Search

The site of in vitro ADP-ribosylation of seminal ribonuclease was determined. Seminal enzyme was found to be a good receptor of [14C]ADP-ribose residues under the reaction conditions used. The recovery of [14C]ADP-ribosylated RNase was about 65% after purification. After tryptic digestion of modified enzyme, a fraction containing [14C]ADP-ribosylated peptides was separated from the others by ion-exchange chromatography on M82 resin. Radioactive peptides were then purified by affinity chromatography on anti-poly(ADP-ribose)IgG-Sepharose. High performance liquid chromatography of a mixture obtained after pronase digestion of purified ADP-ribosylated peptides revealed only one radioactive peptide whose amino acid composition corresponded to a peptide that has equimolar quantities of aspartic acid, serine, and glycine. Carboxypeptidase Y digestion of this peptide showed that its amino acid sequence was Asp-Ser-Gly. Only position 14-16 of seminal RNase corresponded to this sequence. The chemical stability of the ADP-ribose/enzyme linkage indicated that aspartic acid 14 is the modification site in seminal RNase.

Poly(ADPR)polymerase (PARP) is a chromatin-associated enzyme with a presumptive role in DNA repair during replication and recovery from strand breaks caused by genotoxic agents. It catalyses the attachment and elongation of ADPribose polymers (pADPR) to a variety of acceptor proteins (including PARP itself, and histones) and is particularly active in the testis where its expression varies according to the stage of germ cell differentiation. PARP degradation is also one of the classic indicators of apoptosis. In this investigation we have examined the effects of heat stress on the adult rat testis with respect to the concentration and activity of PARP, the nature of the pADRP nuclear acceptor proteins, the length of ADPR polymers and the activity of the ADPR depolymerizing enzyme, poly(ADPR)glycohydrolase (PARG). Our results show a significant reduction in the concentration and activity of PARP 4 and 8 days after artificial cryptorchidism, but no significant changes were observed in PARG activity or in pADPR length. Unexpectedly, the apoptotic degradation of PARP was not detected following heat stress. These results confirm that PARP gene expression is developmentally regulated during spermatogenesis and indicate that it is suppressed coincidentally with the loss of meiotic spermatocytes during artificial cryptorchidism.

Blocking the interaction between the apoptosis-inducing factor (AIF) and cyclophilin A (CypA) by the AIF fragment AIF(370-394) is protective against glutamate-induced neuronal cell death and brain injury in mice. Starting from AIF(370-394), we report the generation of the disulfide-bridged and shorter variant AIF(381-389) and its structural characterization by nuclear magnetic resonance (NMR) in the free and CypA-bound state. AIF(381-389) in both the free and bound states assumes a β-hairpin conformation similar to that of the fragment in the AIF protein and shows a highly reduced conformational flexibility. This peptide displays a similar in vitro affinity for CypA, an improved antiapoptotic activity in cells and an enhanced proteolytic stability compared to the parent peptide. The NMR-based 3D model of the AIF(381-389)/CypA complex provides a better understanding of the binding hot spots on both the peptide and the protein and can be exploited to design AIF/CypA inhibitors with improved pharmacokinetic and pharmacodynamics features.

Integrins are heterodimeric cell-surface proteins that play important roles during developmental and pathological processes. Diverse human pathologies involve integrin adhesion including thrombotic diseases, inflammation, tumour progression, fibrosis, and infectious diseases. Although in the past decade, novel integrin-inhibitor drugs have been developed for integrin-based medical applications, the structural determinants modulating integrin-ligands recognition mechanisms are still poorly understood, reducing the number of integrin subtype exclusive antagonists. In this scenario, we have very recently showed, by means of chemical and biological assays, that a chimeric peptide (named RGDechi), containing a cyclic RGD motif linked to an echistatin C-terminal fragment, is able to interact with the components of integrin family with variable affinities, the highest for αv β3. Here, in order to understand the mechanistic details driving the molecular recognition mechanism of αv β3 by RGDechi, we have performed a detailed structural and dynamics characterization of the free peptide by natural abundance nuclear magnetic resonance (NMR) spectroscopy. Our data indicate that RGDechi presents in solution an heterogeneous conformational ensemble characterized by a more constrained and rigid pentacyclic ring and a largely unstructured acyclic region. Moreover, we propose that the molecular recognition of αv β3 integrin by RGDechi occurs by a combination of conformational selection and induced fit mechanisms. Finally, our study indicates that a detailed NMR characterization, by means of natural abundance 15 N and 13 C, of a mostly unstructured bioactive peptide may provide the molecular basis to get essential structural insights into the binding mechanism to the biological partner.

Seven families of double-stranded DNA viruses have been identified, among which the Fuselloviridae and Rudiviridae are the most well-studied specimens and therefore represent model systems for detailed studies of archaeal virus biology. Two distinct genetic elements, SSV2 and pSSVx, belong to Fuselloviridae and coexist in the same Sulfolobus islandicus REY15/4 host, thus representing one of the few known two-virus systems in Archaea (1). pSSVx is a satellite virus that generates virus particles with the help of SSV2-associated packaging mechanisms. The transcriptional pattern of pSSVx undergoes a temporal variation of gene expression during its own life cycle, thus providing a good model for studying regulation of gene expression in Archaea (2). This genetic element encodes four TFs possibly implicated in the regulation of gene expression, i.e. ORF-c68, ORF51, ORF91 and ORF76. Among these, ORF76, here named Stf76 (Sulfolobus transcription factor 76 aminoacid protein), has homologs in almost all conjugative and cryptic plasmids from Sulfolobus (3), thus suggesting a relevant role for this protein in replication and/or maintenance of the plasmid. In this study, we have performed a detailed structural and functional characterization of Stf76. The corresponding gene has been cloned, expressed in Escherichia coli and the recombinant protein purified to homogeneity. To elucidate its interaction with the identified DNA operator sequence, analyses regarding its DNA binding capabilities by means of EMSA, circular dichroism, spectrofluorimetric and isothermal titration calorimetry experiments have been performed. Moreover, a structural study has been undertaken by Nuclear Magnetic Resonance spectroscopy leading to:(i) the solution structure of Stf76 based on CS-Rosetta approach, (ii) the characterization of the Stf76-DNA interaction by chemical shift perturbation analysis, (iii) a structural model describing the interaction of a single Stf76 monomer with its DNA operator. Altogether these results contribute to elucidate the regulatory mechanism underpinning the role of this protein (4). REFERENCES: 1. ARNOLD, H.P., ET AL.(1999) THE GENETIC ELEMENT PSSVX OF THE EXTREMELY THERMOPHILIC CRENARCHAEON SULFOLOBUS IS A HYBRID BETWEEN A PLASMID AND A VIRUS. MOL MICROBIOL, 34, 217-226. 2. CONTURSI, P., ET.AL. (2010) TRANSCRIPTION TERMINATION IN THE PLASMID/VIRUS HYBRID PSSVX FROM SULFOLOBUS ISLANDICUS. EXTREMOPHILES, 14, 453-463. 3. LIPPS, G. (2006) PLASMIDS AND VIRUSES OF THE THERMOACIDOPHILIC CRENARCHAEOTE SULFOLOBUS. EXTREMOPHILES, 10, 17-28. 4. CONTURSI P., FARINA B., PIRONE L. ET AL. NUCLEIC ACIDS RES. ACCEPTED 28 FEB. 2014.

The specific arrangement of secondary elements in a local motif often totally relies on the formation of coordination bonds between metal ions and protein ligands. This is typified by the -30 amino acid eukaryotic zinc finger motif in which a beta-sheet and an or-helix are clustered around a zinc ion by various combinations of four ligands. The prokaryotic zinc finger domain (found in the Ros protein from Agrobacterium tumefaciens) is different from the eukaryotic counterpart as it consists of 58 amino acids arranged in a beta beta beta alpha alpha topology stabilized by a 15-residue hydrophobic core. Also, this domain tetrahedrally coordinates zinc and unfolds in the absence of the metal ion.The characterization of proteins belonging to the Ros homologs family has however shown that the prokaryotic zinc finger domain can overcome the metal requirement to achieve the same fold and DNA-binding activity. In the present work, two zinc-lacking Ros homologs (MI4 and MI5 proteins) have been thoroughly characterized using bioinformatics, biochemical and NMR techniques.We show how in these proteins a network of hydrogen bonds and hydrophobic interactions surrogate the zinc coordination role in the achievement of the same functional fold.(c) 2013 Published by Elsevier B.V.

The rat testis chromatin fractions (soluble, S, and insoluble, P) were prepared by mild digestion of nuclei with DNAase I. They appeared to be different in specific biochemical features such as their transcriptional competence and protein patterns, the latter indicating according to results previously obtained, that the testis-specific H1t is preferentially associated to the soluble fraction, whereas the other H1 variants are localized in the pellet. S and P chromatins also differed in the distribution of the poly(ADP-ribosyl)ating system, (poly(ADP-ribose)polymerase, reaction product and acceptor proteins), detected by incubating nuclei with 32P-NAD. The 32P-modified H1s and core histones of both fractions, known as specific ADPribose target proteins, were separated by high performance liquid chromatography and it was demonstrated that the H1 variants from S and P are differently ADPribosylated, being H1t always the best acceptor, and that most of the ADPribosylated variants were solubilized after DNase I treatment. The further digestion of P chromatin with the nuclease produced a fraction (pP) devoid of most DNA, but particularly enriched in transcriptionally competent tracts. The low DNA content of pP chromatin, which reflects the typical feature of a nuclear matrix, corresponded to a relevant poly(ADPribosyl)ation, the highest as compared to S and P fractions. Moreover, long and branched chains of poly(ADP-ribose) were found associated to pP sample which resemble the products determined in the soluble chromatin.

In order to study the possible functional relationship between poly(ADP-ribosyl)ation and spermatogenesis, the three main germinal cell types have been isolated and characterized as haploid spermatids and diploid and tetraploid spermatocytes. Purified germinal cell populations and rats of different age were used for activity-, immuno-, and Northern blot experiments, to determine at which level poly(ADPR)polymerase (PARP) is regulated at various stages of spermatogenesis. Poly(ADPR)glycohydrolase (PARG) activity was also determined, as was the subcellular distribution of both PARP and PARG enzymes. The results show that the maximum of both PARP amount and PARP activity can be detected on tetraploid spermatocytes which undergo meiotic division, whereas PARG activity does not differ in germinal cells; the cytoplasmic form of this enzyme is prevalent in testis. Moreover, a difference in timing was observed in maximal level between PARP expression, determined on testis from 60-day-old rats, and PARP activity, detected on testis from 30-day-old animals. It seems that different mechanisms modulate the poly(ADPribosyl)ation system during spermatogenesis. Regulation of the poly(ADPribose) turnover, variations of PARP amount, as well as changes of PARP transcription level, seem to accompany germinal cell differentiation, possibly being implicated in DNA replication, repair, and transcription.

The RNA binding motif protein 5 (RBM5), also known as Luca15 or H37, is a component of prespliceosomal complexes that regulates the alternative splicing of several mRNAs, such as Fas and caspase-2. The RBM5 gene is located at the 2p21.3 chromosomal region, which is strongly associated with lung cancer and many other cancers. Both increased and decreased levels of RBM5 can play a role in tumor progression. In particular, downregulation of rbm5 is involved in lung cancer and other cancers upon Ras activation, and, also, represents a molecular signature associated with metastasis in various solid tumors. On the other hand, upregulation of RBM5 occurs in breast and ovarian cancer. Moreover, RBM5 was also found to be involved in the early stage of the HIV-1 viral cycle, representing a potential target for the treatment of the HIV-1 infection. While the molecular basis for RNA recognition and ubiquitin interaction has been structurally characterized, small molecules binding this zinc finger (ZF) domain that might contribute to characterizing their activity and to the development of potential therapeutic agents have not yet been reported. Using an NMR screening of a fragment library we identified several binders and the complex of the most promising one, compound 1, with the RBM5 ZF1 was structurally characterized in solution. Interestingly, the binding mechanism reveals that 1 occupies the RNA binding pocket and is therefore able to compete with the RNA to bind RBM5 RanBP2-type ZF domain, as indicated by NMR studies. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In this study, we have identified a 28-kDa protein resembling the linker H1 in the testis and prostate of the reproductive system of Octopus vulgaris. This protein, OvH1, was partially purified by reverse phase high-pressure liquid chromatography (HPLC) of the perchloric acid extract from testis nuclei. It showed electrophoretic mobility, CD spectrum and amino acid composition highly comparable with those of the mammalian histone. Moreover, it was microheterogeneous, as resulted from prostate and testis HPLC and mass spectrometry analyses. Such analysis showed that in testis there are two H1 subfractions, which do not appear in the prostate. Amino acid composition of the major testis specific variant (OvH1t) showed high similarity with rat testis specific H1t. The histone-like nature of OvH1 was confirmed by its ability to bind DNA as tested both by circular dichroism and protection of the nucleic acid toward deoxyribonuclease I activity. The circular dichroism spectra of Octopus DNA in the absence and presence of increasing amounts of the protein showed a dose-dependent effect, leading to a progressive compactness of the polynucleotide. OvH1/DNA complexes were also resistant to nuclease digestion. The presence of H1 in the testis and prostate of the reproductive system of Octopus is discussed in light of the fact that there is a similarity between its behavior and that of vertebrates. Mol. Reprod. Dev. 63: 355–365, 2002. © 2002 Wiley-Liss, Inc.

Endogenous levels of poly(ADP-ribose) and betaNAD+ have been determined in rat male germinal cells at different stages of differentiation. The levels of both metabolites decreased progressively from primary spermatocytes to secondary spermatocytes and especially in spermatids. We have also determined the size and complexity of the ADP-ribose polymers synthesized in permeabilized germ cells. Polymers of different chain length and complexity were observed in cells incubated with different concentrations of [32P]betaNAD+; short polymers characterized primary spermatocytes incubated with low betaNAD+ concentration. In all cell fractions, polymers of over 20 residues in size were observed at high betaNAD+ levels. Long polymers were associated with the sulfuric acid-insoluble proteins (nonhistone proteins such as PARP itself). By contrast, oligomers of 20 ADP-ribose units or less were found in the sulfuric acid-soluble proteins (histone proteins). We have also identified the main ADP-ribose protein acceptors formed in each cell type. In all cells examined, PARP appears to be extensively automodified. However, by far, the H1t variant of histone H1 appeared to be the preferred ADP-ribose target among the acid-soluble proteins separated by reverse-phase HPLC. Therefore, we conclude that an active protein-poly(ADP-ribosyl)ation system is concentrated in primary spermatocytes, based on a high level of PARP automodification accompanied by the preferential heteromodification of the histone H1 variant specifically expressed in the cells undergoing the pachytene phase of the meiotic division.

The amino acid sequence of a novel tissueand phasespecific nuclear protein (SNP) has been determined, after purification from the nuclei of the oviduct of the lizard Podarcis sicula Raf. during the reproductive period of the seasonal growth. SNP has a pI of 9.0 and contains 81 amino acid residues with a molecular weight of 9211.88 ± 0.09. It shows a bipartite organization as the first 40 amino acids contain all 8 cysteinyl residues, while the last 41 amino acids contain 16 prolyl residues. Two more components have also been identified and characterized, with the first 79 amino acids matching SNP and missing one or two residues at the Cterminus. They have thus been named [des(Ala[81]) SNP1] and [des(Lys[80]Ala[81]) SNP2], respectively. The molecular weights are 9140.21 ± 0.83 for [des(Ala[81]) SNP1] and 9011 ± 0.09 for [des(Lys[80]Ala[81]) SNP2].

An ADP-ribosylating system was detected in a crude homogenate from Sulfolobus solfataricus, a thermophilic archaeon, optimally growing at 87 degrees C. The archaeal ADP-ribosylation reaction was time-, temperature- and NAD-dependent. It proved to be highly thermostable, with about 30% decrease of 14C incorporation from [14C]NAD on incubation at 80 degrees C for up to 24 h. The main reaction product was found to be mono-ADP-ribose. Testing both [adenine-14C(U)]NAD and [adenine-14C(U)]ADPR as substrates, it was found that acceptor proteins were modified by ADP-ribose both enzymatically, via ADP-ribosylating enzymes, and via chemical attachment of free ADP-ribose, likely produced by NAD glycohydrolase activity. The synthesis of ADP-ribose-protein complexes was shown to involve mainly acceptors with molecular masses in the 40-100 kDa range, as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate.

Poly(ADPribosylation) of nuclear proteins has been investigated in the nuclei from growing oviducts of intact and estrogen-treated spayed females of the lizard Podarcis s. sicula Raf. Isolated nuclei were incubated with [14C]NAD and nuclear proteins extracted in 0.2 M H2SO4. Labeled acid-soluble proteins were analysed by reverse-phase high-performance liquid chromatography (HPLC) and acetic acid-urea gel electrophoresis. The results reported here indicate that the ADPribosylation reaction is involved in modifying, besides histone H2b, tissue specific proteins (SNPs and LMG-O). Moreover, comparable results have been obtained from nuclei prepared from the fully active oviduct of intact animals and spayed lizards stimulated with 17β-estradiol. It is concluded that the poly-(ADPribosylation) of hormone-induced proteins might play a role in the differentiation of the lizard oviduct.

Culture and differentiation parameters of a human thyroid transformed cell line (HuT) were analyzed. Treatment with high concentrations of chemical agents namely dimethyl sulphoxide and retinoic acid, exerted a dramatic cytotoxic effect. The exposure of these cells to the lowest doses of retinoic acid as well as to 8 mM-16 mM 3-aminobenzamide a potent inhibitor of poly(ADPribose)polymerase, resulted in a delay of cell proliferation. Poly(ADPribose)polymerase activity was differently affected by retinoic acid (stimulation) and 3-aminobenzamide (inhibition).

Poly(ADPR)polymerase activity and poly(ADP-ribosyl)ation of nuclear proteins have been investigated in ventral prostate nuclei of different aged rats (14, 28, 60, 180, 360 day old animals), by reverse-phase HPLC and acetic acid-urea polyacrylamide gel electrophoresis. The major ADP-ribose acceptor proteins were identified as histone H1 and H2b. It is concluded that concomitant with major changes to chromatin organization, poly(ADP-ribosyl)ation reaction is progressively inhibited during aging of rat ventral prostate. These results support the hypothesis that prostatic dysfunction in senescent animals is related to a failure of DNA repair mechanisms and deregulated template activity.

Ribonuclease activity in HeLa cell nuclei is markedly inhibited by ADP-ribosylation following incubation of intact isolated nuclei with [14C]NAD. Time course experiments demonstrate that [14C] incorporation into proteins is accompanied by a 50% inhibition of ribonuclease activity on single-strand and double-strand polynucleotides. Inhibition does not occur when 3-aminobenzamide, a potent (ADP-ribose) polymerase inhibitor, is present. Two enzymatic activities that degrade double-strand polynucleotides have been purified and partially characterized. A relevant level of radioactivity resulting from [14C]NAD incubation of nuclei was associated to the purified enzyme. The RNase F1 component, which shows maximal activity on polyU-polyA is demonstrated to be the major ADP-ribose acceptor protein.

Acceptor proteins for poly(ADP-ribose) have been purified from mouse testis nuclei. Nuclear proteins were labelled in vitro with [14C]ribose and [3H]adenine, extracted with 5% (v/v) HClO4 and 0.25 M-HCl and separated by ion-exchange chromatography. Non-histone proteins were found to be the major acceptors in both the 5% (w/v)-HClO4-soluble and 5%-HClO4-insoluble HCl-extractable fractions. Of the two groups of non-histone proteins associated with chromatin, the LMG (low-mobility-group) proteins were preferentially ADP-ribosylated. HMG (high-mobility group) proteins were labelled to lower specific radioactivity. Six LMG proteins were purified to approx. 90% homogeneity and were identified from their mobility on polyacrylamide gels at pH 2.9 and from their amino acid composition. The average length of the poly(ADP-ribose) chain was estimated to be four to six repeating ADP-ribose units. It is suggested that ADP-ribosylation of LMG proteins, a long-neglected group of chromatin-associated proteins, is important during spermatogenesis for the production of spermatozoa with intact and competent DNA.

The thermophilic microorganism Sulfolobus solfataricus is able to grow at low pH (3.5) and high temperature (87°C) and has been isolated from an acidic hot spring in Agnano (Napoli), Italy (1). This bacterium belongs to the archaebacteria, a phylogenetic group of microorganisms that can be distinguished from other bacteria and eukaryotes (2, 3). The archaebacteria group was initially defined on the basis of a partial 16S ribosomal RNA sequence comparison, which has been well supported by other biochemical features (2-4). More recent studies by Woese and collaborators, based on comparison of complete 16S ribosomal RNA sequences, confirmed and extended earlier archaebacteria phylogeny (3). Although archaebacteria taxonomy is complex and controversial, three main phenotypes have been described, characterized by their peculiar ecological niches, namely high salt environments (halophilic and haloalkaliphilic archaebacteria), high temperature environments (thermophilic and sulfur-dependent archaebacteria) and stringent anaerobic conditions (methanogenic archaebacteria). Several features indicate that the sulfur-dependent branch of archaebacteria, to which Sulfolobus solfataricus belongs, is more closely related to eukaryotes than eubacteria or other archaebacteria (3). One example is the diphtheria toxin reaction, which catalyzes the transfer of ADP-ribose to eukaryotic elongation factor 2. Kessel and Klink (5) demonstrated that the toxin is able to catalyze, in several archaebacteria including Sulfolobus solfataricus, the covalent binding of ADP-ribose to elongation factor, EF-2.