Removal of histone H1 from intact nuclei alters the digestion of nucleosome core DNA by staphylococcal nuclease

We have examined the gel profiles of staphylococcal nuclease digests of intact nuclei following different extents of removal of histone H1 by low pH. It was found that the submonomer fragment pattern (i.e. fragments less than 140 base pairs (bp) changed dramatically following removal of H1. The most striking feature of this change was a marked increase in the relative intensity of a band migrating at 102 +/- 4 bp when about 20-50% of the nuclear DNA is rendered acid soluble. All other submonomer bands decreased in relative intensity. There was no evidence for an approximately 100-bp repeat pattern accompanying the enhanced generation of the 102-bp fragment following H1 removal. This result, along with the comparisons of gel profiles for different extents of digestion, suggests that removal of histone H1 from nuclei results in an increased susceptibility of the DNA to staphylococcal nuclease at one or both ends of many of the core particles and that a strong block to further digestion occurs within these core particles resulting in the formation of a relatively stable 102-bp fragment.     

Thyrotropin-releasing activity of histone H2A, H2B and peptide MB35

Histones possess multiple hormone-like activities. We studied the specificity and signal transduction pathways involved in the thyrotrophin (TSH)-releasing activity of histones H2A, H2B and peptide MB35, a H2A fragment, using perifused and incubated dispersed rat pituitary cells and measuring TSH release by a specific R1A. Histones released TSH in a dose- and time-dependent fashion while peptide MB35 behaved as a weaker secretagogue. Trifluoperazine and EGTA blocked histone-stimulated TSH release while forskolin and other cAMP enhancers did not. We conclude that the TSH-releasing activity of histones H2A and H2B is mediated by calcium- and diacylglycerol-associated pathways.     

The presenilin 2 loop domain interacts with the mu-calpain C-terminal region

Presenilin 2 (PS2) is a gene responsible for the early-onset familial Alzheimer's disease (AD). PS2 mutations are considered to be closely related to the pathogenesis of AD. We screened for proteins that interact with PS2 to understand its pathological and physiological functions. Using the PS2 loop domain as the bait, the yeast two-hybrid system was used for screening, and mu-calpain was identified as a PS2 binding protein. In COS-1 cells, the interaction of PS2 with mu-calpain was confirmed by immunoprecipitation. These results suggested that PS2 and mu-calpain interact with each other, and might regulate each other's functions.     

Deposition-related sites K5/K12 in histone H4 are not required for nucleosome deposition in yeast

Histone H4 can be acetylated at N-terminal lysines K5, K8, K12, and K16, but newly synthesized H4 is diacetylated at K5/K12 in diverse organisms. This pattern is widely thought to be important for histone deposition onto replicating DNA. To investigate the importance of K5/K12 we have mutagenized these lysines in yeast and assayed for nucleosome assembly. Assaying was done in the absence of the histone H3 N terminus, which has functions redundant with those of H4 in histone deposition. Nucleosome assembly was assayed by three methods. Because nucleosome depletion may be lethal, we examined cell viability. We also analyzed nucleosome assembly in vivo and in vitro by examining plasmid superhelicity density in whole cells and supercoiling in yeast cell extracts. All three approaches demonstrate that mutagenizing K5 and K12 together does not prevent cell growth and histone deposition in vivo or in vitro. Therefore, K5/K12 cannot be required for nucleosome assembly in yeast. It is only when the first three sites of acetylation-K5, K8, and K12-are mutagenized simultaneously that lethality occurs and assembly is most strongly decreased both in vivo and in vitro. These data argue for the redundancy of sites K5, K8, and K12 in the deposition of yeast histone H4.     

The Leishmania infantum histone H3 possesses an extremely divergent N-terminal domain

The isolation of a Leishmania cDNA clone coding for an antigen identified as the histone H3 is described. The nucleotide sequence of the cDNA predicts that the Leishmania histone H3 contains 129 residues and that it has a molecular mass of 14,620 Da. Comparison of the amino acid sequence with the consensus sequence of the eukaryotic histone H3 shows that the Leishmania protein has a highly conserved globular region and an extremely divergent amino-terminal portion.     

Binding of the von Willebrand factor A1 domain to histone

Human cytochrome P450 2D6 metabolizes more than 50 common drugs and is polymorphically expressed, with 5-10% of the population lacking expression caused by mutant genes. This may result in a defective and toxic response in deficient individuals treated with 2D6 drug substrates. Baculovirus-expressed 2D6 was used to immunize mice for hybridoma production and two clones yielded monoclonal antibodies, that were positive against 2D6 by ELISA and inhibited 2D6 catalysed metabolism of bufuralol, dextromethorphan and phenanthrene by more than 90%. The inhibitory activity was highly specific to 2D6 and the monoclonal antibodies did not bind to 11 other P450s, nor inhibit seven human P450s tested. Analysis of eight human liver microsome samples showed that their basal bufuralol 1'-hydroxylase activity varied from 6.7-83.5 pmol min-1 nmol-1 P450. The monoclonal antibody 512-1-8 inhibited 2D6-dependent bufuralol 1'-hydroxylase in these samples by 10-70% indicating a widely variable role for 2D6 in human liver bufuralol 1'-hydroxylase activity and a role for other P450s in bufuralol metabolism. Independent analysis of several recombinant human P450s showed that 2D6, 2C8, 2C9, 2C19 and 1A2 exhibited bufuralol 1'-hydroxylase activity with 2D6 and 2C19 being the most active. Further analysis of three liver samples was made with individual inhibitory monoclonal antibodies. Inhibitory antibodies to 2D6, 2B6, 2E1, 2C8/9/19, 3A4 and 1A2 were added to the microsomes either singly or additively. Inhibitory activity of bufuralol 1'-hydroxylase was observed with antibodies to 2D6 (14-76%), 2C8/9/19 (24-69%) and 1A2 (2-25%) indicating a variable and different role for each of these P450s in the bufuralol 1'-hydroxylase of human liver. The monoclonal antibodies to 2B6, 2E1 and 3A4 were not inhibitory, indicating that these enzymes play no role in bufuralol 1'-hydroxylase metabolism. When the three antibodies to 2D6, 2C8/9/19 and 1A2, respectively, were all added, the total bufuralol 1'-hydroxylase of the liver samples was inhibited by more than 90%, indicating that the latter P450s catalyse all of liver bufuralol 1'-hydroxylase metabolism. These studies demonstrate that inhibitory monoclonal antibodies offer a simple and precise method for assessing the quantitative role of each P450 in the metabolism of a P450 substrate in a tissue, which include drugs, carcinogens, mutagens, toxic chemicals and endobiotics.     

Negative charges in the C-terminal domain stabilize the alphaB-crystallin complex

alphaB-Crystallin is one of the six known mammalian small heat-shock proteins (sHsps). These are characterized by the presence of a conserved sequence of 80-100 residues, which constitutes the putative C-terminal domain. Like other sHsps, alphaB-crystallin forms multimeric globular complexes, often in combination with related sHsps. Here we show that in a yeast two-hybrid system, alphaB-crystallin can specifically interact with itself as well as with alphaA-crystallin and Hsp27. Analyses of the separate domains show that the conserved C-terminal domain (CalphaB) is essential for this interaction between subunits. To try and detect residues that are important in subunit interaction, the CalphaB domain was used in a two-hybrid screen as bait to select randomly mutated CalphaB mutants. In this way we obtained nine mutants that were still able to interact with wild-type CalphaB despite the presence of up to 15 replacements. Similarly, we obtained 16 mutants that were unable to bind, because of the presence of just three to nine replacements. In binding CalphaB mutants, lysine residues were most often replaced by glutamic acid residues, and in non-binding CalphaB mutants, acidic residues were often found to be replaced by non-charged residues. This indicates that negative charges are important for subunit interaction and we propose a model to explain this role of acidic residues. Furthermore, we observed that two homologs of alphaB-crystallin, alphaA-crystallin and Hsp27, generally interact similarly with the binding and non-binding CalphaB mutants as does alphaB-crystallin. This suggests that interactions involved in the complex formation of these three sHsps are largely comparable.     

Asymmetric linker histone association directs the asymmetric rearrangement of core histone interactions in a positioned nucleosome containing a thyroid hormone response element

We describe histone-DNA cross-linking in a positioned nucleosome containing a thyroid hormone response element (TRE) from the Xenopus laevis thyroid hormone receptor betaA gene (TRbetaA). Histones H3 and H4 are cross-linked to DNA in the nucleosome core within 30 base pairs to either side of the dyad axis. Histone H2A cross-links to DNA in the core at the dyad axis, and histones H2A and H2B have extensive interactions with DNA 40-80 bp away from the dyad axis. Linker histone H5 and the globular domain of Xenopus H1(0) associate asymmetrically with DNA at one edge of the TRbetaA nucleosome. Nevertheless, the asymmetric association of H5 leads to a significant rearrangement of core histone-DNA contacts at the dyad axis of the nucleosome. In the presence of linker histone, cross-linkings of H4 within 15 bp to one side of the dyad axis, of histone H2A at the dyad axis, and of H2A and H2B 40-80 bp to one side of the dyad axis are all reduced. This reduction in cross-linking occurs preferentially on the side of the nucleosome to which H5 is bound. Our results indicate that core histone contacts within mononucleosomes are conformationally dynamic and that linker histone incorporation at the edge of the nucleosome can influence core histone-DNA interactions in an asymmetric way including contacts at the dyad axis.     

DNA sequences coding for the H2B histone of Psammechinus miliaris

Starting from restriction enzyme cleavage sites of known topologies the DNA sequences coding for two thirds of the H2B histone protein, together with some 3 inch extracistronic sequences, have been determined for the sea urchin Psammechinus miliaris. This unambiguously identifies this gene and further reveals its 5 inch-3inch polarity and accurate map position in a cloned histone DNA repeat unit.     

The C-terminal domain of matrilin-2 assembles into a three-stranded alpha-helical coiled coil

Matrilin-2 is a member of von Willebrand factor A containing extracellular matrix proteins in which the cDNA-derived sequence shows similar domain organization to cartilage matrix protein/matrilin-1, but information on the protein structure is limited. Here we studied the oligomerization potential of a synthetic peptide NH2-ENLILFQNVANEEVRKLTQRLEEMTQRMEALENRLKYR-COOH corresponding to the C-terminal sequence of mouse matrilin-2. The central portion of this sequence shows a periodicity of hydrophobic residues occupying positions a and d of a heptad pattern (abcdefg)n, which is characteristic for alpha-helical coiled-coil proteins. Circular dichroism spectroscopy revealed a high alpha-helical content, and the shape of the spectra is indicative for a coiled-coil conformation. Chemical cross-linking and size exclusion chromatography suggest a homotrimeric configuration. Thermal denaturation in benign buffer shows a single cooperative transition with DeltaH0 = -375 kJ/mol. Melting temperatures Tm varied from 38 to 51 degreesC within a concentration range of 10 to 85 microM, which is about 35 degreesC lower than determined for a peptide corresponding to the C-terminal domain of matrilin-1. The data suggest that despite the low sequence identity within this region, matrilin-2 will form a homotrimer as matrilin-1 does.     

Phospholipase C-gamma 1 binds to actin-cytoskeleton via its C-terminal SH2 domain in vitro

Association of phospholipase C (PLC)-gamma 1 with the cytoskeleton has been postulated to be one of the crucial steps for PLC-gamma 1 activation and translocation to the plasma membrane. In this report, direct binding assays were carried out to study which fragment of PLC-gamma 1 Src homology region has been able to bind to the actin-cytoskeleton. Using GST fusion proteins containing various deletions of the PLC-gamma 1 Src homology region, it was found that PLC-gamma 1 binds to the actin-cytoskeleton directly via its C-terminal SH2 domain but not the SH3 domain in vitro. However, the binding of the C-terminal SH2 domain of PLC-gamma 1 to actin did not interfere with the SH2 domain's ability to associate with phosphotyrosine, which suggested that actin and phosphotyrosine residues may bind to different sequences in the C-terminal SH2 domain of PLC-gamma 1.     

A developmental stage-specific histone H1 homolog of Coxiella burnetii

Two DNA-binding proteins have been detected in Coxiella burnetii by southwestern (DNA-protein) blotting. One of these, termed Hq1, is enriched in the small cell variant stage of the developmental cycle and displays compositional and primary amino acid sequence similarities to eukaryotic histone H1. C. burnetii appears to be another example of an intracellular parasite with morphologically distinct developmental forms whose nucleoid structure may be controlled by histone H1 homologs.     

A C-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit

The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication. All other replication proteins, including DNA polymerase alpha-primase (polalpha-primase), are derived from the host cell. We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polalpha-primase subunits. Deletion analysis showed that a C-terminal region of E1 (amino acids [aa] 432 to 583 or 617) is required for E2 binding. HPV-16 E1 was unable to bind the ori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2. Of the polalpha-primase subunits, only p68 bound E1, and binding was competitive with E2. The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues. In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding. Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain. Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases. The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.     

alpha-Crystallin C-terminal domain: on the track of an Ig fold

New results obtained from a two-dimensional sequence analysis of the small heat shock protein (shsp) family are described. It is confirmed that the conserved C-terminal alpha-crystallin domain is essentially made of beta-strands, most probably two groups of beta-strands separated by a large loop. A direct correspondence between the putative beta-strands that have been identified in shsps and the seven beta-strands of a classical immunoglobulin-like fold is proposed. The hypothesis that the shsp family could belong to the immunoglobulin superfamily (IgSF) is consistent with the ubiquitous distribution and the multifunctional properties of the crystallins that are now emerging.