Two active-site tyrosyl residues of protein TrwC act sequentially at the origin of transfer during plasmid R388 conjugation

Protein TrwC is the relaxase-helicase responsible for the initiation and termination reactions of DNA processing during plasmid R388 conjugation. Site-directed mutagenesis was used to change to phenylalanine each of a set of four conserved tyrosyl residues in the sequence of the N-terminal relaxation domain of the protein. Simultaneous mutation of both Y18 and Y26 was required to abolish in vitro cleavage and strand-transfer reactions catalyzed by protein TrwC on oligonucleotides containing the nic site. Thus, both Y18 and Y26 could be involved independently in the formation of oligonucleotide-protein covalent complexes that constitute presumed intermediates of these reactions. This hypothesis was confirmed by the observation of Y18 and Y26-specific peptide-oligonucleotide adducts after protease digestion of TrwC and mutant derivatives. Finally mutation Y18F, but not mutation Y26F, abolished nic-cleavage of a supercoiled DNA containing the R388 origin of transfer (oriT). These data allowed the construction of a model for conjugative DNA processing in which Y18 specifically catalyzes the initial cleavage reaction, while Y26 is used for the second strand-transfer reaction, which terminates conjugation. The model suggests a control mechanism that can be effective at each conjugative replication cycle.     

Genetic and biochemical manipulations of the small ribosomal subunit from Thermus thermophilus HB8

Crystals of the small ribosomal subunit from Thermus thermophilus diffract to 3A and exhibit reasonable isomorphism and moderate resistance to irradiation. A 5A MIR map of this particle shows a similar shape to the part assigned to this particle within the cryo-EM reconstructions of the whole ribosome and contains regions interpretable either as RNA chains or as protein motifs. To assist phasing at higher resolution we introduced recombinant methods aimed at extensive selenation for MAD phasing. We are focusing on several ribosomal proteins that can be quantitatively detached by chemical means. These proteins can be modified and subsequently reconstituted into depleted ribosomal cores. They also can be used for binding heavy atoms, by incorporating chemically reactive binding sites, such as -SH groups, into them. In parallel we are co-crystallizing the ribosomal particles with tailor made ligands, such as antibiotics or cDNA to which heavy-atoms have been attached or diffuse the latter compounds into already formed crystals.     

Effects of phosphinotricin treatment on glutamine synthetase isoforms in Scots pine seedlings

The occurrence of GS isoenzymes has been investigated in Scots pine (Pinus sylvestris) seedlings. A transient increase of glutamine synthetase (GS, EC 6.3.1.2) activity was observed in the cotyledon whorl of plants treated with the herbicide phosphinotricin (PPT). The increase in GS activity was accompanied by a parallel accumulation of GS1 protein, which remained at high levels throughout the PPT treatment. Two-dimensional SDS-PAGE western analysis showed that pine extracts contained two GS1 polypeptides which differ in their corresponding isoelectric points. Analysis of crude extracts by ion-exchange chromatography led to the separation of two GS isoforms. The first peak (GS1-a) eluted from the columns at a low ionic strength (0.15-0.18 M KCl), whereas the second one (GS1-b) was detected at 0.5 M KCl. A detailed molecular study of both GS holoenzymes confirmed that their subunits were similar in size (about 41 kDa) but different in charge. All these data clearly demonstrate the presence of two GS1 forms in Scots pine cotyledons. Moreover, a comparison of isolated GS isoproteins with the recombinantly expressed Scots pine cytosolic subunit suggests that GS1-a corresponds to the previously characterized cDNA (pGSP114) whereas GS1-b is a minor GS isoenzyme with increased relative abundance in phosphinotricin treated plants.     

Some Like It Fat: Comparative Ultrastructure of the Embryo in Two Demosponges of the Genus Mycale (Order Poecilosclerida) from Antarctica and the Caribbean

During embryogenesis, organisms with lecithotrophic indirect development usually accumulate large quantities of energetic reserves in the form of yolk that are necessary for larval survival. Since all sponges have lecithotrophic development, yolk formation is an ineludible step of their embryogenesis. Sponge yolk platelets have a wide range of morphological forms, from entirely lipid or protein platelets to a combined platelet showing both lipids and proteins and even glycogen. So far, there are no comparative studies on the nature and content of yolk in congeneric species of sponges inhabiting contrasting environments, which could have putative effects on the larval adaptation to environmental conditions. Here, we have taken advantage of the worldwide distribution of the sponge genus Mycale, in order to compare the embryogenesis and yolk formation in two species inhabiting contrasting latitudinal areas: M. acerata from Antarctic waters and M. laevis from the Caribbean. We have compared their brooded embryos and larvae using scanning and transmission electron microscopy, and calculated their energetic signatures based on the nature of their yolk. While the general morphological feature of embryos and larvae of both species were very similar, the main difference resided in the yolk nature. The Antarctic species, M. acerata, showed exclusively lipid yolk, whereas the Caribbean species, M. laevis, showed combined platelets of lipids and proteins and less frequently protein yolk platelets. The larvae of M. acerata were estimated to possess a two-fold energetic signature compared to that of M. laevis, which may have important ecological implications for their survival and for maintaining large population densities in the cold waters of the Southern Ocean.     

Regulation of GSK3 isoforms by phosphatases PP1 and PP2A

Dephosphorylation of phospho GSK3 isoforms, from COS-7 cells, was determined in vitro and in cultured cells in the absence or the presence of okadaic acid and lithium. Our results indicate a preferential dephosphorylation of phospho GSK3α by PP2A phosphatase, whereas dephosphorylation of phospho GSK3β mainly takes place by PP1 phosphatase.     

High-yield expression and purification of the Hsp90-associated p23, FKBP52, HOP and SGTα proteins

Hsp90 is a ubiquitous molecular chaperone that plays a key role in the malignant development of hormone-dependent pathologies such as cancer. An important role for Hsp90 is to facilitate the stable binding of steroid hormones to their respective receptors enabling the ligand-based signal to be carried to the nucleus and ultimately resulting in the up-regulation of gene expression. Along with Hsp90, this dynamic and transient process also involves the recruitment of additional proteins and co-chaperones that add further stability to the mature receptor–chaperone complex. In the work presented here, we describe four new protocols for the bacterial over-expression and column chromatographic purification of the human p23, FKBP52, HOP and SGTα proteins. Each of these proteins plays a distinct role in the steroid hormone receptor regulatory cycle. Affinity, ion-exchange and size-exclusion techniques were used to produce target yields greater than 50 mg/L of cultured media, with each purified sample reaching near absolute sample homogeneity. These results reveal a reliable system for the production of p23, FKBP52, HOP and SGTα substrate proteins for use in the investigation of the Hsp90-associated protein interactions of the steroid hormone receptor cycle.     

Immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis

Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad‐active degradome, and the immunoproteome were obtained by using 2‐DE, 2‐D‐zymograms, 2‐D‐Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty‐nine silver‐stained spots were detected in the region of 200–21 kDa of parasite protease‐resistant extracts. A similar proteolytic pattern was observed in the 2‐D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4‐like, TvCP12, TvCPT, TvLEGU‐1, and another legumain‐like CP). The major reactive spots to T. vaginalis‐positive patient sera by 2‐D‐WB corresponded to four papain‐like (TvCP2, TvCP4, TvCP4‐like, TvCPT), and one legumain‐like (TvLEGU‐1) CPs. The genes of TvCP4, TvCPT, and TvLEGU‐1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture‐positive patient sera in 1‐D‐WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.     

Removal of the carboxy terminus of beta-tubulin subunit produces lateral annealing of microtubules with different orientations

1. Tubulin, lacking the carboxy terminus region of its beta subunit assembles into composite microtubule structures showing opposite polarity. 2. Since in these polymers, microtubules are laterally bound, this type of interaction could lead to the generation of microtubules with different polarities, as those found in some cellular types.