Why specific anti‐integrase antibodies from HIV‐infected patients can efficiently hydrolyze 21‐mer oligopeptide corresponding to antigenic determinant of human myelin basic protein

Human immunodeficiency virus‐infected patients possess anti‐integrase (IN) catalytic IgGs and IgMs (abzymes), which, unlike canonical proteases, specifically hydrolyze only intact globular IN. Anti‐myelin MBP abzymes from patients with multiple sclerosis and systemic lupus erythematosus efficiently hydrolyze only intact MBP. Anti‐MBP and anti‐IN abzymes do not hydrolyze several other tested control globular proteins. Here, we show that anti‐IN abzymes efficiently hydrolyze a 21‐mer oligopeptide (OP21) corresponding to one antigenic determinant (AGD) of MBP, whereas anti‐MBP abzymes extremely poorly cleave oligopeptides corresponding to AGDs of IN. All sites of IgG‐mediated and IgM‐mediated proteolysis of OP21 by anti‐IN abzymes were found for the first time by a combination of reverse phase and thin layer chromatography and mass spectrometry. Several clustered sites of OP21 cleavage were revealed and compared with the cleavage sites within the complete IN. Several fragments of OP21 had good homology with many fragments of the IN sequence. The active sites of anti‐IN abzymes are known to be located on their light chains, whereas heavy chains are responsible for the affinity for protein substrates. Interactions of intact IN with both light and heavy chains of the abzymes provide high affinity for IN and the specificity of its hydrolysis. Our data suggest that OP21 interacts mainly with the light chains of polyclonal anti‐IN abzymes, which possess lower affinity and specificity for substrate. The hydrolysis of the non‐cognate OP21 oligopeptide may be also less specific than the hydrolysis of the globular IN because in contrast to previously described serine protease‐like abzymes against different proteins, anti‐IN abzymes possess serine, thiol, acidic, and metal‐dependent protease activities. Copyright © 2013 John Wiley & Sons, Ltd.     

Specific anti‐integrase abzymes from HIV‐infected patients: a comparison of the cleavage sites of intact globular HIV integrase and two 20‐mer oligopeptides corresponding to its antigenic determinants

HIV‐infected patients possess anti‐integrase (IN) IgGs and IgMs that, after isolation by chromatography on IN‐Sepharose, unlike canonical proteases, specifically hydrolyze only IN but not many other tested proteins. Hydrolysis of intact globular IN first leads to formation of many long fragments of protein, while its long incubation with anti‐IN antibodies, especially in the case of abzymes (Abzs) with a high proteolytic activity, results in the formation of short and very short oligopeptides (OPs). To identify all sites of IgG‐mediated proteolysis corresponding to known AGDs of integrase, we have used a combination of reverse‐phase chromatography, matrix‐assisted laser desorption/ionization spectrometry, and thin‐layer chromatography to analyze the cleavage products of two 20‐mer OPs corresponding to these AGDs. Both OPs contained 9–10 mainly clustered major, medium, and minor sites of cleavage. The main superficial cleavage sites of the AGDs in the intact IN and sites of partial or deep hydrolysis of the peptides analyzed do not coincide. The active sites of anti‐IN Abzs are localized on their light chains, whereas the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of Abzs provide high specificity of IN hydrolysis. The affinity of anti‐IN Abzs for intact integrase was ~1000‐fold higher than for the OPs. The data suggest that both OPs interact mainly with the light chains of different monoclonal Abzs of the total pool of IgGs, which possesses lower affinity for substrates; and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific and remarkably different in comparison with the cleavage of intact globular IN. Copyright © 2013 John Wiley & Sons, Ltd.     

HIV-1 integrase-hydrolyzing antibodies from sera of HIV-infected patients

Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether Abs from patients with viral diseases can hydrolyze viral proteins. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of AIDS patients by chromatography on several affinity sorbents. We present evidence showing that 89.5% IgGs purified from the sera of HIV-infected patients using several affinity resins including Sepharose with immobilized integrase specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Several rigid criteria have been applied to show that the IN-hydrolyzing activity is an intrinsic property of AIDS IgGs but not from healthy donors. Similar to autoimmune proteolytic abzymes, IN-hydrolyzing IgGs from some patients were inhibited by specific inhibitors of serine and metal-dependent proteases but a significant inhibition of the activity by specific inhibitors of acidic- and thiol-like proteases was observed for the first time. Although HIV infection leads to formation of Abs to many viral and human antigens, no possible biological role for most of them is known. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for estimation of the immune status in AIDS patients.     

Anti‐histone H1 IgGs from blood serum of systemic lupus erythematosus patients are capable of hydrolyzing histone H1 and myelin basic protein

Novel hydrolytic activity of the anti‐histone H1 antibodies (Ab) toward histone H1 and myelin basic protein (MBP) was shown. Blood serum of ten patients with clinically diagnosed systemic lupus erythematosus (SLE), and nine healthy donors (control) were screened for the anti‐histone H1 antibody‐ and anti‐MBP antibody‐mediated specific proteolytic activity. IgGs were isolated by chromatography on Protein G‐Sepharose, and four of ten SLE patients appeared to possess IgGs that were capable of cleaving both histone H1 and MBP. Such activity was confirmed to be an intrinsic property of the IgG molecule, since it was preserved at gel filtration at alkaline and acidic pH. At the same time, proteolytic activity was absent in the sera‐derived Ab of all healthy donors under control. Anti‐histone IgGs were purified by the affinity chromatography on histone H1‐Sepharose. Their cross‐reactivity toward cationic proteins (histones, lysozyme, and MBP) and their capability of hydrolyzing histone H1 and MBP were detected. However, these IgGs were not cleaving core histones, lysozyme, or albumin. Capability of cleaving histone H1 and MBP was preserved after additional purification of anti‐histone H1 IgGs by the HPLC gel filtration. The protease activity of anti‐histone H1 IgG Ab was inhibited by serine protease inhibitors. Copyright © 2010 John Wiley & Sons, Ltd.     

Multiple sites of the cleavage of 17- and 19-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus

In contrast to canonical proteases, myelin basic protein (MBP)-Sepharose-purified IgG from multiple sclerosis (MS) and systemic lupus erythematosus (SLE) patients efficiently hydrolyze only MBP, but not many other tested proteins. It was shown that anti-MBP SLE IgGs cleave nonspecific tri- and tetrapeptides with an extremely low efficiency and cannot efficiently hydrolyse longer oligopeptides corresponding to antigenic determinants (AGDs) of HIV-1 integrase. To identify all sites of IgG-mediated proteolysis corresponding to two AGDs of MBP, we have used a combination of reverse-phase chromatography (RPhC), MALDI spectrometry, and TLC to analyze the cleavage products of two (17- and 19-mer) encephalytogenic oligopeptides corresponding to these AGDs. Both oligopeptides contained several clustered major and minor sites of cleavage. The active sites of anti-MBP abzymes are localized on their light chains, while the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high specificity of MBP hydrolysis. The affinity of anti-MBP abzymes for intact MBP was ～10(3)-fold higher than for the oligopeptides. The data suggest that both oligopeptides interact mainly with the light chain of different monoclonal abzymes of total pool of IgGs, which possesses lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific.     

IgGs containing light chains of the λ and κ type and of all subclasses (IgG1‐IgG4) from sera of patients with multiple sclerosis hydrolyze DNA

We present the first evidence demonstrating that small fractions of IgGs of all four subclasses (IgG1–IgG4) are catalytically active in the hydrolysis of DNA and on average their relative activity (nM supercoiled DNA/1mg IgG/1 h) increases in the order: IgG1 (0.58) < IgG2 (0.94) < IgG3 (1.4) < IgG4 (4.1), while their approximate relative contribution to the total activity of abzymes increases in the order: IgG1 (6.9%) < IgG3 (9.3%) < IgG2 (18.2%) < IgG4 (65.6%). On average IgGs containing light chains of the λ‐type are severalfold more active in the hydrolysis of DNA than IgGs with light chains of the κ‐type. Using different physicochemical methods of antibody analysis we have shown that the immune system of multiple sclerosis patients generates a variety of anti‐DNA abzymes of different type and with different catalytic properties, which can play an important role in multiple sclerosis pathogenesis. Copyright © 2010 John Wiley & Sons, Ltd.     

Evidence for the nuclear import of histones H3.1 and H4 as monomers

Newly synthesised histones are thought to dimerise in the cytosol and undergo nuclear import in complex with histone chaperones. Here, we provide evidence that human H3.1 and H4 are imported into the nucleus as monomers. Using a tether‐and‐release system to study the import dynamics of newly synthesised histones, we find that cytosolic H3.1 and H4 can be maintained as stable monomeric units. Cytosolically tethered histones are bound to importin‐alpha proteins (predominantly IPO4), but not to histone‐specific chaperones NASP, ASF1a, RbAp46 (RBBP7) or HAT1, which reside in the nucleus in interphase cells. Release of monomeric histones from their cytosolic tether results in rapid nuclear translocation, IPO4 dissociation and incorporation into chromatin at sites of replication. Quantitative analysis of histones bound to individual chaperones reveals an excess of H3 specifically associated with sNASP, suggesting that NASP maintains a soluble, monomeric pool of H3 within the nucleus and may act as a nuclear receptor for newly imported histone. In summary, we propose that histones H3 and H4 are rapidly imported as monomeric units, forming heterodimers in the nucleus rather than the cytosol.     

Catalytic antibodies from HIV-infected patients specifically hydrolyzing viral integrase suppress the enzyme catalytic activities

Human immunodeficiency virus type 1 integrase (IN) catalyzes integration of a DNA copy of the viral genome into the host genome. It was shown previously that IN preincubation with various oligodeoxynucleotides (ODNs) induces formation of dimers and oligomers of different gyration radii; only specific ODNs stimulate the formation of catalytically active dimers. Here we have shown that preincubation of IN with specific and nonspecific ODNs leads to a significant and comparable decrease in its hydrolysis by chymotrypsin, while nonspecific ODNs protect the enzyme from the hydrolysis by trypsin worse than specific ODNs; all ODNs had little effect on the IN hydrolysis by proteinase K. In contrast to canonical proteweases, IgGs from HIV‐infected patients specifically hydrolyze only IN. While d(pT)_n markedly decreased the IgG‐dependent hydrolysis of IN, d(pA)_n and d(pA)_n?d(pT)_n demonstrated no detectable protective effect. The best protection from the hydrolysis by IgGs was observed for specific single‐ and especially double‐stranded ODNs. Although IN was considerably protected by specific ODNs, proteolytic IgGs and IgMs significantly suppressed both 3′‐processing and integration reaction catalyzed by IN. Since anti‐IN IgGs and IgMs can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. Copyright © 2011 John Wiley & Sons, Ltd.     

JMJD5 cleaves monomethylated histone H3 N‐tail under DNA damaging stress

The histone H3 N‐terminal protein domain (N‐tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N‐tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain‐containing protein, is a Cathepsin L‐type protease that mediates histone H3 N‐tail proteolytic cleavage under stress conditions that cause a DNA damage response. JMJD5 clips the H3 N‐tail at the carboxyl side of monomethyl‐lysine (Kme1) residues. In vitro H3 peptide digestion reveals that JMJD5 exclusively cleaves Kme1 H3 peptides, while little or no cleavage effect of JMJD5 on dimethyl‐lysine (Kme2), trimethyl‐lysine (Kme3), or unmethyl‐lysine (Kme0) H3 peptides is observed. Although H3 Kme1 peptides of K4, K9, K27, and K36 can all be cleaved by JMJD5 in vitro, K9 of H3 is the major cleavage site in vivo, and H3.3 is the major H3 target of JMJD5 cleavage. Cleavage is enhanced at gene promoters bound and repressed by JMJD5 suggesting a role for H3 N‐tail cleavage in gene expression regulation.     

Multiple Sites of the Cleavage of 21- and 25-Mer Encephalytogenic Oligopeptides Corresponding to Human Myelin Basic Protein (MBP) by Specific Anti-MBP Antibodies from Patients with Systemic Lupus Erythematosus

IgGs from patients with multiple sclerosis and systemic lupus erythematosus (SLE) purified on MBP-Sepharose in contrast to canonical proteases hydrolyze effectively only myelin basic protein (MBP), but not many other tested proteins. Here we have shown for the first time that anti-MBP SLE IgGs hydrolyze nonspecific tri- and tetrapeptides with an extreme low efficiency and cannot effectively hydrolyze longer 20-mer nonspecific oligopeptides corresponding to antigenic determinants (AGDs) of HIV-1 integrase. At the same time, anti-MBP SLE IgGs efficiently hydrolyze oligopeptides corresponding to AGDs of MBP. All sites of IgG-mediated proteolysis of 21-and 25-mer encephalytogenic oligopeptides corresponding to two known AGDs of MBP were found by a combination of reverse-phase chromatography, TLC, and MALDI spectrometry. Several clustered major, moderate, and minor sites of cleavage were revealed in the case of 21- and 25-mer oligopeptides. The active sites of anti-MBP abzymes are localised on their light chains, while heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high affinity to MBP and specificity of this protein hydrolysis. The affinity of anti-MBP abzymes for intact MBP is approximately 1000-fold higher than for the oligopeptides. The data suggest that all oligopeptides interact mainly with the light chains of different monoclonal abzymes of total pool of IgGs, which possesses a lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific than globular protein and can occur in several sites.     

Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains

Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 ? resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.     

Phosphorylation protects sperm‐specific histones H1 and H2B from proteolysis after fertilization

At intermediate stages of male pronucleus formation, sperm‐derived chromatin is composed of hybrid nucleoprotein particles formed by sperm H1 (SpH1), dimers of sperm H2A‐H2B (SpH2A‐SpH2B), and a subset of maternal cleavage stage (CS) histone variants. At this stage in vivo, the CS histone variants are poly(ADP‐ribosylated), while SpH2B and SpH1 are phosphorylated. We have postulated previously that the final steps of sperm chromatin remodeling involve a cysteine‐protease (SpH‐protease) that degrades sperm histones in a specific manner, leaving the maternal CS histone variants unaffected. More recently we have reported that the protection of CS histones from degradation is determined by the poly(ADP‐ribose) moiety of these proteins. Because of the selectivity displayed by the SpH‐protease, the coexistence of a subset of SpH together with CS histone variants at intermediate stages of male pronucleus remodeling remains intriguing. Consequently, we have investigated the phosphorylation state of SpH1 and SpH2B in relation to the possible protection of these proteins from proteolytic degradation. Histones H1 and H2B were purified from sperm, phosphorylated in vitro using the recombinant α‐subunit of casein kinase 2, and then used as substrates in the standard assay of the SpH‐protease. The phosphorylated forms of SpH1 and SpH2B were found to remain unaltered, while the nonphosphorylated forms were degraded. On the basis of this result, we postulate a novel role for the phosphorylation of SpH1 and SpH2B that occurs in vivo after fertilization, namely to protect these histones against degradation at intermediate stages of male chromatin remodeling. J. Cell. Biochem. 76:173–180, 1999. © 1999 Wiley‐Liss, Inc.     

PELP1 is a reader of histone H3 methylation that facilitates oestrogen receptor‐α target gene activation by regulating lysine demethylase 1 specificity

Histone methylation has a key role in oestrogen receptor (ERα)‐mediated transactivation of genes. Proline glutamic acid and leucine‐rich protein 1 (PELP1) is a new proto‐oncogene that functions as an ERα co‐regulator. In this study, we identified histone lysine demethylase, KDM1, as a new PELP1‐interacting protein. These proteins, PELP1 and KDM1, were both recruited to ERα target genes, and PELP1 depletion affected the dimethyl histone modifications at ERα target genes. Dimethyl‐modified histones H3K4 and H3K9 are recognized by PELP1, and PELP1 alters the substrate specificity of KDM1 from H3K4 to H3K9. Effective demethylation of dimethyl H3K9 by KDM1 requires a KDM1–ERα–PELP1 functional complex. These results suggest that PELP1 is a reader of H3 methylation marks and has a crucial role in modulating the histone code at the ERα target genes.     

Organismal differences in post-translational modifications in histones H3 and H4

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.     

Rapid replacement of somatic linker histones with the oocyte-specific linker histone H1foo in nuclear transfer

The most distinctive feature of oocyte-specific linker histones is the specific timing of their expression during embryonic development. In Xenopus nuclear transfer, somatic linker histones in the donor nucleus are replaced with oocyte-specific linker histone B4, leading to the involvement of oocyte-specific linker histones in nuclear reprogramming. We recently have discovered a mouse oocyte-specific linker histone, named H1foo, and demonstrated its expression pattern in normal preimplantation embryos. The present study was undertaken to determine whether the replacement of somatic linker histones with H1foo occurs during the process of mouse nuclear transfer. H1foo was detected in the donor nucleus soon after transplantation. Thereafter, H1foo was restricted to the chromatin in up to two-cell stage embryos. After fusion of an oocyte with a cell expressing GFP (green fluorescent protein)-tagged somatic linker histone H1c, immediate release of H1c in the donor nucleus was observed. In addition, we used fluorescence recovery after photobleaching (FRAP), and found that H1foo is more mobile than H1c in living cells. The greater mobility of H1foo may contribute to its rapid replacement and decreased stability of the embryonic chromatin structure. These results suggest that rapid replacement of H1c with H1foo may play an important role in nuclear remodeling.     

Acetylation of histones during spermatogenesis in the rat

Acetate was actively incorporated into rat testis histones when testis cells were prepared by the trypsinization technique in the presence of [³H]acetate. The acetylation was enhanced by 10 mm sodium butyrate. Although histones H3 and H4 were the only histones which incorporated high levels of acetate, the testis-specific histones TH2B and TH3 also appeared to incorporate acetate. This was shown by electrophoresis of the histones on polyacrylamide gels containing Triton X-100. Results, obtained from analysis of histones by two-dimensional gel electrophoresis, confirmed a recent report (P. K. Trostle-Weige, M. L. Meistrich, W. A. Brock, K. Nishioka, and J. W. Bremer, (1982) J. Biol. Chem.257, 5560–5567) that TH2A was a testis-specific histone. The results also confirmed the H2A nature of a testis-enriched histone band, previously designated X2. When histones from populations of cells enriched in specific testis cell types, representing various stages of spermatogenesis, were examined, the patterns of acetylation varied dramatically. Very high levels of acetate were incorporated into multiacetylated species of histone H4 from a population of cells enriched in transition stage spermatids (steps 9–12) compared to the levels of acetate incorporated into H4 from round spermatids (steps 1–8) and earlier stages of spermatogenesis, where acetate was incorporated primarily into the monoacetylated species of H4. Thus, a striking correlation exists between the time of hyperacetylation of histone H4 and the time of removal of histones for their replacement by the basic spermatidal transition proteins designated TP, TP2, and TP4. Hyperacetylation of histone H4 may facilitate the removal of the entire histone complement during the protein transition. In any case, it must be an obligatory step in the dramatic process.     

Phosphorylation of H1 histones

The phosphorylation of H1 histones is reviewed. Consideration is given to phosphorylation reactions which occur in both replicating and nonreplicating cells. The available evidence suggests that H1 histones accept phosphate groups at different sites in response to different stimuli. The tentative location of the acceptor sites is summarized, and the effects of site-specific phosphorylation on the conformation of H1 histones in vitro is discussed. The phosphorylation of H1 histones which occurs during cell replication is reviewed in detail, and it is concluded that there is no clocklike mechanism which couples the phosphorylation of a particular site or region in H1 histones to a set point in the cell cycle. There is both species-and cell-specific variability in the phosphorylation of H1 histones during cell replication. Recent studies are discussed which show that an interspecific somatic cell hybrid of mouse and Chinese hamster can replicate the Chinese hamster genome in a stable manner using only mouse H1 histones and their phosphorylated forms. I speculate that H1 histone phosphorylation is a mechanism for the relaxation of long-term structures needed for differential gene activity in order to attain the short-term goal of genome replication.     

A plant‐specific histone H3 lysine 4 demethylase represses the floral transition in Arabidopsis

Histone demethylation regulates chromatin structure and gene expression, and is catalyzed by various histone demethylases. Trimethylation of histone H3 at lysine 4 (H3K4) is coupled to active gene expression; trimethyl H3K4 is demethylated by Jumonj C (JmjC) domain‐containing demethylases in mammals. Here we report that a plant‐specific JmjC domain‐containing protein known as PKDM7B (At4g20400) demethylates trimethyl H3K4. PKDM7B mediates H3K4 demethylation in a key floral promoter, FLOWERING LOCUS T (FT), and an FT homolog, TWIN SISTER OF FT (TSF), and represses their expression to inhibit the floral transition in Arabidopsis. Our findings suggest that there are at least two distinct sub‐families of JmjC domain‐containing demethylases that demethylate the active trimethyl H3K4 mark in eukaryotic genes, and reveal a plant‐specific JmjC domain enzyme capable of H3K4 demethylation.