Publications by authors named "Gianluca Tasco"

22 Publications

  • Page 1 of 1

Genomic and expression analyses of Tursiops truncatus T cell receptor gamma (TRG) and alpha/delta (TRA/TRD) loci reveal a similar basic public γδ repertoire in dolphin and human.

BMC Genomics 2016 08 15;17(1):634. Epub 2016 Aug 15.

Department of Biology, University of Bari, via E. Orabona 4, 70125, Bari, Italy.

Background: The bottlenose dolphin (Tursiops truncatus) is a mammal that belongs to the Cetartiodactyla and have lived in marine ecosystems for nearly 60 millions years. Despite its popularity, our knowledge about its adaptive immunity and evolution is very limited. Furthermore, nothing is known about the genomics and evolution of dolphin antigen receptor immunity.

Results: Here we report a evolutionary and expression study of Tursiops truncatus T cell receptor gamma (TRG) and alpha/delta (TRA/TRD) genes. We have identified in silico the TRG and TRA/TRD genes and analyzed the relevant mature transcripts in blood and in skin from four subjects. The dolphin TRG locus is the smallest and simplest of all mammalian loci as yet studied. It shows a genomic organization comprising two variable (V1 and V2), three joining (J1, J2 and J3) and a single constant (C), genes. Despite the fragmented nature of the genome assemblies, we deduced the TRA/TRD locus organization, with the recent TRDV1 subgroup genes duplications, as it is expected in artiodactyls. Expression analysis from blood of a subject allowed us to assign unambiguously eight TRAV genes to those annotated in the genomic sequence and to twelve new genes, belonging to five different subgroups. All transcripts were productive and no relevant biases towards TRAV-J rearrangements are observed. Blood and skin from four unrelated subjects expression data provide evidence for an unusual ratio of productive/unproductive transcripts which arise from the TRG V-J gene rearrangement and for a "public" gamma delta TR repertoire. The productive cDNA sequences, shared both in the same and in different individuals, include biases of the TRGV1 and TRGJ2 genes. The high frequency of TRGV1-J2/TRDV1- D1-J4 productive rearrangements in dolphins may represent an interesting oligo-clonal population comparable to that found in human with the TRGV9- JP/TRDV2-D-J T cells and in primates.

Conclusions: Although the features of the TRG and TRA/TRD loci organization reflect those of the so far examined artiodactyls, genomic results highlight in dolphin an unusually simple TRG locus. The cDNA analysis reveal productive TRA/TRD transcripts and unusual ratios of productive/unproductive TRG transcripts. Comparing multiple different individuals, evidence is found for a "public" gamma delta TCR repertoire thus suggesting that in dolphins as in human the gamma delta TCR repertoire is accompanied by selection for public gamma chain.
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http://dx.doi.org/10.1186/s12864-016-2841-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986337PMC
August 2016

Computer-based prediction of mitochondria-targeting peptides.

Methods Mol Biol 2015 ;1264:305-20

Biocomputing Group, CIRI Health Sciences & Technologies (HST), University of Bologna, Bologna, Italy.

Computational methods are invaluable when protein sequences, directly derived from genomic data, need functional and structural annotation. Subcellular localization is a feature necessary for understanding the protein role and the compartment where the mature protein is active and very difficult to characterize experimentally. Mitochondrial proteins encoded on the cytosolic ribosomes carry specific patterns in the precursor sequence from where it is possible to recognize a peptide targeting the protein to its final destination. Here we discuss to which extent it is feasible to develop computational methods for detecting mitochondrial targeting peptides in the precursor sequences and benchmark our and other methods on the human mitochondrial proteins endowed with experimentally characterized targeting peptides. Furthermore, we illustrate our newly implemented web server and its usage on the whole human proteome in order to infer mitochondrial targeting peptides, their cleavage sites, and whether the targeting peptide regions contain or not arginine-rich recurrent motifs. By this, we add some other 2,800 human proteins to the 124 ones already experimentally annotated with a mitochondrial targeting peptide.
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http://dx.doi.org/10.1007/978-1-4939-2257-4_27DOI Listing
October 2015

Characteristics of the somatic hypermutation in the Camelus dromedarius T cell receptor gamma (TRG) and delta (TRD) variable domains.

Dev Comp Immunol 2014 Oct 13;46(2):300-13. Epub 2014 May 13.

Department of Biology, University of Bari, via E. Orabona 4, 70125 Bari, Italy.

In previous reports, we had shown in Camelus dromedarius that diversity in T cell receptor gamma (TRG) and delta (TRD) variable domains can be generated by somatic hypermutation (SHM). In the present paper, we further the previous finding by analyzing 85 unique spleen cDNA sequences encoding a total of 331 mutations from a single animal, and comparing the properties of the mutation profiles of dromedary TRG and TRD variable domains. The transition preference and the significant mutation frequency in the AID motifs (dgyw/wrch and wa/tw) demonstrate a strong dependence of the enzymes mediating SHM in TRG and TRD genes of dromedary similar to that of immunoglobulin genes in mammals. Overall, results reveal no asymmetry in the motifs targeting, i.e. mutations are equally distributed among g:c and a:t base pairs and replacement mutations are favored at the AID motifs, whereas neutral mutations appear to be more prone to accumulate in bases outside of the motifs. A detailed analysis of clonal lineages in TRG and TRD cDNA sequences also suggests that clonal expansion of mutated productive rearrangements may be crucial in shaping the somatic diversification in the dromedary. This is confirmed by the fact that our structural models, computed by adopting a comparative procedure, are consistent with the possibility that, irrespective of where (in the CDR-IMGT or in FR-IMGT) the diversity was generated by mutations, both clonal expansion and selection seem to be strictly related to an enhanced structural stability of the γδ subunits.
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http://dx.doi.org/10.1016/j.dci.2014.05.001DOI Listing
October 2014

Frataxin mRNA isoforms in FRDA patients and normal subjects: effect of tocotrienol supplementation.

Biomed Res Int 2013 23;2013:276808. Epub 2013 Sep 23.

Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, 40126 Bologna, Italy.

Friedreich's ataxia (FRDA) is caused by deficient expression of the mitochondrial protein frataxin involved in the formation of iron-sulphur complexes and by consequent oxidative stress. We analysed low-dose tocotrienol supplementation effects on the expression of the three splice variant isoforms (FXN-1, FXN-2, and FXN-3) in mononuclear blood cells of FRDA patients and healthy subjects. In FRDA patients, tocotrienol leads to a specific and significant increase of FXN-3 expression while not affecting FXN-1 and FXN-2 expression. Since no structural and functional details were available for FNX-2 and FXN-3, 3D models were built. FXN-1, the canonical isoform, was then docked on the human iron-sulphur complex, and functional interactions were computed; when FXN-1 was replaced by FXN-2 or FNX-3, we found that the interactions were maintained, thus suggesting a possible biological role for both isoforms in human cells. Finally, in order to evaluate whether tocotrienol enhancement of FXN-3 was mediated by an increase in peroxisome proliferator-activated receptor-γ (PPARG), PPARG expression was evaluated. At a low dose of tocotrienol, the increase of FXN-3 expression appeared to be independent of PPARG expression. Our data show that it is possible to modulate the mRNA expression of the minor frataxin isoforms and that they may have a functional role.
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http://dx.doi.org/10.1155/2013/276808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794619PMC
June 2014

Expression of different forms of transglutaminases by immature cells of Helianthus tuberosus sprout apices.

Amino Acids 2013 Jan 18;44(1):271-83. Epub 2012 Oct 18.

Department of Biology, II University of Rome Tor Vergata, via della Ricerca Scientifica, 00173 Rome, Italy.

Immature cells of etiolated apices of sprouts growing from Helianthus tuberosus (H. t.) tubers showed Ca(2+)-dependent transglutaminase (TG, EC 2.3.2.13) activity on fibronectin (more efficiently) and dimethylcasein as substrates. Three main TG bands of about 85, 75 and 58 kDa were isolated from the 100,000×g apices supernatant through a DEAE-cellulose column at increasing NaCl concentrations and immuno-identified by anti-TG K and anti-rat prostate gland TG antibodies. These three fractions had catalytic activity as catalyzed polyamine conjugation to N-benzyloxycarbonyl-L-γ-glutaminyl-L-leucine (Z-L-Gln-L-Leu) and the corresponding glutamyl-derivatives were identified. The amino acid composition of these TG proteins was compared with those of several sequenced TGs of different origin. The composition of the two larger bands presented great similarities with annotated TGs; in particular, the 75 kDa form was very similar to mammalian inactive EPB42. The 58 kDa form shared a low similarity with other TGs, including a maize sequence of similar molecular mass, which, however, did not present the catalytic triad in the position of all annotated TGs. A 3D model of the H. t. TGs was built adopting TG2 as template. These novel plant TGs are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells. These data suggest that in plants, multiple TG forms are active in the same organ and that plant and animal enzymes probably are very close not only for their catalytic activity but also structurally.
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http://dx.doi.org/10.1007/s00726-012-1411-yDOI Listing
January 2013

Role of the C-terminus of Pleurotus eryngii Ery4 laccase in determining enzyme structure, catalytic properties and stability.

Protein Eng Des Sel 2013 Jan 20;26(1):1-13. Epub 2012 Sep 20.

CNR-Istituto di Scienze delle Produzioni Alimentari (ISPA), Lecce, Italy.

The ERY4 laccase gene of Pleurotus eryngii is not biologically active when expressed in yeast. To explain this finding, we analysed the role of the C-terminus of Ery4 protein by producing a number of its different mutant variants. Two different categories of ERY4 mutant genes were produced and expressed in yeast: (i) mutants carrying C-terminal deletions and (ii) mutants carrying different site-specific mutations at their C-terminus. Investigation of the catalytic properties of the recombinant enzymes indicated that each novel variant acquired different affinities and catalytic activity for various substrates. Our results highlight that C-terminal processing is fundamental for Ery4 laccase enzymatic activities allowing substrate accessibility to the enzyme catalytic core. Apparently, the last 18 amino acids in the C-terminal end of the Ery4 laccase play a critical role in enzyme activity, stability and kinetic and, in particular biochemical and structural data indicate that the K532 residue is fundamental for enzyme activation. These studies shed light on the structure/function relationships of fungal laccases and will enhance the development of biotechnological strategies for the industrial exploitation of these enzymes.
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http://dx.doi.org/10.1093/protein/gzs056DOI Listing
January 2013

Generation of diversity by somatic mutation in the Camelus dromedarius T-cell receptor gamma variable domains.

Eur J Immunol 2012 Dec 25;42(12):3416-28. Epub 2012 Oct 25.

Department of Biology, University of Bari, Bari, Italy.

In jawed vertebrates the V-(D)-J rearrangement is the main mechanism generating limitless variations of antigen-specific receptors, immunoglobulins (IGs), and T-cell receptors (TCRs) from few genes. Once the initial diversity is established in primary lymphoid organs, further diversification occurs in IGs by somatic hypermutation, a mechanism from which rearranged TCR genes were thought to be excluded. Here, we report the locus organization and expression of the T-cell receptor gamma (TCRG) genes in the Arabian camel (Camelus dromedarius). Expression data provide evidence that dromedary utilizes only two TCRG V-J genomic arrangements and, as expected, CDR3 contributes the major variability in the V domain. The data also suggest that diversity might be generated by mutation in the productively rearranged TCRGV genes. As for IG genes, the mutational target is biased toward G and C bases and (A/G/T)G(C/T)(A/T) motif (or DGYW). The replacement and synonymous substitutions (R/S) ratios in TCRGV regions are higher for CDR than for framework region, thus suggesting selection toward amino acid changes in CDR. Using the counterpart human TCR γδ receptor as a template, structural models computed adopting a comparative procedure show that nonconservative mutations contribute to diversity in CDR2 and at the γδ V domain interface.
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http://dx.doi.org/10.1002/eji.201142176DOI Listing
December 2012

A novel subfamily of mitochondrial dicarboxylate carriers from Drosophila melanogaster: biochemical and computational studies.

Biochim Biophys Acta 2011 Mar 3;1807(3):251-61. Epub 2010 Dec 3.

Department of Pharmaco-Biology, University of Calabria, Arcavacata di Rende, 87036 Cosenza, Italy.

The dicarboxylate carrier is an important member of the mitochondrial carrier family, which catalyzes an electroneutral exchange across the inner mitochondrial membrane of dicarboxylates for inorganic phosphate and certain sulfur-containing compounds. Screening of the Drosophila melanogaster genome revealed the presence of a mitochondrial carrier subfamily constituted by four potential homologs of mammalian and yeast mitochondrial dicarboxylate carriers designated as DmDic1p, DmDic2p, DmDic3p, and DmDic4p. In this paper, we report that DmDIC1 is broadly expressed at comparable levels in all development stages investigated whereas DmDIC3 and DmDIC4 are expressed only in the pupal stage, no transcripts are detectable for DmDIC2. All expressed proteins are localized in mitochondria. The transport activity of DmDic1-3-4 proteins has been investigated by reconstitution of recombinant purified protein into liposomes. DmDic1p is a typical dicarboxylate carrier showing similar substrate specificity and inhibitor sensitivity as mammalian and yeast mitochondrial dicarboxylate carriers. DmDic3p seems to be an atypical dicarboxylate carrier being able to transport only inorganic phosphate and certain sulfur-containing compounds. No transport activity was observed for DmDic4p. The biochemical results have been supported at molecular level by computing the protein structures and by structural alignments. All together these results indicate that D. melanogaster dicarboxylate carriers form a protein subfamily but the modifications in the amino acids sequences are indicative of specialized functions.
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http://dx.doi.org/10.1016/j.bbabio.2010.11.013DOI Listing
March 2011

Thermodynamics of binding of regulatory ligands to tissue transglutaminase.

Amino Acids 2010 Jun 22;39(1):297-304. Epub 2009 Dec 22.

Department of Biochemistry and Molecular Biology, University of Ferrara, Via Borsari 46, 44100, Ferrara, Italy.

The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the peptidyl-glutamine substrate. We have analysed binding of these ligands by calorimetric and computational approaches. In the case of GTP we have detected a single high affinity site (K (D) approximately 1 microM), with moderate thermal effects suggestive that binding GTP involves replacement of GDP, normally bound to the protein. On line with this possibility no significant binding was observed during titration with GDP and computational studies support this view. Titration with calcium at a high cation molar excess yielded a complex binding isotherm with a number of "apparent binding sites" in large excess over those detectable by equilibrium dialysis (6 sites). This binding pattern is ascribed to occurrence of additional thermal contributions, beyond those of binding, due to the occurrence of conformational changes and to catalysis itself (with protein self-crosslinking). In contrast only one site for binding calcium with high affinity (K (D) approximately 0.15 microM) is observed with samples of enzyme inactivated by alkylation at the active site (to prevent enzyme crosslinkage and thermal effects of catalysis). These results indicate an intrinsic ability of tissue transglutaminase to bind calcium with high affinity and the necessity of careful reassessment of the enzyme regulatory pattern in relation to the concentrations of ligands in living cells, taking also in account effects of ligands on protein subcellular compartimentation.
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http://dx.doi.org/10.1007/s00726-009-0442-5DOI Listing
June 2010

The prediction of membrane protein structure and genome structural annotation.

Comp Funct Genomics 2003 ;4(4):406-9

Laboratory of Biocomputing, CIRB/Department of Biology, University of Bologna, via Irnerio 42, Bologna 40126, Italy.

New methods, essentially based on hidden Markov models (HMM) and neural networks (NN), can predict the topography of both beta-barrel and all-alpha membrane proteins with high accuracy and a low rate of false positives and false negatives. These methods have been integrated in a suite of programs to filter proteomes of Gram-negative bacteria, searching for new membrane proteins.
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http://dx.doi.org/10.1002/cfg.308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2447372PMC
June 2010

Plant and animal transglutaminases: do similar functions imply similar structures?

Amino Acids 2009 Apr 12;36(4):643-57. Epub 2008 Jul 12.

Unit of Plant Cell Growth and Differentiation, Dipartimento di Biologia Evoluzionistica Sperimentale, Sede Botanica, University of Bologna, via Irnerio 42, 40126, Bologna, Italy.

In plants the post-translational modification of proteins by polyamines catalysed by transglutaminases has been studied since 1987; it was identified by the production of glutamyl-polyamine derivatives, biochemical features, recognition by animal antibodies and modification of typical animal substrates. Transglutaminases are widespread in all plant organs and cell compartments studied until now, chloroplast being the most studied. Substrates are: photosynthetic complexes and Rubisco in chloroplasts, cytoskeleton and cell wall proteins. Roles either specific of plants or in common with animals are related to photosynthesis, fertilisation, stresses, senescence and programmed cell death, showing that the catalytic function is conserved across the kingdoms. AtPng1p, the first plant transglutaminase sequenced shows undetectable sequence homology to the animal enzymes, except for the catalytic triad. It is, however, endowed with a calcium-dependent activity that allowed us to build a three-dimensional model adopting as a template the animal transglutaminase 2.
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http://dx.doi.org/10.1007/s00726-008-0131-9DOI Listing
April 2009

A protein structure prediction service in the ProGenGrid system.

Stud Health Technol Inform 2008 ;138:135-46

SPACI Consortium, University of Salento, Lecce & NNL/CNR-INFM, Lecce, Italy.

This paper describes a protein tertiary structure prediction service implemented in a Grid Environment. The service has been used for predicting the dicarboxylate carrier (DIC) of Saccharomyces cerevisiae by using the homology modelling approach. The visualization of the predicted model is made possible by using an interactive virtual reality environment based on X3D and Ajax3d technologies.
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October 2008

Cloning, functional identification and structural modelling of Vitis vinifera S-adenosylmethionine decarboxylase.

J Plant Physiol 2007 Sep 18;164(9):1208-19. Epub 2006 Sep 18.

Department of Biology e s and Interdepartmental Centre for Biotechnology, University of Bologna, Via Irnerio 42, Bologna, Italy.

In this paper we report the cloning and full sequencing of S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) cDNA from Vitis vinifera L. (VV) leaves, an enzyme belonging to the polyamine biosynthetic pathway, which appears to play an important role in the regulation of plant growth and development. The presence of two overlapping ORFs (tiny ORF and small ORF) upstream of the main ORF is reported in the Vitis cDNA. When the Vitis SAMDC cDNA was expressed in yeast without the two upstream ORFs, the resulting activity was about 50 times higher than the activity obtained with the full cDNA. These results demonstrated the strong regulatory activity of the tiny and small ORFs. RT-PCR expression analysis showed evidence of a similar mRNA level in all the tissues tested, with the exception of the petioles. The VV SAMDC was also modelled using its homologues from Solanum tuberosum and Homo sapiens as template. The present work confirmed, for the first time in a woody plant of worldwide economic interest such as grapevine, the presence of a regulatory mechanism of SAMDC, enzyme that has a well-established importance in the modulation of plant growth and development.
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http://dx.doi.org/10.1016/j.jplph.2006.07.009DOI Listing
September 2007

Thinking the impossible: how to solve the protein folding problem with and without homologous structures and more.

Methods Mol Biol 2007 ;350:305-20

Biocomputing Group, CIRB/Department of Biology, University of Bologna, Bologna, Italy.

Structure prediction of proteins is a difficult task as well as prediction of protein-protein interaction. When no homologous sequence with known structure is available for the target protein, search of distantly related proteins to the target may be done automatically (fold recognition/threading). However, there are difficult proteins for which still modeling on the basis of a putative scaffold is nearly impossible. In the following, we describe that for some specific examples, human expertise was able to derive alignments to proteins of similar function with the aid of machine learning-based methods specifically suited for predicting structural features. The manually curate search of putative templates was successful in generating low-resolution three-dimensional (3D) models in at least two cases: the human tissue transglutaminase and the alcohol dehydrogenase from Sulfolobus solfataricus. This is based on the structural comparison of the model with the 3D protein structure that became available after prediction. For protein-protein interaction, a knowledge-based method can give predictions of putative interaction patches on the protein surface; this feature may help in adding additional weight to specific nodes in nets of interacting proteins.
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http://dx.doi.org/10.1385/1-59745-189-4:305DOI Listing
November 2006

Systematic analysis of human kinase genes: a large number of genes and alternative splicing events result in functional and structural diversity.

BMC Bioinformatics 2005 Dec 1;6 Suppl 4:S20. Epub 2005 Dec 1.

Biomedical Technologies Institute (ITB), National Research Council, Milan, Italy.

Background: Protein kinases are a well defined family of proteins, characterized by the presence of a common kinase catalytic domain and playing a significant role in many important cellular processes, such as proliferation, maintenance of cell shape, apoptosis. In many members of the family, additional non-kinase domains contribute further specialization, resulting in subcellular localization, protein binding and regulation of activity, among others. About 500 genes encode members of the kinase family in the human genome, and although many of them represent well known genes, a larger number of genes code for proteins of more recent identification, or for unknown proteins identified as kinase only after computational studies.

Results: A systematic in silico study performed on the human genome, led to the identification of 5 genes, on chromosome 1, 11, 13, 15 and 16 respectively, and 1 pseudogene on chromosome X; some of these genes are reported as kinases from NCBI but are absent in other databases, such as KinBase. Comparative analysis of 483 gene regions and subsequent computational analysis, aimed at identifying unannotated exons, indicates that a large number of kinase may code for alternately spliced forms or be incorrectly annotated. An InterProScan automated analysis was performed to study domain distribution and combination in the various families. At the same time, other structural features were also added to the annotation process, including the putative presence of transmembrane alpha helices, and the cystein propensity to participate into a disulfide bridge.

Conclusion: The predicted human kinome was extended by identifying both additional genes and potential splice variants, resulting in a varied panorama where functionality may be searched at the gene and protein level. Structural analysis of kinase proteins domains as defined in multiple sources together with transmembrane alpha helices and signal peptide prediction provides hints to function assignment. The results of the human kinome analysis are collected in the KinWeb database, available for browsing and searching over the internet, where all results from the comparative analysis and the gene structure annotation are made available, alongside the domain information. Kinases may be searched by domain combinations and the relative genes may be viewed in a graphic browser at various level of magnification up to gene organization on the full chromosome set.
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http://dx.doi.org/10.1186/1471-2105-6-S4-S20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1866387PMC
December 2005

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study.

Mol Membr Biol 2005 Sep-Oct;22(5):443-52

INFM and Department of Biology, University of Rome "Tor Vergata", via della Ricerca Scientifica, Rome.

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.
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http://dx.doi.org/10.1080/09687860500269335DOI Listing
January 2006

Oligomerization of Sulfolobus solfataricus signature amidase is promoted by acidic pH and high temperature.

Archaea 2005 Dec;1(6):411-23

Dipartimento di Scienze Biochimiche A. Rossi-Fanelli, Università La Sapienza, P.le A. Moro 5, 00185 Roma, Italy.

The recombinant amidase from the hyperthermophylic archaeon Sulfolobus solfataricus (SSAM) a signature amidase, was cloned, purified and characterized. The enzyme is active on a large number of aliphatic and aromatic amides over the temperature range 60-95 degrees C and at pH values between 4.0 and 9.5, with an optimum at pH 5.0. The recombinant enzyme is in the form of a dimer of about 110 kD that reversibly associates into an octamer in a pH-dependent reaction. The pH dependence of the state of association was studied using gel permeation chromatography, analytical ultracentrifugation and dynamic light scattering techniques. At pH 7.0 all three techniques show the presence of two species, in about equal amounts, which is compatible with the existence of a dimeric and an octameric form. In decreasing pH, the dimers formed the octameric species and in increasing pH, the octameric species was converted to dimers. Above pH 8.0, only dimers were present, below pH 3.0 only octamers were present. The association of dimers into octamers decreased in non-polar solvents and increased with temperature. A mutant (Y41C) was obtained that did not show this behavior.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2685583PMC
http://dx.doi.org/10.1155/2005/543789DOI Listing
December 2005

Histone deacetylase 1: a target of 9-hydroxystearic acid in the inhibition of cell growth in human colon cancer.

J Lipid Res 2005 Aug 16;46(8):1596-603. Epub 2005 Feb 16.

Department of Biochemistry G. Moruzzi, University of Bologna, Bologna, Italy.

Recent studies have shown that an endogenous lipoperoxidation product, 9-hydroxystearic acid (9-HSA), acts in colon carcinoma cells (HT29) as a growth inhibitor by inducing p21(WAF1) in an immediate-early, p53-independent manner and that p21(WAF1) is required for 9-HSA-mediated growth arrest in HT29 cells. It is conceivable, therefore, to hypothesize that the cytostatic effect induced by this agent is at least partially associated with a molecular mechanism that involves histone deacetylase 1 (HDAC1) inhibition, as demonstrated for sodium butyrate and other specific inhibitors, such as trichostatin A and hydroxamic acids. Here, we show that, after administration, 9-HSA causes an accumulation of hyperacetylated histones and strongly inhibits the activity of HDAC1. The interaction of 9-HSA with the catalytic site of the enzyme has been highlighted by computational modeling of the human HDAC1, using its homolog from the hyperthermophilic Aquifex aeolicus as a template. Consistent with the experimental data, we find that 9-HSA can bind to the active site of the protein, showing that the inhibition of the enzyme can be explained at the molecular level by the ligand-protein interaction.
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http://dx.doi.org/10.1194/jlr.M400424-JLR200DOI Listing
August 2005

Reduction of active elastase concentration by means of immobilized inhibitors: a novel therapeutic approach.

Biotechnol Prog 2004 May-Jun;20(3):968-74

Department of Experimental Medicine, Second University of Naples, Via S. M. di Costantinopoli 16, 80138 Naples, Italy.

The inhibitory power of three different active Nylon membranes, separately loaded with three different protease inhibitors, was studied with the aim of reducing the increased elastase concentration occurring during hemodialysis or extracorporeal blood circulation in patients undergoing cardiopulmonary bypass. Chemical grafting was carried out to make the inert Nylon membrane suitable for the immobilization of the inhibitors. The behavior of immobilized alpha(1)-antitrypsin, bovine pancreatic trypsin inhibitor (BPTI), or elastatinal was separately studied. alpha(1)-Antitrypsin and BPTI were covalently immobilized by means of a diazotization process, whereas elastatinal was covalently attached via a condensation process mediated by glutaraldehyde. The inhibitory power of each membrane type was studied as a function of the amount of immobilized inhibitor and temperature. All active membranes have shown good inhibitory power. The most efficient membrane was that loaded with alpha(1)-antitrypsin, the less efficient that with BPTI.
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http://dx.doi.org/10.1021/bp034304bDOI Listing
December 2004

Functional characterization of a second porin isoform in Drosophila melanogaster. DmPorin2 forms voltage-independent cation-selective pores.

J Biol Chem 2004 Jun 30;279(24):25364-73. Epub 2004 Mar 30.

Department of Chemical Sciences, University of Catania, 95125 Catania, Italy.

Mitochondrial porins or voltage-dependent anion-selective channels are channel-forming proteins mainly found in the mitochondrial outer membrane. Genome sequencing of the fruit fly Drosophila melanogaster revealed the presence of three additional porin-like genes. No functional information was available for the different gene products. In this work we have studied the function of the gene product closest to the known Porin gene (CG17137 coding for DmPorin2). Its coding sequence was expressed in Escherichia coli. The recombinant DmPorin2 protein is able to form channels similar to those formed by DmPorin1 reconstituted in artificial membranes. Furthermore, DmPorin2 is clearly voltage-independent and cation-selective, whereas its counterpart isoform 1 is voltage-dependent and anion-selective. Sequence comparison of the two porin isoforms indicates the exchange of four lysines in DmPorin1 for four glutamic acids in DmPorin2. We have mutated two of them (Glu-66 and Glu-163) to lysines to investigate their role in the functional features of the pore. The mutants E163K and E66K/E163K are endowed with an almost full inversion of the ion selectivity. Both single mutations partially restore the voltage dependence of the pore. We found that an additional effect with the double mutant E66K/E163K was the restoration of voltage dependence. Protein structure predictions highlight a 16 beta-strand pattern, typical for porins. In a three-dimensional model of DmPorin2, Glu-66 and Glu-163 are close to the rim of the channel, on two opposite sides. DmPorin2 is expressed in all the fly tissues and in all the developmental stages tested. Our main conclusions are as follows. 1) The CG17137 gene may express a porin with a functional role in D. melanogaster. 2) We have identified two amino acids of major relevance for the voltage dependence of the porin pore.
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http://dx.doi.org/10.1074/jbc.M310572200DOI Listing
June 2004

New functions of an old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC).

Ital J Biochem 2003 Mar;52(1):17-24

Lab. of Biochemistry and Molecular Biology, Dept. of Chemical Sciences, University of Catania, Catania, Italy.

Mitochondrial porin or VDAC (Voltage Dependent Anion selective Channels) was identified for the first time in 1976, on the basis of the evolutionary similarity between the gram negative and mitochondrial outer membranes. Since this achievement VDAC has been extensively investigated: its functional features have been sharply defined upon reconstitution in artificial membranes and its sequence has been determined in many genomes. Unfortunately the tertiary structure has not yet been solved, mainly because it proved to be very difficult to get suitable crystals. Despite this established knowledge, in the last few years this protein has attracted renewed interest. There are two main reasons for this interest: the discovery, in most eukaryotes, of a family of genes encoding VDAC isoforms and the claims of VDAC involvement in the intrinsic pathway of apoptosis and in particular in the mechanism of cytochrome c release from mitochondria. We can affirm that nowadays the eukaryotic porin (or VDAC) is studied in a more general cellular contest, looking at the interactions and integration with other molecules, since VDAC is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. In this minireview we will briefly focus our attention onto the following topics: 1) recent advances about the structure of VDAC; 2) the VDAC-related multigene families; 3) the presence, targeting and function of VDAC in various cell membranes.
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March 2003
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