Descripción: Papillomaviruses are small DNA tumor viruses that infect mammalian hosts, with consequences from benign to cancerous lesions. The Early protein 2 is the master regulator for the virus life cycle, participating in gene transcription, DNA replication, and viral episome migration. All of these functions rely on primary target recognition by its dimeric DNA-binding domain. In this work, we performed molecular dynamics simulations in order to gain insights into the structural dynamics of the DNA-binding domains of two prototypic strains, human papillomavirus strain 16 and the bovine papillomavirus strain 1. The simulations underline different dynamic features in the two proteins. The human papillomavirus strain 16 domain displays a higher flexibility of the β2-β3 connecting loop in comparison with the bovine papillomavirus strain 1 domain, with a consequent effect on the DNA-binding helices, and thus on the modulation of DNA recognition. A compact β-barrel is found in human papillomavirus strain 16, whereas the bovine papillomavirus strain 1 protein is characterized by a loose β-barrel with a large number of cavities filled by water, which provides great flexibility. The rigidity of the human papillomavirus strain 16 β-barrel prevents protein deformation, and, as a consequence, deformable spacers are the preferred targets in complex formation. In contrast, in bovine papillomavirus strain 1, a more deformable β-barrel confers greater adaptability to the protein, allowing the binding of less flexible DNA regions. The flexibility data are confirmed by the experimental NMR S2 values, which are reproduced well by calculation. This feature may provide the protein with an ability to discriminate between spacer sequences. Clearly, the deformability required for the formation of the Early protein 2 C-terminal DNA-binding domain-DNA complexes of various types is based not only on the rigidity of the base sequences in the DNA spacers, but also on the intrinsic deformability properties of each domain. © 2007 The Authors.

Descripción: The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol-1), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol-1 from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: Cytoplasmic events depending on RNA-binding proteins contribute to the fine-tuning of gene expression. Sterile α motif-containing RNA-binding proteins constitute a novel family of post-transcriptional regulators that recognize a specific RNA sequence motif known as Smaug recognition element (SRE). The Drosophila member of this family, dSmaug, triggers the translational repression and deadenylation of maternal mRNAs by independent mechanisms, and the yeast homologue Vts1 stimulates degradation of SRE-containing messengers. Two homologous genes are present in the mammalian genome. Here we showed that hSmaug 1, encoded in human chromosome 14, represses the translation of reporter transcripts carrying SRE motifs. When expressed in fibroblasts, hSmaug 1 forms cytoplasmic granules that contain polyadenylated mRNA and the RNA-binding proteins Staufen, TIAR, TIA-1, and HuR. Smaug 1 foci are distinct from degradation foci. The murine protein mSmaug 1 is expressed in the central nervous system and is abundant in post-synaptic densities, a subcellular region where translation is tightly regulated by synaptic stimulation. Biochemical analysis indicated that mSmaug 1 is present in synaptoneurosomal 20 S particles. These results suggest a role for mammalian Smaug 1 in RNA granule formation and translation regulation in neurons. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: The specificity in phosphorylation by kinases is determined by the molecular recognition of the peptide target sequence. In Saccharomyces cerevisiae, the protein kinase A (PKA) specificity determinants are less studied than in mammalian PKA. The catalytic turnover numbers of the catalytic subunits isoforms Tpk1 and Tpk2 were determined, and both enzymes are shown to have the same value of 3 s-1. We analyze the substrate behavior and sequence determinants around the phosphorylation site of three protein substrates, Pyk1, Pyk2, and Nth1. Nth1 protein is a better substrate than Pyk1 protein, and both are phosphorylated by either Tpk1 or Tpk2. Both enzymes also have the same selectivity toward the protein substrates and the peptides derived from them. The three substrates contain one or more Arg-Arg-X-Ser consensus motif, but not all of them are phosphorylated. The determinants for specificity were studied using the peptide arrays. Acidic residues in the position P+1 or in the N-terminal flank are deleterious, and positive residues present beyond P-2 and P-3 favor the catalytic reaction. A bulky hydrophobic residue in position P+1 is not critical. The best substrate has in position P+4 an acidic residue, equivalent to the one in the inhibitory sequence of Bcy1, the yeast regulatory subunit of PKA. The substrate effect in the holoenzyme activation was analyzed, and we demonstrate that peptides and protein substrates sensitized the holoenzyme to activation by cAMP in different degrees, depending on their sequences. The results also suggest that protein substrates are better co-activators than peptide substrates. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: CO recombination kinetics has been investigated in the type II truncated hemoglobin from Thermobifida fusca (Tf-trHb) over more than 10 time decades (from 1 ps to ~100 ms) by combining femtosecond transient absorption, nanosecond laser flash photolysis and optoacoustic spectroscopy. Photolysis is followed by a rapid geminate recombination with a time constant of ~2 ns representing almost 60% of the overall reaction. An additional, small amplitude geminate recombination was identified at ~100 ns. Finally, CO pressure dependent measurements brought out the presence of two transient species in the second order rebinding phase, with time constants ranging from ~3 to ~100 ms. The available experimental evidence suggests that the two transients are due to the presence of two conformations which do not interconvert within the time frame of the experiment. Computational studies revealed that the plasticity of protein structure is able to define a branched pathway connecting the ligand binding site and the solvent. This allowed to build a kinetic model capable of describing the complete time course of the CO rebinding kinetics to Tf-trHb. © 2012 Marcelli et al.

Descripción: DNA size polymorphisms were utilized in a study of 24 natural populations of Ceratitis capitata Wiedemann (Diptera: Tephritidae) from Argentina. The first intron of alcohol dehydrogenase 1 gene (Adh1) was amplified using exon priming intron crossing-polymerase chain reaction. Three size variants were detected among the 307 samples analyzed. To better differentiate the size variants, further digestion of PCR products with the EcoRI restriction enzyme was carried out. Complete nucleotide sequences of the three-allele variants were obtained and single changes, insertions, deletions, and EcoRI recognition sites were located. Population allele frequencies were analyzed and a global mean heterozygosity (He) of 0.33 was obtained. In most populations, observed allelic frequencies conformed to Hardy-Weinberg expectations. Significant differences between provinces and sampling sites within these provinces, and among some populations were found. The average number of insects exchanged among populations (Nm) was estimated and high values were observed between Argentina and populations from two African countries (Morocco and Kenya), Australia, and Hawaii (Kauai). Pest introduction sources and dispersion patterns in Argentina are discussed based on these results as well as on available bibliographical data. © 2010 The Authors. Journal compilation © 2010 The Netherlands Entomological Society.

Descripción: In order to clarify the relationships among southern South American (sSA) representatives of the genus Ilex, an amplified fragment length polymorphism (AFLP) analysis was accomplished. In addition, the phylogenetic relationships of the species were studied using ribosomal internal transcribed spacer (ITS) sequence data alone and in combination with AFLP data, taking into account the possible existence of paralogous sequences and the influence of alignment parameters. To explore stability of phylogenetic hypotheses, a sensitivity analysis was performed using 15 indel-substitution models. Within each species assayed, the AFLPs allowed the recognition of several diagnostic bands. Furthermore, the AFLP analysis revealed that individuals belonging to the same morpho-species formed coherent clades. In addition, some cases of geographical association were noted. Studies on ITS sequences revealed divergence between data obtained herein and sequence data downloaded from GenBank. The sensitivity analyses yielded different interspecific hypotheses of relationships. Notwithstanding, analyses of the ITS data alone and in combination with AFLPs, rendered clades stable to variation in the analytical parameters. Topologies obtained for the AFLPs, the ITS data alone and the combined analyses, demonstrated the existence of a group formed by I. argentina, I. brasiliensis, I. brevicuspis, I. integerrima, and I. theezans, and that I. dumosa and I. paraguariensis were distantly related to the former. Incongruence with traditional taxonomical treatments was found.

Descripción: Protein modification by conjugation of small ubiquitin-related modifier (SUMO) is involved in diverse biological functions, such as transcription regulation, subcellular partitioning, stress response, DNA damage repair, and chromatin remodeling. Here, we show that the serine/arginine-rich protein SF2/ASF, a factor involved in splicing regulation and other RNA metabolism-related processes, is a regulator of the sumoylation pathway. The overexpression of this protein stimulates, but its knockdown inhibits SUMO conjugation. SF2/ASF interacts with Ubc9 and enhances sumoylation of specific substrates, sharing characteristics with already described SUMO E3 ligases. In addition, SF2/ASF interacts with the SUMO E3 ligase PIAS1 (protein inhibitor of activated STAT-1), regulating PIAS1-induced overall protein sumoylation. The RNA recognition motif 2 of SF2/ASF is necessary and sufficient for sumoylation enhancement. Moreover, SF2/ASF has a role in heat shock-induced sumoylation and promotes SUMO conjugation to RNA processing factors. These results add a component to the sumoylation pathway and a previously unexplored role for the multifunctional SR protein SF2/ASF.

Descripción: By taking advantage of the extreme stability of a protein-DNA complex, we have obtained two highly specific monoclonal antibodies against a predetermined palindromic DNA sequence corresponding to the binding site of the E2 transcriptional regulator of the human papillomavirus (HPV-16). The purified univalent antibody fragments bind to a double-stranded DNA oligonucleotide corresponding to the E2 binding site in solution with dissociation constants in the low and subnanomolar range. This affinity matches that of the natural DNA binding domain and is severalfold higher than the affinity of a homologous bovine E2 C-terminal domain (BPV-1) for the same DNA. These antibodies discriminate effectively among a number of double- and single-stranded synthetic DNAs with factors ranging from 125-to 20,000-fold the dissociation constant of the specific DNA sequence used in the immunogenic protein-DNA complex. Moreover, they are capable of fine specificity tuning, since they both bind less tightly to another HPV-16 E2 binding site, differing in only 1 base pair in a noncontact flexible region. Beyond the relevance of obtaining a specific anti-DNA response, these results provide a first glance at how DNA as an antigen is recognized specifically by an antibody. The accuracy of the spectroscopic method used for the binding analysis suggests that a detailed mechanistic analysis is attainable.

Descripción: Background: Human papillomavirus (HPV) is the main causative agent of cervical cancer, particularly high risk strains such us HPV-16, -18 and -31. The viral encoded E2 protein acts as a transcriptional modulator and exerts a key role in viral DNA replication. Thus, E2 constitutes an attractive target for developing antiviral agents. E2 is a homodimeric protein that interacts with the DNA target through an α-helix of each monomer. However, a peptide corresponding to the DNA recognition helix of HPV-16 E2 binds DNA with lower affinity than its full-length DNA binding domain. Therefore, in an attempt to promote the DNA binding of the isolated peptide, we have designed a conjugate compound of the E2 α-helix peptide and a derivative of the antibiotic distamycin, which involves simultaneous minor- and major-groove interactions. Methodology/Principal Findings: An E2 α-helix peptide-distamycin conjugate was designed and synthesized. It was characterized by NMR and CD spectroscopy, and its DNA binding properties were investigated by CD, DNA melting and gel shift experiments. The coupling of E2 peptide with distamycin does not affect its structural properties. The conjugate improves significantly the affinity of the peptide for specific DNA. In addition, stoichiometric amounts of specific DNA increase meaningfully the helical population of the peptide. The conjugate enhances the DNA binding constant 50-fold, maintaining its specificity. Conclusions/Significance: These results demonstrate that peptide-distamycin conjugates are a promising tool to obtain compounds that bind the E2 target DNA-sequences with remarkable affinity and suggest that a bipartite major/minor groove binding scaffold can be a useful approach for therapeutic treatment of HPV infection. © 2011 Wetzler et al.

Descripción: Chromatin structure is an important factor in the functional coupling between transcription and mRNA processing, not only by regulating alternative splicing events, but also by contributing to exon recognition during constitutive splicing. We observed that depolarization of neuroblastoma cell membrane potential, which triggers general histone acetylation and regulates alternative splicing, causes a concentration of SR proteins in nuclear speckles. This prompted us to analyze the effect of chromatin structure on splicing factor distribution and dynamics. Here, we show that induction of histone hyper-acetylation results in the accumulation in speckles of multiple splicing factors in different cell types. In addition, a similar effect is observed after depletion of the heterochromatic protein HP1α, associated with repressive chromatin. We used advanced imaging approaches to analyze in detail both the structural organization of the speckle compartment and nuclear distribution of splicing factors, as well as studying direct interactions between splicing factors and their association with chromatin in vivo. The results support a model where perturbation of normal chromatin structure decreases the recruitment efficiency of splicing factors to nascent RNAs, thus causing their accumulation in speckles, which buffer the amount of free molecules in the nucleoplasm. To test this, we analyzed the recruitment of the general splicing factor U2AF65 to nascent RNAs by iCLIP technique, as a way to monitor early spliceosome assembly. We demonstrate that indeed histone hyper-acetylation decreases recruitment of U2AF65 to bulk 3′ splice sites, coincident with the change in its localization. In addition, prior to the maximum accumulation in speckles, ~20% of genes already show a tendency to decreased binding, while U2AF65 seems to increase its binding to the speckle-located ncRNA MALAT1. All together, the combined imaging and biochemical approaches support a model where chromatin structure is essential for efficient co-transcriptional recruitment of general and regulatory splicing factors to pre-mRNA. © 2012 Schor et al.