Structure of an integrin-ligand complex deduced from solution x-ray scattering and site-directed mutagenesis

The structural basis of the interaction of integrin heterodimers with their physiological ligands is poorly understood. We have used solution x-ray scattering to visualize the head region of integrin alpha 5 beta 1 in an inactive (Ca2+-occupied) state, and in complex with a fragment of fibronectin containing the RGD and synergy recognition sequences. Shape reconstructions of the data have been interpreted in terms of appropriate molecular models. The scattering data suggest that the head region undergoes no gross conformational changes upon ligand binding but do lend support to a proposed outward movement of the hybrid domain in the beta subunit. Fibronectin is observed to bind across the top of the head region, which contains an alpha subunit beta-propeller and a beta subunit vWF type A domain. The model of the complex indicates that the synergy region binds on the side of the beta-propeller domain. In support of this suggestion, mutagenesis of a prominent loop region on the side of the propeller identifies two residues (Tyr208 and Ile210) involved in recognition of the synergy region. Our data provide the first view of a complex between an integrin and a macromolecular ligand in solution, at a nominal resolution of approximately 10 A.     

Structural basis of type VI collagen dimer formation

We have determined the interactive sites required for dimer formation in type VI collagen. Despite the fact that type VI collagen is a heterotrimer composed of alpha1(VI), alpha2(VI), and alpha3(VI) chains, the formation of dimers is determined principally by interactions of the alpha2(VI) chain. Key components of this interaction are the metal ion-dependent adhesion site (MIDAS) motif of the alpha2C2 A-domain and the GER sequence in the helical domain of another alpha2(VI) chain. Replacement of the alpha2(VI) C2 domain with the alpha3(VI) domain abolished dimer formation, whereas alterations in the alpha2(VI) C1 domain did not disrupt dimer formation. When the helical sequences were investigated, replacement of the alpha2(VI) sequence GSPGERGDQ with the alpha3(VI) sequence GEKGERGDV abolished dimer formation. Mutating the Pro-108 to a Lys-108 in this alpha2(VI) sequence did not influence dimer formation and suggests that, unlike the integrin I-domain/triple-helix interaction, hydroxyproline is not required in collagen VI A-domain/helix interaction. These results demonstrate that the alpha2(VI) chain position in the assembled triple-helical molecule is critical for antiparallel dimer formation and identify the interacting collagenous and MIDAS sequences involved. These interactions underpin the subsequent assembly of type VI collagen.     

Plasmid maintenance and recombinant cell fitness explored in bacterial colonies

During growth of recombinant bacteria, irregular plasmid partitioning generates non-productive, plasmid-free cells whose proportion usually increases in the culture. For Escherichia coli producing engineered -galactosidases, we have shown a coincidence between plasmid stability and the extension of white/blue areas within individual colonies on X-gal plates. In this context, a good correlation between plasmid permanence in colonies and parameters accurately describing the dynamics of plasmid-free cell population in liquid cultures has been observed. Moreover, the impact of lacZ gene engineering and the metabolic burden imposed by the encoded proteins has been evaluated through plasmid stability by simple image analysis, revealing an enhanced plasmid loss rate as the cells enter into the stationary phase that is modulated by the expression of particular recombinant genes.     

The Interaction of Gibberellins and Photoperiod in the Control of Potato Tuberization

Solanum tuberosum ssp. andigena plants require a short-day (SD) photoperiod for tuber formation, a process that is also affected by gibberellins (GAs). Grafting experiments have confirmed that the photoperiod is perceived in the leaves. Tuber formation, however, usually takes place in the underground stolons. In this review, photoperiod-dependent tuberization has been divided into five chronological events: SD photoperiod perception, short-term adaptive responses to SD conditions, generation and transport of tuber-inducing signal(s), tuber formation, and long-term adaptive responses to tuber growth. Within this frame of study, the interaction of GAs and photoperiod is revised. Similar to the flowering process in Arabidopsis, we suggest the existence of two independent pathways that control tuber formation: a photoperiod-dependent pathway and a GA-dependent pathway. Nevertheless, photoperiod-dependent tuber formation requires the action of GAs at specific stages to orchestrate this complex process of development.     

spalt-induced specification of distinct dorsal and ventral domains is required for Drosophila tracheal patterning

Morphogenesis of the Drosophila tracheal system relies on different signalling pathways that have distinct roles in specifying both the migration of the tracheal cells and the particular morphological features of the primary branches. The current view is that the tracheal cells are initially specified as an equivalent group of cells whose diversification depends on signals from the surrounding cells. In this work, we show that the tracheal primordia are already specified as distinct dorsal and ventral cell populations. This subdivision depends on the activity of the spalt (sal) gene and occurs prior to the activity of the signalling pathways that dictate the development of the primary branches. Finally, we show that the specification of these two distinct cell populations, which are not defined by cell lineage, are critical for proper tracheal patterning. These results indicate that tracheal patterning depends not only on signalling from surrounding cells but also in the different response of the tracheal cells depending on their allocation to the dorsal or ventral domains.     

Trachynilysin,a neurosecretory protein isolated from stonefish (Synanceia trachynis) venom,forms nonselective pores in the membrane of NG108-15 cells

Trachynilysin, a protein toxin isolated from the venom of the stonefish Synanceia trachynis, has been reported to elicit massive acetylcholine release from motor nerve endings of isolated neuromuscular preparations and to increase both cytosolic Ca2+ and catecholamine release from chromaffin cells. In the present study, we used the patch clamp technique to investigate the effect of trachynilysin on the cytoplasmic membrane of differentiated NG108-15 cells in culture. Trachynilysin increased membrane conductance the most when the negativity of the cell holding membrane potential was reduced. The trachynilysin-induced current was carried by cations and reversed at about -3 mV in standard physiological solutions, which led to strong membrane depolarization and Ca2+ influx. La3+ blocked the trachynilysin current in a dose-, voltage-, and time-dependent manner, and antibodies raised against the toxin antagonized its effect on the cell membrane. The inside-out configuration of the patch clamp technique allowed the recording of single channel activity from which various multiples of 22 pS elementary conductance were resolved. These results indicate that trachynilysin forms pores in the NG108-15 cell membrane, and they advance our understanding of the toxin's mode of action on motor nerve endings and neurosecretory cells.     

Regulation of c-Ret,GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta in a rat model of Parkinson's disease

Glial cell line-derived neurotrophic factor (GDNF) family members have been proposed as candidates for the treatment of Parkinson's disease because they protect nigral dopaminergic neurons against various types of insult. However, the efficiency of these factors depends on the availability of their receptors after damage. We evaluated the changes in the expression of c-Ret, GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta in a rat model of Parkinson's disease by in situ hybridization. Intrastriatal injection of 6-hydroxydopamine (6-OHDA) transiently increased c-Ret and GFRalpha1 mRNA levels in the substantia nigra pars compacta at 1 day postlesion. At later time points, 3 and 6 days, the expression of c-Ret and GFRalpha1 was downregulated. GFRalpha2 expression was differentially regulated, as it decreased only 6 days after 6-OHDA injection. Triple-labeling studies, using in situ hybridization for the GDNF family receptors and immunohistochemistry for neuronal or glial cell markers, showed that changes in the expression of c-Ret, GFRalpha1, and GFRalpha2 in the substantia nigra pars compacta were localized to neurons. In conclusion, our results show that nigral neurons differentially regulate the expression of GDNF family receptors as a transient and compensatory response to 6-OHDA lesion.     

Critical residues for structure and catalysis in short-chain dehydrogenases/reductases

Short-chain dehydrogenases/reductases form a large, evolutionarily old family of NAD(P)(H)-dependent enzymes with over 60 genes found in the human genome. Despite low levels of sequence identity (often 10-30%), the three-dimensional structures display a highly similar alpha/beta folding pattern. We have analyzed the role of several conserved residues regarding folding, stability, steady-state kinetics, and coenzyme binding using bacterial 3beta/17beta-hydroxysteroid dehydrogenase and selected mutants. Structure determination of the wild-type enzyme at 1.2-A resolution by x-ray crystallography and docking analysis was used to interpret the biochemical data. Enzyme kinetic data from mutagenetic replacements emphasize the critical role of residues Thr-12, Asp-60, Asn-86, Asn-87, and Ala-88 in coenzyme binding and catalysis. The data also demonstrate essential interactions of Asn-111 with active site residues. A general role of its side chain interactions for maintenance of the active site configuration to build up a proton relay system is proposed. This extends the previously recognized catalytic triad of Ser-Tyr-Lys residues to form a tetrad of Asn-Ser-Tyr-Lys in the majority of characterized short-chain dehydrogenases/reductase enzymes.     

A green nonsulfur bacterium,Dehalococcoides ethenogenes,with the LexA binding sequence found in gram-positive organisms

Dehalococcoides ethenogenes is a member of the physiologically diverse division of green nonsulfur bacteria. Using a TBLASTN search, the D. ethenogenes lexA gene has been identified, cloned, and expressed and its protein has been purified. Mobility shift assays revealed that the D. ethenogenes LexA protein specifically binds to both its own promoter and that of the uvrA gene, but not to the recA promoter. Our results demonstrate that the D. ethenogenes LexA binding site is GAACN(4)GTTC, which is identical to that found in gram-positive bacteria. In agreement with this fact, the Bacillus subtilis DinR protein binds specifically to the D. ethenogenes LexA operator. This constitutes the first non-gram-positive bacterium exhibiting a LexA binding site identical to that of B. subtilis.