Structural properties of the linkers connecting the n- and c- terminal domains in the mocr bacterial transcriptional regulators

Peptide inter-domain linkers are peptide segments covalently linking two adjacent domains within a protein. Linkers play a variety of structural and functional roles in naturally occurring proteins. In this work we analyze the sequence properties of the predicted linker regions of the bacterial transcriptional regulators belonging to the recently discovered MocR subfamily of the GntR regulators. Analyses were carried out on the MocR sequences taken from the phyla Actinobacteria, Firmicutes, Alpha-, Beta- and Gammaproteobacteria. The results suggest that MocR linkers display phylum-specific characteristics and unique features different from those already described for other classes of inter-domain linkers. They show an average length significantly higher: 31.8 ± 14.3 residues reaching a maximum of about 150 residues. Compositional propensities displayed general and phylum-specific trends. Pro is dominating in all linkers. Dyad propensity analysis indicate Pro–Pro as the most frequent amino acid pair in all linkers. Physicochemical properties of the linker regions were assessed using amino acid indices relative to different features: in general, MocR linkers are flexible, hydrophilic and display propensity for β-turn or coil conformations. Linker sequences are hypervariable: only similarities between MocR linkers from organisms related at the level of species or genus could be found with sequence searches. The results shed light on the properties of the linker regions of the new MocR subfamily of bacterial regulators and may provide knowledge-based rules for designing artificial linkers with desired properties. © 2016 The Author(s

Metabolism of the EGFR tyrosin kinase inhibitor gefitinib by cytochrome P450 1A1 enzyme in EGFR-wild type non small cell lung cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Gefitinib is a tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) especially effective in tumors with activating EGFR gene mutations while EGFR wild-type non small cell lung cancer (NSCLC) patients at present do not benefit from this treatment.</p> <p>The primary site of gefitinib metabolism is the liver, nevertheless tumor cell metabolism can significantly affect treatment effectiveness.</p> <p>Results</p> <p>In this study, we investigated the intracellular metabolism of gefitinib in a panel of EGFR wild-type gefitinib-sensitive and -resistant NSCLC cell lines, assessing the role of cytochrome P450 1A1 (CYP1A1) inhibition on gefitinib efficacy. Our results indicate that there is a significant difference in drug metabolism between gefitinib-sensitive and -resistant cell lines. Unexpectedly, only sensitive cells metabolized gefitinib, producing metabolites which were detected both inside and outside the cells. As a consequence of gefitinib metabolism, the intracellular level of gefitinib was markedly reduced after 12-24 h of treatment. Consistent with this observation, RT-PCR analysis and EROD assay showed that mRNA and activity of CYP1A1 were present at significant levels and were induced by gefitinib only in sensitive cells. Gefitinib metabolism was elevated in crowded cells, stimulated by exposure to cigarette smoke extract and prevented by hypoxic condition. It is worth noting that the metabolism of gefitinib in the sensitive cells is a consequence and not the cause of drug responsiveness, indeed treatment with a CYP1A1 inhibitor increased the efficacy of the drug because it prevented the fall in intracellular gefitinib level and significantly enhanced the inhibition of EGFR autophosphorylation, MAPK and PI3K/AKT/mTOR signalling pathways and cell proliferation.</p> <p>Conclusion</p> <p>Our findings suggest that gefitinib metabolism in lung cancer cells, elicited by CYP1A1 activity, might represent an early assessment of gefitinib responsiveness in NSCLC cells lacking activating mutations. On the other hand, in metabolizing cells, the inhibition of CYP1A1 might lead to increased local exposure to the active drug and thus increase gefitinib potency.</p

Unbalanced Occlusion Modifies the Pattern of Brain Activity During Execution of a Finger to Thumb Motor Task

In order to assess possible influences of occlusion on motor performance, we studied by functional magnetic resonance imaging (fMRI) the changes in the blood oxygenation level dependent (BOLD) signal induced at brain level by a finger to thumb motor task in a population of subjects characterized by an asymmetric activation of jaw muscles during clenching (malocclusion). In these subjects, appropriate occlusal correction by an oral orthotic (bite) reduced the masticatory asymmetry. The finger to thumb task was performed while the subject’s dental arches were touching, in two conditions: (a) with the teeth in direct contact (Bite OFF) and (b) with the bite interposed between the arches (Bite ON). Both conditions required only a very slight activation of masticatory muscles. Maps of the BOLD signal recorded during the movement were contrasted with the resting condition (activation maps). Between conditions comparison of the activation maps (Bite OFF/Bite ON) showed that, in Bite OFF, the BOLD signal was significantly higher in the trigeminal sensorimotor region, the premotor cortex, the cerebellum, the inferior temporal and occipital cortex, the calcarine cortex, the precuneus on both sides, as well as in the right posterior cingulate cortex. These data are consistent with the hypothesis that malocclusion makes movement performance more difficult, leading to a stronger activation of (a) sensorimotor areas not dealing with the control of the involved body part, (b) regions planning the motor sequence, and (c) the cerebellum, which is essential in motor coordination. Moreover, the findings of a higher activation of temporo-occipital cortex and precuneus/cingulus, respectively, suggest that, during malocclusion, the movement occurs with an increased visual imagery activity, and requires a stronger attentive effort

L'antica festività dei Consualia e il ruolo degli animali da trasporto nella fase più antica della storia di Roma

The study compares through texts from Denys of Halicamassus, Plutarch and Ausonius the festivals dedicated in Rome to the gods Consus and Neptune, and more specifically the association of each of them to a horse. Of the complex yet unfortunately incomplete dossier which has been sent us the outstanding points are as follows : 1) Neptune Equester long remained an "earthly" god of transportation ; 2) Consus, god of the public granaries, connected at a very early date with the ritual of horse races in the Grand Circus, may well have seen his powers "absorbed" into those of Neptune, prior to his rediscovery by Ausonius ; 3) Plutarch, in his tentative explanation of the "vacations" of horses and donkeys during the Consualia (QR. 48), forgets the part played by those animals in funeral ceremonies as bearers of impurities.L'étude compare, en s' appuyant sur des textes de Denys d'Halicarnasse, de Plutarque et d'Ausone, les fêtes dédiées à Rome aux dieux Consus et Neptune, et plus spécialement l'association de chacun d'eux avec le cheval. Du dossier complexe, quoique malheureusement incomplet, qu'on peut réunir, ressortent les points suivants : 1) Neptune Equester est longtemps resté un dieu "terrestre" des transports ; 2) Consus, dieu des greniers publics, lié à date très haute au rituel des courses de chevaux dans le Grand Cirque, a pu voir ses pouvoirs "absorbés" par ceux de Neptune, avant sa redécouverte par Ausone ; 3) Plutarque, dans sa tentative d'explication des "vacances" des chevaux et des ânes pendant les Consualia (Q.R. 48), oublie le rôle joué par ces animaux dans les cérémonies funéraires comme porteurs d'impuretés.Tramonti Stefano. L'antica festività dei Consualia e il ruolo degli animali da trasporto nella fase più antica della storia di Roma. In: Pallas, 44/1996. L'antiquité à la page. pp. 101-107

The MocR‐like transcription factors: pyridoxal 5′‐phosphate‐dependent regulators of bacterial metabolism

Many biological functions played by current proteins were not created by evolution from scratch, rather they were obtained combining already available protein scaffolds. This is the case of MocR-like bacterial transcription factors (MocR-TFs), a subclass of GntR transcription regulators, whose structure is the outcome of the fusion between DNA-binding proteins and pyridoxal 5'-phosphate (PLP)-dependent enzymes. The resultant chimeras can count on the properties of both protein classes, i.e. the capability to recognize specific DNA sequences and to bind PLP and amino-compounds; it is the modulation of such binding properties to confer to MocR-TFs chimeras the ability to interact with effector molecules and DNA so as to regulate transcription. MocR-TFs control different metabolic processes involving vitamin B6and amino acids, which are canonical ligands of PLP-dependent enzymes. However, MocR-TFs are also implicated in the metabolism of compounds that are not substrates of PLP-dependent enzymes, such as rhizopine and ectoine. Genomic analyses show that MocR-TFs are widespread among eubacteria, implying an essential role in their metabolism and highlighting the scarcity of our knowledge on these important players in microbial metabolism. Although MocR-TFs have been discovered 15 years ago, the research activity on these transcriptional regulators has only recently intensified, producing a wealth of information that needs to be brought back to general principles. This is the main task of this review, which reports and analyses the available information concerning MocR-TFs functional role, structural features, interaction with effector molecules and the characteristics of DNA transcriptional factor-binding sites of MocR-based regulatory systems

Allosteric communication of tryptophan synthase: functional and regulatory properties of the betaSer178Pro mutant

The alpha (2)beta (2) tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the beta S178P mutant. Ser-178 is located at the end of helix 6 of the beta subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of beta Ser-178 is hydrogen bonded to Gly-181 of loop 6 of the alpha subunit only when alpha subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the beta S178P mutation suggests that the hydrogen bond involving alpha Gly-181 is disrupted as a result of localized structural perturbations. The ratio of alpha to beta subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight alpha-beta complex. Both the activity of the alpha subunit and the inhibitory effect of the alpha subunit ligands indole-3-acetylglycine and D,L-alpha -glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the beta subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effecters indole-3-acetylglycine and D,L-alpha -glycerol-3-phosphate do not affect the beta activity. Accordingly, the distribution of L-serine intermediates at the beta -site, dominated by the alpha -aminoacrylate, is only slightly influenced by alpha subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the alpha -aminoacrylate tautomer absorbing at 450 nm, Crystals of the beta S178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the alpha and beta activity in the alpha2 beta2 complex with respect to the isolated subunits results from interactions that involve residues different from beta Ser-178 and (ii) beta Ser178 is a critical residue in ligand-triggered signals between alpha and beta active sites

The MocR-like transcription factors: pyridoxal 5'-phosphate-dependent regulators of bacterial metabolism

Many biological functions played by current proteins were not created by evolution from scratch, rather they were obtained combining already available protein scaffolds. This is the case of MocR-like bacterial transcription factors (MocR-TFs), a subclass of GntR transcription regulators, whose structure is the outcome of the fusion between DNA-binding proteins and pyridoxal 5'-phosphate (PLP)-dependent enzymes. The resultant chimeras can count on the properties of both protein classes, i.e. the capability to recognize specific DNA sequences and to bind PLP and amino-compounds; it is the modulation of such binding properties to confer to MocR-TFs chimeras the ability to interact with effector molecules and DNA so as to regulate transcription. MocR-TFs control different metabolic processes involving vitamin B6and amino acids, which are canonical ligands of PLP-dependent enzymes. However, MocR-TFs are also implicated in the metabolism of compounds that are not substrates of PLP-dependent enzymes, such as rhizopine and ectoine. Genomic analyses show that MocR-TFs are widespread among eubacteria, implying an essential role in their metabolism and highlighting the scarcity of our knowledge on these important players in microbial metabolism. Although MocR-TFs have been discovered 15 years ago, the research activity on these transcriptional regulators has only recently intensified, producing a wealth of information that needs to be brought back to general principles. This is the main task of this review, which reports and analyses the available information concerning MocR-TFs functional role, structural features, interaction with effector molecules and the characteristics of DNA transcriptional factor-binding sites of MocR-based regulatory systems

Salmonella typhimurium PtsJ is a novel MocR-like transcriptional repressor involved in regulating the vitamin B6 salvage pathway

The vitamin B6 salvage pathway, involving pyridoxine 5′-phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B6 vitamers from nutrients and protein turnover to produce pyridoxal 5′-phosphate (PLP), the catalytically active form of the vitamin. Regulation of this pathway, widespread in living organisms including humans and many bacteria, is very important to vitamin B6 homeostasis but poorly understood. Although some information is available on the enzymatic regulation of PNPOx and PLK, little is known on their regulation at the transcriptional level. In the present work, we identified a new MocR-like regulator, PtsJ from Salmonella typhimurium, which controls the expression of the pdxK gene encoding one of the two PLKs expressed in this organism (PLK1). Analysis of pdxK expression in a ptsJ knockout strain demonstrated that PtsJ acts as a transcriptional repressor. This is the first case of a MocR-like regulator acting as repressor of its target gene. Expression and purification of PtsJ allowed a detailed characterisation of its effector and DNA-binding properties. PLP is the only B6 vitamer acting as effector molecule for PtsJ. A DNA-binding region composed of four repeated nucleotide sequences is responsible for binding of PtsJ to its target promoter. Analysis of binding stoichiometry revealed that protein subunits/DNA molar ratio varies from 4 : 1 to 2 : 1, depending on the presence or absence of PLP. Structural characteristics of DNA transcriptional factor-binding sites suggest that PtsJ binds DNA according to a different model with respect to other characterised members of the MocR subgroup. © 2016 Federation of European Biochemical Societie

Data from computational analysis of the peptide linkers in the MocR bacterial transcriptional regulators

Detailed data from statistical analyses of the structural properties of the inter-domain linker peptides of the bacterial regulators of the family MocR are herein reported. MocR regulators are a recently discovered subfamily of bacterial regulators possessing an N-terminal domain, 60 residue long on average, folded as the winged-helix-turn-helix architecture responsible for DNA recognition and binding, and a large C-terminal domain (350 residue on average) that belongs to the fold type-I pyridoxal 5′-phosphate (PLP) dependent enzymes such aspartate aminotransferase. Data show the distribution of several structural characteristics of the linkers taken from bacterial species from five different phyla, namely Actinobacteria, Alpha-, Beta-, Gammaproteobacteria and Firmicutes. Interpretation and discussion of reported data refer to the article “Structural properties of the linkers connecting the N- and C- terminal domains in the MocR bacterial transcriptional regulators” (T. Milano, S. Angelaccio, A. Tramonti, M. L. Di Salvo, R. Contestabile, S. Pascarella, 2016) [1]

A Bioinformatics Analysis Reveals a Group of MocR Bacterial Transcriptional Regulators Linked to a Family of Genes Coding for Membrane Proteins

The MocR bacterial transcriptional regulators are characterized by an N-terminal domain, 60 residues long on average, possessing the winged-helix-turn-helix (wHTH) architecture responsible for DNA recognition and binding, linked to a large C-terminal domain (350 residues on average) that is homologous to fold type-I pyridoxal 5′-phosphate (PLP) dependent enzymes like aspartate aminotransferase (AAT). These regulators are involved in the expression of genes taking part in several metabolic pathways directly or indirectly connected to PLP chemistry, many of which are still uncharacterized. A bioinformatics analysis is here reported that studied the features of a distinct group of MocR regulators predicted to be functionally linked to a family of homologous genes coding for integral membrane proteins of unknown function. This group occurs mainly in the Actinobacteria and Gammaproteobacteria phyla. An analysis of the multiple sequence alignments of their wHTH and AAT domains suggested the presence of specificity-determining positions (SDPs). Mapping of SDPs onto a homology model of the AAT domain hinted at possible structural/functional roles in effector recognition. Likewise, SDPs in wHTH domain suggested the basis of specificity of Transcription Factor Binding Site recognition. The results reported represent a framework for rational design of experiments and for bioinformatics analysis of other MocR subgroups