The non-autonomous retrotransposon SVA is trans-mobilized by the human LINE-1 protein machinery

SINE-VNTR-Alu (SVA) elements are non-autonomous, hominid-specific non-LTR retrotransposons and distinguished by their organization as composite mobile elements. They represent the evolutionarily youngest, currently active family of human non-LTR retrotransposons, and sporadically generate disease-causing insertions. Since preexisting, genomic SVA sequences are characterized by structural hallmarks of Long Interspersed Elements 1 (LINE-1, L1)-mediated retrotransposition, it has been hypothesized for several years that SVA elements are mobilized by the L1 protein machinery in trans. To test this hypothesis, we developed an SVA retrotransposition reporter assay in cell culture using three different human-specific SVA reporter elements. We demonstrate that SVA elements are mobilized in HeLa cells only in the presence of both L1-encoded proteins, ORF1p and ORF2p. SVA trans-mobilization rates exceeded pseudogene formation frequencies by 12- to 300-fold in HeLa-HA cells, indicating that SVA elements represent a preferred substrate for L1 proteins. Acquisition of an AluSp element increased the trans-mobilization frequency of the SVA reporter element by ~25-fold. Deletion of (CCCTCT)(n) repeats and Alu-like region of a canonical SVA reporter element caused significant attenuation of the SVA trans-mobilization rate. SVA de novo insertions were predominantly full-length, occurred preferentially in G+C-rich regions, and displayed all features of L1-mediated retrotransposition which are also observed in preexisting genomic SVA insertions.     

A highly specialized flavin mononucleotide riboswitch responds differently to similar ligands and confers roseoflavin resistance to Streptomyces davawensis

Streptomyces davawensis is the only organism known to synthesize the antibiotic roseoflavin, a riboflavin (vitamin B(2)) analog. Roseoflavin is converted to roseoflavin mononucleotide (RoFMN) and roseoflavin adenine dinucleotide in the cytoplasm of target cells. (Ribo-)Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for the biosynthesis and transport of riboflavin. Streptomyces davawensis is roseoflavin resistant, and the closely related bacterium Streptomyces coelicolor is roseoflavin sensitive. The two bacteria served as models to investigate roseoflavin resistance of S. davawensis and to analyze the mode of action of roseoflavin in S. coelicolor. Our experiments demonstrate that the ribB FMN riboswitch of S. davawensis (in contrast to the corresponding riboswitch of S. coelicolor) is able to discriminate between the two very similar flavins FMN and RoFMN and shows opposite responses to the latter ligands.     

Rate and irregularity of electrical activation during atrial fibrillation affect myocardial NGF expression via different signalling routes

An irregular ventricular response during atrial fibrillation (AF) has been shown to mediate an increase in sympathetic nerve activity in human subjects. The molecular mechanisms remain unclear. This study aimed to investigate the impact of rate and irregularity on nerve growth factor (NGF) expression in cardiomyocytes, since NGF is known to be the main contributor to cardiac sympathetic innervation density. Cell cultures of neonatal rat ventricular myocytes were electrically stimulated for 48 h with increasing rates (0, 5 and 50 Hz) and irregularity (standard deviation (SD) = 5%, 25% and 50% of mean cycle length). Furthermore, we analyzed the calcineurin-NFAT and the endothelin-1 signalling pathways as possible contributors to NGF regulation during arrhythmic stimulation. We found that the increase of NGF expression reached its maximum at the irregularity of 25% SD by 5 Hz (NGF: 5 Hz 0% SD = 1 vs. 5 Hz 25% SD = 1.57, P < 0.05). Specific blockade of the ET-A receptor by BQ123 could abolish this NGF increase (NGF: 5 Hz 25% SD + BQ123 = 0.66, P < 0.05). High frequency electrical field stimulation (HFES) with 50 Hz decreased the NGF expression in a significant manner (NGF: 50 Hz = 0.55, P < 0.05). Inhibition of calcineurin-NFAT signalling with cyclosporine-A or 11R-VIVIT abolished the HFES induced NGF down-regulation (NGF: 50 Hz + CsA = 1.14, P < 0.05). In summary, this study reveals different signalling routes of NGF expression in cardiomyocytes exposed to increasing rates and irregularity. Whether this translates into different degrees of NGF expression and possibly neural sympathetic growth in various forms of ventricular rate control during AF remains to be elucidated in further studies.     

The transport of wear particles in the prosthetic hip joint: a computational fluid dynamics investigation

The joint fluid mechanics and transport of wear particles in the prosthetic hip joint were analyzed for subluxation and flexion motion using computational fluid dynamics (CFD). The entire joint space including a moving capsule boundary was considered. It was found that particles suspended in the joint space are drawn into the joint gap between prosthesis cup and head during subluxation, which was also documented by Lundberg et al. (2007; Journal of Biomechanics 40, 1676-1685), however, wear particles remain in the joint gap. Wear particles leave the joint gap during flexion and can finally migrate to the proximal boundaries including the acetabular bone, where the particle deposition can cause osteolysis according to the established literature. Thus, the present study supports the theory of polyethylene wear particle induced osteolysis of the acetabular bone as a major factor in the loosening of hip prosthesis cups.     

Properties of photosystem I antenna protein complexes of the diatom Cyclotella meneghiniana

Analysis of photosystem I (PSI) complexes from Cyclotella meneghiniana cultured under different growth conditions led to the identification of three groups of antenna proteins, having molecular weights of around 19, 18, and 17 kDa. The 19-kDa proteins have earlier been demonstrated to be more peripherally bound to PSI, and their amount in the PSI complexes was significantly reduced when the iron supply in the growth medium was lowered. This polypeptide was almost missing, and thus the total amount of fucoxanthin-chlorophyll proteins (Fcps) bound to PSI was reduced as well. When treating cells with high light in addition, no further changes in antenna polypeptide composition were detected. Xanthophyll cycle pigments were found to be bound to all Fcps of PSI. However, PSI of high light cultures had a significantly higher diatoxanthin to diadinoxanthin ratio, which is assumed to protect against a surplus of excitation energy. PSI complexes from the double-stressed cultures (high light plus reduced iron supply) were slightly more sensitive against destruction by the detergent treatment. This could be seen as a higher 674-nm emission at 77 K in comparison to the PSI complexes isolated from other growth conditions. Two major emission bands of the Fcps bound to PSI at 77 K could be identified, whereby chlorophyll a fluorescing at 697 nm was more strongly coupled to the PSI core than those fluorescing at 685 nm. Thus, the build up of the PSI antenna of several Fcp components enables variable reactions to several stress factors commonly experienced by the diatoms in vivo, in particular diatoxanthin enrichment under high light and reduction of antenna size under reduced iron conditions.     

The Claudin Megatrachea Protein Complex

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.     

Acquisition of structure-guiding and structure-forming properties during maturation from the pro-silicatein to the silicatein form

Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and results in the release of the 23 kDa mature silicatein, a process which very likely proceeds by autocatalysis. Almost in parallel with its formation, the mature enzyme precipitates as pure 23 kDa protein. When the precipitate is dissolved in an urea buffer, the solubilized protein displays its full enzymatic activity which is enhanced multi-fold in the presence of the silicatein interactor silintaphin-1 or of poly(ethylene glycol) (PEG). The biosilica product formed increases its compactness if silicatein is supplemented with silintaphin-1 or PEG. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa (silicatein) via 61 MPa (silicatein:silintaphin-1) to 101 MPa (silicatein:silintaphin-1 and PEG). These data show that the maturation process from the pro-silicatein state to the mature form is the crucial step during which silicatein acquires its structure-guiding and structure-forming properties.     

High Selectivity of the γ-Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-based Approach

The solute carrier 6 (SLC6) is a family of ion-dependent transporters that mediate uptake into the cell of osmolytes such as neurotransmitters and amino acids. Four SLC6 members transport GABA, a key neurotransmitter that triggers inhibitory signaling pathways via various receptors (e.g., GABA_A). The GABA transporters (GATs) regulate the concentration of GABA available for signaling and are thus targeted by a variety of anticonvulsant and relaxant drugs. Here, we characterize GAT-2, a transporter that plays a role in peripheral GABAergic mechanisms, by constructing comparative structural models based on crystallographic structures of the leucine transporter LeuT. Models of GAT-2 in two different conformations were constructed and experimentally validated, using site-directed mutagenesis. Computational screening of 594,166 compounds including drugs, metabolites, and fragment-like molecules from the ZINC database revealed distinct ligands for the two GAT-2 models. 31 small molecules, including high scoring compounds and molecules chemically related to known and predicted GAT-2 ligands, were experimentally tested in inhibition assays. Twelve ligands were found, six of which were chemically novel (e.g., homotaurine). Our results suggest that GAT-2 is a high selectivity/low affinity transporter that is resistant to inhibition by typical GABAergic inhibitors. Finally, we compared the binding site of GAT-2 with those of other SLC6 members, including the norepinephrine transporter and other GATs, to identify ligand specificity determinants for this family. Our combined approach may be useful for characterizing interactions between small molecules and other membrane proteins, as well as for describing substrate specificities in other protein families.