Biomedical subjects

C Turano

Publications and source records attributed to C Turano.

At least 19 recordsLinked to original sources

PubMed1999

Binding of the protein disulfide isomerase isoform ERp60 to the nuclear matrix-associated regions of DNA.

Protein ERp60, previously found in the internal nuclear matrix in chicken liver nuclei, is a member of the protein disulfide isomerase family. It binds DNA and double helical polynucleotides in vitro with a preferential recognition toward the matrix-associated regions of DNA and poly(dA) x poly(dT), and its binding is inhibited by distamycin. ERp60 can be cross-linked chemically to DNA in the intact nuclei, suggesting that its association with DNA is present in vivo. As a whole, these results indicate that ERp60 is a component of the subset of nuclear matrix proteins that are responsible for the attachment of DNA to the nuclear matrix and for the formation of DNA loops. A distinctive feature of this protein, which has two thioredoxin-like sites, is that its affinity to poly(dA) x poly(dT) is strongly dependent on its redox state. Only its oxidized form, in fact, does it bind poly(dA) x poly(dT). The hypothesis can be made that through the intervention of ERp60, the redox state of the nucleus influences the formation or the stability of some selected nuclear matrix-DNA interactions.

Animals↗

PubMed1999

Cross-linked telomere-protein complexes from chicken erythrocyte nuclei: isolation by a new procedure.

DNA-protein cross-linkages were produced in intact nuclei of chicken erythrocytes by the action of cis-diammine dichloroplatinum. The telomeric DNA-protein cross-linked complexes were then isolated by hybridization with a biotinylated oligonucleotide and selective binding on immobilized streptavidin. Two main nonhistone proteins were present in the purified complexes, migrating in SDS-gel electrophoresis with apparent molecular masses of 66 and 58 kDa, respectively. Although the identity of these two proteins is still unknown, it is significant that two proteins with similar electrophoretic behavior have been described as constituents of the human telomeric complexes. This procedure could also be applied to the isolation of DNA-protein cross-linked complexes containing any chosen DNA sequence.

Animals↗

PubMed1997

Glycosylation of RNA polymerase II from wheat germ.

RNA polymerase II from wheat germ was analyzed for the presence of sugars. The two largest subunits and the 27 and 25 kDa subunits were found to be glycosylated by a variety of sugars. However, no N-acetylglucosamine was detected, which was found by Kelly et al. (J. Biol. Chem. (1993) 268, 10416-10424) in the largest subunit of RNA polymerase II from calf thymus. Thus it appears that the regulatory function of this sugar, postulated by Kelly et al., is performed in the wheat germ enzyme by other monosaccharides. Carbohydrate analysis of the two largest subunits of the calf thymus enzyme also revealed the presence, beside N-acetylglucosamine, of other sugars. Some similarities in the features of glycosylation of the two polymerases, isolated from very different organisms, suggest that the sugar moieties have an important role in the structure and/or function of these enzymes.

Animals↗

PubMed1996

Comparison of DNA-protein interactions in intact nuclei from avian liver and erythrocytes: a cross-linking study.

DNA-protein cross-linkages were formed in intact nuclei of chicken erythrocytes and liver cells by the action of cis-diammine dichloroplatinum (II). Most cross-linked proteins were components of the nuclear matrix, and their heterogeneity reflected the different complexity of liver and erythrocytes matrices, respectively. Some basic proteins, including histones, were also cross-linked, particularly in erythrocyte nuclei. South-Western blotting revealed that a variety of proteins isolated from the cross-linked liver nuclei recognized DNA specifically. In this group of proteins two relatively abundant, acidic, species of 38 and 66 kDa, respectively, might represent novel DNA-binding proteins from the nuclear matrix. In the case of erythrocytes, only the basic proteins showed a DNA-recognition capacity, and among them there were some unidentified species, absent from liver. Lamin B2 was cross-linked but was unable to recognize DNA, and the same was true for other abundant, cross-linked proteins from both types of nuclei. This led to the hypothesis that for some DNA-nuclear matrix interactions the aggregation typical of matrix proteins is essential for the specificity of DNA recognition. Hybridization analysis of the DNA isolated from the cross-linked complexes showed that SARs (scaffold attachment regions) and telomeric sequences were well represented in the cross-linked fragments, that the cross-linked DNA of liver was partially different from that of erythrocytes and that two defined SAR sequences were found to be present only in the cross-linked DNA. These results are in agreement with the present views on DNA-nuclear matrix interactions, which are usually studied on isolated nuclear matrices or purified proteins. Instead, our results provide experimental evidence obtained directly from intact nuclei.

Animals↗

PubMed1996

Nuclear matrix localization of annexin V in chicken liver.

Fractionation of internal matrix proteins from chicken liver nuclei led to the isolation of a 32 kDa protein which was identified by partial amino acid sequence and immunological analysis as annexin V, an unreported nuclear matrix component. Our results showed that this protein is preferentially associated with the internal nuclear matrix fraction, since this is the only nuclear fraction where the protein can be immunodetected. Immunostaining on cultured cells also revealed a nuclear distribution with the exclusion of the nucleolar compartment and an association with cytosolic filamentous structures most likely corresponding to the cytoskeleton. Moreover, immunostaining on extracted cells to reveal the nuclear matrix showed a network-like distribution. Since annexin V has been reported as an inhibitor of protein kinase C, its nuclear localization in association with the internal matrix, which plays an important role in several nuclear processes, indicates its involvement in the regulation of signal transduction.

3T3 Cells↗

PubMed1996

Influence of the carbohydrate moiety on the stability of glycoproteins.

To study the role of oligosaccharides on the properties of glycoproteins, five glycoproteins (yeast external invertase, bovine serum fetuin, glucoamylase from Aspergillus niger, and chicken egg white ovotransferrin and avidin) of previously established glycan patterns were purified to homogeneity and deglycosylated with endo- and exo-glycosidases in native conditions. Thermal stability and conformational changes were measured by high-resolution differential scanning microcalorimetry and circular dicroism spectroscopy before and after they were deglycosylated. It was found that deglycosylation decreases protein thermal stability, as judged by the decrease in denaturation temperature and denaturation enthalpy, while it does not affect substantially the conformation as indicated by the CD spectra in the far UV range. The destabilization effect of deglycosylation seems to depend on the carbohydrate content, i.e., the maximum effect was observed for the most heavily glycosylated protein, irrespective of the types (N-linked or O-linked) or patterns (mono- or multi-branched) of the covalently attached carbohydrate chains. In addition, studies of the reversibility to heat denaturation revealed that deglycosylated proteins have a poorer thermal reversibility in calorimetric scans than their native counterparts and tend to aggregate during thermal inactivation at acidic pH. These results suggest that carbohydrate moieties, in addition to the apparent stabilizing effect, may prevent the unfolded or partially folded protein molecules from aggregation. Our results support the hypothesis that the general function of protein glycosylation is to aid in folding of the nascent polypeptide chain and in stabilization of the conformation of the mature glycoprotein.

Animals↗

PubMed1996

Purification of a 60-kDa protein from chicken liver associated with the internal nuclear matrix and closely related to carboxylesterases.

A 60-kDa protein was purified from chicken liver internal nuclear matrix and its nuclear localization was confirmed by immunofluorescence analysis. Structural information acquired from sequence analysis of the intact protein and of fragments obtained from enzymatic and chemical cleavages strongly suggests that it belongs to the carboxylesterases family, even if with some very peculiar features. The N-terminal sequence of the 60-kDa protein is completely different from the other carboxylesterases, but is similar to a region that is normally internal to all mammalian esterase sequences and localized after the serine residue at the active site. This suggests that the protein may be derived from a gene duplication and/or rearrangement. Since the 60-kDa protein shows a low esterase activity of about 0.2 micromol x min(-1) x mg(-1) using either p-nitrophenyl acetate or p-nitrophenyl butyrate as substrates, it is not possible to rule out that the protein shares only a sequence similarity with carboxylesterases and is not a true esterase. Otherwise it could be an esterase which has developed different properties, i.e. a special substrate specificity, the requirement of additional factors or a different stability in solution. In the latter case, this protein could be related to the physiological control of hydrolysis of exogenous and endogenous esters which can act on nuclear functions and/or metabolism.

Amino Acid Sequence↗

PubMed1995

DNA-nuclear matrix interactions analyzed by cross-linking reactions in intact nuclei from avian liver.

To detect the interactions of DNA with the nuclear matrix proteins, DNA-protein cross-linkages were induced in intact nuclei from chicken liver by the use of cis-diammine dichloroplatinum. Methods have been devised for fast purification both of the proteins and of the DNA fragments involved in the cross-linked complexes. By Southern-Western blotting a number of matrix proteins isolated from the complexes have been shown to recognize specifically DNA sequences present in the cross-linked DNA fragments. This experimental approach not only allows to identify the nuclear matrix-DNA interactions existing in the nucleus before its disruption, but also provides a preparation of matrix proteins enriched in those species which are involved in such interactions and which can therefore be detected with high sensitivity.

Animals↗

PubMed1995

Protein-DNA interactions at the nuclear scaffold regions of DNA loops.

DNA in cell nuclei is organized in large loops, which are formed by the binding of DNA to proteins present in a nuclear structure called matrix or scaffold. There is ample evidence that these interactions have not only a structural role, but also a functional one. Studies of these interactions have so far been carried out mainly in vitro. Their relevance, therefore, for the in vivo situation is not proven. We have analyzed the DNA-protein interactions directly in the intact nucleus by means of cross-linking reactions. Cross-linking by UV irradiation and by cis-diamine dichloro platinum (II) have been performed on nuclei from chicken liver and compared. The platinum complex has been found to be more efficient. The proteins complexed to DNA have been isolated and analyzed, and have been found to be enriched in species present in the nuclear scaffold, independently from its method of preparation.

Animals↗

PubMed1994

RNA polymerase II from wheat germ: a cross-linking study of subunits topography.

RNA polymerase II purified from wheat germ has been treated with a series of cleavable bifunctional reagents and the resulting crosslinked products have been analyzed by diagonal electrophoresis. The results indicate that the three largest subunits (220, 140, and 42,40 kDa, respectively) form a core around which the smaller subunits are bound. The 220- and 140-kDa subunits can be also crosslinked together in the absence of bifunctional reagents by disulfide bond(s) formation. The 27-, 16.3- and 16-kDa subunits appear to be close to the largest subunit (220 kDa). The 21-kDa subunit is close to the 27- and 25-kDa subunits. The reaction of monofunctional reagents with the enzyme shows that the 42,40-kDa subunit is partially hidden in the interior of the protein molecule. On the basis of these results a model of the quaternary structure of the enzyme is proposed.

Cross-Linking Reagents↗

PubMed1994

Glycoproteins of the nuclear matrix from chicken liver cells.

The nuclear matrix from chicken liver cells contains a small amount of glycoproteins recognized by Concanavalin A. These proteins are present not only in the peripheral matrix, but also in the internal one. In this latter localization many glycoprotein species appear, by cross-linking experiments, to be placed in the proximity of DNA. The effect of a partial enzymatic deglycosylation of matrix preparations suggests that these proteins contribute to the stabilization of the native matrix structure.

Animals↗

PubMed1994

Specific recognition sites for oligosaccharides in avian liver nuclei.

The presence of glycoproteins and sugar-binding sites in the nucleus is well ascertained. In order to verify the existence of specific nuclear protein-carbohydrate interactions, oligosaccharides were released from nuclear or non-nuclear glycoproteins by Peptide-N-glycosidase F, labeled by reduction with NaB3H4 and added to whole chicken liver nuclei and to subnuclear insoluble fractions (nucleoli, nuclear matrix and nuclear envelope). The analysis of the binding of the oligosaccharides to the nuclear fractions, performed in the presence or absence of competitor sugars, suggest that some rare oligosaccharides species, present only among the nuclear carbohydrates, are specifically recognized by the nuclear matrix.

Animals↗

PubMed1993

Purification of a 57kDa nuclear matrix protein associated with thiol:protein-disulfide oxidoreductase and phospholipase C activities.

Proteins of the internal nuclear matrix from chicken liver were fractionated, by chromatographic procedures, in non denaturing conditions. At least two fractions were present with phosphatidylinositol-specific phospholipase C and three with thiol:protein-disulfide oxidoreductase activity. A 57kDa protein was isolated which copurified with both these activities. Partial amino acid sequences showed a high degree of homology with a cytosolic protein previously identified as a phospholipase C and with a microsomal protein identified as a thiol:protein-disulfide oxidoreductase. Our finding leaves the question still unanswered of the real function of this protein, which for the first time has been isolated from the nuclear matrix.

Amino Acid Sequence↗

PubMed1993

The effect of polyols on the stability of duplex DNA.

It has been observed that double stranded DNA in solution is thermally destabilized by the presence of several organic molecules, among which are alcohols and polyols. This finding, and the fact that sugar moieties are components of some nuclear proteins and DNA binding drugs, may suggest a role for oligosaccharides in DNA recognition and/or interaction. In order to investigate this possibility, the effect of several sugars and other polyols on the thermal stability and on the conformation of DNA has been studied by high sensitivity differential scanning calorimetry and circular dichroism. While addition of small size polyols does not influence appreciably the melting enthalpy, it always results in a decrease in the DNA melting temperature. The magnitude of the destabilizing effect depends on the concentration of additives, reaching 13.5 degrees C in 50% (5.4M) glycerol. It also depends on the nature of the additives, e.g., sorbitol is more efficient than inositol, while dextran has no effect on the DNA melting temperature. According to circular dichroism results, DNA undergoes a significant structural change in the presence of small sugars or polyols: a continuous loss in 'B' character is observed as the glycerol concentration is gradually increased. This conformation change appears to be related to the decrease in thermostability.

Animals↗

PubMed1992

Differential scanning calorimetry of chicken erythrocyte nuclei.

Investigation of structural features of native chromatin requires the use of intact nuclei, a turbid material which cannot be analyzed by optical methods. Differential scanning calorimetry does not require optically clear samples and has been proved by a number of authors to be a powerful tool in this field of study. By this technique, chicken erythrocyte nuclei were found to undergo at least four thermal transitions, centered at 59, 74, 88 and 98 degrees C. The highest temperature transition is strongly dependent on age and storage conditions of the nuclei. Adequate storage conditions overcame this problem and reproducible scans were obtained over a period of several months. This technical improvement has permitted the reconsideration of the occurrence of the fourth calorimetric transition, previously believed to be displayed only in replicating nuclei. Evidence gathered in the presence of perturbants and possible ligands allows the assignment of the four transitions to a nuclear protein scaffold, histones, nucleosomal DNA and a superstructured form of DNA. Moreover, it suggests that the higher-order structure is stabilized by fibronectin-like proteins.

Animals↗

PubMed1992

Crosslinking of nuclear proteins to DNA by cis-diamminedichloroplatinum in intact cells. Involvement of nuclear matrix proteins.

In order to detect the nuclear matrix proteins involved in DNA binding, avoiding possible artifacts derived from the disruption of nuclei, proteins were crosslinked to DNA by the action of cis-diamminedichloroplatinum on intact chicken liver cells and analyzed by two-dimensional gel electrophoresis. At least eleven species of crosslinked proteins were found to derive from the nuclear matrix prepared from the same cell type, and five of these were found also among the proteins crosslinked to DNA in intact liver cells from ox and pig. This subset of common proteins, conserved in different animal species, is likely to have a fundamental role for the anchorage of DNA to the nuclear matrix.

Animals↗

PubMed1992

Isolation of a novel nuclear glycoprotein from pig kidney.

A nuclear glycoprotein with an apparent Mr of 66,000 Da has been isolated from pig kidney chromatin after extraction with urea, guanidine-HCl and 2 M NaCl, and some of its structural features have been characterized. It belongs to the group of N-glycosylated proteins, which in the nucleus has so far received little attention. From its monosaccharide composition and recognition by lectins its oligosaccharides appear to be of high mannose and/or hybrid types. Some properties of its protein moiety suggest that it has a role in the packing of the DNA loops in the condensed chromatin.

Amino Acid Sequence↗

PubMed1991

The presence of N-glycosylated proteins in cell nuclei.

The protein-DNA crosslinking capability of cis-dichloro diammineplatinum has been exploited to check the intranuclear location of N-glycosylated proteins. When intact liver cells were treated with this reagent, a number of glycoproteins, recognized by Concanavalin A, have been shown to become crosslinked to DNA; many of them have been recognized as nuclear matrix components. The recognition by this lectin was abolished by treatment with N-glycosidase F, showing the presence of N-glycosidic bonds between the sugar moiety and the protein. Most of the glycoproteins appeared to have high mannose oligosaccharide chains, but sialic acid containing oligosaccharides were also identified.

Animals↗