Biomedical subjects

Mosè Rossi

Publications and source records attributed to Mosè Rossi.

At least 19 recordsLinked to original sources

PubMed2006

The human GINS complex binds to and specifically stimulates human DNA polymerase alpha-primase.

The eukaryotic GINS complex has an essential role in the initiation and elongation phases of genome duplication. It is composed of four paralogous subunits--Sld5, Psf1, Psf2 and Psf3--which are ubiquitous and evolutionarily conserved in eukaryotic organisms. Here, we report the biochemical characterization of the human GINS complex (hGINS). The four hGINS subunits were coexpressed in Escherichia coli in a highly soluble form and purified as a complex. hGINS was shown to interact directly with the heterodimeric human DNA primase, by using either surface plasmon resonance measurements or by immunoprecipitation experiments carried out with anti-hGINS antibodies. The DNA polymerase alpha-primase synthetic activity was specifically stimulated by hGINS on various primed DNA templates. The significance of these findings is discussed in view of the molecular dynamics at the human replication fork.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Use of an inhibitor to identify members of the hormone-sensitive lipase family.

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.

Amino Acid Sequence↗

PubMed2006

Biochemical evidence of a physical interaction between Sulfolobus solfataricus B-family and Y-family DNA polymerases.

The hyper-thermophilic archaeon Sulfolobus solfataricus possesses two functional DNA polymerases belonging to the B-family (Sso DNA pol B1) and to the Y-family (Sso DNA pol Y1). Sso DNA pol B1 recognizes the presence of uracil and hypoxanthine in the template strand and stalls synthesis 3-4 bases upstream of this lesion ("read-ahead" function). On the other hand, Sso DNA pol Y1 is able to synthesize across these and other lesions on the template strand. Herein we report evidence that Sso DNA pol B1 physically interacts with DNA pol Y1 by surface plasmon resonance measurements and immuno-precipitation experiments. The region of DNA pol B1 responsible for this interaction has been mapped in the central portion of the polypeptide chain (from the amino acid residue 482 to 617), which includes an extended protease hyper-sensitive linker between the N- and C-terminal modules (amino acid residues Asn482-Ala497) and the alpha-helices forming the "fingers" sub-domain (alpha-helices R, R' and S). These results have important implications for understanding the polymerase-switching mechanism on the damaged template strand during genome replication in S. solfataricus.

Archaeal Proteins↗

PubMed2006

Characterization of a multifunctional protein disulfide oxidoreductase from Sulfolobus solfataricus.

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been ascribed to a new family of protein disulfide oxidoreductases (PDOs). We report on the characterization of SsPDO, isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. SsPDO was cloned and expressed in Escherichia coli. We revealed that SsPDO is the substrate of a thioredoxin reductase in S. solfataricus (K(M) 0.3 microm) and not thioredoxins (TrxA1 and TrxA2). SsPDO/S. solfataricus thioredoxin reductase constitute a new thioredoxin system in aerobic thermophilic archaea. While redox (reductase, oxidative and isomerase) activities of SsPDO point to its central role in the biochemistry of cytoplasmic disulfide bonds, chaperone activities also on an endogenous substrate suggest a potential role in the stabilization of intracellular proteins. Northern and western analysis have been performed in order to analyze the response to the oxidative stress.

Amino Acid Sequence↗

PubMed2006

Lack of strand-specific repair of UV-induced DNA lesions in three genes of the archaeon Sulfolobus solfataricus.

In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to UV light. In both Eucarya and Bacteria, UV-induced pyrimidine dimers in the transcribed strand of active genes are repaired at a faster rate compared to the non-transcribed strand and the rest of the genome. Preferential repair of transcribed strands requires the Transcription-Repair Coupling Factor in Escherichia coli and the CSA and CSB proteins in humans. These factors are needed for coupling of transcription to nucleotide excision repair (NER), a major pathway for repair of UV-induced lesions. Whereas transcription-coupled NER (TC-NER) is an evolutionary conserved process, not all active genes show preferential repair of transcribed strands. The existence of a NER pathway in the Archaea has not been demonstrated directly, yet it is suggested by the presence and properties of homologues of NER nucleases and helicases. However, none of the proteins responsible for the lesion recognition steps or for TC-NER has been found in archaeal genomes. Moreover, the kinetics of gene or strand-specific repair has never been investigated in any organism of this domain. We have analysed the kinetics of repair of UV-induced DNA damage in the transcribed and non-transcribed strands of three genes of the hyperthermophilic archaeon Sulfolobus solfataricus. We found that in all three genes the two strands are repaired with the same efficiency with each other and with the genome in general, thus providing no evidence of strand bias or transcription coupling of the repair process in the genes analysed. Further studies will be required to test the existence of a transcription-coupled repair pathway in other archaeal genes and to elucidate the mechanism of UV lesion recognition and repair in Archaea.

Bacteria↗

PubMed2006

Structural, kinetic, and thermodynamic analysis of glucoimidazole-derived glycosidase inhibitors.

Inhibition of glycosidases has great potential in the quest for highly potent and specific drugs to treat diseases such as diabetes, cancer, and viral infections. One of the most effective ways of designing such compounds is by mimicking the transition state. Here we describe the structural, kinetic, and thermodynamic dissection of binding of two glucoimidazole-derived compounds, which are among the most potent glycosidase inhibitors reported to date, with two family 1 beta-glycosidases. Provocatively, while inclusion of the phenethyl moiety improves binding by a factor of 20-80-fold, this does not appear to result from better noncovalent interactions with the enzyme; instead, improved affinity may be derived from significantly better entropic contributions to binding displayed by the phenethyl-substituted imidazole compound.

Archaeal Proteins↗

PubMed2006

Pressure affects the structure and the dynamics of the D-galactose/D-glucose-binding protein from Escherichia coli by perturbing the C-terminal domain of the protein.

The effect of the pressure on the structure and stability of the D-galactose/D-glucose binding protein from Escherichia coli in the absence (GGBP) and in the presence (GGBP/Glc) of glucose was studied by Fourier transform infrared (FT-IR) spectroscopy and molecular dynamic (MD) simulations. FT-IR spectroscopy experiments showed that the protein beta-structures are more resistant than alpha-helices structures to pressure value increases. In addition, the infrared data indicated that the binding of glucose stabilizes the protein structure against high pressure values, and the protein structure does not completely unfold up to pressure values close to 9000 bar. MD simulations allow a prediction of the most probable configuration of the protein, consistent with the increasing pressures on the two systems. The detailed analysis of the structures at molecular level confirms that, among secondary structures, alpha-helices are more sensitive than beta-structures to the destabilizing effect of high pressure and that glucose is able to preserve the structure of the protein in the complex. Moreover, the evidence of the different resistance of the two domains of this protein to high pressure is investigated and explained at a molecular level, indicating the importance of aromatic amino acid in protein stabilization.

Escherichia coli↗

PubMed2006

A spreadable, non-integrative and high copy number shuttle vector for Sulfolobus solfataricus based on the genetic element pSSVx from Sulfolobus islandicus.

The pSSVx genetic element from Sulfolobus islandicus REY15/4 is a hybrid between a plasmid and a fusellovirus, able to be maintained in non-integrative form and to spread when the helper SSV2 virus is present in the cells. In this work, the satellite virus was engineered to obtain an Escherichia coli-Sulfolobus solfataricus shuttle vector for gene transfer and expression in S.solfataricus by fusing site-specifically the pSSVx chromosome with an E.coli plasmid replicon and the ampicillin resistance gene. The pSSVx-based vector was proven functional like the parental virus, namely it was able to spread efficiently through infected S.solfataricus cells. Moreover, the hybrid plasmid stably transformed S.solfataricus and propagated with no rearrangement, recombination or integration into the host chromosome. The high copy number of the artificial genetic element was found comparable with that calculated for the wild-type pSSVx in the new host cells, with no need of genetic markers for vector maintenance in the cells and for transfomant enrichment. The newly constructed vector was also shown to be an efficient cloning vehicle for the expression of passenger genes in S.solfataricus. In fact, a derivative plasmid carrying an expression cassette of the lacS gene encoding the beta-glycosidase from S.solfataricus under the control of the Sulfolobus chaperonine (thermosome tf55) heat shock promoter was also able to drive the expression of a functional enzyme. Complementation of the beta-galactosidase deficiency in a deletion mutant strain of S.solfataricus demonstrated that lacS gene was an efficient marker for selection of single transformants on solid minimal lactose medium.

DNA Replication↗

PubMed2006

The gene of an archaeal alpha-L-fucosidase is expressed by translational frameshifting.

The standard rules of genetic translational decoding are altered in specific genes by different events that are globally termed recoding. In Archaea recoding has been unequivocally determined so far only for termination codon readthrough events. We study here the mechanism of expression of a gene encoding for a alpha-l-fucosidase from the archaeon Sulfolobus solfataricus (fucA1), which is split in two open reading frames separated by a -1 frameshifting. The expression in Escherichia coli of the wild-type split gene led to the production by frameshifting of full-length polypeptides with an efficiency of 5%. Mutations in the regulatory site where the shift takes place demonstrate that the expression in vivo occurs in a programmed way. Further, we identify a full-length product of fucA1 in S.solfataricus extracts, which translate this gene in vitro by following programmed -1 frameshifting. This is the first experimental demonstration that this kind of recoding is present in Archaea.

Archaea↗

PubMed2006

A novel member of the protein disulfide oxidoreductase family from Aeropyrum pernix K1: structure, function and electrostatics.

The formation of disulfide bonds between cysteine residues is a rate-limiting step in protein folding. To control this oxidative process, different organisms have developed different systems. In bacteria, disulfide bond formation is assisted by the Dsb protein family; in eukarya, disulfide bond formation and rearrangement are catalyzed by PDI. In thermophilic organisms, a potential key role in disulfide bond formation has recently been ascribed to a new cytosolic Protein Disulphide Oxidoreductase family whose members have a molecular mass of about 26 kDa and are characterized by two thioredoxin folds comprising a CXXC active site motif each. Here we report on the functional and structural characterization of ApPDO, a new member of this family, which was isolated from the archaeon Aeropyrum pernix K1. Functional studies have revealed that ApPDO can catalyze the reduction, oxidation and isomerization of disulfide bridges. Structural studies have shown that this protein has two CXXC active sites with fairly similar geometrical parameters typical of a stable conformation. Finally, a theoretical calculation of the cysteine pK(a) values has suggested that the two active sites have similar functional properties and each of them can impart activity to the enzyme. Our results are evidence of functional similarity between the members of the Protein Disulphide Oxidoreductase family and the eukaryotic enzyme PDI. However, as the different three-dimensional features of these two biological systems strongly suggest significantly different mechanisms of action, further experimental studies will be needed to make clear how different three-dimensional structures can result in systems with similar functional behavior.

Aeropyrum↗

PubMed2006

A novel thermostable sulfite oxidase from Thermus thermophilus: characterization of the enzyme, gene cloning and expression in Escherichia coli.

A novel sulfite oxidase has been identified from Thermus thermophilus AT62. Despite this enzyme showing significant amino-acid sequence homology to several bacterial and eukaryal putative and identified sulfite oxidases, the kinetic analysis, performed following the oxidation of sulfite and with ferricyanide as the electron acceptor, already pointed out major differences from representatives of bacterial and eukaryal sources. Sulfite oxidase from T. thermophilus, purified to homogeneity, is a monomeric enzyme with an apparent molecular mass of 39.1 kDa and is almost exclusively located in the periplasm fraction. The enzyme showed sulfite oxidase activity only when ferricyanide was used as electron acceptor, which is different from most of sulfite-oxidizing enzymes from several sources that use cytochrome c as co-substrate. Spectroscopic studies demonstrated that the purified sulfite oxidase has no cytochrome like domain, normally present in homologous enzymes from eukaryotic and prokaryotic sources, and for this particular feature it is similar to homologous enzyme from Arabidopsis thaliana. The identified gene was PCR amplified on T. thermophilus AT62 genome, expressed in Escherichia coli and the recombinant protein identified and characterized.

Amino Acid Sequence↗

PubMed2006

pIT3, a cryptic plasmid isolated from the hyperthermophilic crenarchaeon Sulfolobus solfataricus IT3.

The plasmid pIT3 (4,967 bp) was isolated from the hyperthermophilic archaeon Sulfolobus solfataricus, strain IT3. The completely sequenced plasmid contains six open reading frames (ORFs), the largest (ORF915) spanning more than half of the plasmid and encoding a putative protein with significant similarity to the helicase domain of viral and plasmid primase proteins, as well as to the newly described archaeal primase-polymerase domain. A small ORF, (ORF80), located upstream of this putative polymerase, encodes a putative copy number control protein. Specific transcripts corresponding to the ORF80 and ORF915, were detected by Northern blot analyses, and their transcriptional start sites were determined by primer extension. Moreover, the transfer and the maintenance of the plasmid in other Sulfolobus strains were demonstrated to be effective and stable.

Amino Acid Sequence↗

PubMed2006

Selective degradation of reverse gyrase and DNA fragmentation induced by alkylating agent in the archaeon Sulfolobus solfataricus.

Reverse gyrase is a peculiar DNA topoisomerase, specific of hyperthermophilic Archaea and Bacteria, which has the unique ability of introducing positive supercoiling into DNA molecules. Although the function of the enzyme has not been established directly, it has been suggested to be involved in DNA protection and repair. We show here that the enzyme is degraded after treatment of Sulfolobus solfataricus cells with the alkylating agent MMS. MMS-induced reverse gyrase degradation is highly specific, since (i) neither hydroxyurea (HU) nor puromycin have a similar effect, and (ii) topoisomerase VI and two chromatin components are not degraded. Reverse gyrase degradation does not depend on protein synthesis. Experiments in vitro show that direct exposure of cell extracts to MMS does not induce reverse gyrase degradation; instead, extracts from MMS-treated cells contain some factor(s) able to degrade the enzyme in extracts from control cells. In vitro, degradation is blocked by incubation with divalent metal chelators, suggesting that reverse gyrase is selectively degraded by a metal-dependent protease in MMS-treated cells. In addition, we find a striking concurrence of extensive genomic DNA degradation and reverse gyrase loss in MMS-treated cells. These results support the hypothesis that reverse gyrase plays an essential role in DNA thermoprotection and repair in hyperthermophilic organisms.

Alkylating Agents↗

PubMed2006

Characterization of a beta-glycosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius.

In cell free extracts of the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius ATCC27009, we have identified beta-gluco- and galactosidase activities showing a specific activity of 0.1 and 12 U/mg, respectively. The two enzymatic activities are associated with different polypeptides and we show here the functional cloning, the expression in Escherichia coli and the characterisation of the beta-glucosidase (Aabeta-gly). The enzyme, which is optimally active and stable at temperatures above 65 degrees C, belongs to glycoside hydrolase family 1 (GH1) and shows wide substrate specificity on different aryl-glycosides and cello-oligosaccharides with k (cat)/K (M) for 4-nitrophenyl-beta-D-glucoside and cellobiose of 2,976 and 185 s(-1)mM(-1), respectively. Interestingly, upstream to the beta-glycosidase gene, we identified a second ORF homologous to the ATPase subunit of the bacterial ABC transporters (abc1) that is co-transcribed with the beta-glycosidase gene glyB and that could be involved in the carbohydrate import. The activity of the enzyme on cello-oligosaccharides of up to five glucose units strongly indicates that the enzyme could be involved in vivo in the degradation of glucans together with endoglucanase enzymes previously described. This, together with the co-expression of the two genes, suggests a role for the glyB-abc1 cluster in A. acidocaldarius in the degradation of cellulose and hemicelluloses.

Amino Acid Sequence↗

PubMed2006

Pressure effect on the stability and the conformational dynamics of the D-Galactose/D-Glucose-binding protein from Escherichia coli.

The effect of the pressure on the structure and stability of the D-Galactose/D-Glucose binding protein (GGBP) from Escherichia coli was studied by steady-state and time-resolved fluorescence spectroscopy, and the ability of glucose ligand to stabilize the GGBP structure was also investigated. Steady-state fluorescence experiments showed a marked quenching of fluorescence emission of GGBP in the absence of glucose. Instead, the presence of glucose seems to stabilize the structure of GGBP at low and moderate pressure values. Time-resolved fluorescence measurements showed that the GGBP taumean in the absence of glucose varies significantly up to 600 bar, while in the presence of the ligand it is almost unaffected by pressure increase up to 600 bar. The effect of the pressure on GGBP was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results and confirm that the presence of glucose is able to contrast the negative effects of pressure on the protein structure. Taken together, the spectroscopic and computer simulation studies suggest that at pressure values up to 2000 bar the structure of GGBP in the absence of glucose remains folded, but a significant perturbation of the protein secondary structures can be detected. The binding of glucose reduces the negative effect of pressure on protein structure and confers protection from perturbation especially at moderate pressure values.

Computer Simulation↗

PubMed2006

Interrupted genes in extremophilic archaea: mechanisms of gene expression in early organisms.

Extremophilic Archaea populate biotopes previously considered inaccessible for life. This feature, and the possibility that they are the extant forms of life closest to the last common ancestor, make these organisms excellent candidates for the study of evolution on Earth and stimulate the exobiological research in planets previously considered totally inhospitable. Among the other aspects of the physiology of these organisms, the study of the molecular genetics of extremophilic Archaea can give hints on how the genetic information is transmitted and propagated in ancient forms of life. We review here the expression of interrupted genes in a recently discovered nanoarchaeon and the mechanisms of reprogrammed genetic decoding in Archaea.

Amino Acid Sequence↗

PubMed2006

Glutamine-binding protein from Escherichia coli specifically binds a wheat gliadin peptide allowing the design of a new porous silicon-based optical biosensor.

In this work, the binding of the recombinant glutamine-binding protein (GlnBP) from Escherichia coli to gliadin peptides, toxic for celiac patients, was investigated by mass spectrometry experiments and optical techniques. Mass spectrometry experiments demonstrated that GlnBP binds the following amino acid sequence: XXQPQPQQQQQQQQQQQQL, present only into the toxic prolamines. The binding of GlnBP to gliadin suggested us to design a new optical biosensor based on nanostructured porous silicon (PSi) for the detection of trace amounts of gliadin in food. The GlnBP, which acts as a molecular probe for the gliadin, was covalently linked to the surface of the PSi wafer by a proper passivation process. The GlnBP-gliadin interaction was revealed as a shift in wavelength of the fringes in the reflectivity spectrum of the PSi layer. The GlnBP, covalently bonded to the PSi chip, selectively recognized the toxic peptide. Finally, the sensor response to the protein concentration was measured in the range 2.0-40.0 microg/L and the sensitivity of the sensor was determined.

Amino Acid Sequence↗

PubMed2006

Binding of glucose to the D-galactose/D-glucose-binding protein from Escherichia coli restores the native protein secondary structure and thermostability that are lost upon calcium depletion.

The effect of the depletion of calcium on the structure and thermal stability of the D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli was studied by fluorescence spectroscopy and Fourier-transform infrared spectroscopy. The calcium-depleted protein (GGBP-Ca) was also studied in the presence of glucose (GGBP-Ca/Glc). The results show that calcium depletion has a small effect on the secondary structure of GGBP, and, in particular it affects a population of alpha-helices with a low exposure to solvent. Alternatively, glucose-binding to GGBP-Ca eliminates the effect induced by calcium depletion by restoring a secondary structure similar to that of the native protein. In addition, the infrared and fluorescence data obtained reveal that calcium depletion markedly reduces the thermal stability of GGBP. In particular, the spectroscopic experiments show that the depletion of calcium mainly affects the stability of the C-terminal domain of the protein. However, the binding of glucose restores the thermal stability of GGBP-Ca. The thermostability of GGBP and GGBP-Ca was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results. New insights into the role of calcium in the thermal stability of GGBP contribute to a better understanding of the protein function and constitute important information for the development of biotechnological applications of this protein. Mutations and/or labelling of amino acid residues located in the protein C-terminal domain may affect the stability of the whole protein structure.

Binding Sites↗