A cis-acting element in the promoter of human ether à go-go 1 potassium channel gene mediates repression by calcitriol in human cervical cancer cells.

The human ether à go-go 1 potassium channel (hEAG1) is required for cell cycle progression and proliferation of cancer cells. Inhibitors of hEAG1 activity and expression represent potential therapeutic drugs in cancer. Previously, we have shown that hEAG1 expression is downregulated by calcitriol in a variety of cancer cells. Herein, we provided evidence on the regulatory mechanism involved in such repressive effect in cells derived from human cervical cancer. Our results indicate that repression by calcitriol occurs at the transcriptional level and involves a functional negative vitamin D response element (nVDRE) E-box type in the hEAG1 promoter. The described mechanism in this work implies that a protein complex formed by the vitamin D receptor-interacting repressor, the vitamin D receptor, the retinoid X receptor, and the Williams syndrome transcription factor interact with the nVDRE in the hEAG1 promoter in the absence of ligand. Interestingly, all of these transcription factors except the vitamin D receptor-interacting repressor are displaced from hEAG1 promoter in the presence of calcitriol. Our results provide novel mechanistic insights into calcitriol mode of action in repressing hEAG1 gene expression.

Novel splice donor site mutation in MERTK gene associated with retinitis pigmentosa.

BACKGROUND/AIM: Mutations in MERTK, a member of the MER/AXL/TYRO3 receptor kinase family, have been associated with disruption of the Retinal Pigment Epithelium (RPE) phagocytosis pathway and settling of autosomal recessive RP (arRP) in humans. This study reports a novel MERTK mutation (IVS16+1G>T) in a Spanish consanguineous family presenting arRP. METHODS: 21 genes were screened by high-throughput SNP multiplexing assay. Subsequent direct sequencing was performed in exons and intronic boundaries of the cosegregating gene. The effect of the mutation in mRNA splicing was confirmed by cDNA analysis. RESULTS: Haplotypic data revealed MERTK cosegregation with RP in affected individuals. MERTK sequencing showed a G-to-T substitution at the first nucleotide of intron 16. Finally, cDNA analysis confirmed the lack of exon 16 in the mRNA splicing process. CONCLUSIONS: IVS16+1G>T disrupts the splice donor site causing exon 16 skipping. Absence of exon 16 causes a frameshift and, subsequently, the introduction of a premature termination codon into exon 17 creating an altered mRNA transcript with a seriously affected tyrosine kinase domain.

The ubiquitin-specific protease USP25 interacts with three sarcomeric proteins.

The biological functions of the more than one hundred genes coding for deubiquitinating enzymes in the human genome remain mostly unknown. The USP25 gene, located at 21q11.2, encodes three protein isoforms produced by alternative splicing. While two of the isoforms are expressed nearly ubiquituously, the expression of the longer USP25 isoform (USP25m) is restricted to muscular tissues and is upregulated during myogenesis. USP25m interacts with three sarcomeric proteins: actin alpha-1 (ACTA1), filamin C (FLNC), and myosin binding protein C1 (MyBPC1), which are critically involved in muscle differentiation and maintenance, and have been implicated in the pathogenesis of severe myopathies. Biochemical analyses demonstrated that MyBPC1 is a short-lived proteasomal substrate, and its degradation is prevented by over-expression of USP25m but not by other USP25 isoforms. In contrast, ACTA1 and FLNC appear to be stable proteins, indicating that their interaction with USP25m is not related to their turnover rate.

Merging protein, gene and genomic data: the evolution of the MDR-ADH family.

Multiple members of the MDR-ADH (MDR: Medium-chain dehydrogenases/reductases; ADH: alcohol dehydrogenase) family are found in vertebrates, although the enzymes that belong to this family have also been isolated from bacteria, yeast, plant and animal sources. Initial understanding of the physiological roles and evolution of the family relied on biochemical studies, protein alignments and protein structure comparisons. Subsequently, studies at the genetic level yielded new information: the expression pattern, exon-intron distribution, in silico-derived protein sequences and murine knockout phenotypes. More recently, genomic and EST databases have revealed new family members and the chromosomal location and position in the cluster of both the first and new forms. The data now available provide a comprehensive scenario, from which a reliable picture of the evolutionary history of this family can be made.

Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, is under-expressed in Down syndrome fetal brains.

Suppression subtractive hybridization performed on Down syndrome (DS) versus control fetal brains revealed differential expression of peroxiredoxin 2 (PRDX2), mapped at 13q12. Peroxiredoxins are antioxidant enzymes involved in protein and lipid protection against oxidative injury and in cellular signalling pathways regulating apoptosis. The under-expression of PRDX2 observed in DS samples was confirmed by real-time PCR (0.73-fold). To test whether decreased expression is associated with enhanced sensitivity of DS neurons to reactive oxygen species, we down-regulated PRDX2 through stable transfections of SH-SY5Y neuroblastoma cells with antisense contructs of the complete PRDX2 coding sequence. In addition, we over-expressed SOD1 and compared the effects of the two genes on cell viability. Cells transfected with either construct showed similar sensitivity to oxidative stress in addition to increased apoptosis under basal conditions and after treatment with oxidative cytotoxic agents. This suggests that the decreased expression of PRDX2 may contribute to the altered redox state in DS at levels comparable to that of the increased expression of SOD1.

The first non-LTR retrotransposon characterised in the cephalochordate amphioxus, BfCR1, shows similarities to CR1-like elements.

BfCR1 is the first non-long terminal repeat retrotransposon to be characterised in the amphioxus genome. Sequence alignment of the predicted translation product reveals that BfCR1 belongs to the CR1-like retroposon class, a family widely distributed in vertebrate and invertebrate lineages. Structural analysis shows conservation of the specific motifs of the ORF2-CR1 elements: the N-terminal endonuclease, the reverse transcriptase and the C-terminal domains. The BfCR1 element possesses an atypical 3' terminus consisting of the tandem repeat (AAG)6. We gathered evidence supporting the mobility of this element and report an estimated 15 copies of BfCR1, mostly truncated, per haploid genome, a remarkably low number when compared to that of vertebrates. Phylogenetic analysis, including the amphioxus element, seems to indicate that (i) CR1-like retroposons cluster in a monophyletic group and (ii) the CR1-like family was already present in the chordate ancestor. Our data provide further support for the horizontal transmission of CR1-like elements during early vertebrate evolution.

Characterization of the amphioxus presenilin gene in a high gene-density genomic region illustrates duplication during the vertebrate lineage.

Molecular studies on the pathology of Alzheimer's disease (AD) have revealed that mutations in presenilin genes (PS) account for some familial cases. Although the contribution of these genes to the etiology is clear, their biological function remains obscure. Approaches using model organisms have been hampered by the fact that rodents contain two PS copies in the genome and do not develop the hallmark features associated with AD upon aging. To understand PS function and evolution, we have searched for PS homologous sequences in the genome of a lower chordate, Branchiostoma floridae. We report the structure of a single copy Branchiostoma floridae PS gene, named BfPS, and describe new features at the molecular level. Moreover, molecular phylogenetic analysis suggests that BfPS is orthologous to the vertebrate PS-1 and PS-2 forms. Finally, the analysis of more than 16 kb of genomic DNA encompassing BfPS identified three novel genes, which cluster with BfPS in a high gene-density region.

Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25.

BACKGROUND: The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis. RESULTS: Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR. CONCLUSIONS: On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes.

Secretion of mouse-metallothionein by engineered E. coli cells in metal-enriched culture media.

Heterologous Escherichia coli expression systems were designed and assayed for the synthesis of functional mouse metallothionein (MT) as a secreted fusion protein. MT secretion was compared among different systems, and the optimum vector/host/medium combination was tested for metal removal. In this case, the Cu content of the medium decreased by up to 34% after growth of recombinant bacteria. The potential use of these genetically-engineered bacteria for water bioremediation is discussed as an alternative to cytoplasmic MT or membrane-bound MT heterologous expression systems.

Homodimerization of presenilin N-terminal fragments is affected by mutations linked to Alzheimer's disease.

Mutations on human presenilins 1 and 2 cause dominant early-onset familial Alzheimer's disease (FAD). Presenilins are polytopic transmembrane proteins endoproteolytically processed in vivo to N- and C-terminal fragments (NTFs and CTFs). The functional presenilin unit consists of a high molecular weight complex that contains both fragments. Here we show NTF:NTF, CTF:CTF and NTF:CTF interactions by yeast two-hybrid and in vivo endoplasmic reticulum split-ubiquitin assays. Our results also highlight the involvement of HL1--the hydrophilic loop between TMI and TMII--in the NTF:NTF binding site. Besides, nine FAD-linked presenilin mutations substantially affected HL1:HL1 binding. From the evidence of NTF and CTF homodimerization, we propose the contribution of two NTFs and two CTFs, instead of a single NTF:CTF heterodimer, to the functional presenilin-gamma-secretase complex and that FAD mutations affect the assembly or stability of this complex.

Endogenous beta-galactosidase activity in amphioxus: a useful histochemical marker for the digestive system.

Endogenous beta-galactosidase activity has been shown in the digestive tract of amphioxus from the larval to the adult stage and it can be easily followed as a histochemical marker. Enzymatic activity first appeared in 30-h larvae, became evident in 36-h larvae and remained in adults. In situ detection of beta-galactosidase activity was used to monitor morphological and functional differentiation of the digestive system and the posteriorization of the endodermal structures in retinoic-acid treated embryos. The endogenous beta-galactosidase activity was distinguished from the bacterial lacZ reporter by incubation at low pH.

A new insight into metallothionein (MT) classification and evolution. The in vivo and in vitro metal binding features of Homarus americanus recombinant MT.

We report the synthesis and characterization of a Homarus americanus MT-cDNA (MTH) through retrotranscription of MTH-mRNA from metal-injected lobsters. Heterologous Escherichia coli expression in zinc- and copper-supplemented medium was achieved for MTH, the two domains betabetaMTH and betaalphaMTH and three site-directed mutants, betabetaC9H, betaalphaC37H, and betaalphaE31C/T34C. The in vivo conformed metal complexes and the in vitro substituted cadmium aggregates were characterized. Major stoichiometries of M(II)6-MTH for the entire MTH and M(II)3-betabetaMTH and M(II)3-betaalphaMTH for the independent domains fully validated our expression system. A low affinity binding site for a seventh Zn(II) in the in vivo synthesized MTH was located in the betaalpha domain. Additionally, minor M(II)4 species were found for each domain. Both single Cys to His mutations exhibited a similar reduction of their in vivo zinc binding ability but differed in their cadmium binding behavior when compared with the wild-type forms. Conversely, the double mutant showed an enhanced zinc and cadmium binding capacity. In vivo synthesis of MTH and of its independent domains in the presence of copper only afforded heterometallic copper-zinc species. These findings allow consideration of MTH as a zinc thionein and question the view of all crustacea MT structures as copper thioneins. Furthermore, a new approach for the evolutionary and functional classification of MT is proposed, based on the stoichiometry of metal-MT species and molecular phylogenetic analysis.

Spectrum of ABCA4 (ABCR) gene mutations in Spanish patients with autosomal recessive macular dystrophies.

The ABCA4 gene has been involved in several forms of inherited macular dystrophy. In order to further characterize the complex genotype-phenotype relationships involving this gene, we have performed a mutation analysis of ABCA4 in 14 Spanish patients comprising eight STGD (Stargardt), four FFM (fundus flavimaculatus), and two CRD (Cone-rod dystrophy) patients. SSCP (single-strand conformation polymorphism) analysis and DNA sequencing of the coding and 5' upstream regions of this gene allowed the identification of 16 putatively pathogenic alterations, nine of which are novel. Most of these were missense changes, and no patient was found to carry two null alleles. Overall, the new data agree with a working model relating the different pathogenic phenotypes to the severity of the mutations. When considering the information presented here together with that of previous reports, a picture of the geographic distribution of three particular mutations emerges. The R212C change has been found in French, Italian, Dutch, German, and Spanish but not in British patients. In the Spanish collection, R212C was found in a CRD patient, indicating that it may be a rather severe change. In contrast, c.2588G>C, a very common mild allele in the Dutch population, is rarely found in Southern Europe. Interestingly, the c.2588G>C mutation has been found in a double mutant allele together with the missense R1055W. Finally, the newly described L1940P was found in two unrelated Spanish patients, and may be a moderate to severe allele.

Characterization of a microsomal retinol dehydrogenase gene from amphioxus: retinoid metabolism before vertebrates.

Amphioxus, a member of the subphylum Cephalochordata, is thought to be the closest living relative to vertebrates. Although these animals have a vertebrate-like response to retinoic acid, the pathway of retinoid metabolism remains unknown. Two different enzyme systems - the short chain dehydrogenase/reductases and the cytosolic medium-chain alcohol dehydrogenases (ADHs) - have been postulated in vertebrates. Nevertheless, recent data show that the vertebrate-ADH1 and ADH4 retinol-active forms originated after the divergence of cephalochordates and vertebrates. Moreover, no data has been gathered in support of medium-chain retinol active forms in amphioxus. Then, if the cytosolic ADH system is absent and these animals use retinol, the microsomal retinol dehydrogenases could be involved in retinol oxidation. We have identified the genomic region and cDNA of an amphioxus Rdh gene as a preliminary step for functional characterization. Besides, phylogenetic analysis supports the ancestral position of amphioxus Rdh in relation to the vertebrate forms.

Genesis of Drosophila ADH: the shaping of the enzymatic activity from a SDR ancestor.

Drosophila alcohol dehydrogenase (ADH) is an NAD(H)-dependent oxidoreductase that catalyzes the oxidation of alcohols and aldehydes. Structurally and biochemically distinct from all the reported ADHs (typically, the mammalian medium-chain dehydrogenase/reductase-ethanol-metabolizing enzyme), it stands as the only small-alcohol transforming system that has originated from a short-chain dehydrogenase/reductase (SDR) ancestor. The crystal structures of the apo, binary (E.NAD(+)) and three ternary (E.NAD(+).acetone, E.NAD(+).3-pentanone and E.NAD(+).cyclohexanone) forms of Drosophila lebanonensis ADH have allowed us to infer the structural and kinetic features accounting for the generation of the ADH activity within the SDR lineage.

Amphioxus alcohol dehydrogenase is a class 3 form of single type and of structural conservation but with unique developmental expression.

The coding region of amphioxus alcohol dehydrogenase class 3 (ADH3) has been characterized from two species, Branchiostoma lanceolatum and Branchiostoma floridae. The species variants have residue differences at positions that result in only marginal functional distinctions. Activity measurements show a class 3 glutathione-dependent formaldehyde dehydrogenase, with kcat/Km values about threefold those of the human class 3 ADH enzyme. Only a single ADH3 form is identified in each of the two amphioxus species, and no ethanol activity ascribed to other classes is detectable, supporting the conclusion that evolution of ethanol-active ADH classes by gene duplications occurred at early vertebrate radiation after the formation of the amphioxus lineage. Similarly, Southern blot analysis indicated that amphioxus ADH3 is encoded by a single gene present in the methylated fraction of the amphioxus genome and northern blots revealed a single 1.4-kb transcript. In situ experiments showed that amphioxus Adh3 expression is restricted to particular cell types in the embryos. Transcripts were first evident at the neurula stage and then located at the larval ventral region, in the intestinal epithelium. This tissue-specific pattern contrasts with the ubiquitous Adh3 expression in mammals.

Identification of foetal brain proteins by two-dimensional gel electrophoresis and mass spectrometry comparison of samples from individuals with or without chromosome 21 trisomy.

Protein expression in foetal brain with or without chromosome 21 trisomy (Down's syndrome) was analyzed by two-dimensional gel electrophoresis and mass spectrometry. Data generated by in-gel digestion and matrix-assisted laser desorption/ionization mass spectrometry allowed identification of 40 proteins. Most of these are common to syndrome and healthy subjects and represent different types of protein. However, a few proteins, identified as truncated structural proteins (tubulin, actin), were present in part of the trisomy samples but absent from the controls. This is interpreted to indicate increased proteolysis in the syndrome samples but could also reflect some altered expression or processing. Independent of the apparently increased proteolysis in the syndrome samples, and in spite of the use of total brain tissues, the results show that two-dimensional protein separation patterns are largely similar between the syndrome and control samples upon silver-staining, but that differences associated with structural components can be detected and identified.

Structure-function relationships in Drosophila melanogaster alcohol dehydrogenase allozymes ADH(S), ADH(F) and ADH(UF), and distantly related forms.

Drosophila melanogaster alcohol dehydrogenase (ADH), a paradigm for gene-enzyme molecular evolution and natural selection studies, presents three main alleloforms (ADHS, ADHF and ADHUF) differing by one or two substitutions that render different biochemical properties to the allelozymes. A three-dimensional molecular model of the three allozymes was built by homology modeling using as a template the available crystal structure of the orthologous D. lebanonensis ADH, which shares a sequence identity of 82.2%. Comparison between D. lebanonensis and D. melanogaster structures showed that there is almost no amino-acid change near the substrate or coenzyme binding sites and that the hydrophobic active site cavity is strictly conserved. Nevertheless, substitutions are not distributed at random in nonconstricted positions, or located in external loops, but they appear clustered mainly in secondary structure elements. From comparisons between D. melanogaster allozymes and with D. simulans, a very closely related species, a model based on changes in the electrostatic potential distribution is presented to explain their differential behavior. The depth of knowledge on Drosophila ADH genetics and kinetics, together with the recently obtained structural information, could provide a better understanding of the mechanisms underlying molecular evolution and population genetics.

Engineering outer-membrane proteins in Pseudomonas putida for enhanced heavy-metal bioadsorption.

Metallothioneins (MTs) are small, cysteine-rich proteins with a strong metal-binding capacity that are ubiquitous in the animal kingdom. Recombinant expression of MT fused to outer-membrane components of gram-negative bacteria may provide new methods to treat heavy-metal pollution in industrial sewage. In this work, we have engineered Pseudomonas putida, a per se highly robust microorganism able to grow in highly contaminated habitats in order to further increase its metal-chelating ability. We report the expression of a hybrid protein between mouse MT and the beta domain of the IgA protease of Neisseria in the outer membrane of Pseudomonas cells. The metal-binding capacity of such cells was increased three-fold. The autotranslocating capacity of the beta domain of the IgA protease of Neisseria, as well as the correct anchoring of the transported protein into the outer membrane, have been demonstrated for the first time in a member of the Pseudomonas genus.