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Target validation using protein aptamers enables the characterization of a specific function of a target protein in an environment that resembles native conditions as closely as possible. A major obstacle to the use of this technology has been the generation of bioactive aptamers, which is dependent on the choice of scaffold. Constraining binding peptides within a particular scaffold does not necessarily result in binding aptamers, as suboptimal presentation of peptides can occur. It is therefore understandable that different peptides might require different scaffolds for optimal presentation. In this article, we describe a novel scaffold protein that bypasses the conventional requirement for scaffolds to have known rigid structures and yet successfully presents several peptides that need to adopt a wide range of conformations for binding to their target protein. Using an unstructured protein, 4EBP1, as scaffold, we successfully construct binding aptamers to three different target proteins: Mdm2, proliferating cell nuclear antigen, and cyclin A. The Mdm2-binding aptamer constructed using 4EBP1 as scaffold demonstrates better stability and bioactivity compared to that constructed using thioredoxin as scaffold. This new scaffold protein, which makes it relatively easy to create bioactive aptamers based on known interaction sequences, will greatly facilitate the aptamer approach to target validation. 相似文献
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E. V. Sheval O. A. Dudnik S. S. Abramchuk V. Y. Polyakov 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2009,3(2):168-183
According to the radial loop model of chromosome organization, a major role in the formation and maintenance of chromosomes
is played by the residual structures (the nuclear matrix in interphase nuclei and the chromosome scaffold in metaphase chromosomes).
However, in vivo microscopy has recently revealed that the components of these “static” structures are highly mobile and continuously
exchanged between specific target sites and the nucleoplasm or cytoplasm. This contradiction between predicted stability and
observed dynamics led us to reexamine the principles underlying the association of proteins with residual structures. In the
present paper, we have analyzed the association of two perichromosomal layer proteins, pKi-67 and B23, with the residual structures.
The results show that these two proteins are associated with residual structures throughout the cell cycle; only those structures
change that contain proteins precipitated by 2 M NaCl (nucleoli, perichromosomal layer, prenucleolar bodies, cytoplasm of
mitotic cells). Both pKi-67 and B23 remain associated with the nuclear matrix even when they are translocated to nucleoplasmic
foci due to inhibitor action or hypotonic treatment. However, in most cases it remains possible to extract a structurally
visible protein fraction with 2 M NaCl (protein distributed in nucleoplasm). One may suppose that the protein fraction associated
with residual structures includes molecules interacting with their binding sites at the moment of permeabilization, while
the free proteins are extracted (i.e., during the interaction with binding sites, these proteins form salt-resistant complexes;
however, on diffusion the same proteins are extractable by the high-salt solution). The residual structures may be considered
as a “snapshot” of all proteins transiently (or statically) bound to their target sites at the moment of permeabilization.
The article is published in the original. 相似文献
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Specific inhibition of DNA binding to nuclear scaffolds and histone H1 by distamycin. The role of oligo(dA).oligo(dT) tracts 总被引:24,自引:0,他引:24
Scaffold-associated regions (SARs) are A + T-rich sequences defined by their specific interaction with the nuclear scaffold. These sequences also direct highly specific binding to purified histone H1, and are characterized by the presence of oligo(dA).oligo(dT) tracts, which are a target for the drug distamyin, an antibiotic with a wide range of biological activities. The interaction of distamycin with SAR sequences results in the complete suppression of binding to either scaffolds or histone H1, suggesting that (dA.dT)n tracts play a direct role in mediating these specific interactions and that histone H1 and nuclear scaffold proteins may recognize a characteristic minor groove width or conformation. The effect of distamycin on these specific DNA-protein interactions in vitro suggests that binding of SARs to the nuclear scaffold and SAR-dependent nucleation of H1 assembly might be important targets of the drug in vivo. 相似文献
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Antinuclear autoantibodies (ANAs) derived from patients with systemic autoimmune diseases have proven to be powerful tools in cell and molecular biology. The availability of these autoantibodies has been instrumental in the identification and characterization of a wide range of intracellular proteins involved in essential cellular activities. Recently, these autoantibodies have been used in molecular studies of apoptosis, particularly in the identification of substrates cleaved by proteases of the ICE/CED-3 family during this cell death pathway. The identification of these substrates may help to understand the role of proteolysis in apoptosis. Examples of nuclear autoantigens whose cleavage during apoptosis have been defined using ANAs include the 70 kD protein of the U1 small nuclear ribonucleoprotein particle (U1-70 kD), the nuclear mitotic apparatus protein (NuMA), DNA topoisomerase I, the RNA polymerase I upstream binding factor (UBF), and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). The use of ANAs as probes for defining proteolytic events associated with apoptosis promises to yield important insights into the mechanisms driving this cell death pathway.Abbreviations ANAs
Antinuclear autoantibodies
- DNA-PKcs
DNA-dependent protein kinase
- ICE
interleukin-1 (IL-1)-converting enzyme
- MARs or SARs
matrix or scaffold attachment regions
- NuMA
nuclear mitotic apparatus protein
- PARP
poly (ADP) ribose polymerase
- snRNPs
small nuclear ribonucleoproteins
- SLE
systemic lupus erythematosus
- UBF
upstream binding factor 相似文献
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Heterogeneous nuclear ribonucleoproteins (hnRNPs) are thought to be involved in pre-mRNA processing. hnRNP-U, also termed scaffold attachment factor A (SAF-A), binds to pre-mRNA and nuclear matrix/scaffold attachment region DNA elements. However, its role in the regulation of gene expression is as yet poorly understood. In the present study, we show that hnRNP-U specifically enhances the expression of tumor necrosis factor alpha mRNA by increasing its stability, possibly through binding to the 3' untranslated region. We also show that hnRNP-U enhances the expression of several other genes as well, including GADD45A, HEXIM1, HOXA2, IER3, NHLH2, and ZFY, by binding to and stabilizing these mRNAs. These results suggest that hnRNP-U enhances the expression of specific genes by regulating mRNA stability. 相似文献
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Characterization of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements. 总被引:6,自引:0,他引:6
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We identified four proteins in nuclear extracts from HeLa cells which specifically bind to a scaffold attachment region (SAR) element from the human genome. Of these four proteins, SAF-A (scaffold attachment factor A), shows the highest affinity for several homologous and heterologous SAR elements from vertebrate cells. SAF-A is an abundant nuclear protein and a constituent of the nuclear matrix and scaffold. The homogeneously purified protein is a novel double stranded DNA binding protein with an apparent molecular weight of 120 kDa. SAF-A binds at multiple sites to the human SAR element; competition studies with synthetic polynucleotides indicate that these sites most probably reside in the multitude of A/T-stretches which are distributed throughout this element. In addition we show by electron microscopy that the protein forms large aggregates and mediates the formation of looped DNA structures. 相似文献
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ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure 总被引:17,自引:5,他引:12
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《The Journal of cell biology》1994,127(2):303-318
Here, we describe the cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified. ScII is structurally related to a protein, Smc1p, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae. ScII and the other members of the emerging family of SMC1-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an alpha-helical coiled-coil. Analysis of the ScII B site predicted that ScII might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme. ScII is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes. However, ScII appears not to be associated with the interphase nuclear matrix. ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II. 相似文献
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A nuclear matrix protein binds very tightly to DNA in the avian beta-globin gene enhancer 总被引:3,自引:0,他引:3
Current evidence suggests that DNA is covalently attached to proteins in the nuclear matrix of eukaryotic cells and that specific DNA sequences are tightly associated with the nuclear matrix. However, it has not been documented that specific DNA sequences can become covalently attached to nuclear matrix protein. We have examined the binding of cloned DNA sequences that contain the avian beta-globin gene enhancer, a region previously shown to be matrix associated in erythroid cells in vivo, with nuclear matrices from several avian tissue sources to determine if covalent DNA-protein bonds are formed. Our results indicate that sequence-specific DNA-protein complexes that are resistant to denaturation by SDS, boiling, and phenol and disulfide reduction are formed. Excess protein, capable of forming very tight bonds with DNA that contains the beta-globin gene enhancer, is present in cells in which matrix attachment of this DNA sequence is not detected in vivo. Evidence is presented that suggests that the protein to which DNA forms very tight bonds is not topoisomerase II. These results are discussed in relation to current models of the nuclear matrix and the utility of in vitro assays of matrix attachment regions using cloned DNA. 相似文献
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SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA
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Kipp M Göhring F Ostendorp T van Drunen CM van Driel R Przybylski M Fackelmayer FO 《Molecular and cellular biology》2000,20(20):7480-7489
SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold. 相似文献
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Mitotic phosphorylation of chromosomal protein HMGN1 inhibits nuclear import and promotes interaction with 14.3.3 proteins
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Prymakowska-Bosak M Hock R Catez F Lim JH Birger Y Shirakawa H Lee K Bustin M 《Molecular and cellular biology》2002,22(19):6809-6819
Progression through mitosis is associated with reversible phosphorylation of many nuclear proteins including that of the high-mobility group N (HMGN) nucleosomal binding protein family. Here we use immunofluorescence and in vitro nuclear import studies to demonstrate that mitotic phosphorylation of the nucleosomal binding domain (NBD) of the HMGN1 protein prevents its reentry into the newly formed nucleus in late telophase. By microinjecting wild-type and mutant proteins into the cytoplasm of HeLa cells and expressing these proteins in HmgN1(-/-) cells, we demonstrate that the inability to enter the nucleus is a consequence of phosphorylation and is not due to the presence of negative charges. Using affinity chromatography with recombinant proteins and nuclear extracts prepared from logarithmically growing or mitotically arrested cells, we demonstrate that phosphorylation of the NBD of HMGN1 promotes interaction with specific 14.3.3 isotypes. We conclude that mitotic phosphorylation of HMGN1 protein promotes interaction with 14.3.3 proteins and suggest that this interaction impedes the reentry of the proteins into the nucleus during telophase. Taken together with the results of previous studies, our results suggest a dual role for mitotic phosphorylation of HMGN1: abolishment of chromatin binding and inhibition of nuclear import. 相似文献
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A method of nuclear matrix and chromosomal scaffold preparation from cultured animal cells was developed. After the high-salt extraction, interphase and mitotic cells were not detached from the coverslips that enabled us to analyse the nuclear matrix and chromosomal scaffold in cells at all mitotic phases. Morphological methods (phase contrast microscopy and electron microscopy of ultrathin sections) did not reveal any structures that could be identified as a chromosomal scaffold. However, after staining with antibodies to XCAP-E and topoisomerase IIalpha some structures were revealed in metaphase cells having both localization and morphology of a chromosomal scaffold. The cell residuals were not stained with antibodies to XCAP-E and topoisomerase IIalpha, if the nuclear matrix and chromosomal scaffold were destabilized by addition of beta-mercaptoethanol. 相似文献
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In vivo binding of wild-type and mutant human immunodeficiency virus type 1 Rev proteins: implications for function.
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The Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1) is required for protein expression from the HIV-1 RNAs which contain a binding site for the Rev protein, termed the Rev-responsive element (RRE). This transactivator acts both at the level of splicing/transport of nuclear RNAs and at the level of translation of cytoplasmic RNAs. We used a monoclonal antibody specific for the HIV-1 Rev protein to immunoprecipitate cellular extracts from HIV-1-infected and -transfected cells. High levels of specific binding of wild-type Rev to the RRE-containing RNAs were found in cytoplasmic, but not nuclear, extracts from these cells. A Rev mutant which lacked both nuclear and cytoplasmic Rev function but retained RNA binding in vivo was generated. This binding was detectable with both nuclear and cytoplasmic extracts. These results verify the existence of direct binding of Rev to HIV-1 RNAs in vivo and conclusively prove that binding of Rev is not sufficient for nuclear or cytoplasmic Rev function. The results also support a direct role for Rev in the nuclear export and translation of HIV-1 RNAs. 相似文献
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Parra-Palau JL Scheper GC Wilson ML Proud CG 《The Journal of biological chemistry》2003,278(45):44197-44204
Eukaryotic initiation factor eIF4E binds to the 5'-cap structure of the mRNA and also to the molecular scaffold protein eIF4G. eIF4E is a phosphoprotein, and the kinases that act on it have been identified as the MAPK-interacting kinases Mnk1 and Mnk2. Mnk1/2 also bind to the scaffold protein eIF4G. The N-terminal region of Mnk1 has previously been shown to bind to importin alpha, a component of the nuclear transport machinery, although Mnk1 itself is cytoplasmic. Here we identify a CRM1-type nuclear export motif in the C-terminal part of Mnk1. Substitution of hydrophobic residues in this motif results in Mnk1 becoming nuclear. This has allowed us to study the features of Mnk1 that are involved in its transport to the nucleus. This process requires part, but not all, of a polybasic region near the N terminus of Mnk1. Residues required for nuclear transport are also required for its interaction with importin alpha. This polybasic region also serves a second function in that it is required for the binding of Mnk1 to eIF4G, although the residues involved in this interaction are not identical to those involved in the binding of Mnk1 to importin alpha. Interaction of Mnk1 with eIF4G promotes the phosphorylation of eIF4E. Mutations that reduce the binding of Mnk1 to eIF4G in vivo and in vitro also decrease the ability of Mnk1 to enhance eIF4E phosphorylation in vivo, underlining the importance of the eIF4G-Mnk1 interaction in this process. 相似文献
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AHM1, a novel type of nuclear matrix-localized, MAR binding protein with a single AT hook and a J domain-homologous region
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Interactions between the nuclear matrix and special regions of chromosomal DNA called matrix attachment regions (MARs) have been implicated in various nuclear functions. We have identified a novel protein from wheat, AT hook-containing MAR binding protein1 (AHM1), that binds preferentially to MARs. A multidomain protein, AHM1 has the special combination of a J domain-homologous region and a Zn finger-like motif (a J-Z array) and an AT hook. For MAR binding, the AT hook at the C terminus was essential, and an internal portion containing the Zn finger-like motif was additionally required in vivo. AHM1 was found in the nuclear matrix fraction and was localized in the nucleoplasm. AHM1 fused to green fluorescent protein had a speckled distribution pattern inside the nucleus. AHM1 is most likely a nuclear matrix component that functions between intranuclear framework and MARs. J-Z arrays can be found in a group of (hypothetical) proteins in plants, which may share some functions, presumably to recruit specific Hsp70 partners as co-chaperones. 相似文献
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