Characterization of mouse profilaggrin: Evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N-terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal anti-body raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

Copper transfer from the Cu(I) chaperone, CopZ, to the repressor, Zn(II)CopY: Metal coordination environments and protein interactions

Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(l) per CopZ and two copper(l) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(1)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(1)(2)CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(l)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper. from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(l) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange: a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.

Matching SOX: partner proteins and co-factors of the SOX family of transcriptional regulators

SOX transcription factors perform a remarkable variety of important roles in vertebrate development, either activating or repressing specific target genes through interaction with different partner proteins. Surprisingly, these interactions are often mediated by the conserved, DNA-binding HMG domain, raising questions as to how each factor's specificity is generated. We propose a model whereby non-HMG domains may influence partner protein selection and/or binding stability.

The AF-1 Domain of the Orphan Nuclear Receptor NOR-1 Mediates Trans-activation, Coactivator Recruitment, and Activation by the Purine Anti-metabolite 6-Mercaptopurine

NOR-1/NR4A3 is an orphan member of the nuclear hormone receptor superfamily. NOR-1 and its close relatives Nurr1 and Nur77 are members of the NR4A subgroup of nuclear receptors. Members of the NR4A subgroup are induced through multiple signal transduction pathways. They have been implicated in cell proliferation, differentiation, T-cell apoptosis, chondrosarcomas, neurological disorders, inflammation, and atherogenesis. However, the mechanism of transcriptional activation, coactivator recruitment, and agonist-mediated activation remain obscure. Hence, we examined the molecular basis of NOR-1-mediated activation. We observed that NOR-1 trans-activates gene expression in a cell- and target-specific manner; moreover, it operates in an activation function (AF)-1-dependent manner. The N-terminal AF-1 domain delimited to between amino acids 1 and 112, preferentially recruits the steroid receptor coactivator (SRC). Furthermore, SRC-2 modulates the activity of the AF-1 domain but not the C-terminal ligand binding domain (LBD). Homology modeling indicated that the NOR-1 LBD was substantially different from that of hRORbeta, a closely related AF-2-dependent receptor. In particular, the hydrophobic cleft characteristic of nuclear receptors was replaced with a very hydrophilic surface with a distinct topology. This observation may account for the inability of this nuclear receptor LBD to efficiently mediate cofactor recruitment and transcriptional activation. In contrast, the N-terminal AF-1 is necessary for cofactor recruitment and can independently conscript coactivators. Finally, we demonstrate that the purine anti-metabolite 6-mercaptopurine, a widely used antineoplastic and anti-inflammatory drug, activates NOR-1 in an AF-1-dependent manner. Additional 6-mercaptopurine analogs all efficiently activated NOR-1, suggesting that the signaling pathways that modulate proliferation via inhibition of de novo purine and/or nucleic acid biosynthesis are involved in the regulation NR4A activity. We hypothesize that the NR4A subgroup mediates the genotoxic stress response and suggest that this subgroup may function as sensors that respond to genotoxicity.

Analysis of a 17.9 kb region from Saccharomyces cerevisiae chromosome VII reveals the presence of eight open reading frames, including BRF1 (TFIIIB70) and GCN5 genes

We report the nucleotide sequence of a 17,893 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII. This fragment begins at 482 kb from the centromere. The sequence includes the BRF1 gene, encoding TFIIIB70, the 5' portion of the GCN5 gene, an open reading frame (ORF) previously identified as ORF MGA1, whose translation product shows similarity to heat-shock transcription factors and five new ORFs. Among these, YGR250 encodes a polypeptide that harbours a domain present in several polyA binding proteins. YGR245 is similar to a putative Schizosaccharomyces pombe gene, YGR248 shows significant similarity with three ORFs of S. cerevisiae situated on different chromosomes, while the remaining two ORFs, YGR247 and YGR251, do not show significant similarity to sequences present in databases.

Gold electrode modified by self-assembled monolayers of thiols to determine DNA sequences hybridization

The process of immobilization of biological molecules is one of the most important steps in the construction of a biosensor. In the case of DNA, the way it exposes its bases can result in electrochemical signals to acceptable levels. The use of self-assembled monolayer that allows a connection to the gold thiol group and DNA binding to an aldehydic ligand resulted in the possibility of determining DNA hybridization. Immobilized single strand of DNA (ssDNA) from calf thymus pre-formed from alkanethiol film was formed by incubating a solution of 2-aminoethanothiol (Cys) followed by glutaraldehyde (Glu). Cyclic voltammetry (CV) was used to characterize the self-assembled monolayer on the gold electrode and, also, to study the immobilization of ssDNA probe and hybridization with the complementary sequence (target ssDNA). The ssDNA probe presents a well-defined oxidation peak at +0.158 V. When the hybridization occurs, this peak disappears which confirms the efficacy of the annealing and the DNA double helix performing without the presence of electroactive indicators. The use of SAM resulted in a stable immobilization of the ssDNA probe, enabling the hybridization detection without labels. This study represents a promising approach for molecular biosensor with sensible and reproducible results.

Computational design with flexible backbone sampling for protein remodeling and scaffolding of complex binding sites

Dissertation presented to obtain the Doutoramento (Ph.D.) degree in Biochemistry at the Instituto de Tecnologia Qu mica e Biol ogica da Universidade Nova de Lisboa

On the track of β-lactam resistance: Studies on the regulation of methicillinresistance in Staphylococcus aureus

Dissertação para obtenção do Grau de Doutor em Biologia

Synthesis, characterization and applications of new schiff base fluorescent chemosensors for metal and DNA interactions: conventional and "green" approaches

Dissertação apresentada para a obtenção do Grau de Doutor em Química Sustentável, especialidade de Química-Física Inorgânica, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Pathfinding of corticothalamic axons relies on a rendezvous with thalamic projections.

Major outputs of the neocortex are conveyed by corticothalamic axons (CTAs), which form reciprocal connections with thalamocortical axons, and corticosubcerebral axons (CSAs) headed to more caudal parts of the nervous system. Previous findings establish that transcriptional programs define cortical neuron identity and suggest that CTAs and thalamic axons may guide each other, but the mechanisms governing CTA versus CSA pathfinding remain elusive. Here, we show that thalamocortical axons are required to guide pioneer CTAs away from a default CSA-like trajectory. This process relies on a hold in the progression of cortical axons, or waiting period, during which thalamic projections navigate toward cortical axons. At the molecular level, Sema3E/PlexinD1 signaling in pioneer cortical neurons mediates a "waiting signal" required to orchestrate the mandatory meeting with reciprocal thalamic axons. Our study reveals that temporal control of axonal progression contributes to spatial pathfinding of cortical projections and opens perspectives on brain wiring.

Visualisation of human rad52 protein and its complexes with hRad51 and DNA.

The human Rad52 protein stimulates joint molecule formation by hRad51, a homologue of Escherichia coli RecA protein. Electron microscopic analysis of hRad52 shows that it self-associates to form ring structures with a diameter of approximately 10 nm. Each ring contains a hole at its centre. hRad52 binds to single and double-stranded DNA. In the ssDNA-hRad52 complexes, hRad52 was distributed along the length of the DNA, which exhibited a characteristic "beads on a string" appearance. At higher concentrations of hRad52, "super-rings" (approximately 30 nm) were observed and the ssDNA was collapsed upon itself. In contrast, in dsDNA-hRad52 complexes, some regions of the DNA remained protein-free while others, containing hRad52, interacted to form large protein-DNA networks. Saturating concentrations of hRad51 displaced hRad52 from ssDNA, whereas dsDNA-Rad52 complexes (networks) were more resistant to hRad51 invasion and nucleoprotein filament formation. When Rad52-Rad51-DNA complexes were probed with gold-conjugated hRad52 antibodies, the presence of globular hRad52 structures within the Rad51 nucleoprotein filament was observed. These data provide the first direct visualisation of protein-DNA complexes formed by the human Rad51 and Rad52 recombination/repair proteins.

Sumoylated PPARalpha mediates sex-specific gene repression and protects the liver from estrogen-induced toxicity in mice.

As most metabolic studies are conducted in male animals, understanding the sex specificity of the underlying molecular pathways has been broadly neglected; for example, whether PPARs elicit sex-dependent responses has not been determined. Here we show that in mice, PPARalpha has broad female-dependent repressive actions on hepatic genes involved in steroid metabolism and immunity. In male mice, this effect was reproduced by the administration of a synthetic PPARalpha ligand. Using the steroid oxysterol 7alpha-hydroxylase cytochrome P4507b1 (Cyp7b1) gene as a model, we elucidated the molecular mechanism of this sex-specific PPARalpha-dependent repression. Initial sumoylation of the ligand-binding domain of PPARalpha triggered the interaction of PPARalpha with GA-binding protein alpha (GABPalpha) bound to the target Cyp7b1 promoter. Histone deacetylase and DNA and histone methylases were then recruited, and the adjacent Sp1-binding site and histones were methylated. These events resulted in loss of Sp1-stimulated expression and thus downregulation of Cyp7b1. Physiologically, this repression conferred on female mice protection against estrogen-induced intrahepatic cholestasis, the most common hepatic disease during pregnancy, suggesting a therapeutic target for prevention of this disease.

Structure of a multisubunit complex that promotes DNA branch migration.

The RuvA and RuvB proteins of Escherichia coli, which are induced in response to DNA damage, are important in the formation of heteroduplex DNA during genetic recombination and related recombinational repair processes. In vitro studies show that RuvA binds Holiday junctions and acts as a specificity factor that targets the RuvB ATPase, a hexameric ring protein, to the junction. Together, RuvA and RuvB promote branch migration, an ATP-dependent reaction that increases the length of the heteroduplex DNA. Electron microscopic visualization of RuvAB now provides a new insight into the mechanism of this process. We observe the formation of a tripartite protein complex in which RuvA binds the crossover and is sandwiched between two hexameric rings of RuvB. The Holliday junction within this complex adopts a square-planar structure. We propose a molecular model for branch migration, a unique feature of which is the role played by the two oppositely oriented RuvB ring motors.

Nuclear Factor I genomic binding associates with chromatin boundaries.

BACKGROUND: The Nuclear Factor I (NFI) family of DNA binding proteins (also called CCAAT box transcription factors or CTF) is involved in both DNA replication and gene expression regulation. Using chromatin immuno-precipitation and high throughput sequencing (ChIP-Seq), we performed a genome-wide mapping of NFI DNA binding sites in primary mouse embryonic fibroblasts. RESULTS: We found that in vivo and in vitro NFI DNA binding specificities are indistinguishable, as in vivo ChIP-Seq NFI binding sites matched predictions based on previously established position weight matrix models of its in vitro binding specificity. Combining ChIP-Seq with mRNA profiling data, we found that NFI preferentially associates with highly expressed genes that it up-regulates, while binding sites were under-represented at expressed but unregulated genes. Genomic binding also correlated with markers of transcribed genes such as histone modifications H3K4me3 and H3K36me3, even outside of annotated transcribed loci, implying NFI in the control of the deposition of these modifications. Positional correlation between + and - strand ChIP-Seq tags revealed that, in contrast to other transcription factors, NFI associates with a nucleosomal length of cleavage-resistant DNA, suggesting an interaction with positioned nucleosomes. In addition, NFI binding prominently occurred at boundaries displaying discontinuities in histone modifications specific of expressed and silent chromatin, such as loci submitted to parental allele-specific imprinted expression. CONCLUSIONS: Our data thus suggest that NFI nucleosomal interaction may contribute to the partitioning of distinct chromatin domains and to epigenetic gene expression regulation.NFI ChIP-Seq and input control DNA data were deposited at Gene Expression Omnibus (GEO) repository under accession number GSE15844. Gene expression microarray data for mouse embryonic fibroblasts are on GEO accession number GSE15871.

Nucleic acid-binding properties of the RRM-containing protein RDM1.

RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L119GF --> AAA mutation affects the mode of RDM1 binding to single-stranded DNA.