hnRNP proteins and B23 are the major proteins of the internal nuclear matrix of HeLa S3 cells

The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa S3 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA. © 1996 Wiley-Liss, Inc.     

Alteration of Nuclear Matrix Protein Composition during Apoptosis in Rat Embryo Cells

Alteration of the nuclear matrix protein composition during active cell death was investigated by high resolution 2-dimensional gel electrophoresis and computer-assisted image analysis. Nuclear matrices were isolated from purified nuclei of a rat embryo cell line showing an immediate apoptotic response to serum reduction. While cell shrinkage and cytoplasmic compaction, characteristic features of apoptosis, were induced, the nuclear matrix protein pattern was not altered 1 h after induction of apoptosis. However, two sets of novel nuclear matrix protein spots appeared with differing kinetics within the following 5 h of apoptosis. They consisted of five and six protein spots, respectively. In addition, the intensity of five nuclear matrix protein spots that had already been present in the uninduced cells increased continuously within an observation period of 12 h. These coincidences point to a potential involvement of the described nuclear matrix proteins in the apoptotic process.     

Interaction of MAR-sequences with nuclear matrix proteins.

The recent discovery of DNA sequences responsible for the specific attachment of chromosomal DNA to the nuclear skeleton (MARs/SARs) was an important step towards our understanding of the functional and structural organization of eukaryotic chromatin [Mirkovitch et al.: Cell 44:273-282, 1984; Cockerill and Garrard: Cell 44:273-282, 1986]. A most important question, however, remains the nature of the matrix proteins involved in the specific binding of the MARs. It has been shown that topoisomerase II and histone H1 were capable of a specific interaction with SARs by the formation of precipitable complexes [Adachi et al.: EMBO J8:3997-4006, 1989; Izaurralde et al.: J Mol Biol 210:573-585, 1989]. Here, applying a different approach, we were able to "visualize" some of the skeletal proteins recognizing and specifically binding MAR-sequences. It is shown that the major matrix proteins are practically the same in both salt- and LIS-extracted matrices. However, the relative MAR-binding activity of the individual protein components may be different, depending on the method of matrix preparation. The immunological approach applied here allowed us to identify some of the individual MAR-binding matrix proteins. Histone H1 and nuclear actin are shown to be not only important components of the matrix, but to be involved in a highly efficient interaction with MAR-sequences as well. Evidence is presented that proteins recognized by the anti-HMG antibodies also participate in MAR-interactions.     

Changes of nuclear matrix proteins following the differentiation of human osteosarcoma MG-63 cells

Human osteosarcoma MG-63 cells were induced into differentiation by 5 mmol/L hexamethylene bisacetamide （HMBA）. Their nuclear matrix proteins （NMPs） were selectively extracted and subjected to two-dimensional gel electrophoresis analysis. The results of protein patterns were analyzed by Melanie software. The spots of differentially expressed NMPs were excised and subjected to in situ digestion with trypsin. The maps of peptide mass fingerprinting were obtained by MALDI-TOF-MS analysis, and were submitted for NCBI database searches by Mascot tool. There were twelve spots changed remarkably during the differentiation induced by HMBA, nine of which were identified. The roles of the regulated proteins during the MG-63 differentiation were analyzed. This study suggests that the induced differentiation of cancer cells is accompanied by the changes of NMPs, and confirms the presence of some specific NMPs related to the cancer cell proliferation and differentiation. The changed NMPs are potential markers for cancer diagnosis or targets for cancer therapy.     

Analysis of EDTA-chelatable proteins from DNA-protein crosslinks induced by a carcinogenic chromium(VI) in cultured intact human cells

DNA-protein crosslinks (DPCs) were induced in intact human leukemic T-lymphocyte MOLT4 cells or isolated nuclei by treatment with potassium chromate, chromium(III) chloride hexahydrate or x-rays. The proteins complexed to DNA were analyzed by two-dimensional SDS-polyacrylamide gel electrophoresis (PAGE). A group of identical non-histone proteins was crosslinked to DNA by any of the three treatments, except that a 51 kDa basic protein was additionally complexed to DNA when either potassium chromate or chromium(III) chloride hexahydrate was the crosslinking agent. Treatment of chromate-induced DNA-protein crosslinks with EDTA or thiourea followed by ultracentifugation dissociated the major proteins from the complex indicating that these proteins were crosslinked to DNA by direct participation of a EDTA-chelatable form of chromium such as Cr(III) through sulfur containing amino acid residues. The 51 kDa protein was not seen in the post-EDTA pellet but was present in the post-thiourea pellet, indicating that it was also crosslinked to DNA by Cr(III) through non-sulfur-containing amino acids. Digestion of x-rays-induced DPCs by DNase I also revealed this protein on two-dimensional gels indicating that the same protein was also crosslinked by oxidative mechanisms. The involvement of oxidative mechanisms in the crosslinking process was indicated as the majority of the proteins in chromate-induced DPCs were resistant to EDTA and thiourea treatment, and were found to crosslink to DNA when x-rays were used as the crosslinking agent. These results suggest that the chromate-induced DPCs are formed by the generation of reactive oxygen species during the intracellular chromate reduction as well as by the biologically generated Cr(III). About 19% of DNA-protein crosslinks actually involve Cr(III) crosslinking DNA to proteins, about 14% involve Cr(III) crosslinking DNA to proteins through non-sulfhydryl containing moieties and about 5% involve Cr(III) crosslinking DNA to sulfhydryl groups on proteins. The remaining 81% of DNA-protein crosslinks appear to be oxidatively crosslinked out of which about 45% appear to be through sulfhydryl groups and another 36% appear to be through non-sulfhydryl groups.     

Cleavage of cell surface proteins by thrombin

This study was based on our previous findings that the mitogenic action of thrombin on cultured fibroblasts can result from interaction of thrombin with the cell surface in the absence of internalization, and that the proteolytic activity of thrombin is required for stimulation of cell division. This prompted us to look for thrombin-mediated cleavages using 2-dimensional gel electrophoresis of labeled cell surface proteins. Surface membrane components were labeled by 3 procedures: (1) proteins were labeled by lactoperoxidase-catalyzed iodination using ¹²⁵I^?; (2) galactose and galactosamine residues of glycoproteins were oxidized with galactose oxidase and reduced with ³H-NaBH₄; and (3) glycoproteins were metabolically labeled by incubating cells with ³H-fucose. Labeling with the first 2 procedures was carried out after thrombin treatment; in contrast, cells metabolically labeled with ³H-fucose were subsequently treated with thrombin to look for proteolytic cleavages. Collectively, these studies indicated that only about 5 cell surface proteins were thrombin-sensitive, consistent with the high specificity of this protease. Each of the labeling procedures revealed a thrombin-sensitive cell surface glycoprotein which was identified as fibronectin by immunoprecipitation experiments. In addition, cell surface proteins of about 140K and 55K daltons were thrombin-sensitive. However, cell surface proteins of about 45K daltons and 130K to 1 50K daltons were increased after thrombin treatment. These experiments were conducted on an established line of Chinese hamster lung cells with the eventual goal of studying thrombin-mediated cleavages of cell surface proteins in a large number of cloned populations derived from this line that are either responsive or unresponsive to the mitogenic action of thrombin. This approach should permit identification of proteolytic cleavages that are necessary for thrombin-stimulated cell division.     

Hydroxylamine cleavage of proteins in polyacrylamide gels

A modification of the hydroxylamine cleavage of proteins is presented in which proteins were cleaved while immobilized in the matrix of a polyacrylamide gel. The reaction under these conditions retains its high specificity for Asn-Gly bonds and has the advantage that the gel matrix, acting as a carrier, facilitates simultaneous treatment of many samples, and contributes to a high recovery efficiency (60-90%) of the cleavage products. The cleavage is performed with individual protein bands excised from dried slab gels after detection by staining, autoradiography, or fluorography. The procedure can be easily combined with other techniques to further characterize the cleavage fragments. Also a two-dimensional version of the cleavage method was developed, which allows rapid recognition of interrelationships between proteins in a complicated mixture. The versatility of the procedure is demonstrated in a number of applications. Highly related strains of murine leukemia viruses were easily distinguished from one another by the unique cleavage patterns of their gag- and env-precursor polypeptides. Comparing the env-precursor gPr82env synthesized in the presence or absence of tunicamycin with its cell-free synthesized counterpart, revealed the presence of an amino-terminal signal sequence. Cleavage patterns of pro-opiomelanocortin (POMC) from three different species revealed a high degree of homology between rat and mouse POMC, whereas Xenopus POMC was very different. Regions to which carbohydrates are attached could be identified by comparing glycosylated and unglycosylated forms of POMC. Combining the hydroxylamine cleavage procedure with immunological characterization of the fragments showed a small but significant difference between the amino-terminal sequences of the recombinant transforming protein P120 of Abelson murine leukemia virus and of its parent molecule Pr65gag of Moloney murine leukemia virus.     

Ionic interactions between proteins in nonequilibrium pH gradient electrophoresis: histones affect the migration of high mobility group nonhistone chromatin proteins

In two-dimensional gel electrophoresis of the high mobility group (HMG) proteins, it has proved necessary to use nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension rather than isoelectric focusing, because of the basic character of most of the HMG proteins [D. Tyrell, P. J. Isackson, and G. R. Reeck (1982) Anal. Biochem. 119, 433-439]. In this paper it is reported that in samples that contain histones, the mobilities of HMG proteins (particularly HMG-1, HMG-2, and HMG-E) are severely distorted in NEPHGE. This presumably results from formation of complexes between histones and HMG proteins through ionic interactions. Analysis of HMG proteins by NEPHGE/sodium dodecyl sulfate-gel electrophoresis is thus precluded in samples containing histones. Our results raise the possibility of similar artifacts occurring in NEPHGE (or isoelectric focusing) analysis of other proteins with regions of high charge density.     

Dynamic continuity of nuclear and mitotic matrix proteins in the cell cycle

The eukaryotic cell nucleus is a membrane-enclosed compartment containing the genome and associated molecules supported by a highly insoluble filamentous network known as the nucleoskeleton or nuclear matrix. The nuclear matrix is believed to play roles in maintaining nuclear architecture and organizing nuclear metabolism. Recently, advances in microscopic techniques and the availability of new molecular probes have made it possible to localize functional domains within the nuclear matrix and demonstrate dynamic interactions between both soluble and insoluble components involved in the control of multiple nuclear transactions. Like the cytoplasm and its skeleton, the nucleoplasm is highly structured and very crowded with an equally complex skeletal framework. In fact, there is growing evidence that the two skeletal systems are functionally contiguous, providing a dynamic cellular matrix connecting the cell surface with the genome. If we impose cell cycle dynamics upon this skeletal organization, it is obvious that the genome and associated nuclear matrix must undergo a major structural transition during mitosis, being disassembled and/or reorganized in late G2 and reassembled again in daughter nuclei. However, recent evidence from our laboratory and elsewhere suggests that much of the nuclear matrix is used to form the mitotic apparatus (MA). Indeed, both facultative and constitutive matrix-associated proteins such as NuMA, CENP-B, CENP-F, and the retinoblastoma protein (Rb) associate within and around the MA. During mitosis, the nuclear matrix proteins may either become inert “passengers” or assume critical functions in partitioning the genome into newly formed G1 nuclei. Therefore, we support the view that the nuclear matrix exists as a dynamic architectural continuum, embracing the genome and maintaining cellular regulation throughout the cell cycle. © 1996 Wiley-Liss, Inc.     

Organization of the lamin scaffold in the internal nuclear matrix of normal and transformed hepatocytes

Nuclear lamins are among the more abundant proteins making up the internal nuclear matrix, but very little is known about their structure in the nucleoplasm. Using immunoelectron microscopy, we demonstrate the organization of lamins in the nuclear matrix isolated from rat hepatocytes for the first time. Lamin epitopes are arrayed both in locally ordered clusters and in quasi-regular rows. Fourier filtering of the images demonstrates that the epitopes are placed at the nodes and halfway between the nodes of square or rhombic lattices that are about 50 nm on each side, as well as along rows at regular ∼25-nm intervals. In addition, we have compared this structure with that of the internal nuclear matrix isolated from persistent hepatocyte nodules. In transformed hepatocytes, the islands of lamin lattice are lost, and only a partial regularity in the rows of gold particles remains. We suggest that orthogonal lattice assembly might be an intrinsic property of lamin molecules, and that the disassembly may be triggered by simple molecular events such as phosphorylation.     

Polymorphic proteins in rice seed embryo revealed by two-dimensional gel electrophoresis and their application for subspecies identification

Two-dimensional polyacrylamide gel electrophoresis revealed 10 polymorphic proteins in seed embryos of 29 cultivated rices (Oryza sativa L.) including 16 japonica cultivars, three so-called ‘Javanica’ ones and 10 indica ones. We attempted to use these polymorphic proteins to identify rice subspecies by scoring the polymorphisms. Since all japonica cultivars examined showed the same pattern of protein spots, we considered it to be a standard one with a score of zero, and the protein polymorphisms of other cultivars were given scores of 0.0, 0.5 or 1.0 according to spot density. This scoring method gave characteristic scores for indica and ‘Javanica’ cultivars, i.e. typical japonica cultivars selected as standards presumed the score of 0.0 whereas ‘Javanica’ cultivars and indica ones had the scores of 2.5–4.0 and of 5.0–8.0, respectively. By using this scoring method and the subspecies-specific proteins previously reported, 19 cultivars of unknown subspecies were classified as three indica cultivars and 16 japonica ones including four so-called ‘Javanica’ ones. This scoring method also detected a difference between the perennial wild rice Oryza rufipogon and the annual one O. nivara at the protein level.     

A nuclear matrix protein related to intermediate filaments proteins is a member of the complex binding alphoid DNA in vitro

A complex of three proteins (of 80, 70, 58 kDa-p80, p70, and p58, respectively) with the ability to bind alphoid DNA (alpha-satDNA) was revealed by gel mobility shift assay (GMSA) in human nuclear matrix. The probes of the alpha-satDNA bound in the GMSA with the greatest specificity, but the complex was capable of binding human satellite 3 fragment. According to ion exchange and affinity chromatography, the complex includes two DNA-binding proteins, p70 and p80, and a non-DNA-binding one, p58, which enhances the specificity of binding to the alpha-satDNA. GMSA, SDS-PAGE and immunoblotting showed that the lamins, as well as constitutive centromeric proteins (CENP-A, CENP-B, CENP-C, CENP-G), were not incorporated into the complex. It was demonstrated by immunoprecipitation assay that p70 and, probably p58, share a common antigen determinant with the rod domain of intermediate filaments (IF) proteins. The results obtained indicate that the nuclear matrix contains at least one IF-related protein that is able to bind specifically to alpha-satDNA in vitro and that this protein is distinct from the lamins.     

Differential expression of nuclear matrix proteins during the differentiation of human neuroblastoma SK‐N‐SH cells induced by retinoic acid

To investigate the alteration of nuclear matrix proteins (NMPs) during the differentiation of neuroblastoma SK‐N‐SH cells induced by retinoic acid (RA), differentiation markers were detected by immunocytochemistry and NMPs were selectively extracted and subjected to two‐dimensional gel electrophoresis analysis. Immunocytochemical observation demonstrated that the expression of neuronal markers was up‐regulated in SK‐N‐SH cells following RA treatment. Meanwhile, 52 NMPs (41 of which were identified) changed significantly during SK‐N‐SH differentiation; four of these NMPs were further confirmed by immunoblotting. This study suggests that the differentiation of neuroblastoma cells was accompanied by the altered expression of neuronal markers and NMPs. The presence of some differentially expressed NMPs was related to the proliferation and differentiation of neuroblastomas. Our results may help to reveal the relationship between NMPs and neuroblastoma carcinogenesis and reversion, as well as elucidate the regulatory principals driving neural cell proliferation and differentiation. J. Cell. Biochem. 106: 849–857, 2009. © 2009 Wiley‐Liss, Inc.     

Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.     

Nonchromatin nuclear proteins of mammalian lens epithelial cells

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determine possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [³⁵S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development. J. Cell. Biochem. 64:644–650. © 1997 Wiley-Liss, Inc.     

An improved method for separation of low-molecular-weight polypeptides by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel

An improved system for SDS-polyacrylamide gel electrophoresis, capable of analyzing polypeptides having molecular weights from 1500 to 100,000 (especially showing high resolving power in the 1500 to 25,000 molecular weight range) is described. The 10 to 18% linear gradient gel containing 7 M urea with an acrylamide:bisacrylamide ratio of 20:1 and the Laemmli discontinuous buffer was used. The use of the gel with a high crosslinkage ratio is shown to be effective in lowering the leakage of low-molecular-weight polypeptides from the gel. This method has facilitated rapid detection of small amounts of low-molecular-weight polypeptides in body fluids by the use of silver stain. A procedure is presented for the elimination of false bands on the gel frequently encountered during silver staining. The separation patterns of enzymatic cleavage products of proteins, uremic plasma, and urines from nephropathy patients are illustrated. This system is also applicable in the separation of lipopolysaccharides and also for the detection of phospholipids.