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PubMed2002

A new possible mechanism of human immunodeficiency virus type 1 infection of neural cells.

To study the mechanism by which HIV-1 infects neurons we have used human neuroblastoma cell lines (NB). NB (SK-N-SH and SK-N-MC) were found to be susceptible to productive infection by X4 or R5 HIV-1, as detected by viral load and Ag-p24. To identify the putative receptor, we tested the cell surface expression of previously described receptors such as CD4, nucleolin, galactosylceramide, and CCR1, CCR5, and CXCR4 by cytometry and RT-PCR. NB express no CD4 and low levels of galactosylceramide or nucleolin. Furthermore, antibodies to any of these molecules did not affect NB infection. NB express variable levels of CCR5, CCR1, and CXCR4. Interestingly, exogenous heparan sulfate alone was able to substantially inhibit HIV-1 infection, an effect which was potentiated by RANTES or SDF-1 in the HIV-1-infection with R5 or X4 isolates. Besides, anti-CCR5 and anti-CXCR4 significantly blocked HIV-1 infection of R5 and X4 isolates. Our results suggest that HIV-1 entry involves a chemokine-receptor-dependent but CD4-independent entry in neural cells.

CD4 Antigens↗
PubMed1990

Identification of major nucleolar proteins as candidate mitotic substrates of cdc2 kinase.

Following the identification of the cdc2 kinase as a major element controlling entry of cells into mitosis, it is important to define the physiological target range of this enzyme. Here, we demonstrate that two major nucleolar proteins, nucleolin and NO38, are highly phosphorylated during mitosis. Importantly, the two nucleolar proteins are also phosphorylated by highly purified starfish cdc2 kinase in vitro, on sites that correspond to those observed specifically during mitosis in vivo. A repeated motif (TPXKK) is identified as the likely mitotic phosphoacceptor site in nucleolin, in that a synthetic peptide mimicking this site functions as both a substrate and a competitive inhibitor of cdc2 kinase. These results identify two novel candidate substrates for cdc2 kinase, and they implicate protein phosphorylation in controlling mitotic changes in nucleolar structure and activity.

Amino Acid Sequence↗
PubMed2005

Inhibition of NF-kappaB stabilizes gadd45alpha mRNA.

Growth arrest- and DNA damage-inducible protein alpha (gadd45alpha) is an important regulator for cell cycle, genomic stability, and cell apoptosis. In the present report, we demonstrated that NF-kappaB inhibition due to Ikkbeta deficiency enhanced the stability of gadd45alpha mNRA. Using embryo fibroblast cells derived from wild type (wt) or Ikkbeta gene knockout (Ikkbeta(-/-)) mice, reverse transcription-polymerase chain reaction revealed a three- to fourfold increase of gadd45alpha mRNA in Ikkbeta(-/-) cells compared with wt cells. The deficiency in Ikkbeta substantially decreased basal NF-kappaB activity and increased accumulation of reactive oxygen species (ROS). However, such deficiency had no effect on the basal expression or activity of Akt, FoxO3a, p53, and c-myc that regulate the transcription of gadd45alpha gene positively or negatively. Analysis of gadd45alpha mRNA stability showed a ROS-dependent increase in the half-life of gadd45alpha mRNA in Ikkbeta(-/-) cells. Immunoprecipitation experiments indicated an increased binding of a RNA stabilizing protein, nucleolin, to gadd45alpha mRNA in Ikkbeta(-/-) cells. The binding of nucleolin to gadd45alpha mRNA could be prevented by the antioxidant, N-acetyl-cysteine. Thus, these data are the first to suggest that inhibition of Ikkbeta-NF-kappaB signaling up-regulates the expression of gadd45alpha mNRA through a post-transcriptional, rather than a transcriptional, mechanism.

Animals↗
PubMed2003

Intracellular redistribution of nuclear and nucleolar proteins during differentiation of 32D murine hemopoietic cells.

We have investigated the intracellular localization of four proteins in murine hemopoietic 32D and 32D-derived cells during exponential growth and after induction of differentiation. The four proteins studied were the insulin receptor substrate-1 (IRS-1), the ID2 protein, nucleolin, and the upstream binding factor (UBF), all of which are involved directly or indirectly in the differentiation program. These four proteins were found to be predominantly nuclear (and/or nucleolar) during exponential growth, as expected. In three models of induced differentiation along the granulocytic pathway, IRS-1, ID2, and nucleolin shifted in part to the cytoplasm, where their levels eventually decreased. UBF also disappeared during differentiation, but we could not detect a cytoplasmic shift in this protein. These experiments indicate that induction of granulocytic differentiation in 32D and 32D-derived cells is accompanied by intracellular redistribution of proteins. This nucleo-cytoplasmic shuttle may play a significant role in the changes in gene expression that occur during differentiation.

Active Transport, Cell Nucleus↗
PubMed2004

Characterization of Staufen 1 ribonucleoprotein complexes.

In Drosophila oocytes and neuroblasts, the double-stranded RNA binding protein Staufen assembles into ribonucleoprotein particles, which mediate cytoplasmic mRNA trafficking and translation. Two different mammalian orthologues also appear to reside in distinct RNA-containing particles. To date, relatively little is known about the molecular composition of Staufen-containing ribonucleoprotein complexes. Here, we have used a novel one-step affinity purification protocol to identify components of Staufen 1-containing particles. Whereas the nucleocytoplasmic RNA-binding protein nucleolin is linked to Staufen in an RNA-dependent manner, the association of protein phosphatase 1, the microtubule-dependent motor protein kinesin and several components of the large and small ribosomal subunits with Staufen ribonucleoprotein complexes is RNA-independent. Notably, all these components do not co-purify with a second RNA-binding protein, hnRNPK (heterogeneous ribonucleoprotein K), demonstrating the high specificity of the purification protocol. Furthermore, pull-down and immunoprecipitation experiments suggest a direct interaction between Staufen 1 and the ribosomal protein P0 in vitro as well as in cells. In cell fractionation and sucrose gradient assays, Staufen co-fractionates with intact ribosomes and polysomes, but not with the isolated 40 S ribosomal subunit. Taken together, these findings imply that, in the cytoplasm of mammalian cells, an association with the ribosomal P-stalk protein P0 recruits Staufen 1 into ribosome-containing ribonucleoprotein particles, which also contain kinesin, protein phosphatase 1 and nucleolin.

Animals↗
PubMed1998

Identification of apoptosis-associated proteins in a human Burkitt lymphoma cell line. Cleavage of heterogeneous nuclear ribonucleoprotein A1 by caspase 3.

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified proteins that are involved in anti-IgM antibody-mediated apoptosis using a subclone of the human Burkitt lymphoma cell line BL60. Apoptosis-associated proteins were detected by high resolution two-dimensional gel electrophoresis on a micropreparative scale. Comparison of the high resolution two-dimensional gel electrophoresis protein patterns from apoptotic and non-apoptotic cells showed differences in approximately 80 spots including protein modifications. Analysis of the predominantly altered proteins was performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. Analysis was significantly improved by using new micropreparative high resolution two-dimensional gels employing high protein concentrations. The following 12 apoptosis-associated proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A1, hnRNP C1/C2, FUSE-binding protein, dUTPase, lymphocyte-specific protein LSP1, UV excision repair protein RAD23 homologue B (HHR23B), 60 S acidic ribosomal protein P0 (L10E), heterochromatin protein 1 homologue alpha (HP1alpha), nucleolin, lamin, neutral calponin, and actin. Fragmentation of actin, hnRNP A1, hnRNP C1/C2, 60 S acidic ribosomal protein P0, lamin, and nucleolin could be inhibited by benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone, a selective irreversible inhibitor of CPP32 (caspase 3).

Apoptosis↗
PubMed2002

Molecular basis for the loss of CD28 expression in senescent T cells.

CD28(null) T cells are the most consistent biological indicator of the aging immune system in humans and are predictors of immunoincompetence in the elderly. The loss of CD28 is the result of an inoperative transcriptional initiator (INR), which consists of two nonoverlapping alpha and beta motifs that have distinct protein binding profiles but function as a unit. In CD28(null) T cells, there is a coordinate loss of alpha-/beta-bound complexes, hence the alphabeta-INR is inactive. In the present work therefore, studies were conducted to identify the components of such complexes that may account for the trans-activation of the alphabeta-INR. By affinity chromatography and tandem mass spectrometry, two proteins, namely, nucleolin and the A isoform of heterogeneous nuclear ribonucleoprotein-D0 (hnRNP-D0A), were identified to be among the key components of the site alpha complex. In DNA binding assays, specific antibodies indicated their antigenic presence in alpha-bound complexes. Transcription assays showed that they are both required in the trans-activation of alphabeta-INR-driven DNA templates. Because CD28 is T cell-restricted, and nucleolin and hnRNP-D0A are ubiquitous proteins, these results support the notion that cell-specific functions can be regulated by commonly expressed proteins. The present data also provide evidence for INR-regulated transcription that is independent of the known components of the basal transcription complex.

Base Sequence↗
PubMed1998

Presence of pre-rRNAs before activation of polymerase I transcription in the building process of nucleoli during early development of Xenopus laevis.

During the early development of Xenopus laevis, we followed in individual nuclei the formation of a nucleolus by examining simultaneously its structural organization and its transcriptional competence. Three distinct situations were encountered with different frequencies during development. During the first period of general transcriptional quiescence, the transcription factor UBF of maternal origin, was present in most nuclei at the ribosomal gene loci. In contrast, fibrillarin, a major protein of the processing machinery, was found in multiple prenucleolar bodies (PNBs) whereas nucleolin was dispersed largely in the nucleoplasm. During the second period, for most nuclei these PNBs had fused into two domains where nucleolin concentrated, generating a structure with most features expected from a transcriptionally competent nucleolus. However, RNA polymerase I-dependent transcription was not detected using run-on in situ assays whereas unprocessed ribosomal RNAs were observed. These RNAs were found to derive from a maternal pool. Later, during a third period, an increasing fraction of the nuclei presented RNA polymerase I-dependent transcription. Thus, the structural organization of the nucleolus preceded its transcriptional competence. We conclude that during the early development of X. laevis, the organization of a defined nucleolar structure, is not associated with the transcription process per se but rather with the presence of unprocessed ribosomal RNAs.

Animals↗
PubMed2003

S100C/A11 is a key mediator of Ca(2+)-induced growth inhibition of human epidermal keratinocytes.

An increase in extracellular Ca2+ induces growth arrest and differentiation of human keratinocytes in culture. We examined possible involvement of S100C/A11 in this growth regulation. On exposure of the cells to high Ca2+, S100C/A11 was specifically phosphorylated at 10Thr and 94Ser. Phosphorylation facilitated the binding of S100C/A11 to nucleolin, resulting in nuclear translocation of S100C/A11. In nuclei, S100C/A11 liberated Sp1/3 from nucleolin. The resulting free Sp1/3 transcriptionally activated p21CIP1/WAF1, a representative negative regulator of cell growth. Introduction of anti-S100C/A11 antibody into the cells largely abolished the growth inhibition induced by Ca2+ and the induction of p21CIP1/WAF1. In the human epidermis, S100C/A11 was detected in nuclei of differentiating cells in the suprabasal layers, but not in nuclei of proliferating cells in the basal layer. These results indicate that S100C/A11 is a key mediator of the Ca(2+)-induced growth inhibition of human keratinocytes in culture, and that it may be possibly involved in the growth regulation in vivo as well.

Active Transport, Cell Nucleus↗
PubMed1997

Coiled bodies without coilin.

Nuclei assembled in vitro in Xenopus egg extract contain coiled bodies that have components from three different RNA processing pathways: pre-mRNA splicing, pre-rRNA processing, and histone pre-mRNA 3'-end formation. In addition, they contain SPH-1, the Xenopus homologue of p80-coilin, a protein characteristic of coiled bodies. To determine whether coilin is an essential structural component of the coiled body, we removed it from the egg extract by immunoprecipitation. We showed that nuclei with bodies morphologically identical to coiled bodies (at the light microscope level) formed in such coilin-depleted extract. As expected, these bodies did not stain with antibodies against coilin. Moreover, they failed to stain with an antibody against the Sm proteins, although Sm proteins associated with snRNAs were still present in the extract. Staining of the coilin- and Sm-depleted coiled bodies was normal with antibodies against two nucleolar proteins, fibrillarin and nucleolin. Similar results were observed when Sm proteins were depleted from egg extract: staining of the coiled bodies with antibodies against the Sm proteins and coilin was markedly reduced but bright nucleolin and fibrillarin staining remained. These immunodepletion experiments demonstrate an interdependence between coilin and Sm snRNPs and suggest that neither is essential for assembly of nucleolar components in coiled bodies. We propose that coiled bodies are structurally heterogeneous organelles in which the components of the three RNA processing pathways may occur in separate compartments.

Animals↗
PubMed1997

An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo.

Amino Acid Sequence↗
PubMed2002

A novel association of the SMN protein with two major non-ribosomal nucleolar proteins and its implication in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by the loss of functional survival motor neuron 1 (SMN1) protein. This ubiquitously expressed protein is a component of a novel complex immunodetected in both the cytoplasm and the nucleus, which is associated with complexes involved in mRNA splicing, ribosome biogenesis and transcription. Here, we study a mutant protein corresponding to the N-terminal half of the protein that is encoded by the SMA frameshift mutation SMN 472del5. We show by confocal microscopy that the resulting mutant protein exhibits various distribution patterns in different transiently transfected COS cells. The mutant distributes into the nucleoplasm and/or the nucleolus, whereas the normal SMN protein accumulates at discrete nucleocytoplasmic dot-like structures previously named gems/Cajal bodies. The cell population with the nucleolar distribution is enriched upon treatment with mimosine, a synchronizing drug in late G(1) phase. Co-immunoprecipitation studies carried out on nuclear extracts reveal that both the endogenous SMN and mutant proteins are associated with complexes containing two major non-ribosomal nucleolar proteins, namely nucleolin and protein B23, and that the association is mediated, by among other things, RNA moieties. Both the association of the SMN protein with nucleolin-containing complexes and the nucleolin/B23 complex are disrupted in fibroblasts derived from a type I SMA patient harboring a homozygous SMN1 gene deletion. These findings suggest that altered assembly and/or stability of ribonucleoprotein complexes may contribute to the pathophysiological processes in SMA.

Animals↗
PubMed2001

RNA-binding strategies common to cold-shock domain- and RNA recognition motif-containing proteins.

Numerous RNA-binding proteins have modular structures, comprising one or several copies of a selective RNA-binding domain generally coupled to an auxiliary domain that binds RNA non-specifically. We have built and compared homology-based models of the cold-shock domain (CSD) of the Xenopus protein, FRGY2, and of the third RNA recognition motif (RRM) of the ubiquitous nucleolar protein, nucleolin. Our model of the CSD(FRG)-RNA complex constitutes the first prediction of the three-dimensional structure of a CSD-RNA complex and is consistent with the hypothesis of a convergent evolution of CSD and RRM towards a related single-stranded RNA-binding surface. Circular dichroism spectroscopy studies have revealed that these RNA-binding domains are capable of orchestrating similar types of RNA conformational change. Our results further show that the respective auxiliary domains, despite their lack of sequence homology, are functionally equivalent and indispensable for modulating the properties of the specific RNA-binding domains. A comparative analysis of FRGY2 and nucleolin C-terminal domains has revealed common structural features representing the signature of a particular type of auxiliary domain, which has co-evolved with the CSD and the RRM.

Amino Acid Motifs↗
PubMed2002

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell.

Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.

Animals↗
PubMed2004

Nucleolar localization of human hepatitis B virus capsid protein.

Wild-type human hepatitis B virus (HBV) exhibits selective export of virions containing mature genomes. In contrast, changing an isoleucine to a leucine at amino acid 97 (I97L) of the HBV core antigen (HBcAg) causes it to release immature genomes. To elucidate the structure-function relationship of HBcAg at amino acid 97, we systematically replaced the isoleucine residue at this position with 18 other amino acids via mutagenesis. Twelve of the 18 mutants exhibited no significant phenotype, while five new mutants displayed strong phenotypes. The I97D mutant had a near lethal phenotype, the I97P mutant exhibited a significantly reduced level of virion secretion, and the I97G mutant lacked the full-length relaxed circular form of viral DNA. The tip of the spike of the capsid particle is known to contain a predominant B-cell epitope. However, the recognition of this exposed epitope by an anti-HBc antibody appeared to be affected by the I97E mutation or by histidine tagging at the C terminus of mutant HBcAg, which is presumably in the capsid interior. Surprisingly, the nuclear HBcAg of mutants I97E and I97W, produced from either a replicon or an expression vector, was found to be colocalized with nucleolin and B23 at a frequency of nearly 100% by confocal immunofluorescence microscopy. In contrast, this colocalization occurred with wild-type HBcAg only to a limited extent. We also noted that nucleolin-colocalizing cells were often binucleated or apoptotic, suggesting that the presence of HBcAg in the nucleolus may perturb cytokinesis. The mechanism of this phenomenon and its potential involvement in liver pathogenesis are discussed. To our knowledge, this is the first report of nucleolar HBcAg in culture.

Amino Acid Sequence↗
PubMed2006

Perturbations of cell cycle control in T cells contribute to the different outcomes of simian immunodeficiency virus infection in rhesus macaques and sooty mangabeys.

In contrast to human immunodeficiency virus (HIV) infection of humans and experimental simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs), SIV infection of sooty mangabeys (SMs), a natural host African monkey species, is typically nonpathogenic and associated with preservation of CD4+ T-cell counts despite chronic high levels of viral replication. In previous studies, we have shown that the lack of SIV disease progression in SMs is related to lower levels of immune activation and bystander T-cell apoptosis compared to those of pathogenic HIV/SIV infection (G. Silvestri, D. Sodora, R. Koup, M. Paiardini, S. O'Neil, H. M. McClure, S. I. Staprans, and M. B. Feinberg, Immunity 18:441-452, 2003; G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. M. McClure, M. B. Feinberg, and S. I. Staprans, J. Virol. 79:4043-4054, 2005). In HIV-infected patients, increased T-cell susceptibility to apoptosis is associated with a complex cell cycle dysregulation (CCD) that involves increased activation of the cyclin B/p34-cdc2 complex and abnormal nucleolar structure with dysregulation of nucleolin turnover. Here we report that CCD is also present during pathogenic SIV infection of RMs, and its extent correlates with the level of immune activation and T-cell apoptosis. In marked contrast, naturally SIV-infected SMs show normal regulation of cell cycle control (i.e., normal intracellular levels of cyclin B and preserved nucleolin turnover) and a low propensity to apoptosis in both peripheral blood- and lymph node-derived T cells. The absence of significant CCD in the AIDS-free, non-immune-activated SMs despite high levels of viral replication indicates that CCD is a marker of disease progression during lentiviral infection and supports the hypothesis that the preservation of cell cycle control may help to confer the disease-resistant phenotype of SIV-infected SMs.

Animals↗
PubMed1995

Amount variability of total and individual Ag-NOR proteins in cells stimulated to proliferate.

Ribosomal genes are associated with a subset of acidic proteins called Ag-NOR proteins. The amount of nucleolar Ag-NOR proteins varies, depending on nucleolar activity and/or cell proliferation. To understand the linkage between the amount of Ag-NOR proteins, ribosome biogenesis, and cell proliferation, we investigated the variability of Ag-NOR proteins in rRNA-stimulated cells maintained in G1 and in rRNA-stimulated cells entering the mitotic cycle. Rat hepatocytes were stimulated with cortisol for rRNA synthesis (1, 4, and 8 hr) and the cell cycle was induced by hepatectomy in regenerating hepatocytes (3-21 hr). In non-stimulated hepatocytes, nucleolin and protein B23 were the two major Ag-NOR proteins, corresponding to 70% of total Ag-NOR staining. In hepatocytes stimulated for rRNA synthesis in G1, the amount of Ag-NOR proteins was only slightly increased, whereas in cycle-stimulated cells it was increased 3.04-fold. This is the consequence of a differential increase of the major Ag-NOR proteins that appears earlier and is proportionally more important for nucleolin (3.5-fold) than for protein B23 (twofold) and also for the increase of several minor Ag-NOR proteins. We conclude that, in dividing cells, the mean value of the Ag-NOR proteins measured reflects the percentage of cells in the different phases. This could explain why the amount of Ag-NOR proteins can be used as a marker of cell proliferation.

Animals↗
PubMed1994

Cell cycle redistribution of U3 snRNA and fibrillarin. Presence in the cytoplasmic nucleolus remnant and in the prenucleolar bodies at telophase.

The distribution of the U3 small nuclear RNA during the cell cycle of the CHO cell line was studied by in situ hybridization using digoxigenin-labelled oligonucleotide probes. The location of the hybrids by immunofluorescence microscopy and at the ultrastructural level was correlated with the distribution of two nucleolar proteins, nucleolin and fibrillarin. The U3 snRNA molecules persist throughout mitosis in close association with the nucleolar remnant. U3 snRNA is present in the prenucleolar bodies (PNBs) and could participate in nucleologenesis in association with several nucleolar proteins such as nucleolin and fibrillarin. The interaction of U3 snRNP with the 5' external spacer of pre-RNA newly synthesized by active NORs is proposed to be the promoting event of nucleologenesis.

Animals↗