Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have characterized the hydrodynamic behavior of nucleosome arrays in which the N- and C-terminal "tails" of the histone H2A-H2B and H3-H4 domains have been selectively removed by digestion with immobilized trypsin. The sedimentation coefficient of the polynucleosome fibers lacking the histone H2A-H2B tails exhibited a salt dependence close to that of the non-trypsinized nucleosome arrays. In contrast, the salt-dependent behavior of the H3-H4-trypsinized polynucleosome fibers was found to be closer to that observed for the nucleosome arrays on which all the histones were trypsinized. This indicates that the N- and C-terminal domains of histones H3-H4 play a major role in the folding of the chromatin fiber. Magnesium titration of the polynucleosome fibers consisting of these trypsinized histone octamer hybrids at low ionic strength indicates that the histone H3-H4 tails also play an important role in the association of the polynucleosome fibers. These findings suggest that, after linker histones (histones of the H1 family), the tails of the histone H3-H4 domains are the major players in the processes that lead to the intra-association (folding) and inter-association of the chromatin fiber.
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PMID:Major role of the histones H3-H4 in the folding of the chromatin fiber. 902 30

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from a plasmid containing a promoter and a terminator for this polymerase, (H3 x H4)2 tetramers deprived of their tail domains, and H2A x H2B dimers. Histone (H3 x H4)2 tetramers lacking their terminal domains were obtained from trypsin-digested nucleosomal cores. The oligonucleosomal templates containing (H3 x H4)2 tetramers lacking their tail domains, like the control templates with intact core histone octamers, protect approximately 146 base pairs of DNA against micrococcal nuclease digestion. The transcriptional inhibition caused by the association of DNA with core histone octamers is significantly reduced upon elimination of the tail domains of the (H3 x H4)2 tetramers. Apparently, the terminal domains of (H3 x H4)2 must be present to block transcription efficiently. These results show the important inhibitory role played by the tail domains of the histone (H3 x H4)2 tetramers, suggesting the involvement of these regions in transcriptional regulation.
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PMID:Transcriptional inhibitory role of the tail domains of histone (H3 x H4)2 tetramers. 975 Jan 70

Reconstitution of a DNA fragment containing a Xenopus borealis somatic type 5S rRNA gene into a nucleosome greatly restricts the binding of transcription factor IIIA (TFIIIA) to its cognate DNA sequence within the internal promoter of the gene. Removal of all core histone tail domains by limited trypsin proteolysis or acetylation of the core histone tails significantly relieves this inhibition and allows TFIIIA to exhibit high-affinity binding to nucleosomal DNA. Since only a single tail or a subset of tails may be primarily responsible for this effect, we determined whether removal of the individual tail domains of the H2A-H2B dimer or the H3-H4 tetramer affects TFIIIA binding to its cognate DNA site within the 5S nucleosome in vitro. The results show that the tail domains of H3 and H4, but not those of H2A and/or H2B, directly modulate the ability of TFIIIA to bind nucleosomal DNA. In vitro transcription assays carried out with nucleosomal templates lacking individual tail domains show that transcription efficiency parallels the binding of TFIIIA. In addition, we show that the stoichiometry of core histones within the 5S DNA-core histone-TFIIIA triple complex is not changed upon TFIIIA association. Thus, TFIIIA binding occurs by displacement of H2A-H2B-DNA contacts but without complete loss of the dimer from the nucleoprotein complex. These data, coupled with previous reports (M. Vettese-Dadey, P. A. Grant, T. R. Hebbes, C. Crane-Robinson, C. D. Allis, and J. L. Workman, EMBO J. 15:2508-2518, 1996; L. Howe, T. A. Ranalli, C. D. Allis, and J. Ausio, J. Biol. Chem. 273:20693-20696, 1998), suggest that the H3/H4 tails are the primary arbiters of transcription factor access to intranucleosomal DNA.
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PMID:The H3-H4 N-terminal tail domains are the primary mediators of transcription factor IIIA access to 5S DNA within a nucleosome. 1068 63

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.
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PMID:Linker DNA destabilizes condensed chromatin. 1146 48

We have investigated the role of the labile terminal domains of the core histones on the stability of the subunits of the protein core of the nucleosome by studying the thermodynamic behavior of the products of limited trypsin digestion of these subunits. The thermal stabilities of the truncated H2A-H2B dimer and the truncated (H3-H4)/(H3-H4)(2) system were studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. The thermal denaturation of the truncated H2A-H2B dimer at pH 6.0 and low ionic strength is centered at 47.3 degrees C. The corresponding enthalpy change is 35 kcal/mol of 11.5 kDa monomer unit, and the heat capacity change upon unfolding is 1.2 kcal/(K mol of 11.5 kDa monomer unit). At pH 4.5 and low ionic strength, the truncated (H3-H4)/(H3-H4)(2) system, like its full-length counterpart, is quantitatively dissociated into two truncated H3-H4 dimers. The thermal denaturation of the truncated H3-H4 dimer is characterized by the presence of a single calorimetric peak centered at 60 degrees C. The enthalpy change is 25 kcal/mol of 10 kDa monomer unit, and the change in heat capacity upon unfolding is 0.5 kcal/(K mol of 10 kDa monomer unit). The thermal stabilities of both types of truncated dimers exhibit salt and pH dependencies similar to those of the full-length proteins. Finally, like their full-length counterparts, both truncated core histone dimers undergo thermal denaturation as highly cooperative units, without the involvement of any significant population of melting intermediates. Therefore, removal of the histone "tails" does not generally affect the thermodynamic behavior of the subunits of the core histone complex, indicating that the more centrally located regions of the histone fold and the extra-fold structured elements are primarily responsible for their stability and responses to parameters of their chemical microenvironment.
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PMID:Thermodynamic studies of the core histones: stability of the octamer subunits is not altered by removal of their terminal domains. 1166 50

A histone mixture (H1, H2A, H2B, H3, and H4) derived from calf thymus stimulated IgM production by human-human hybridoma HB4C5 cells. On the contrary, the histone mixture did not increase IgM production by the human Burkitt's lymphoma cell line NAT-30, IgG production by the human B lymphoblastoid cell line HMy-2, and IgE production by the human myeloma cell line U266. The immunoglobulin production-stimulating activity of the histone mixture was inactivated by trypsin or chymotrypsin digestion. In addition, confocal laser microscopic analysis had shown that HB4C5 cells incorporated a lot of histone but other cell lines did not incorporate it as much. These facts strongly suggest that histone acts as an immunoglobulin production-stimulating factor (IPSF) after internalization into the human B cell lines and the native structure of histone is required for the IPSF activity.
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PMID:Increase of immunoglobulin productivity of human-human hybridoma HB4C5 cells by histone. 1216 44

Histone acetylation and methylation are processes that are generally considered to play crucial roles in the chromatin-based regulatory mechanism. Characterization of the histones as well as their modification sites has become increasingly important. In this paper, the use of LC-MS and peptide mapping methods to analyze chicken core histones and identify the modification sites is reported. Microbore C(4) HPLC separated the core histones into H2A, H2B, H3 and H4 using HFBA as the ion-pairing agent. The four subclasses of histones and their putative acetylated or methylated isoforms were identified by LC-MS simultaneously. MALDI-TOF and tandem mass spectrometry provided peptide mapping of the modification sites of the histones through trypsin digestion of the HPLC eluents. This approach is straightforward and prospective for further application in the field of chromatin research.
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PMID:Analysis of core histones by liquid chromatography-mass spectrometry and peptide mapping. 1245 May 36

Histones play important role in DNA packaging, replication and gene expression. Here, we describe the isolation and characterization of histone 2B (PvH2B) gene from the most common but non-cultivable human malaria parasite Plasmodium vivax. The isolated cDNA clone of PvH2B was allowed to express in Escherichia coli and the recombinant protein was purified by affinity chromatography. The expressed PvH2B protein showed DNA-binding properties on the South-Western analysis and the confocal microscopy localized it in the parasite nucleus. This gene is actively expressed during blood stages of the parasite and all P. vivax patients produced antibodies against the protein. The mRNA of PvH2B was found to contain a poly(A) tail at its 3' end, unlike abundant mRNA of human H2B. The encoded polypeptide is 118 amino acid long contains a nuclear targeting site, a signature motif of H2B and showed 74% homology to its host molecule. The structure of PvH2B showed that it has certain differences from that of its host at critical functional sites (viz acetylation, methylation, trypsin cleavage, DNA-binding and inter-histone interaction) which are required for general gene expression and DNA packaging. The distinctive structural features of P. vivax H2B described here may help in designing the specific antimalarial drugs.
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PMID:Identification, expression, modeled structure and serological characterization of Plasmodium vivax histone 2B. 1527 99

Daunomycin is an anticancer drug that is well-known to interact with DNA in chromatin. Using a compositionally defined chicken erythrocyte chromatin fraction, we have obtained conclusive evidence that the drug is also able to interact with chromatin-bound linker histones without any noticeable binding to core histones. The drug can interact in an equal fashion with both histone H1 and H5 and to a greater extent with core histones H3/H4 and H2A/H2B as free proteins in solution. Thus, the binding of daunomycin to linker histones in the chromatin fiber is most likely due to the well-known higher accessibility of these histones to the surrounding environment of the fiber. Binding of daunomycin to linker histones appears to primarily involve the trypsin-resistant (winged-helix) domain of these proteins. The studies described here reveal the occurrence of a previously undisclosed mechanism for the antitumor activity of anthracycline drugs at the chromatin level.
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PMID:Binding of antitumor antibiotic daunomycin to histones in chromatin and in solution. 1561 44

SCN- binds to the charged amino group of lysines, inducing local changes in the electrostatic free energy of histones. We exploited this property to selectively perturb the histone-DNA interactions involved in the stabilization of eu and heterochromatin. Differential scanning calorimetry (DSC) was used as leading technique in combination with trypsin digestion that selectively cleaves the histone end domains. Euchromatin undergoes progressive destabilization with increasing KSCN concentration from 0 to 0.3 M. Trypsin digestion in the presence of 0.2 M KSCN show that the stability of the linker decreases as a consequence of the competitive binding of SCN- to the amino groups located in the C and N-terminal domain of H1 and H3, respectively; likewise, the release of the N-terminal domain of H4 induces an appreciable depression in both the temperature and enthalpy of melting of core particle DNA. Unfolding of heterochromatin requires, in addition to further cleavage of H4, extensive digestion of H2A and H2B, strongly suggesting that these histones stabilize the higher order structure by forming a protein network which extends throughout the heterochromatin domain.
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PMID:SCN- binding to the charged lysines of histones end domains mimics acetylation and shows the major histone-DNA interactions involved in eu and heterochromatin stabilization. 1625


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