Functional characterization of EhADH112: an Entamoeba histolytica Bro1 domain-containing protein

EhADH112 is part of the EhCPADH complex, a protein involved in key events of the Entamoeba histolytica host invasion. EhADH112 participates in trophozoite adherence to target cells and in phagocytosis. We report here the finding of two EhADH112 homologues in the E. histolytica genome (EhADH112-like proteins). EhADH112 and its relatives have a Bro1 domain at their amino-terminus and a consensus context for phosphorylation by Src-tyrosine kinases, both involved in signal transduction processes in other organisms. Our findings associate EhADH112 to supplementary functions related to those reported for the Alix/AIP1 family. To elucidate the precise function of EhADH112, we studied the phenotypes displayed by trophozoites transfected with the Ehadh112 full gene. Transfected trophozoites overexpressed a 78 kDa protein, which was mainly targeted to the EhCPADH complex. Moreover, these trophozoites exhibited enhanced phagocytic rates, providing further evidence of EhADH112 contribution to adhesion and phagocytosis activities.     

Molecular cloning and sequencing of metallothionein in squamates: New insights into the evolution of the metallothionein genes in vertebrates

Metallothioneins are cysteine-rich, metal-binding proteins ubiquitously expressed in living organisms. In the last past years, a plethora of vertebrate metallothionein sequences have become available, but so far there has been an almost absolute lack of data about sequences of metallothionein of non-avian diapsida. In the framework of the investigations on structural and functional properties of non-mammalian metallothioneins, we have cloned and sequenced the cDNAs encoding for metallothioneins of 10 squamate reptiles, belonging to 5 different infraorders. These sequences have been used to gain insight into the evolutionary history of metallothioneins in reptiles. Phylogenetic analysis shows that reptilian metallothionein phylogeny is inconsistent with the species phylogeny. Such findings allow us to hypothesize that the identified metallothionein in each squamate species used for this study might be considered a paralogous gene derived from more events of gene duplication and losses occurred during the diversification of the squamate species. Finally, through vertebrate metallothionein comparisons and phylogenetic analysis, we also add a novel contribution to the understanding of the evolution of metallothionein genes along the major vertebrate lineages.     

Exclusive paternal expression and novel alternatively spliced variants of epsilon-sarcoglycan mRNA in mouse brain

Mutations of SGCE encoding epsilon-sarcoglycan cause myoclonus-dystonia. SGCE is paternally expressed; however, 5-10% of patients show maternal inheritance of the disease. We found Sgce was exclusively paternally expressed in mice by using a novel polymorphism marker. The result was confirmed in Sgce heterozygous knockout mice. This finding suggests that maternally inherited myoclonus-dystonia may not result from maternal expression of SGCE. Furthermore, we report a new family of alternatively spliced Sgce mRNA expressed in the brain coding for different C-terminal sequences possessing a PDZ-binding motif. Our results provide a better basis for diagnosis and understanding of the pathogenesis of myoclonus-dystonia.     

Molecular insights into the binding of coenzyme F420 to the conserved protein Rv1155 from Mycobacterium tuberculosis

Coenzyme F₄₂₀ is a deazaflavin hydride carrier with a lower reduction potential than most flavins. In Mycobacterium tuberculosis (Mtb), F₄₂₀ plays an important role in activating PA-824, an antituberculosis drug currently used in clinical trials. Although F₄₂₀ is important to Mtb redox metabolism, little is known about the enzymes that bind F₄₂₀ and the reactions that they catalyze. We have identified a novel F₄₂₀-binding protein, Rv1155, which is annotated in the Mtb genome sequence as a putative flavin mononucleotide (FMN)-binding protein. Using biophysical techniques, we have demonstrated that instead of binding FMN or other flavins, Rv1155 binds coenzyme F₄₂₀. The crystal structure of the complex of Rv1155 and F₄₂₀ reveals one F₄₂₀ molecule bound to each monomer of the Rv1155 dimer. Structural, biophysical, and bioinformatic analyses of the Rv1155–F₄₂₀ complex provide clues about its role in the bacterium.     

Molecular Polymorphism and Morphometrics of Species of the Heterodera avenae Group in Syria and Turkey

Molecular characterization of the three most common cereal cyst nematode species of the Heterodera avenae group (H. avenae, H. filipjevi, and H. latipons), originating from various locations in major cereal-cultivating areas in Syria and Turkey, showed distinct restriction fragment patterns of the ITS-rDNA following PCR amplification and RFLP digestion with four endonucleases (Hae III, Hinf I, Ita I, and Pst I). Genetic dissimilarity within H. avenae group populations increased in comparison with H. avenae and other species; it was 0.164 with H. filipjevi and 0.354 with H. latipons populations. No intraspecific polymorphism was observed within H. latipons or H. filipjevi populations. Principal component analysis revealed contrasted correlations among 12 morphological parameters of cysts and juveniles of the three Heterodera species that separated them and distinguished differences within populations of H. latipons. Our results showed a clear separation of the three cyst nematode species on cereal using a conventional method for classification and molecular tests, and confirmed the congruence between genetics and morphological traits.     

cDNA cloning and the response to overfeeding in the expression of stearoyl-CoA desaturase 1 gene in Landes goose

Stearoyl-CoA desaturase 1 (SCD1) is a rate limiting enzyme in the biosynthesis of monounsaturated fatty acids. It has been cloned from several species: Rattus norvegicus, Mus musculus, Homo Sapiens and Gallus gallus, but not from Anser anser. This study was conducted to isolate the SCD1 cDNA sequence and investigate the effect of overfeeding on SCD1 gene tissue expression in Landes goose. The complete cDNA is 3294 bp in length, with an ORF of 1.083 bp encoding a predicted polypeptide of 360 amino acids and 5′/3′-UTR of 74 and 2137 bp, respectively. Quantitative real time PCR (qPCR) was used to examine SCD1 expression in heart, liver, spleen, lung, kidney, gizzard, glandular stomach, intestine, crureus, pectoral muscle, hypothalamus and adipose tissue (abdominal fat) in both the overfed and control group. SCD1 mRNA was highly expressed in goose fatty liver, and the expression levels of SCD1 in liver and fat of overfeeding group were more than double that of the control group. During the overfeeding period, SCD1 expression in liver and adipose tissue reached the highest level after 70 days, but declined at 79 days. In the control group, after fasting 24 h, the expression level of SCD1 gene in tissues declined sharply. However, SCD1 gene expression in hypothalamus was unaffected. The results of this study provide a theoretical basis to study the relationship between SCD1 gene expression and the formation of fatty liver of Landes goose in response to overfeeding.     

The role of reactive oxygen and nitrogen species in cellular iron metabolism

The catalytic role of iron in the Haber-Weiss chemistry, which results in propagation of damaging reactive oxygen species (ROS), is well established. In this review, we attempt to summarize the recent evidence showing the reverse: That reactive oxygen and nitrogen species can significantly affect iron metabolism. Their interaction with iron-regulatory proteins (IRPs) seems to be one of the essential mechanisms of influencing iron homeostasis. Iron depletion is known to provoke normal iron uptake via IRPs, superoxide and hydrogen peroxide are supposed to cause unnecessary iron uptake by similar mechanism. Furthermore, ROS are able to release iron from iron-containing molecules. On the contrary, nitric oxide (NO) appears to be involved in cellular defense against the iron-mediated ROS generation probably mainly by inducing iron removal from cells. In addition, NO may attenuate the effect of superoxide by mutual reaction, although the reaction product—peroxynitrite—is capable to produce highly reactive hydroxyl radicals.     

Splicing of intron 3 of human BACE requires the flanking introns 2 and 4

Regulation of proteolytic cleavage of the amyloid precursor protein by the aspartic protease BACE may occur by alternative splicing and the generation of enzymatically inactive forms. In fact, the presence of exonic donor and acceptor sites for intron 3 generates the two deficient variants BACE457 and BACE476. In HEK293 cells, when introns are inserted separately in the BACE cDNA, we found that whilst introns 2 and 4 are efficiently spliced out, intron 3 is not removed. On the other hand, splicing to wild-type BACE is restored when intron 3 is flanked by the two other introns. The presence of all three introns also leads to alternative splicing of intron 3 and the generation of BACE476. In contrast, BACE457 expression takes place only after mutating the donor splice site of intron 3, indicating that additional regulatory elements are necessary for the use of the splicing site within exon 4. Overall, our data demonstrate that a complex splicing of intron 3 regulates the maturation of the BACE mRNA. This appears orchestrated by domains present in the exons and introns flanking intron 3. Excessive BACE activity is a risk factor for Alzheimer’s disease, therefore this complex regulation might guarantee low neuronal BACE activity and disease prevention.     

Molecular characterization and expression patterns of the actinin-associated LIM protein (ALP) subfamily genes in porcine skeletal muscle

The actinin-associated LIM protein (ALP) subfamily has important functions in cell signal transduction, cell proliferation, and integration of cytoskeletal architecture. To detect their functions in pig skeletal muscle, we cloned and characterized the pig ALP subfamily genes, drew their genomic structure maps, and detected their tissue expression patterns. We identified a new spliced variant of PDLIM3 in pig skeletal muscle and named it as PDLIM3-4, which was only expressed in the heart and skeletal muscle. Our results showed that PDLIM3-4 was expressed in adult pig skeletal muscle with the highest expression level, and both PDLIM3-4 isoform and PDLIM4 had different expression profiles during the prenatal and postnatal stages of skeletal muscle development among the three pig breeds. These studies provide useful information for further research on the functions of pig ALP subfamily genes in skeletal muscle development.     

Properties of the inulinase gene levH1 of Lactobacillus casei IAM 1045; cloning, mutational and biochemical characterization

Though some genetic features of lactobacillar fructan hydrolases were elucidated, information about their enzymology or mutational analyses were scarce. Lactobacillus casei IAM1045 exhibits extracellular activity degrading inulin. After partial purification of the inulin-degrading protein from the spent culture medium, several fragments were obtained by protease digestion. Based on their partial amino-acid sequences, oligonucleotide primers were designed, and its structural gene (levH1) was determined using the gene library constructed in the E. coli system. The levH1 gene encoded a protein (designated as LevH1), of which calculated molecular mass and pI were 138.8-kDa and 4.66, respectively. LevH1 (1296 amino-acids long) was predicted to have a four-domain structure, containing (i) an N-terminal secretion signal of 40 amino-acids, (ii) variable domain of about 140 residues whose function is unclear, (iii) a catalytic domain of about 630 residues with glycoside-hydrolase activity consisting of two modules, a five-blade β-propeller module linked to a β-sandwich module, (iv) a C-terminal domain of about 490 residues comprising five nearly perfect repeat sequences of 80 residues homologous to equivalents of other hypothetical cell surface proteins, followed by 37-residues rich in Ser/Thr/Pro/Gly, a pentad LPQAG (the LPXTG homologue). When overproduced in E. coli, the putative variable-catalytic domain region of about 770 residues exhibited exo-inulinase activity. Deletion analyses demonstrated that the variable-catalytic domain region containing two modules is important for enzymatic activity. Presence of eight conserved motifs (I-VIII) was suggested in the catalytic domain by comparative analysis, among which motif VIII was newly identified in the β-sandwich module in this study. Site-directed mutagenesis of conserved amino-acids in these motifs revealed that D198, R388, D389 and E440, were crucial for inulinase activity. Moreover, mutations of D502A and D683A in motif VI and VIII respectively caused significant decrease in the activity. These results suggested that the variable domain and β-sandwich module, besides the β-propeller module, are important for inulin-degrading activity of LevH1.     

Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~ 165 K) than that of the low spin atom (~ 207 K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17 meV) and higher (17-25 meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~ 6 meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q_A and Q_B) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.     

Developmental cis-regulatory analysis of the cyclin D gene in the sea urchin Strongylocentrotus purpuratus

Cyclin D genes regulate the cell cycle, growth and differentiation in response to intercellular signaling. While the promoters of vertebrate cyclin D genes have been analyzed, the cis-regulatory sequences across an entire cyclin D locus have not. Doing so would increase understanding of how cyclin D genes respond to the regulatory states established by developmental gene regulatory networks, linking cell cycle and growth control to the ontogenetic program. Therefore, we conducted a cis-regulatory analysis on the cyclin D gene, SpcycD, of the sea urchin, Strongylocentrotus purpuratus, during embryogenesis, identifying upstream and intronic sequences, located within six defined regions bearing one or more cis-regulatory modules each.