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PubMed2002

Nuclear localization and DNA interaction of protein disulfide isomerase ERp57 in mammalian cells.

Protein disulfide isomerase ERp57 is localized predominantly in the endoplasmic reticulum, but is also present in the cytosol and, according to preliminary evidence, in the nucleus of avian cells. Conclusive evidence of its nuclear localization and of its interaction with DNA in vivo in mammalian cells is provided here on the basis of DNA-protein cross-linking experiments performed with two different cross-linking agents on viable HeLa and 3T3 cells. Nuclear ERp57 could also be detected by immunofluorescence in HeLa cells, where it showed an intracellular distribution clearly different from that of an homologous protein, located exclusively in the endoplasmic reticulum. Mammalian ERp57 resembles the avian protein in its recognition of S/MAR-like DNA sequences and in its association with the nuclear matrix. It can be hypothesized that ERp57, which is known to associate with other proteins, in particular STAT3 and calreticulin, may contribute to their nuclear import, DNA binding, or other functions that they fulfil inside the nucleus.

3T3 Cells↗
PubMed2005

Nuclear actin is partially associated with Cajal bodies in human cells in culture and relocates to the nuclear periphery after infection of cells by adenovirus 5.

Cajal bodies are intra-nuclear structures enriched in proteins involved in transcription and mRNA processing. In this study, immunofluorescence microscopy experiments using a highly specific antibody to actin revealed nuclear actin spots that colocalized in part with p80 coilin-positive Cajal bodies. Actin remained associated with Cajal bodies in cells extracted to reveal the nuclear matrix. Adenovirus infection, which is known to disassemble Cajal bodies, resulted in loss of actin from these structures late in infection. In infected cells, nuclear actin was observed to relocate to structures at the periphery of the nucleus, inside the nuclear envelope. Based on these findings, it is suggested that actin may play an important role in the organization or function of the Cajal body.

Actins↗
PubMed1999

A human papillomavirus E2 transcriptional activator. The interactions with cellular splicing factors and potential function in pre-mRNA processing.

The human papillomavirus (HPV) E2 protein plays an important role in transcriptional regulation of viral genes as well as in viral DNA replication. Unlike most types of HPV, the E2 protein of epidermodysplasia verruciformis (EV)-associated HPVs harbors a relatively long hinged region between the terminal, conserved transactivation and DNA binding/dimerization domains. The sequence of EV-HPV E2 hinge contains multiple arginine/serine (RS) dipeptide repeats which are characteristic of a family of pre-messenger RNA splicing factors, called SR proteins. Here we show that the HPV-5 (an EV-HPV) E2 protein can specifically interact with cellular splicing factors including a set of prototypical SR proteins and two snRNP-associated proteins. Transiently expressed HPV-5 E2 protein colocalizes with a nuclear matrix associated-splicing coactivator in nuclear speckled domains. The RS-rich hinge is essential for E2 transactivator interaction with splicing factors and for its subnuclear localization. Moreover, we present functional evidence for the HPV-5 E2 transactivator, which shows that the RS-rich hinge domain of the E2 protein can facilitate the splicing of precursor messenger RNA made via transactivation by E2 itself. Our results, therefore, suggest that a DNA binding transactivator containing an RS-rich sequence can play a dual role in gene expression.

Gene Expression Regulation↗
PubMed2005

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins.

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.

Animals↗
PubMedDate not supplied

Intermediate filament proteins: many unanswered questions, few unquestioned answers.

Major constituents of the cytoskeleton and the nuclear matrix, cytoplasmic intermediate filament subunits and nuclear lamins belong to a multigene family of proteins whose function is poorly understood. It has now become a general contention that important clues to the physiological roles of these proteins may reside in their developmental and tissue-specific expression patterns, as well as their cellular organization. The present review brings into focus experimental strategies that have been developed, over the past few years, to gain insights into the cellular mechanisms regulating the molecular polymorphism and supramolecular assembly of intermediate filaments. In this context new concepts are discussed that may be pivotal for the orientation of future studies on intermediate filament proteins.

Animals↗
PubMed1986

Spermine inhibits the phosphorylation of the 11,000- and 10,000-dalton nuclear proteins catalyzed by nuclear protein kinase NI in NB-15 mouse neuroblastoma cells.

The nuclear protein kinase NI (NI kinase) was purified from NB-15 mouse neuroblastoma cells by phosphocellulose column and casein affinity column chromatography. The purified NI kinase exhibited (i) an apparent subunit molecular weight of about 37,000, (ii) autophosphorylation, and (iii) insensitivity to inhibition by heparin. When NI kinase was added to heat-treated neuroblastoma nuclei in the presence of [gamma-32P] ATP, two proteins with apparent subunit molecular weights of 11,000 and 10,000 were prominently phosphorylated. Other protein kinases tested including the nuclear protein kinase NII, Type I cAMP-dependent protein kinase, and protein kinase C did not catalyze the phosphorylation of these two proteins. The NI kinase-catalyzed phosphorylation of these two proteins was completely inhibited by 1 mM spermine. In contrast, 10 mM putrescine, 2 mM spermidine, 5 mM arginine, and 10 mM NH4Cl, had no inhibitory effect on this phosphorylation reaction. Our study also indicated that the phosphorylation of the 11,000- and 10,000-dalton proteins occurred in the nuclear matrix fraction but not in heterogeneous nuclear ribonucleoproteins, high mobility group proteins, or histone fractions. We have previously reported that spermine specifically inhibits the endogenous phosphorylation of an 11,000-dalton nuclear protein in various mammalian cell lines (Chen, K. Y., and Verma, R. (1984) Biochem. Biophys. Res. Commun. 118, 710-716). The present study suggests that the 11,000- and 10,000-dalton nuclear proteins may be native substrates of nuclear protein kinase NI and that their phosphorylation can be affected by physiological concentrations of spermine.

Animals↗
PubMed2003

Scapinin, a putative protein phosphatase-1 regulatory subunit associated with the nuclear nonchromatin structure.

It is thought that the nuclear nonchromatin structures, such as the nuclear matrix and lamina, play regulatory roles in gene expression. In this study, we identified an insoluble protein that was associated with the chromatin-depleted nuclear structure of proliferating human leukemia HL-60 cells. Preparation of the chromatin-depleted nuclear structure, referred to as the nuclear matrix-intermediate filament scaffold (Fey, E., Krochmalnic, G., and Penman, S. (1986) J. Cell. Biol. 102, 1654-1665), involved cell extraction using a series of buffers containing Triton X-100, DNase I, and 2 M NaCl. A yeast two-hybrid assay revealed that this protein bound to the catalytic subunit of protein phosphatase-1 (PP1). Furthermore, it inhibited PP1 activity in vitro. We therefore named it scapinin (scaffold-associated PP1 inhibiting protein). cDNA cloning revealed that scapinin had two splicing variants of 448 amino acids (scapinin-S) and 518 amino acids (scapinin-L). Scapinin was down-regulated by differentiation in HL-60 cells. These results suggest that scapinin is a putative regulatory subunit of PP1 and is involved in transformed or immature phenotypes of HL-60 cells. We also describe the presence of scapinin family proteins from worm to human.

Amino Acid Sequence↗
PubMed1997

Matrix attachment regions and transcription units in a polygenic mammalian locus overlapping two isochores.

Eukaryotic chromosomes are ponctuated by specialized DNA sequences (MARs) characterized by their ability to bind the network of nonhistone proteins that form the nuclear matrix or scaffold. We previously described an amplifiable cluster of genes with different tissue-specific expression patterns, located on Chinese hamster chromosome 1q. This model is especially appropriate to study the relationships between MARs and transcription units. We show here that four attachment regions, with sequences exhibiting motifs specific to MARs, are present within the 100 kb of screened DNA. Three of them are relatively short sequences localized in intergenic regions. The last one extends over one of the transcription units and contains a region previously identified as a recombination hot spot. Moreover, the analysis of a DNA sequence extending over some 50 Kb of this region and spanning at least four genes, disclosed a strikingly sharp change in G + C content. This strongly suggests that the studied region contains the boundary of two isochores. We propose that the frequency and the size of MARs are correlated to their localization in G + C rich or poor domains.

Amino Acid Sequence↗
PubMed1992

A novel nonhistone protein (MENT) promotes nuclear collapse at the terminal stage of avian erythropoiesis.

The terminal stage of differentiation of nucleated chicken erythrocytes is associated with an overall gene repression and a condensation of the repressed chromatin portion. Two-dimensional DNP electrophoresis has been used to separate transcriptionally active and repressed chromatin of mature chicken erythrocytes. The repressed chromatin fraction is shown to be enriched with histone H5 as well as with a 42-kDa nonhistone chromosomal protein. The 42-kDa protein designated here as MENT (mature erythrocyte nuclear termination stage-specific protein) is hyperexpressed at the terminal stage of chicken erythropoiesis and is accumulated in adult chicken erythrocyte nuclei. This protein was purified by ion-exchange chromatography from 0.4 M NaCl extracts of the erythrocyte nuclei. It appeared to be a basic polypeptide (pI 9.2) which, however, precipitated at low pH. When reconstituted in vitro with immature erythrocyte nuclei, MENT promoted condensation of intact nuclear chromatin and enhanced the solubilization of nuclease-digested polynucleosomes, thus mimicking the processes occurring in vivo at the final stage of erythrocyte maturation. The extent of dissociation of specific gene sequences from the nuclear matrix in MENT-treated nuclei is in striking correlation with their transcriptional activity. No other basic proteins (H5, cytochrome c, RNase A) added to the nuclear preparation at the same level as MENT (protein/DNA = 0.005) caused any effect on nuclear organization. No alterations were observed when MENT was mixed with erythroblasts and nonerythroid nuclei having little or no histone H5. We propose that MENT cooperates with histone H5 to complete the nuclear collapse in mature nucleated erythrocytes.

Animals↗
PubMed1998

The SURF-6 protein is a component of the nucleolar matrix and has a high binding capacity for nucleic acids in vitro.

The recently identified novel protein SURF-6 is shown to be a component of the nucleolar matrix. Immunofluorescence analysis demonstrated that SURF-6 was localized in residual nucleoli of in situ nuclear matrix preparations of mouse fibroblast cells (NIH 3T3), which were depleted of soluble and chromatin related proteins. Immunoblot analysis of biochemical nucleolar subfractions confirmed that SURF-6 was present in the nucleolar matrix fraction, and was absent from the fractions of soluble proteins released by DNase or RNase. The capacity of SURF-6 to bind nucleic acids was investigated in vitro. Both endogenous SURF-6 from nuclear extracts and recombinant SURF-6 exhibited a strong binding capacity for nucleic acids. It was shown that SURF-6 bound to both DNA and RNA, however, it showed stronger binding to RNA. The presence and nuclear distribution of SURF-6 during the cell cycle was explored by immunofluorescence analysis. It was shown that SURF-6 was always found in the nucleolus regardless of the phase of the cell cycle suggesting that it is a structural protein constitutively present in nucleolar substructures. The colocalization of SURF-6 with the major nucleolar proteins B23 and fibrillarin, which are known to be involved in the processing of ribosomal RNA (rRNA), was examined both in interphase and mitosis by double immunolabeling of cells. SURF-6 was found to be largely coincident with both proteins in interphase and it was distributed in the same cellular locations, namely the perichromosomal layer, the cytoplasm and prenucleolar bodies, in mitosis. However, colocalization of SURF-6 with fibrillarin and B23 was only partial in interphase, and the dynamics of its localization was not completely the same as those of either fibrillarin or B23 during mitosis. Taken together, these results indicate that SURF-6 is a novel nucleolar matrix component and imply that SURF-6 might support nucleolar matrix structure and function(s) via its association with nucleic acids. We propose that SURF-6 may be involved in processing of rRNA, based on its cytological characteristics, but at stages in ribosomal biogenesis which are different from those for fibrillarin and B23.

3T3 Cells↗
PubMed1994

6-Iodoacetamidofluorescein labelling to assess the state of sulphhydril groups after thermal stabilization of isolated nuclei.

Isolated nuclei and nuclear matrices, prepared from mouse erythroleukaemia cells, were reacted with the sulphhydryl-specific dye 6-iodoacetamidofluorescein. To determine whether in vitro formation of disulphide bonds might play a role in the nuclear matrix stabilization triggered by exposure of isolated nuclei to the physiological temperature of 37 degrees C, a variety of techniques were employed to assess the state of cysteinyl residues after such an incubation. Both flow cytometry and confocal microscopy quantitative analysis did not reveal major differences in the fluorescence intensity of nuclei incubated at 37 degrees C in comparison with those maintained at 0 degrees C. Confocal scanning laser microscopy revealed that 6-iodoacetamidofluorescein labelled a fibrogranular network in isolated nuclei. The fluorescent pattern of the network was not affected by a 37 degrees C exposure of nuclei. However, such a network was not detectable in isolated nuclear matrices, thus suggesting a possible protein re-arrangement during matrix preparation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of fluorescent-labelled nuclear proteins showed no difference between heat-exposed and control samples. We conclude that oxidation of cysteinyl residues is not a major factor leading to the stabilization of nuclei incubated at 37 degrees C.

Animals↗
PubMed1995

Rethinking cell structure.

Cell structure, emerging from behind the veil of conventional electron microscopy, appears far more complex than formerly realized. The standard plastic-embedded, ultrathin section can image only what is on the section surface and masks the elaborate networks of the cytoplasm and nucleus. Embedment-free electron microscopy gives clear, high-contrast micrographs of cell structure when combined with removal of obscuring material such as soluble proteins. The resinless ultrathin section is the technique of choice; it is simple and inexpensive, and it uses ordinary electron microscopes. The resulting pictures reveal a world of complex cell structure and function. These images necessarily change our conception of the cytoskeleton, nuclear matrix, mitosis, and the relation of membranes to cytostructure.

Animals↗
PubMed1991

Presence of calmodulin and calmodulin-binding proteins in the nuclei of brain cells.

The nuclear calmodulin levels have been measured in rat neurons and glial cells. The values are 1.0 and 1.1 micrograms/mg of protein, respectively. These levels are about threefold higher than those in the nuclei of rat liver cells. We have also investigated the presence of several calmodulin-binding proteins in the nuclei of both brain cellular types. As similarly observed in the nuclei of liver cells, we detected the presence of alpha-spectrin and a 62-kDa calmodulin-binding protein (p62) in the nuclei of neurons and glial cells by immunoblotting and immunocytochemical methods. Both proteins are enriched in the purified nuclear matrix samples from both cellular types. In contrast to that occurring in rat hepatocytes, we have not been able to detect, by immunoblotting methods, caldesmon in the nuclear matrices of neurons and glial cells. The immunocytochemical studies suggest, however, that caldesmon can be present in the nuclei but in a fraction distinct from the nuclear matrices.

Animals↗
PubMed2001

Plasmids with a mammalian replication origin and a matrix attachment region initiate the event similar to gene amplification.

Gene amplification plays a crucial role in the development of many human malignancies. Amplified genes are frequently localized on double minutes (DMs). We show here that plasmids bearing both a mammalian replication origin and a nuclear matrix attachment region were able to integrate into DMs if transfected to cells having DMs (COLO 320DM). Furthermore, these plasmids triggered the events leading to the de novo formation of the structure similar to DMs if transfected to the cells without DMs (COLO 320HSR or HeLa). Autonomous replication of these plasmids was suggested to be a prerequisite for these events to occur, because the presence of the origin sequences in the plasmids was required. The presence of matrix attachment region in the plasmids is also required for these events to occur, suggesting that matrix attachment plays an indispensable role in extrachromosomal replication. This model system will allow us to investigate the mechanism of gene amplification as well as to analyze the autonomous replication of the plasmid with mammalian replication origins.

Colorectal Neoplasms↗
PubMed1999

[Theory of integrating cellular tension and consequences from it].

Tensegrity model and implications from it have been reviewed. Experimental data confirming the model are shown. While signal vectorization is well confirmed and well depicted by the model, the vectorization of expression implied by the model has not yet been elaborated. Molecular mechanisms of functional connection between the nuclear matrix and the cytoskeleton need further observations.

Cytoskeleton↗
PubMed2003

A significant fraction of conserved noncoding DNA in human and mouse consists of predicted matrix attachment regions.

Noncoding DNA in the human-mouse orthologous intergenic regions contains "islands" of conserved sequences, the functions of which remain largely unknown. We hypothesized that some of these regions might be matrix-scaffold attachment regions, MARs (or S/MARs). MARs comprise one of the few classes of eukaryotic noncoding DNA with an experimentally characterized function, being involved in the attachment of chromatin to the nuclear matrix, chromatin remodeling and transcription regulation. To test our hypothesis, we analyzed the co-occurrence of predicted MARs with highly conserved noncoding DNA regions in human-mouse genomic alignments. We found that 11% of the conserved noncoding DNA consists of predicted MARs. Conversely, more than half of the predicted MARs co-occur with one or more independently identified conserved sequence blocks. An excess of conserved predicted MARs is seen in intergenic regions preceding 5' ends of genes, suggesting that these MARs are primarily involved in transcriptional control.

Animals↗
PubMed2000

Mini review: form and function in the human interphase chromosome.

A key feature of interphase chromosomes is their compaction into discrete "territories" in the nucleus. In this review, we focus on the compartmentalization of the genome conferred by this organization and evaluate our current understanding of the role of large-scale chromatin folding in the regulation of gene expression. We examine evidence for the hypothesis that transcription occurs at the external surfaces of chromosomes and follow its evolution to include transcription at the surfaces of chromatin-rich domains within chromosomes. We also present prevailing views regarding the details of large-scale chromatin folding and the functional relationship between chromatin and the enigmatic nuclear matrix.

Chromatin↗
PubMed2004

Characterization of the tight junction protein ZO-2 localized at the nucleus of epithelial cells.

ZO-2 is a MAGUK protein that in confluent epithelial sheets localizes at tight junctions (TJ) whereas in sparse cultures accumulates in clusters at the nucleus. Here, we have characterized several nuclear properties of ZO-2. We observe that ZO-2 is present in the nuclear matrix and co-immunoprecipitates with lamin B(1) and actin from the nuclei of sparse cultures. We show that ZO-2 presents several NLS at its amino region, that when deleted, diminish the nuclear import of the ZO-2 amino segment and impair the ability of the region to regulate the transcriptional activity of promoters controlled by AP-1. Several RS repeats are detected in the ZO-2 amino segment, however, their deletion does not preclude the display of a speckled nuclear pattern. ZO-2 displays two putative NES. However, only the second one appears to be functional, as when conjugated to ovalbumin (OV), it is able to translocate this protein from the nucleus to the cytoplasm in a leptomycin B-sensitive way.

Actins↗