, from which it has been purified. It has a tripartite structure in which each domain accounts for different functions. Despite its multifunctionality, the best characterized role of nucleolin is in the primary cleavage of pre-rRNA, an early step of ribosome biogenesis. In the nucleolus of proliferating cells, nucleolin is mostly located in the dense fibrillar component, following a vectorial pattern, from the periphery of fibrillar centers outwards. This pattern is lost in quiescent cells in which nucleolin is present in low levels. Nucleolin is the most phosphorylated protein of the soluble nuclear extract. It is phosphorylated by casein kinase II and CDKA, and phosphorylation is closely associated with the role of nucleolin in proliferating cells. During mitosis, nucleolin is transported from the mother to the daughter cell nucleolus in the form of processing particles, together with pre-rRNA precursors and other nucleolar proteins. It forms part of prenucleolar bodies and plays a role in nucleologenesis. Recent studies on the nucleolin function, carried out on samples with inactivated nucleolin genes (siRNA downregulated or mutants) have evidenced that nucleolin is absolutely essential for cell proliferation, for the organization of the nucleolus and for transcription and processing of pre-rRNA. In plants, nucleolin controls the auxin responsiveness, thus being involved in the regulation of plant development.