Heat shock-induced redistribution of a 160-kDa nuclear matrix protein.

In this paper we describe a 160-kDa protein (p160) which is present in the nuclear matrix of rat, mouse, and human cells. Biochemical and ultrastructural analysis shows that p160 is associated with the internal matrix and is not present in the lamina-pore complex. Immunoelectron microscopy shows that the protein is part of the extranucleolar, fibrogranular network of the nuclear matrix. During an in vivo 42 degrees C heat treatment of HeLa cells, A431 human epidermoid cells, and T24 human bladder carcinoma cells, p160 transiently formed large clusters inside the nucleus. These p160 clusters are associated with the nuclear matrix network, as judged by immunolabeling on isolated nuclear matrices. The percentage of cells showing p160 clusters increased proportionally with longer heat treatments, reaching a maximum after a period of 3 h. At this time 70 +/- 5% of the cells displayed these clusters. Clustering decreased after longer heat treatments and the anti-p160 staining pattern became diffuse granular again. Other nuclear components, such as the A1 antigen of hnRNP (ribonucleoprotein), the Sm antigen of snRNPs, and lamins A and C, did not cluster during the 42 degrees C treatment, indicating that this reallocation is characteristic for the p160 matrix protein. These results demonstrate that p160 is an internal nuclear matrix element with a dynamic spatial distribution.     

Interactions of a laminin-binding peptide from a 33-kDa protein related to the 67-kDa laminin receptor with laminin and melanoma cells are heparin-dependent.

A laminin-binding peptide (peptide G), predicted from the cDNA sequence for a 33-kDa protein related to the 67-kDa laminin receptor, specifically inhibits binding of laminin to heparin and sulfatide. Since the peptide binds directly to heparin and inhibits interaction of another heparin-binding protein with the same sulfated ligands, this inhibition is due to direct competition for binding to sulfated glycoconjugates rather than an indirect effect of interaction with the binding site on laminin for the 67-kDa receptor. Direct binding of laminin to the peptide is also inhibited by heparin. This interaction may result from contamination of the laminin with heparan sulfate, as binding is enhanced by the addition of substoichiometric amounts of heparin but inhibited by excess heparin and two heparin-binding proteins. Furthermore, laminin binds more avidly to a heparin-binding peptide derived from thrombospondin than to the putative receptor peptide. Adhesion of A2058 melanoma cells on immobilized peptide G is also heparin-dependent, whereas adhesion of the cells on laminin is not. Antibodies to the beta 1-integrin chain or laminin block adhesion of the melanoma cells to laminin but not to peptide G. Thus, the reported inhibition of melanoma cell adhesion to endothelial cells by peptide G may result from inhibition of binding of laminin or other proteins to sulfated glycoconjugate receptors rather than from specific inhibition of laminin binding to the 67-kDa receptor.     

Purification and properties of a 90-kDa nuclear actin-binding protein

A 90 kDa actin-binding nuclear protein (ABNP) with a pI of 5.2 has been purified from the 0.7 M NaCl extracted residue fraction of chromatin prepared from Novikoff hepatoma cell nuclei. This residue fraction was previously shown to contain nuclear actin. Although twice the size, similar in pI, and similar in amino acid composition to actin, the tryptic peptide map for ABNP is distinct and contains the appropriate number of tyrosine-containing tryptic peptides for a protein of 90,000 molecular weight. A comparison of the amino acid composition of ABNP with those reported in the literature for gelsolin and villin, using a calculation of S delta Q as an indication of relatedness, results in values of 30 and 27, respectively. Actin-binding activity, however, was demonstrated for both crude and gel purified ABNP using a gel-overlay technique that employs 125I-G-actin to detect specific actin-binding proteins.     

In vitro binding of purified NahR regulatory protein with promoter Psal

The NahR regulatory protein activates the naphthalene catabolic operon through binding to the Psal promoter in the presence of salicylate. Here, we investigated in vitro binding interaction between NahR and Psal using purified functional recombinant NahR. The T7-tagged NahR was shown to exist as a monomer in solution. Electrophoretic mobility shift assay (EMSA) showed that purified NahR bound to Psal in 3 different forms, whereas surface plasmon resonance (SPR) showed on an SPR chip at ratios ranging from 1:1 (at 0.42 microM NahR) to 8:1 (at 6.8 microM NahR). The binding was slightly inhibited by salicylate, suggesting that salicylate may not be involved in the binding of NahR to the promoter, but rather may be important in the activation of prebound NahR. An examination of the binding kinetics by SPR for the interaction between NahR and Psal revealed that the equilibrium dissociation constant was approximately 2.44 x 10(-6) M and the association and dissociation rates were 7.82 x 10(4) M(-1) s(-1) and 0.191 s(-1), respectively. These results demonstrate for the first time that purified NahR binds as a monomer to Psal and undergoes multimerization. In addition, we present novel data on the kinetics of NahR binding.     

In silico characterization of a RNA binding protein of cattle filarial parasite Setaria digitata

Human lymphatic filariasis (HLF) is a neglected tropical disease which threatens nearly 1.4 billion people in 73 countries worldwide. Wuchereria bancrofti is the major causative agent of HLF and it closely resembles cattle filarial parasite Setaria digitata. Due to difficulties in procuring W. bancrofti parasite material, S. digitata cDNA library has been constructed to identify novel drug targets against HLF and many of the cDNA sequences are yet to be assigned structure and function. In this study, a 549 bp long cDNA (sdrbp) has been sequenced and characterized in silico. The shortest ORF of 249 bp from the isolated cDNA encodes a polypeptide of 82 amino acids and shows an amino acid identity of 54% with the RRM domain of human cleavage stimulation factor-64 kDa subunit (CstF-64). Structure of the protein (sdRBP) obtained by homology modelling using RRM of CstF-64 as template adopts classical RRM topology (β1α1β2β3α2β4). sdRBP model built was validated by superimposition tools and Ramachandran plot analysis. CstF-64 plays an important role in pre-mRNA polyadenylation by interacting with specific GU-rich downstream sequence element. Molecular docking studies of sdRBP with different RNA molecules revealed that sdRBP has greater binding affinity to GU-rich RNA and comparable results were obtained upon similar docking of RRM of CstF-64 with the same RNA molecules. Therefore, sdRBP is likely to perform homologous function in S. digitata. This study brings new dimensions to the functional analysis of RNA binding proteins of S. digitata and their evaluation as new drug targets against HLF.     

RFP is a DNA binding protein associated with the nuclear matrix.

We reported that the RFP gene encodes a protein with putative zinc finger domains and was involved in the activation of the ret proto-oncogene. To further characterize the RFP protein, we developed a polyclonal antibody against the product synthesized from a fragment of the RFP cDNA expressed in Escherichia coli. Western blot analysis showed that RFP was identified as a 58 kDa protein in cell lysates from four human and rodent cell lines and from mouse testis. In addition, a unique 68 kDa protein was detected in the testis. Using AH7974 (rat ascites hepatoma) and Raji (human Burkitt lymphoma) cells, we demonstrated strong association of RFP with the nuclear matrix. Furthermore, RFP solubilized from the nuclear matrix had DNA-binding activity although it appears to bind more preferentially to double-stranded DNA than to single-stranded DNA. These results thus suggest that RFP may play a role in molecular processes which occur in the nuclear matrix.     

Pressure-exploration of the 33-kDa protein from the spinach photosystem II particle.

The 33-kDa protein isolated from the spinach photosystem II particle is an ideal model to explore high-pressure protein-unfolding. The protein has a very low free energy as previously reported by chemical unfolding studies, suggesting that it must be easy to modulate its unfolding transition by rather mild pressure. Moreover, the protein molecule consists of only one tryptophan residue (Trp241) and eight tyrosine residues, which can be conveniently used to probe the protein conformation and structural changes under pressure using either fluorescence spectroscopy or fourth derivative UV absorbance spectroscopy. The different experimental methods used in the present study indicate that at 20 degrees C and pH 6, the 33-kDa protein shows a reversible two-state unfolding transition from atmospheric pressure to about 180 MPa. This value is much lower than those found for the unfolding of most proteins studied so far. The unfolding transition induces a large red shift of the maximum fluorescence emission of 34 nm (from 316 nm to 350 nm). The change in standard free energy (DeltaGo) and in volume (DeltaV) for the transition at pH 6.0 and 20 degrees C are -14.6 kJ.mol-1 and -120 mL.mol-1, respectively, in which the DeltaGo value is consistent with that obtained by chemical denaturation. We found that pressure-induced protein unfolding is promoted by elevated temperatures, which seem largely attributed to the decrease in the absolute value of DeltaGo (only a minor variation was observed for the DeltaV value). However, the promotion of the unfolding by alkaline pH seems mainly related to the increase in DeltaV without any significant changes in DeltaGo. It was also found that NaCl significantly protects the protein from pressure-induced unfolding. In the presence of 1 M NaCl, the pressure needed to induce the half-unfold of the protein is shifted to a higher value (shift of 75 MPa) in comparison with that observed without NaCl. Interestingly, in the presence of NaCl, the value of DeltaV is significantly reduced whilst that of DeltaGo remains as before. The unfolding-refolding kinetics of the protein has also been studied by pressure-jump, in which it was revealed that both reactions are a two-state transition process with a relatively slow relaxation time of about 102 s.     

Phosphorylation of bovine brain 81-kDa acidic calmodulin binding protein (ACAMP-81) in vitro.

We found a novel 81-kDa acidic protein (ACAMP-81) in the bovine brain membrane fraction, which bound to calmodulin in a Ca(2+)-dependent manner. The present study reveals physicochemical properties and phosphorylation of this protein with various protein kinases in vitro. The Stokes radius and sedimentation coefficient were calculated to be 52 A and 2.05 S, respectively, suggesting that the structure of ACAMP-81 is highly elongated. Purified Ca2+/phospholipid-dependent protein kinase (protein kinase C), cAMP-dependent protein kinase, and Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) catalyzed the incorporation of 1.46, 0.72, and 0.44 mol of phosphate/mol of ACAMP-81, respectively. The amino acid residues of ACAMP-81 phosphorylated by either protein kinase C or cAMP-dependent protein kinase were almost exclusively on serine. Sequential phosphorylation of ACAMP-81 by cAMP-dependent protein kinase and protein kinase C resulted in the additional incorporation of 1.15 mol of [32P]phosphate into ACAMP-81. Comparison of phosphopeptide maps of ACAMP-81 phosphorylated by each kinase revealed that there are two classes of phosphorylatable polypeptide, one is phosphorylatable by both protein kinases which contained two polypeptides and the others are specific sites for protein kinase C.     

Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein

The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation.     

In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins

An in vitro system was developed that provides a quick microscopic assay for nuclear transport. The assay uses an extract of Xenopus eggs, normal or synthetic nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. This in vitro system accurately mimics in vivo nuclear transport, both in exclusivity and in the amount of accumulation observed (up to 17-fold). Selective accumulation of fluorescent nucleoplasmin is observed microscopically within 30 min with rat liver nuclei, Xenopus embryonic nuclei, regrown Xenopus sperm nuclei, or nuclei reconstituted in vitro from bacteriophage lambda DNA. This transport requires the signal domain of nucleoplasmin. Furthermore, the ability of nuclei to accumulate nucleoplasmin directly correlates with their ability to exclude the fluorescent non-nuclear proteins, FITC-immunoglobulin and phycoerythrin. An active transport model would predict that nuclear transport be temperature- and energy- dependent and that inhibition of transport by either low temperature or energy depletion would be reversible. Both predictions were confirmed in our system. Nucleoplasmin accumulation increases with temperature, while the protein is completely excluded at 0 degrees C. The effects of low temperature are reversible. As found for 125I-labeled nucleoplasmin (Newmeyer, D. D., J. M. Lucocq, T. R. Burglin, and E. M. De Robertis, 1986, EMBO (Eur. Mol. Biol. Organ.) J., 5:501-510), transport of fluorescent nucleoplasmin is inhibited by ATP depletion. This effect is reversed by later ATP addition. Under ATP-depleted conditions non- nuclear proteins continue to be excluded. These results argue for a direct role of ATP in transport rather than for a simple role in preserving envelope integrity. In a first step towards defining the minimum requirements for a transport medium, egg extracts were depleted of membrane vesicles. Membrane-depleted extracts neither support transport nor maintain the integrity of the nuclear envelope.     

Intracellular distribution of a nuclear localization signal binding protein.

The transport of proteins into the nucleus requires the recognition of a nuclear localization signal sequence. Several proteins that interact with these sequences have been identified, including one of about 66 kDa. We have prepared antibodies that recognize the 66-kDa nuclear localization signal binding protein (NLSBP) and inhibit nuclear localization in vitro. By immunofluorescence, it is seen that the NLSBP is predominantly cytoplasmic and is distributed peripherally around the nucleus and the microtubule organizing center. There is also a weak punctate staining of the surface of the nucleus. Methanol-fixed cells can also be stained directly with fluorescently labeled karyophilic proteins. These stains reveal the same cytoplasmic structures as anti-NLSBP. The expression of the NLSBP is growth dependent. When cells grown to confluence are examined, the cytoplasmic staining is greatly reduced, leaving the punctate nuclear staining as the predominant feature. In serum-starved cells, very little staining of either the cytoplasm or the nucleus can be seen. Upon simulation by the addition of serum, the original cytoplasmic and nuclear envelope staining is restored. Cells grown in the presence of colchicine or taxol have an altered NLSBP distribution but apparently normal cytoplasmic nuclear transport.     

A 35-kDa protein binding to a cytosine-rich strand of hypervariable minisatellite DNA.

A minisatellite-binding protein, Msbp-4, with a molecular mass of 35 kDa has been purified from mouse tumor cells that binds to hypervariable Pc-1 and Pc-2 minisatellites. The binding is much more efficient than that to genetically stable minisatellite homologues. As assayed by Southwestern analysis, Msbp-4 favors multiple copies of the Pc-2 repeat sequence GGCAGGA and requires the cytosine-rich single strand for the binding. The activity is also present in extracts from mouse testis but not from liver. The phosphatase treatment revealed that Msbp-4 is phosphorylated and may have a regulatory function, because dephosphorylation affects the activity and specificity of the binding. Sequence preference is demonstrated by a competition experiment using single-base substitution mutants. Thus, the binding properties of Msbp-4 observed here lead to an implication that the protein-DNA complexes result in formation of a single-stranded DNA loop of the G-rich strand in the minisatellite which may enhance the ability of the minisatellite to undergo recombination.     

In vitro phosphorylation of S-100 protein by brain nuclear protein kinases

A three-fold increased ³²P incorporation was observed when S-100 protein was added to a nuclear protein kinase preparation (NPKP) from brain. The specificity of the reaction was indicated by two observations: an increase in ³²P incorporation was not found either with 14–302 protein or when S-100 was added to liver NPKP. SDS-gel analysis shows prominent incorporation of ³²P by brain NPKP into an endogenous brain protein having a molecular weight near 45000 daltons, and, in the presence of S-100, predominantly into S-100 protein itself. Liver NPKP in the presence of S-100, showed an increased incorporation of ³²P into endogenous proteins without any phosphorylation of S-100.     

In vitro binding of retinoids to the nuclear retinoic acid receptor alpha

We describe a rapid method for measuring in vitro binding properties of new synthetic retinoids to the recently identified nuclear receptor RAR alpha. Transfection of cos-7 cells with the expression vector RAR alpha O produces a 100-fold increase in intracellular RAR alpha concentration which allows us to perform accurate determination of binding parameters of various retinoids. Cytosol and nuclear extracts obtained after freeze drying of the transfected cells are incubated with a new stable tritiated analog of retinoic acid, [3H]CD367. Complete separation between RAR alpha and endogenous cellular retinoic acid binding protein is achieved by high-performance size-exclusion chromatography. These improved techniques provide a useful method for determining binding affinities of analogs to RAR alpha.     

In vitro fertilization in cattle: a review

In vitro maturation and fertilization of cattle oocytes and subsequent in vitro culture of zygotes and embryos is discussed in the context of recent encouraging data. Mass production of embryos produced in this way in the future will have a great impact on animal production and animal breeding plans, for example the so-called MO-ET (multiple ovulation and embryo transfer) plan which aims at establishing nucleus breeding herds.     

In vitro binding of an IgE protein to human platelets

Bronchoconstriction in extrinsic asthma is initiated by mediators released from IgE-sensitized leukocytes after contact with polyvalent antigen. Because platelets also contain soluble mediators that can cause bronchoconstriction, platelet activation and release of the contents of platelet granules may play a role in IgE-mediated responses under some circumstances. We therefore sought to determine if platelets are capable of binding IgE and if cross-linking this cell-bound IgE initiates secretion of platelet granule contents. Platelets from 10 normal donors were studied by using automated fluorescence analysis and fluorescence microscopy. We detected binding of a purified myeloma IgE protein to 24.1 +/- 9.6% (mean +/- 2 SD) of the gel-filtered platelets from these normal individuals. Although we could detect the binding of IgE and anti-IgE to a minority of cells, every normal individual had a population of platelets that bound IgE. The amount of IgE that bound to normal platelets appeared to be distributed heterogeneously among the IgE-positive platelet population. Platelets from two individuals with type II Glanzmann's thrombasthenia bound normal amounts of heat-aggregated IgG, but less than 3% of the platelets bound detectable IgE. Moreover, a combination of monoclonal antibodies to glycoproteins IIb and IIIa inhibited the binding of the IgE protein to normal platelets but did not affect the binding of aggregated IgG. Thus, the binding of IgE to human platelets appeared to require the presence of the glycoprotein IIb-IIIa complex. Binding of monomeric IgE to platelets, by itself, did not initiate either platelet aggregation or release of 14C-serotonin. However, both aggregation and secretion of serotonin followed the addition of anti-IgE to IgE-sensitized platelets. These studies indicate that human platelets can bind an IgE myeloma protein in vitro and that cross-linking of surface-bound IgE with anti-IgE initiates aggregation and secretion. If platelets have a similar capacity to bind normal IgE in vivo, it is possible that platelets may participate directly in several atopic or inflammatory disorders in man mediated by this class of antibody.     

Molecular cloning of a 25-kDa high affinity rapamycin binding protein, FKBP25.

Two FK506 binding proteins of molecular mass 12 kDa (FKBP12) and 13 kDa (FKBP13) have been identified as common cellular receptors of the immunosuppressants FK506 and rapamycin. Here we report the molecular cloning and overexpression of a 25-kDa rapamycin and FK506 binding protein (termed FKBP25) with peptidylprolyl cis-trans-isomerase (PPIase) activity. The amino acid sequence, predicted from the FKBP25 cDNA, shares identity with FKBP12 (44%) and FKBP13 (47%) in the C-terminal 97 amino acids. Unlike either FKBP12 or FKBP13, the nucleotide sequence of FKBP25 contains a number of putative nuclear localization sequences. The PPIase activity of recombinant FKBP25 was comparable with that of FKBP12. The PPIase activity of FKBP25 was far more sensitive to inhibition by rapamycin (IC50 = 50 nM) than FK506 (IC50 = 400 nM). PPIase activity of 100 nM FKBP25 was almost completely inhibited by 150 nM rapamycin while only 90% inhibition was achieved by 4 microM FK506. These data demonstrate that FKBP25 has a higher affinity for rapamycin than for FK506 and suggest that this cellular receptor may be an important target molecule for immunosuppression by rapamycin.     

Midkine binds to 37-kDa laminin binding protein precursor, leading to nuclear transport of the complex

Midkine (MK) is a heparin binding multifunctional protein that promotes cell survival and cell migration. MK was found to bind to 37-kDa laminin binding protein precursor (LBP), a precursor of 67-kDa laminin receptor, with K(d) of 1.1 nM between MK and LBP-glutathione-S-transferase fusion protein. The binding was inhibited by laminin, anti-LBP, amyloid beta-peptide, and heparin; the latter two are known to bind to MK. In CMT-93 mouse rectal carcinoma cells, LBP was mostly located in the cytoplasm as revealed by immunostaining with anti-LBP antibody. That a portion of LBP or 67-kDa laminin receptor was located at the surface of these cells was verified by inhibition of cell attachment to laminin-coated dishes by anti-LBP antibody. When MK was added to culture medium of these cells, a part of LBP migrated to the nucleus. The movement occurred concomitantly with nuclear transport of biotin-labeled MK. These findings suggested that the binding of MK to LBP caused nuclear translocation of the molecular complex.     

33-kDa C-terminal heparin binding fragment of fibronectin promotes attachment and spreading of hepatocytes

The possibility of interaction of hepatocytes with the heparin binding domain of Fibronectin was examined. Rat hepatocytes adhered to coverslips coated with the 33-kDa heparin binding fragment of the C-terminal region of plasma fibronectin. When different concentrations of the heparin binding fragment were used to coat coverslips and used as substratum, cell attachment showed saturation kinetics. Half the maximum attachment was observed at 30–40 min after seeding of cells. The cells became flat after 2–3 h indicating that they spread on the heparin binding domain as they do on intact fibronectin. Among the different glycosaminoglycans tested, maximum inhibition of attachment was observed for heparin. However it was not possible to completely inhibit attachment even at high concentrations. These results indicate that hepatocytes interact with fibronectin not only through the Arg-Gly-Asp-containing cell binding fragment, but also through the heparin binding domain of fibronectin and, further, that there exist heparin-dependent and heparin-independent mechanisms of interaction of cells with the 33-kDa heparin binding fragment of fibronectin