Post-translational cleavage of a histone H1-like protein in the sperm of Mytilus

Starting with total RNA from spermatogenic cells of Mytilus trossulus and using random priming, we have cloned and sequenced the c-DNAs corresponding to two variants of the sperm-specific protein PLII* (phi 2B). DNA sequencing in conjunction with mass spectrometry and protein sequence data have allowed us to establish that of the three sperm-specific proteins present in the sperm of Mytilus (PL-II*(phi 2B), PL-III (phi 1), PL-IV (phi 3)), the first and the last one are the result of post-translational cleavage of a common precursor. This common precursor is a member of the histone H1 family, and it exhibits inter- and intraspecific microheterogeneity.     

The chlamydial EUO gene encodes a histone H1-specific protease

Chlamydia trachomatis is an obligate intracellular pathogen, long recognized as an agent of blinding eye disease and more recently as a common sexually transmitted infection. Recently, two eukaryotic histone H1-like proteins, designated Hc1 and Hc2, have been identified in Chlamydia. Expression of Hc1 in recombinant Escherichia coli produces chromatin condensation similar to nucleoid condensation observed late in the parasite's own life cycle. In contrast, chromatin decondensation, observed during the early life cycle, accompanies down-regulation and nondetection of Hc1 and Hc2 among internalized organisms. We reasoned that the early upstream open reading frame (EUO) gene product might play a role in Hc1 degradation and nucleoid decondensation since it is expressed very early in the chlamydial life cycle. To explore this possibility, we fused the EUO coding region between amino acids 4 and 177 from C. trachomatis serovar Lz with glutathione S-transferase (GST) and examined the effects of fusion protein on Hc1 in vitro. The purified fusion protein was able to digest Hc1 completely within 1 h at 37 degrees C. However, GST alone exhibited no Hc1-specific proteolytic activity. The chlamydial EUO-GST gene product also cleaves very-lysine-rich calf thymus histone H1 and chicken erythrocyte histone H5 but displays no measurable activity towards core histones H2A, H2B, H3, and H4 or chlamydial RNA polymerase alpha-subunit. This proteolytic activity appears sensitive to the serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) and aspartic protease inhibitor pepstatin but resistant to high temperature and other broad-spectrum protease inhibitors. The proteolytic activity specified by the EUO-GST fusion product selectively digested the C-terminal portion of chlamydial Hc1, the domain involved in DNA binding, while leaving the N terminus intact. At a molar equivalent ratio of 1:1 between Hc1 and DNA, the EUO gene product cleaves Hc1 complexed to DNA and this cleavage appears sufficient to initiate dissociation of DNA-Hc1 complexes. However, at a higher molar equivalent ratio of Hc1/DNA (10:1), there is partial protection conferred upon Hc1 to an extent that prevents dissociation of DNA-Hc1 complexes.     

Association of the human papillomavirus type 11 E1 protein with histone H1

The E1 and E2 proteins are the only virus-encoded factors required for human papillomavirus (HPV) DNA replication. The E1 protein is a DNA helicase responsible for initiation of DNA replication at the viral origin. Its recruitment to the origin is facilitated by binding to E2, for which specific recognition elements are located at the origin. The remaining replication functions for the virus, provided by the host cell's replication machinery, may be mediated by further interactions with E1 and E2. Histone H1 was identified as an HPV type 11 (HPV-11) E1-binding protein by far-Western blotting and by microsequence analyses of a 34-kDa protein purified by E1 affinity chromatography. E1 also bound in vitro to H1 isolated under native conditions in association with intact nucleosomes. In addition, E1 and H1 were coimmunoprecipitated by an E1 antiserum from a nuclear extract prepared from cells expressing recombinant E1. Bound H1 was displaced from HPV-11 DNA by the addition of E1, suggesting that E1 can promote replication initiation and elongation by alteration of viral chromatin structure and disruption of nucleosomes at the replication fork. Furthermore, a region of the HPV-11 genome containing the origin of replication was identified which had weaker affinity for H1 than that of the remaining genome. This result suggests that the presence of a DNA structure at or near the HPV origin facilitates initiation of DNA replication by exclusion of H1. These results are similar to those of studies of simian virus 40 DNA replication, in which a large T antigen-H1 interaction and an H1-resistant region at the origin of DNA replication have also been demonstrated.     

Interaction of histone H1 with cis-platinum modified DNA

Cis-diamminedichloroplatinum(II) (cis-DDP) is known as an effective anticancer drug. Its therapeutic effect is supposed to be a consequence of the covalent binding to DNA. A number of cellular proteins were found to bind selectively to DNA modified by cis-DDP (but not by its isomer trans-DDP). Here we present our observations on interaction of the linker histone H1 with cis- and trans-DDP modified DNA fragments. The results afford new experimental information about the preferential binding of histone H1 to cis-DDP-distorted DNAs versus trans-DDP modified ones.     

An unusual histone H3 specific for early macronuclear development in Euplotes crassus

A bacterial strain (CF06) that mineralized both the carbonyl group and the aromatic ring of the insecticide carbofuran and that is capable of using carbofuran as a sole source of carbon and nitrogen was isolated from a soil in Washington state. Phospholipid fatty acid and 16S rRNA sequencing analysis indicate that CF06 is a Sphingomonas sp. CF06 contains five plasmids, at least some of which are required for metabolism of carbofuran. Loss of the plasmids induced by growth at 42 degrees C resulted in the inability of the cured strain to grow on carbofuran as a sole source of carbon. Introduction of the plasmids confers on Pseudomonas fluorescens M480R the ability to use carbofuran as a sole source of carbon for growth and energy. Of the five plasmids, four are rich in insertion sequence elements and contain large regions of overlap. Rearrangements, deletions, and loss of individual plasmids that resulted in the loss of the carbofuran-degrading phenotype were observed following introduction of Tn5.     

In vitro binding of H1 histone subtypes to nucleosomal organized mouse mammary tumor virus long terminal repeat promotor

The binding of all known linker histones, named H1a through H1e, including H1(0) and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8-16 nM to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nM. Most of the H1 histones, namely H1a, H1c, H1d/e, and H1(0), completely aggregate polynucleosomes (1.3 kilobase pairs, 6 nucleosomes) at 270-360 nM, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles.     

Expression of murine H1 histone genes during postnatal development

Connective tissue diseases encompass a wide group of nosologic entities of unknown etiology, characterized by multisystemic organ involvement, sharing an immunologic pathogenetic mechanism, producing a variety of inflammatory manifestations, and whose primary lesion is always a diffuse vasculitis. Any part of the cardiovascular system may be involved, including the pericardium, the myocardium, the endocardium and valves, the coronary arteries, the aorta, the pulmonary vasculature, the peripheral arteries, veins, arterioles, venules, and the capillary beds of almost every organ subsystem. Pathologic studies disclose a high prevalence of heart involvement, but the presence and extent of pathologic findings correlate poorly with clinical manifestations. With the advent of echocardiography-Doppler, milder and earlier cases are now recognized. Although these patients continue under the care of rheumatologists and internists, when cardiac involvement arises, cardiologists must be aware of the characteristics, outcome and management of connective tissue diseases.     

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.     

Organic solvents identify specific ligand binding sites on protein surfaces

Over many years, a great deal of attention has focused on the growth regulatory effects of androgens in prostate cells. This, has also prompted widespread interest in the role of these steroid hormones in prostate cancer pathogenesis. Even so, no-one has so far been able to identify the exact relationship between androgenic hormone levels and the risk of these diseases though differences in hormonal patterns amongst racial ethnic groups has been reported to reflect diversities in prostate cancer incidence. One of the difficulties stems from the fact that serum hormone levels do not reflect the changes observed in prostate tissue androgen concentrations as the normal prostate progresses to a disease state. In this article efforts will be directed towards understanding some of the intra-prostate-specific mechanisms responsible for activating and/or repressing the androgen-dependent gene network associated with the gradual transition to a hormone refractive neoplastic state.     

Prothymosin alpha modulates the interaction of histone H1 with chromatin

Prothymosin alpha (ProTalpha) is an abundant acidic nuclear protein that may be involved in cell proliferation. In our search for its cellular partners, we have recently found that ProTalpha binds to linker histone H1. We now provide further evidence for the physiological relevance of this interaction by immunoisolation of a histone H1-ProTalpha complex from NIH 3T3 cell extracts. A detailed analysis of the interaction between the two proteins suggests contacts between the acidic region of ProTalpha and histone H1. In the context of a physiological chromatin reconstitution reaction, the presence of ProTalpha does not affect incorporation of an amount of histone H1 sufficient to increase the nucleosome repeat length by 20 bp, but prevents association of all further H1. Consistent with this finding, a fraction of histone H1 is released when H1-containing chromatin is challenged with ProTalpha. These results imply at least two different interaction modes of H1 with chromatin, which can be distinguished by their sensitivity to ProTalpha. The properties of ProTalpha suggest a role in fine tuning the stoichiometry and/or mode of interaction of H1 with chromatin.     

Sequence and tissue distribution of chicken cellular nucleic acid binding protein cDNA

H+,K+-ATPase preparations from pig stomach were modified with a sulfhydryl fluorescence reagent, N-[p-(2-benzimidazolyl)phenyl] maleimide (BIPM). The addition of ATP to the modified enzyme preparations in the presence of Mg2+ decreased the BIPM fluorescence but increased the Trp fluorescence. After exhaustion of ATP, the fluorescence intensities increased and decreased to the original levels, respectively. The results of stopped flow and rapid quenching experiments suggested that the decrease in BIPM fluorescence (36/s) was accompanied by binding of Mg2+ and ATP or phosphorylation (35 36/s) which was followed by slower increases in Trp fluorescence (24/s) and light scattering (20/s). Tosylphenylalanyl chloromethyl ketone-trypsin treatment of the modified preparations, which showed an about 1% decrease in BIPM fluorescence accompanying phosphorylation, gave one major fluorescent peptide peak on reverse-phase chromatography. Amino acid sequence analysis of the peptide revealed the following sequence, Ser-Pro-Glu-X-Thr-His-Glu-Ser-Pro-Leu-Glu-Thr-Arg. On comparison with the amino acid sequence deduced from cDNA from pig stomach [Maeda, M., Ishizaki, J., and Futai, M. (1988) Biochem. Biophys. Res. Commun. 157, 203-209], X was shown to correspond to Cys241 of the alpha-chain in H+,K+-ATPase. These data and others suggest that the decrease in BIPM fluorescence at Cys241 reflects some molecular event triggered by the binding of ATP with Mg2+ and/or phosphorylation, whereas the increases in the intrinsic Trp fluorescence and light scattering reflect one after phosphorylation.     

The vertebrate linker histones H1 zero, H5, and H1M are descendants of invertebrate "orphon" histone H1 genes

We investigated the evolutionary history of the divergent vertebrate linker histones H1 zero, H5, and H1M. We observed that the sequence of the central conserved domain of these vertebrate proteins shares characteristic features with histone H1 proteins of plants and invertebrate animals which otherwise never appear in any vertebrate histone H1 protein. A quantitative analysis of 58 linker histone sequences also reveals that these proteins are more similar to invertebrate and plant histone H1 than to histone H1 of vertebrates. A phylogenetic tree deduced from an alignment of the central domain of all known linker histones places H1 zero, H5, and H1M in close vicinity to invertebrate sperm histone H1 proteins and to invertebrate histone H1 proteins encoded by polyadenylated mRNAs. We therefore conclude that the ancestors of the vertebrate linker histones H1 zero, H5, and H1M diverged from the main group of histone H1 proteins before the vertebrate type of histone H1 was established in evolution. We discuss this observation in the general context of linker histone evolution.     

Complement receptor 3 of macrophages is associated with galectin-1-like protein

We have previously identified a 16-kDa protein with a pI of 5.1 (P16/5.1) that is associated with macrophage CR3. Microsequencing of P16/5.1 indicated exclusive homology to the beta-galactoside-binding lectin, galectin-1. Abs specific to a galectin-1 unique peptide reacted with P16/5.1. The association of P16/5.1 with CR3 was specifically inhibited by lactose, which binds with high affinity to galectin-1. These data together with similarities in molecular mass and pI suggest that P16/5.1 is galectin-1. Two-color immunofluorescence staining revealed the expression of galectin-1 on the macrophage surface and its colocalization with CR3. However, a surplus of CR3 was free of galectin-1, and some galectin-1 molecules were associated with cell surface receptors other than CR3. Based on these results we propose two models depicting the functional significance of CR3-galectin-1 association: 1) homodimeric galectin-1 possessing a divalent sugar binding site may act as an extracellular adapter molecule that cross-links CR3 with other receptors; and 2) association of galectin-1 with beta-galactosides on the extracellular domain of CR3 may modify the binding affinity of the receptor to its ligand. These possibilities are not mutually exclusive and can clarify the mode by which CR3 transmits signals in macrophages.