Tandem B1 SINE retro-elements may provide a basis for natural antisense transcription in the Magi1 locus of the mouse (Mus musculus)

Transposable elements, which are DNA sequences that can move between different sites in genomes, comprise approximately 40% of the genome of mammals and are emerging as important contributors to biological diversity. Here we report a transcription unit lying within intron 1 of the murine Magi1 (membrane associated guanylate kinase inverted 1) gene that codes for a cell-cell junction scaffolding protein. The transcription unit, termed Magi1OS (Magi1 Opposite Strand), originates from a region with tandem B1 short interspersed nuclear elements (SINEs) and is an antisense gene to Magi1. Mag1OS transcription initiates in a proximal B1 element that shows only 4% divergence from the consensus sequence, indicating that it has been recently inserted into the mouse genome and could be replication competent. Moreover, a chimaeric transcript may result from intra-chromosomal interaction and trans-splicing of the Magi1 antisense transcript (Magi1OS) and Ghrl, which codes for the multifunctional peptide hormone ghrelin. These two genes are 20 megabases apart on chromosome 6 and are transcribed in opposite directions. We propose that the Magi1OS locus may serve as a useful model system to study exaptation and retrotransposition of B1 SINEs, as well as to examine the mechanisms of intra-chromosomal trans-splicing.

Secreted products and extracellular matrix from testicular peritubular myoid cells influence androgen-binding protein secretion by Sertoli cells in culture

Metabolic cooperation mediated by secreted factors between Sertoli cells and peritubular myoid cells has been well documented. We have confirmed that factors secreted by peritubular myoid cells modulate androgen-binding protein (ABP) secretion by Sertoli cells and shown further that this can also be achieved with peritubular myoid cell extracellular matrix (ECM). While peritubular myoid cell ECM potentiated the stimulatory effect of dibutyryl cyclic AMP on Sertoli cell ABP secretion, secreted factors did not, suggesting that the two components influence Sertoli cells through distinct mechanisms. We also tested other factors and other cell lines for effects on ABP production by Sertoli cells. The addition of human plasma fibronectin or conditioned medium from the basement membrane-producing Englebreth-Holm- Swarm sarcoma also stimulated ABP secretion by Sertoli cells. Cocultures of epithelial Sertoli cells with the cells of mesenchymal origin, such as testicular peritubular myoid cells, embryonic skin fibroblasts, and bladder smooth muscle cells, significantly stimulated ABP secretion by Sertoli cells, but co-culture with the epithelial-derived Martin-Darby canine kidney cell line had no effect on Sertoli cell-secreted ABP levels. Our data further define the epithelial-mesenchymal cell interaction that exists between Sertoli cells and peritubular myoid cells in the mammalian testis.

Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1

Two BRCA2-like sequences are present in the Arabidopsis genome. Both genes are expressed in flower buds and encode nearly identical proteins, which contain four BRC motifs. In a yeast two-hybrid assay, the Arabidopsis Brca2 proteins interact with Rad51 and Dmc1. RNAi constructs aimed at silencing the BRCA2 genes at meiosis triggered a reproducible sterility phenotype, which was associated with dramatic meiosis alterations. We obtained the same phenotype upon introduction of RNAi constructs aimed at silencing the RAD51 gene at meiosis in dmc1 mutant plants. The meiotic figures we observed strongly suggest that homologous recombination is highly disturbed in these meiotic cells, leaving aberrant recombination events to repair the meiotic double-strand breaks. The 'brca2' meiotic phenotype was eliminated in spo11 mutant plants. Our experiments point to an essential role of Brca2 at meiosis in Arabidopsis. We also propose a role for Rad51 in the dmc1 context.

Interaction between Arabidopsis Brca2 and its partners Rad51, Dmc1, and Dss1

The Arabidopsis (Arabidopsis thaliana) orthologs of Brca2, a protein whose mutations are involved in breast cancer in humans, were previously shown to be essential at meiosis. In an attempt to better understand the Brca2-interacting properties, we examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms). The two Brca2 and the two Dss1 isoforms are named AtBrca2(IV), AtBrca2(V), AtDss1(I), and AtDss1(V) after their chromosomal localization. We first show that both AtBrca2 proteins can interact with either AtRad51 or AtDmc1 in vitro, and that the N-terminal region of AtBrca2 is responsible for these interactions. More specifically, the BRC motifs (so called because iterated in the Brca2 protein) in Brca2 are involved in these interactions: BRC motif number 2 (BRC2) alone can interact with AtDmc1, whereas BRC motif number 4 (BRC4) recognizes AtRad51. The human Rad51 and Dmc1 proteins themselves can interact with either the complete (HsRad51) or a shorter version of AtBrca2 (HsRad51 or HsDmc1) that comprises all four BRC motifs. We also identified two Arabidopsis isoforms of Dss1, another known partner of Brca2 in other organisms. Although all four Brca2 and Dss1 proteins are much conserved, AtBrca2(IV) interacts with only one of these AtDss1 proteins, whereas AtBrca2(V) interacts with both of them. Finally, we show for the first time that an AtBrca2 protein could bind two different partners at the same time: AtRad51 and AtDss1(I), or AtDmc1 and AtDss1(I).

RAD51-associated Protein 1 (RAD51AP1) Interacts with the Meiotic Recombinase DMC1 through a Conserved Motif

Homologous recombination (HR) reactions mediated by the RAD51 recombinase are essential for DNA and replication fork repair, genome stability, and tumor suppression. RAD51-associated protein 1 (RAD51AP1) is an important HR factor that associates with and stimulates the recombinase activity of RAD51. We have recently shown that RAD51AP1 also partners with the meiotic recombinase DMC1, displaying isoform-specific interactions with DMC1. Here, we have characterized the DMC1 interaction site in RAD51AP1 by a series of truncations and point mutations to uncover a highly conserved WVPP motif critical for DMC1 interaction but dispensable for RAD51 association. This RAD51AP1 motif is reminiscent of the FVPP motif in the tumor suppressor protein BRCA2 that mediates DMC1 interaction. These results further implicate RAD51AP1 in meiotic HR via RAD51 and DMC1.

A variant of the breast cancer type 2 susceptibility protein (BRC) repeat is essential for the RECQL5 Helicase to interact with RAD51 Recombinase for genome stabilization

The BRC repeat is a structural motif in the tumor suppressor BRCA2 (breast cancer type 2 susceptibility protein), which promotes homologous recombination (HR) by regulating RAD51 recombinase activity. To date, the BRC repeat has not been observed in other proteins, so that its role in HR is inferred only in the context of BRCA2. Here, we identified a BRC repeat variant, named BRCv, in the RECQL5 helicase, which possesses anti-recombinase activity in vitro and suppresses HR and promotes cellular resistance to camptothecin-induced replication stress in vivo. RECQL5-BRCv interacted with RAD51 through two conserved motifs similar to those in the BRCA2-BRC repeat. Mutations of either motif compromised functions of RECQL5, including association with RAD51, inhibition of RAD51-mediated D-loop formation, suppression of sister chromatid exchange, and resistance to camptothecin-induced replication stress. Potential BRCvs were also found in other HR regulatory proteins, including Srs2 and Sgs1, which possess anti-recombinase activities similar to that of RECQL5. A point mutation in the predicted Srs2-BRCv disrupted the ability of the protein to bind RAD51 and to inhibit D-loop formation. Thus, BRC is a common RAD51 interaction module that can be utilized by different proteins to either promote HR, as in the case of BRCA2, or to suppress HR, as in RECQL5.

Identification of theta-class glutathione S-transferase in liver cytosol of the marmoset monkey

The presence of theta-class glutathione S-transferase (GST) in marmoset monkey liver cytosol was investigated. An anti-peptide antibody targeted against the C-terminus of rGSTT1 reacted with a single band in marmoset liver cytosol that corresponded to a molecular weight of 28 kDa. The intensity of the immunoreactive band was not affected by treatment of marmoset monkeys with 2,3,7,8-tetrachlorodibenzo-p-dioxin, phenobarbitone, rifampicin or clofibric acid. Similarly, activity towards methyl chloride (MC) was unaffected by these treatments. However, GST activity towards 1,2-epoxy3-(p- nitrophenoxy)-propane (EPNP) was increased in marmosets treated with phenobarbitone (2.6-fold) and rifampicin (2.6-fold), activity towards dichloromethane (DCM) was increased by 50% after treatment of marmosets with clofibric acid, and activity towards 1-chloro-2,4-dinitrobenzene (CDNB) was raised slightly (30-42% increases) after treatment with phenobarbitone, rifampicin or clofibric acid. Compared with humans, marmoset liver cytosol GST activity towards DCM was 18-fold higher, activity towards MC was 7 times higher and activity towards CDNB was 4 times higher. Further, EPNP activity was clearly detectable in marmoset liver cytosol samples, but was undetectable in human samples. Immunoreactive marmoset GST was partially purified by affinity chromatography using hexylglutathione-Sepharose and Orange A resin. The interaction of immunoreactive marmoset GST was similar to that found previously for rat and human GSTT1, suggesting that this protein is also a theta class GST. However, unlike rat GSTT1, the marmoset enzyme was not the major catalyst of EPNP conjugation. Instead, immunoreactivity was closely associated with activity towards MC. In conclusion, these results provide evidence for the presence of theta-class GST in the marmoset monkey orthologous to rGSTT1 and hGSTT1.

Novel approaches for SER spectroscopic analysis of protein cofactors

Biomimetic systems employed for biotechnological applications i.e. as biosensors or bio fuel cells, require initial formation of conducting support/protein complexes with controlled properties. The specific interaction of the protein with the support determines important qualities of the device such as electrical communication, long-term stability and catalytic efficiency. In this respect the system parameters have to be chosen in a way that high protein loading on the support is achieved while protein denaturation upon adsorption is prevented. The conditions on the surface have to be adjusted in such a way that the desired surface reaction of the protein i.e. electron transfer to either the electrode or a second redox partner, is still guaranteed. Hence the choice of support, its functionlisation as well as the right adjustment of solution parameters play a crucial role in the rational design of these support/protein constructs.

Mapping Hydrophobicity on the Protein Molecular Surface at Atom-Level Resolution

A precise representation of the spatial distribution of hydrophobicity, hydrophilicity and charges on the molecular surface of proteins is critical for the understanding of the interaction with small molecules and larger systems. The representation of hydrophobicity is rarely done at atom-level, as this property is generally assigned to residues. A new methodology for the derivation of atomic hydrophobicity from any amino acid-based hydrophobicity scale was used to derive 8 sets of atomic hydrophobicities, one of which was used to generate the molecular surfaces for 35 proteins with convex structures, 5 of which, i.e., lysozyme, ribonuclease, hemoglobin, albumin and IgG, have been analyzed in more detail. Sets of the molecular surfaces of the model proteins have been constructed using spherical probes with increasingly large radii, from 1.4 to 20 A˚, followed by the quantification of (i) the surface hydrophobicity; (ii) their respective molecular surface areas, i.e., total, hydrophilic and hydrophobic area; and (iii) their relative densities, i.e., divided by the total molecular area; or specific densities, i.e., divided by property-specific area. Compared with the amino acid-based formalism, the atom-level description reveals molecular surfaces which (i) present an approximately two times more hydrophilic areas; with (ii) less extended, but between 2 to 5 times more intense hydrophilic patches; and (iii) 3 to 20 times more extended hydrophobic areas. The hydrophobic areas are also approximately 2 times more hydrophobicity-intense. This, more pronounced "leopard skin"-like, design of the protein molecular surface has been confirmed by comparing the results for a restricted set of homologous proteins, i.e., hemoglobins diverging by only one residue (Trp37). These results suggest that the representation of hydrophobicity on the protein molecular surfaces at atom-level resolution, coupled with the probing of the molecular surface at different geometric resolutions, can capture processes that are otherwise obscured to the amino acid-based formalism.

The harnessing of peptide-monolith constructs for single step plasmid DNA purification

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10-8 M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide-monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach.

Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

Peptides constructed from α-helical subunits of the Lac repressor protein (LacI) were designed then tailored to achieve particular binding kinetics and dissociation constants for plasmid DNA purification and detection. Surface plasmon resonance was employed for quantification and characterization of the binding of double stranded Escherichia coli plasmid DNA (pUC19) via the lac operon (lacO) to "biomimics" of the DNA binding domain of LacI. Equilibrium dissociation constants (K D), association (k a), and dissociation rates (k d) for the interaction between a suite of peptide sequences and pUC19 were determined. K D values measured for the binding of pUC19 to the 47mer, 27mer, 16mer, and 14mer peptides were 8.8 ± 1.3 × 10 -10 M, 7.2 ± 0.6 × 10 -10 M, 4.5 ± 0.5 × 10 -8 M, and 6.2 ± 0.9 × 10 -6 M, respectively. These findings show that affinity peptides, composed of subunits from a naturally occurring operon-repressor interaction, can be designed to achieve binding characteristics suitable for affinity chromatography and biosensor devices.

Single step purification of plasmid DNA using peptide ligand affinity chromatography

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels. © 2008 Elsevier B.V. All rights reserved.

Large-scale interlaboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens-to-hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma, and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and 7 control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to sub-nanogram/mL sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and inter-laboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy isotope labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an inter-laboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality c`ontrol measures, enables sensitive, specific, reproducible and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Differential interaction between the C2B domain of synaptotagmin-I and the Drosophila stonedA and stonedB proteins

The stoned locus in Drosophila encodes two proteins StonedA (STNA) and StonedB (STNB), both of which have been suggested to act as adaptins in mediating synaptic vesicle recycling. A combination of immunological, genetic and biochemical studies have shown an interaction of STNA and STNB with the C2B domain of Synaptotagmin-I (SYT-1), an integral synaptic vesicle protein that mediates Ca2+-dependent exocytosis, as well as endocytosis. The C2B domain of SYT-1 contains an AP-2 binding site that controls the size of recycled vesicles, and a C-terminal tryptophan-containing motif that acts as an internalization signal. Investigation of SYT-1 mutations in Drosophila has shown that altering the Ca2+ binding region of the C2B domain, results in a reduction in the rate of vesicle recycling, implicating this region in SYT-I endocytosis. In this poster, we report the molecular dissection of the interactions between the STNA and STNB proteins and the C2B domain of SYT-1. Deletion of the AP-2 binding site decreased the binding of both STNA and STNB. However, C-terminal deletions of the C2B domain significantly increased STNB binding. In contrast, the same C-terminal deletions reduced the affinity of the C2B domain for STNA. The possible interactions of both STNB and STNA with the Ca2+ binding region of SYT-1 will be also investigated.

Letter: Brain natriuretic peptide is a potentially useful screening tool for the detection of cardiovascular disease in patients with rheumatoid arthritis

Patients with rheumatoid arthritis (RA) have a significantly higher risk of coronary heart disease, despite being less likely to report symptoms of angina, and are more likely to experience unrecognised myocardial infarction and sudden cardiac death than non-RA controls.1 Furthermore, left ventricular diastolic dysfunction has been described in up to 40% of patients with RA.2...