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

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 β-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

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

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-\uf067 (PPARG), PPARG expression was evaluated. At 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

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

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, apoptosys. 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 perfomed 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 identifiying 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

Oligomerization of Sulfolobus solfataricus

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 °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

Protein structure prediction in the genomic era: Annotation-facilitated remote homology detection

As a result of large sequencing projects, databases of protein sequences and structures are growing rapidly. The number of protein sequences, however, is orders of magnitude larger than the number of structures known at the atomic level. This is true despite efforts to accelerate procedures to resolve protein structures. Tools have been developed to bridge the gap between protein sequence and 3D structure, based on retrieval of information from databases and the use of knowledge-based methods to provide solutions to the protein folding problem. Several machine learning approaches are available to address various sub-problems of protein structure prediction. These include secondary structure, recognition of domains, motifs, ligand binding sites, and the topology of membrane proteins. In protein design, all these features can help to compute a putative protein model. This chapter reviews the state of the art of protein structure prediction, and describes a recent non-hierarchical clustering procedure implemented to fully exploit current knowledge in the databases on sequences, structures and functions. This procedure increases the number of sequences that can be annotated (inferring function of sequence from database information) by transfer of annotation from a set of 599 genomes, including Homo sapiens. When in a given cluster, distantly related sequences from different genomes co-exist the procedure allows safe transfer of annotation, for both structure and function, independently of the level of sequence identity. In some specific cases of functional annotation, human sequences can be safely modeled on prokaryotic templates. In computing these models, machine learning approaches to sequence analysis may help in constraining the optimal alignment of the distantly related sequences. Our analysis addresses the problem of structural transfer among distantly related proteins and permits solutions that increase the structure of the human proteome b ysome 6,000 models

Computer-based prediction of mitochondrial-targeting peptides

Computational methods are invaluable when protein sequences, directly derived from genomic data, need functional and structural annotation. This is the case of subcellular localisation, a feature necessary for understanding the protein role and the compartment where the mature protein is active. Mitochondrial proteins encoded on the cytosolic ribosomes carry specific patterns in the precursor sequence from where it is computationally possible to recognise a peptide targeting the protein to its final destination. Here we discuss to which extent it is feasible to develop computational methods for detecting targeting peptides in the precursor sequences and benchmark our and other methods on the human mitochondrial proteins endowed with an experimentally characterised targeting peptide. Furthermore we illustrate our web server and its usage on the human poorly characterised proteome in order to infer targeting peptides, their cleavage sites and whether the targeting peptide regions contains or not arginine rich recurrent motifs. By this, we add some other 2800 human proteins to the 124 ones already experimentally annotated with a targeting peptide

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

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

Immature cells of sprout apices of Helianthus tuberosus express different forms of transglutaminases homologous to human transglutaminases

Immature cells of sprout apices of Helianthus tuberosus express different forms of transglutaminases homologous to human transglutaminases Simone Beninati1, Rosa Anna Iorio2, Gianluca Tasco2, Donatella Serafini-Fracassini2, Rita Casadio2 and Stefano Del Duca2 1Department of Biology, II University of Roma "Tor Vergata",via della Ricerca Scientifica, 00173, Roma, Italy. 2Department of Biology E. S., University of Bologna, via Irnerio 42, 40126, Bologna, Italy. e-mail: [email protected] Immature cells of etiolated apices growing from germinating sprouts of tubers of Helianthus tuberosus (H. t.) showed Ca2+-dependent TGase (TGase, EC 2.3.2.13) activity with fibronectin as substrate, dimethylcasein being substrate only at high Ca2+-concentrations. In three fractions of the 100,000g supernatant of extracted apices eluted at increased NaCl concentrations through a DEAE-cellulose column, three main transglutaminase bands of around 85, 75 and 58 kDa apparent molecular weight were immuno-identified by anti-TGase K type I and rat prostate gland TGase 4. These fractions catalysed the polyamine conjugation at the carboxamide group of N-benzyloxycarbonyl-L-leucine-bound -glutaminyl-residues. The amino acid composition of these TGase protein bands were compared to those of several sequenced TGases of different origin. The composition of the H.t. enzyme with a molecular range of 85 kDa showed a difference of about 2.5% with the average composition of four mammalian TGases 2 and a 3D model was built adopting TGase 2 as template. The H. t. band of 75 kDa showed a difference of about 0.3% with the average composition of three mammalian inactive EPB42, whereas this plant enzyme is active as demonstrated by the formation of glutamyl-derivatives when incubated with lebelled PAs. The 58 kDa form shared a low similarity with the human TGases 2, included their proteolytic fragments, and those of the Arabidopsis recombinant one (24-28 % difference), and even more with that of Streptoverticillium (41 % difference) and of the two maize sequences of similar molecular mass (51-52 % difference), which however are very dissimilar from all other known TGases. By comparison to known plant TGases, these novel TGases are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells of the sprout apices. These data also confirm that in plants there are multiple active forms of TGases in the same organ and that plant and animal enzymes probably are similar also structurally and not only, as already known, for their catalytic activity