Biogeographic and Evolutionary Patterns of Trace Element Utilization in Marine Microbial World.

Trace elements are required by all organisms, which are key components of many enzymes catalyzing important biological reactions. Many trace element-dependent proteins have been characterized; however, little is known about their occurrence in microbial communities in diverse environments, especially the global marine ecosystem. Moreover, the relationships between trace element utilization and different types of environmental stressors are unclear. In this study, we used metagenomic data from the Global Ocean Sampling expedition project to identify the biogeographic distribution of genes encoding trace element-dependent proteins (for copper, molybdenum, cobalt, nickel, and selenium) in a variety of marine and non-marine aquatic samples. More than 56,000 metalloprotein and selenoprotein genes corresponding to nearly 100 families were predicted, becoming the largest dataset of marine metalloprotein and selenoprotein genes reported to date. In addition, samples with enriched or depleted metalloprotein/selenoprotein genes were identified, suggesting an active or inactive usage of these micronutrients in various sites. Further analysis of interactions among the elements showed significant correlations between some of them, especially those between nickel and selenium/copper. Finally, investigation of the relationships between environmental conditions and metalloprotein/selenoprotein families revealed that many environmental factors might contribute to the evolution of different metalloprotein and/or selenoprotein genes in the marine microbial world. Our data provide new insights into the utilization and biological roles of these trace elements in extant marine microbes, and might also be helpful for the understanding of how these organisms have adapted to their local environments.

OsmiR528 regulates rice-pollen intine formation by targeting an uclacyanin to influence flavonoid metabolism.

The intine, the inner layer of the pollen wall, is essential for the normal development and germination of pollen. However, the composition and developmental regulation of the intine in rice (Oryza sativa) remain largely unknown. Here, we identify a microRNA, OsmiR528, which regulates the formation of the pollen intine and thus male fertility in rice. The mir528 knockout mutant aborted pollen development at the late binucleate pollen stage, significantly decreasing the seed-setting rate. We further demonstrated that OsmiR528 affects pollen development by directly targeting the uclacyanin gene OsUCL23 (encoding a member of the plant-specific blue copper protein family of phytocyanins) and regulating intine deposition. OsUCL23 overexpression phenocopied the mir528 mutant. The OsUCL23 protein localized in the prevacuolar compartments (PVCs) and multivesicular bodies (MVBs). We further revealed that OsUCL23 interacts with a member of the proton-dependent oligopeptide transport (POT) family of transporters to regulate various metabolic components, especially flavonoids. We propose a model in which OsmiR528 regulates pollen intine formation by directly targeting OsUCL23 and in which OsUCL23 interacts with the POT protein on the PVCs and MVBs to regulate the production of metabolites during pollen development. The study thus reveals the functions of OsmiR528 and an uclacyanin during pollen development.

Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology: Enzymatic Cascades and Directed Evolution.

Artificial metalloenzymes (ArMs) result from anchoring a metal-containing moiety within a macromolecular scaffold (protein or oligonucleotide). The resulting hybrid catalyst combines attractive features of both homogeneous catalysts and enzymes. This strategy includes the possibility of optimizing the reaction by both chemical (catalyst design) and genetic means leading to achievement of a novel degree of (enantio)selectivity, broadening of the substrate scope, or increased activity, among others. In the past 20 years, the Ward group has exploited, among others, the biotin-(strept)avidin technology to localize a catalytic moiety within a well-defined protein environment. Streptavidin has proven versatile for the implementation of ArMs as it offers the following features: (i) it is an extremely robust protein scaffold, amenable to extensive genetic manipulation and mishandling, (ii) it can be expressed in E. coli to very high titers (up to >8 g·L-1 in fed-batch cultures), and (iii) the cavity surrounding the biotinylated cofactor is commensurate with the size of a typical metal-catalyzed transition state. Relying on a chemogenetic optimization strategy, varying the orientation and the nature of the biotinylated cofactor within genetically engineered streptavidin, 12 reactions have been reported by the Ward group thus far. Recent efforts within our group have focused on extending the ArM technology to create complex systems for integration into biological cascade reactions and in vivo. With the long-term goal of complementing in vivo natural enzymes with ArMs, we summarize herein three complementary research lines: (i) With the aim of mimicking complex cross-regulation mechanisms prevalent in metabolism, we have engineered enzyme cascades, including cross-regulated reactions, that rely on ArMs. These efforts highlight the remarkable (bio)compatibility and complementarity of ArMs with natural enzymes. (ii) Additionally, multiple-turnover catalysis in the cytoplasm of aerobic organisms was achieved with ArMs that are compatible with a glutathione-rich environment. This feat is demonstrated in HEK-293T cells that are engineered with a gene switch that is upregulated by an ArM equipped with a cell-penetrating module. (iii) Finally, ArMs offer the fascinating prospect of "endowing organometallic chemistry with a genetic memory." With this goal in mind, we have identified E. coli's periplasmic space and surface display to compartmentalize an ArM, while maintaining the critical phenotype-genotype linkage. This strategy offers a straightforward means to optimize by directed evolution the catalytic performance of ArMs. Five reactions have been optimized following these compartmentalization strategies: ruthenium-catalyzed olefin metathesis, ruthenium-catalyzed deallylation, iridium-catalyzed transfer hydrogenation, dirhodium-catalyzed cyclopropanation and carbene insertion in C-H bonds. Importantly, >100 turnovers were achieved with ArMs in E. coli whole cells, highlighting the multiple turnover catalytic nature of these systems.

Evolutionary relationship between the cysteine and histidine rich domains (CHORDs) and Btk-type zinc fingers.

Summary: Cysteine and histidine rich domains (CHORDs), implicated in immunity and disease resistance signaling in plants, and in development and signal transduction in muscles and tumorigenesis in animals, are seen to have a cylindrical three-dimensional structure stabilized by the tetrahedral chelation of two zinc ions. CHORDs are regarded as novel zinc-binding domains and classified independently in Pfam and ECOD. Our sequence and structure analysis reveals that both the zinc-binding sites in CHORD possess a zinc ribbon fold and are likely related to each other by duplication and circular permutation. Interestingly, we also detect an evolutionary relationship between each of the CHORD zinc fingers (ZFs) and the Bruton's tyrosine kinase (Btk)-type ZF of the zinc ribbon fold group. Btk_ZF is found in eukaryotic Tec kinase family proteins that are also implicated in signaling pathways in several lineages of hematopoietic cells involved in mammalian immunity. Our analysis suggests that the unique zinc-stabilized fold seen only in the CHORD and Btk_ZFs likely emerged specifically in eukaryotes to mediate diverse signaling pathways. Supplementary information: Supplementary data are available at Bioinformatics online.

Structural Determinants for Substrate Binding and Catalysis in Triphosphate Tunnel Metalloenzymes.

Triphosphate tunnel metalloenzymes (TTMs) are present in all kingdoms of life and catalyze diverse enzymatic reactions such as mRNA capping, the cyclization of adenosine triphosphate, the hydrolysis of thiamine triphosphate, and the synthesis and breakdown of inorganic polyphosphates. TTMs have an unusual tunnel domain fold that harbors substrate- and metal co-factor binding sites. It is presently poorly understood how TTMs specifically sense different triphosphate-containing substrates and how catalysis occurs in the tunnel center. Here we describe substrate-bound structures of inorganic polyphosphatases from Arabidopsis and Escherichia coli, which reveal an unorthodox yet conserved mode of triphosphate and metal co-factor binding. We identify two metal binding sites in these enzymes, with one co-factor involved in substrate coordination and the other in catalysis. Structural comparisons with a substrate- and product-bound mammalian thiamine triphosphatase and with previously reported structures of mRNA capping enzymes, adenylate cyclases, and polyphosphate polymerases suggest that directionality of substrate binding defines TTM catalytic activity. Our work provides insight into the evolution and functional diversification of an ancient enzyme family.

Cloning and functional validation of molybdenum cofactor sulfurase gene from Ammopiptanthus nanus.

KEY MESSAGE: The molybdenum cofactor sulfurase gene ( AnMCSU ) was cloned from xerophytic desert plant Ammopiptanthus nanus and validated for its function of tolerance toward abiotic stresses by heterologous expression in Arabidopsis thaliana. Molybdenum cofactor sulfurase participates in catalyzing biosynthesis of abscisic acid, which plays a crucial role in the response of plants to abiotic stresses. In this study, we cloned molybdenum cofactor sulfurase gene (AnMCSU) from a super-xerophytic desert plant, Ammopiptanthus nanus, by using rapid amplification of cDNA ends method. This gene has a total length of 2544 bp, with a 5'- and a 3'-untranslated region of 167 and 88 bp, and an open reading frame of 2289 bp, which encodes an 84.85 kDa protein of 762 amino acids. The putative amino acid sequence shares high homology and conserved amino acid residues crucial for the function of molybdenum cofactor sulfurases with other leguminous species. The encoded protein of the AnMCSU gene was located in the cytoplasm by transient expression in Nicotiana benthamiana. The result of real-time quantitative PCR showed that the expression of the AnMCSU gene was induced by heat, dehydration, high salt stresses, and ABA induction, and inhibited by cold stress. The heterologous expression of the AnMCSU gene significantly enhanced the tolerance of Arabidopsis thaliana to high salt, cold, osmotic stresses, and abscisic acid induction. All these results suggest that the AnMCSU gene might play a crucial role in the adaptation of A. nanus to abiotic stress and has potential to be applied to transgenic improvement of commercial crops.

Molecular cloning, sequencing, and expression analysis of cDNA encoding metalloprotein II (MP II) induced by single and combined metals (Cu(II), Cd(II)) in polychaeta Perinereis aibuhitensis.

We amplified and analyzed the complete cDNA of metalloprotein II (MP II) from the somatic muscle of the polychaete Perinereis aibuhitensis, the full length cDNA is 904 bp encoding 119 amino acids. The MP II cDNA sequence was subjected to BLAST searching in NCBI and was found to share high homology with hemerythrin of other worms. MP II expression of P. aibuhitensis exposed to single and combined metals (Cu(II), Cd(II)) was analyzed using real time-PCR. MP II mRNA expression increased at the start of Cu(II) exposure, then decreased and finally return to the normal level. Expression pattern of MP II under Cd(II) exposure was time- and dose-dependent. MP II expression induced by a combination of Cd(II) and Cu(II) was similar to that induced by Cd(II) alone.

An unconventional copper protein required for cytochrome c oxidase respiratory function under extreme acidic conditions.

Very little is known about the processes used by acidophile organisms to preserve stability and function of respiratory pathways. Here, we reveal a potential strategy of these organisms for protecting and keeping functional key enzymes under extreme conditions. Using Acidithiobacillus ferrooxidans, we have identified a protein belonging to a new cupredoxin subfamily, AcoP, for "acidophile CcO partner," which is required for the cytochrome c oxidase (CcO) function. We show that it is a multifunctional copper protein with at least two roles as follows: (i) as a chaperone-like protein involved in the protection of the Cu(A) center of the CcO complex and (ii) as a linker between the periplasmic cytochrome c and the inner membrane cytochrome c oxidase. It could represent an interesting model for investigating the multifunctionality of proteins known to be crucial in pathways of energy metabolism.

Characterization of a mutant of Chlamydomonas reinhardtii deficient in the molybdenum cofactor.

Molybdenum (Mo) is an essential micronutrient for almost all organisms. In eukaryotes, it forms a part of the molybdenum cofactor (Moco), which is required for numerous enzymes involved in carbon, nitrogen and sulfur metabolism. Mo is taken up by cells in the form of molybdate and recently molybdate transporters have been identified in Arabidopsis thaliana and Chlamydomonas reinhardtii. Here, we report the characterization of a novel mutant (DB6) of C. reinhardtii generated by random insertional mutagenesis that is unable to assimilate nitrate as a nitrogen source because it lacks functional nitrate reductase (NR). Besides lacking NR, DB6 also lacks xanthine dehydrogenase activity; a common requirement of both enzymes is Moco. DB6 displays a 'molybdate-repairable' phenotype--growth on nitrate is partially restored by supplementing media with high levels of molybdate. This phenotype is typically associated with mutants defective in either molybdate transport or insertion of Mo into the pterin precursor of Moco. Mo content was found to be significantly lower in DB6 than in the wild-type strain, AOXR1, which suggests that DB6 is defective in Mo uptake. Genetic complementation with a variety of candidate genes that include the known molybdate transporter MOT1 and DNA that spans the site of mutation was unable to recover the wild-type phenotype. Taken together, our results indicate that DB6 is a novel molybdate transport-deficient mutant.

A histidine-rich and cysteine-rich metal-binding domain at the C terminus of heat shock protein A from Helicobacter pylori: implication for nickel homeostasis and bismuth susceptibility.

HspA, a member of the GroES chaperonin family, is a small protein found in Helicobacter pylori with a unique histidine- and cysteine-rich domain at the C terminus. In this work, we overexpressed, purified, and characterized this protein both in vitro and in vivo. The apo form of the protein binds 2.10 +/- 0.07 Ni(2+) or 1.98 +/- 0.08 Bi(3+) ions/monomer with a dissociation constant (K(d)) of 1.1 or 5.9 x 10(-19) microm, respectively. Importantly, Ni(2+) can reversibly bind to the protein, as the bound nickel can be released either in the presence of a chelating ligand, e.g. EDTA, or at an acidic pH (pH((1/2)) 3.8 +/- 0.2). In contrast, Bi(3+) binds almost irreversibly to the protein. Both gel filtration chromatography and native electrophoresis demonstrated that apo-HspA exists as a heptamer in solution. Unexpectedly, binding of Bi(3+) to the protein altered its quaternary structure from a heptamer to a dimer, indicating that bismuth may interfere with the biological functions of HspA. When cultured in Ni(2+)-supplemented M9 minimal medium, Escherichia coli BL21(DE3) cells expressing wild-type HspA or the C-terminal deletion mutant clearly indicated that the C terminus might protect cells from high concentrations of external Ni(2+). However, an opposite phenomenon was observed when the same E. coli hosts were grown in Bi(3+)-supplemented medium. HspA may therefore play a dual role: to facilitate nickel acquisition by donating Ni(2+) to appropriate proteins in a nickel-deficient environment and to carry out detoxification via sequestration of excess nickel. Meanwhile, HspA can be a potential target of the bismuth antiulcer drug against H. pylori.

Long-term rescue of a lethal inherited disease by adeno-associated virus-mediated gene transfer in a mouse model of molybdenum-cofactor deficiency.

Molybdenum cofactor (MoCo) deficiency is a progressive neurological disorder that inevitably leads to early childhood death because of the lack of any effective therapy. In a mouse model of MoCo deficiency type A, the most frequent form of this autosomal recessively inherited disease, the affected animals show the biochemical characteristics of sulphite and xanthine intoxication and do not survive >2 wk after birth. We have constructed a recombinant-expression cassette for the gene MOCS1, which, via alternative splicing, facilitates the expression of the proteins MOCS1A and MOCS1B, both of which are necessary for the formation of a first intermediate, cyclic pyranopterin monophosphate (cPMP), within the biosynthetic pathway leading to active MoCo. A recombinant adeno-associated virus (AAV) vector was used to express the artificial MOCS1 minigene, in an attempt to cure the lethal MOCS1-deficient phenotype. The vector was used to transduce Mocs1-deficient mice at both 1 and 4 d after birth or, after a pretreatment with purified cPMP, at 40 d after birth. We report here that all Mocs1-deficient animals injected with a control AAV-enhanced green fluorescent protein vector died approximately 8 d after birth or after withdrawal of cPMP supplementation, whereas AAV-MOCS1-transduced animals show significantly increased longevity. A single intrahepatic injection of AAV-MOCS1 resulted in fertile adult animals without any pathological phenotypes.

Porcine purple acid phosphatase: heterologous expression, characterization, and proteolytic analysis.

Uteroferrin is an iron-binding glycoprotein, which is abundantly synthesized in porcine uterine glandular endometrium and believed to be involved in maternal/fetal iron transport. In the present study, uteroferrin has been cloned and functionally expressed using baculovirus-infected insect host cells Spodoptera frugiperda. The work also addresses the possible role of proteolytic cleavage to facilitate the release of uteroferrin-bound iron. The enzyme secreted in culture medium exhibits a molecular mass and catalytic properties similar to native porcine uteroferrin. The specific activity was estimated at 233 U/mg using p-nitrophenyl phosphate as substrate. Partial cleavage of the enzyme with trypsin resulted in a 1.7-fold enhancement in specific activity and a two-subunit polypeptide as observed in preparations of most mammalian purple acid phosphatases. Digestion with the aspartic protease pepsin resulted in a 2.5-fold enzyme inactivation correlated with the appearance of low molecular weight polypeptide fragments and the release of enzyme-bound iron.

Solution structures of a cyanobacterial metallochaperone: insight into an atypical copper-binding motif.

The Atx1 copper metallochaperone from Synechocystis PCC 6803, ScAtx1, interacts with two P(1)-type copper ATPases to supply copper proteins within intracellular compartments, avoiding ATPases for other metals en route. Here we report NMR-derived solution structures for ScAtx1. The monomeric apo form has a betaalphabetabetaalpha fold with backbone motions largely restricted to loop 1 containing Cys-12 and Cys-15. The tumbling rate of Cu(I)ScAtx1 (0.1-0.8 mm) implies dimers. Experimental restraints are satisfied by symmetrical dimers with Cys-12 or His-61, but not Cys-15, invading the copper site of the opposing subunit. A full sequence of copper ligands from the cell surface to thylakoid compartments is proposed, considering in vitro homodimer liganding to mimic in vivo liganding in ScAtx1-ATPase heterodimers. A monomeric high resolution structure for Cu(I)ScAtx1, with Cys-12, Cys-15, and His-61 as ligands, is calculated without violations despite the rotational correlation time. (2)J(NH) couplings in the imidazole ring of His-61 establish coordination of N(epsilon2) to copper. His-61 is analogous to Lys-65 in eukaryotic metallochaperones, stabilizing Cu(I)S(2) complexes but by binding Cu(I) rather than compensating charge. Cys-Cys-His ligand sets are an emergent theme in some copper metallochaperones, although not in related Atx1, CopZ, or Hah1. Surface charge (Glu-13) close to the metal-binding site of ScAtx1 is likely to support interaction with complementary surfaces of copper-transporting ATPases (PacS-Arg-11 and CtaA-Lys-14) but to discourage interaction with zinc ATPase ZiaA and so inhibit aberrant formation of copper-ZiaA complexes.

Combination of all-trans retinoic acid and lithium chloride surmounts a retinoid differentiation block induced by expression of Scl and Rbtn2 transcription factors in myeloid leukemia cells.

We have previously shown that forced expression of the transcription factor Scl in WEHI-3B D(+) cells prevents ATRA-induced cell differentiation. We now find that the overexpression of Rbtn2 also interferes with induction of differentiation by ATRA. Addition of LiCl to ATRA treatment restored the capacity of both Scl- and Rbtn2-expressing cells to respond to the retinoid in a synergistic manner. Similar results were obtained with Scl-transfected HL60 cells where its expression diminished responsiveness to ATRA. These findings suggest that if Scl and/or Rbtn2 are involved in the non-responsiveness of AML patients to ATRA-induced differentiation, addition of LiCl may reverse insensitivity.

A nickel-cobalt-sensing ArsR-SmtB family repressor. Contributions of cytosol and effector binding sites to metal selectivity.

NmtR from Mycobacterium tuberculosis is a new member of the ArsR-SmtB family of metal sensor transcriptional repressors. NmtR binds to the operator-promoter of a gene encoding a P(1) type ATPase (NmtA), repressing transcription in vivo except in medium supplemented with nickel or, to some extent, cobalt. In a cyanobacterial host, Synechococcus PCC 7942 strain R2-PIM8(smt), NmtR-mediated repression is alleviated by cobalt but not nickel or zinc addition, while the related sensor SmtB responds exclusively to zinc. Quantification of the number of atoms of nickel per cell shows that NmtR nickel sensitivity correlates with cytosolic nickel contents. Differential metal discrimination in a common cytosol by SmtB (zinc) and NmtR (cobalt) is not simply explained by affinities at equilibrium; although NmtR does bind nickel substantially more tightly than SmtB, it has a higher affinity for zinc than for cobalt and binds cobalt more weakly than SmtB. SmtB is known to bind and sense zinc at interhelical four-coordinate, tetrahedral sites across the C-terminal alpha 5 helices, while absorption spectroscopy of Co(II)- and Ni(II)-substituted NmtR reveals five- and six-coordinate metal complexes. Site-directed mutagenesis identifies six potential cobalt/nickel ligands that are obligatory for inducer recognition but not repression by NmtR, four of which (Asp(91), His(93), His(104), His(107)) align with alpha 5 ligands of SmtB with two additional His provided by a carboxyl-terminal "extension" (designated alpha 5C). Gel retardation assays reveal that zinc does not allosterically regulate NmtR-DNA binding at concentrations where lower affinity cobalt does. These data suggest that two additional ligands form hexacoordinate metal complexes and are crucial for driving allosteric regulation of DNA binding by NmtR, thereby allowing NmtR to preferentially sense metals that favor higher coordination numbers relative to SmtB.

Identification of metallopanstimulin-1 as a member of a tumor associated antigen in patients with breast cancer.

The immunological screening of breast cancer was performed with IgG autoantibodies by the serological analysis of recombinant cDNA expression library methods to explore novel tumor associated antigens. We have focused on a small zinc finger protein metallopanstimulin-1 (MPS-1). MPS-1 mRNA was ubiquitously expressed in normal human tissues except the brain and the placenta. In Western blot analysis, MPS-1 was easily detected strongly in actively proliferating cells and three breast cancer cell lines. In the tissue the protein of MPS-1 in cancer cells was more abundant than that of surrounding normal cells. Screening of tissue specimens by immunohistochemistry revealed 50.4% positive for MPS-1 in 125 cancer patients. These data suggest that MPS-1 could be applicable to the immunotherapy of breast cancer.

Fighting iron deficiency anemia with iron-rich rice.

OBJECTIVE: Iron deficiency is estimated to affect about 30% of the world population. Iron supplementation in the form of tablets and food fortification has not been successful in developing countries, and iron deficiency is still the most important deficiency related to malnutrition. Here we present experiments that aim to increase the iron content in rice endosperm and to improve its absorption in the human intestine by means of genetic engineering. METHODS: We first introduced a ferritin gene from Phaseolus vulgaris into rice grains, increasing their iron content up to twofold. To increase iron bioavailability, we introduced a thermo-tolerant phytase from Aspergillus fumigatus into the rice endosperm. In addition, as cysteine peptides are considered major enhancers of iron absorption, we over-expressed the endogenous cysteine-rich metallothionein-like protein. RESULTS: The content of cysteine residues increased about sevenfold and the phytase level in the grains about one hundred and thirtyfold, giving a phytase activity sufficient to completely degrade phytic acid in a simulated digestion experiment. CONCLUSIONS: This rice, with higher iron content, rich in phytase and cysteine-peptide has a great potential to substantially improve iron nutrition in those populations where iron deficiency is so widely spread.

Characterization of the subunit structure of the catalytically active type I iodothyronine deiodinase.

Type I iodothyronine deiodinase is a approximately 50-kDa, integral membrane protein that catalyzes the outer ring deiodination of thyroxine. Despite the identification and cloning of a 27-kDa selenoprotein with the catalytic properties of the type I enzyme, the composition and the physical nature of the active deiodinase are unknown. In this report, we use a molecular approach to determine holoenzyme composition, the role of the membrane anchor on enzyme assembly, and the contribution of individual 27-kDa subunits to catalysis. Overexpression of an immunologically unique rat 27-kDa protein in LLC-PK1 cells that contain abundant catalytically active 27-kDa selenoprotein decreased deiodination by approximately 50%, and > 95% of the LLC-PK1 derived 27-kDa selenoprotein was specifically immune precipitated by the anti-rat enzyme antibody. The hybrid enzyme had a molecular mass of 54 kDa and an s(20,w) of approximately 3.5 S indicating that every native 27-kDa selenoprotein partnered with an inert rat 27-kDa subunit in a homodimer. Enzyme assembly did not depend on the presence of the N-terminal membrane anchor of the 27-kDa subunit. Direct visualization of the deiodinase dimer showed that the holoenzyme was sorted to the basolateral plasma membrane of the renal epithelial cell.

Expression and regulatory function of the transcription factor Sp1 in the uterine endometrium at early pregnancy: implications for epithelial phenotype.

The uterus during early pregnancy synthesizes a complex array of signaling molecules with specific spatial and temporal modes of expression and which are critical for embryo implantation and subsequent development. The mechanism(s) underlying the differential pattern of synthesis of these pregnancy-associated proteins is not understood very well. The present study evaluated the expression and trans-activation potential of the transcription factor Sp1 in the early pregnancy porcine endometrium to determine its temporal and functional association with the endometrial epithelial-specific genes encoding the transplacental iron-transport protein uteroferrin (UF) and an Sp-family member, basic transcription element-binding (BTEB) protein. Two identical Sp1 clones (717 bp) were isolated from a porcine endometrial cDNA library by polymerase chain reaction (PCR). The nucleotide sequence of these clones encodes a partial protein sequence of 238 amino acids encompassing the Zn-finger region and had significant identities with the corresponding regions in the rat and human proteins. By using a specific antibody raised against human Sp1, porcine endometrial Sp1 was found to exhibit a molecular weight of 110 kDa, was localized predominantly in the nuclei of glandular and luminal epithelial cells, and appeared to exist as a phosphorylated protein. Northern blot analysis demonstrated three distinct size transcripts of approximately 3.5, 5, and 8 kb for endometrial Sp1. The expression of Sp1 mRNA and protein, determined by RT-PCR and by its ability to bind Sp1 consensus motif in gel mobility shift assays, respectively, overlapped with, but did not parallel that of UF mRNA during early pregnancy. The effect of increased Sp1 expression on UF gene promoter activity was examined using a human Sp1 expression vector that was transiently transfected into primary cultures of pig endometrial glandular epithelial cells. Sp1 increased (P < 0.05) the promoter activities of various UF promoter-Luciferase reporter constructs by 2 to 4-fold, over those transfected with empty expression vector. Co-transfection of a BTEB expression vector with the Sp1 expression vector modified the effect of Sp1 on UF promoter activity in the shortest construct. These results suggest that Sp1 mediates the regulation of endometrial epithelial gene expression during pregnancy, and that this function is likely altered in vivo by co-expression of other family members, including BTEB.

Patología molecular de los gliomas / Molecular biology of gliomas

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