The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation

The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants. In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA). SLEEPY1 (SLY1) encodes an F-box-containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA. These findings suggested that SLY1 recruits RGA to the SCFSLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome. In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response. By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain. The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCFSLY1 targets both RGA and GAI for degradation. The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation. However, we found that this DELLA domain is not required for protein-protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins. We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants. This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins.     

Mucin secretion and PKC isoforms in SPOC1 goblet cells: differential activation by purinergic agonist and PMA

SPOC1 cells, which are a mucin-secreting model of rat airway goblet cells, possess a luminal P2Y2 purinoceptor through which UTP, ATP, and ATPgammaS stimulate secretion with EC50 values of approximately 3 microM. PMA elicits mucin secretion with high EC50 (75 nM) and saturation (300 nM) values. For the first time in airway mucin-secreting cells, the PKC isoforms expressed and activated by a secretagogue were determined using RT-PCR/restriction-enzyme mapping and Western blotting. Five isoforms were expressed: cPKCalpha, nPKCdelta and -eta, and aPKCzeta and -iota/lambda. PMA caused cPKCalpha and nPKCdelta to translocate to the membrane fraction of SPOC1 cells; only nPKCdelta so responded to ATPgammaS. Membrane-associated nPKCdelta and mucin secretion increased in parallel with ATPgammaS concentration and yielded EC50 values of 2-3 microM and maximum values of 100 microM. Effects of PMA to increase membrane-associated cPKCalpha and nPKCdelta saturated at 30 nM, whereas mucin secretion saturated at 300 nM, which suggests a significant PKC-independent effect of PMA on mucin secretion. A prime alternate phorbol ester-receptor candidate is the C1-domain protein MUNC13. RT-PCR revealed the expression of ubiquitous (ub)MUNC13-2 and its binding partner, DOC2-gamma. Hence, P2Y2 agonists activate nPKCdelta in SPOC1 cells. PMA activates cPKCalpha and nPKCdelta at high affinity and stimulates a lower affinity PKC-independent pathway that leads to mucin secretion.     

The tertiary structure and backbone dynamics of human prolactin

Human prolactin is a 199-residue (23 kDa) protein closely related to growth hormone and placental lactogen with properties and functions resembling both a hormone and a cytokine. As a traditional hormone, prolactin is produced by lactotrophic cells in the pituitary and secreted into the bloodstream where it acts distally to regulate reproduction and promote lactation. Pituitary cells store prolactin in secretory granules organized around large prolactin aggregates, which are produced within the trans layer of the Golgi complex. Extrapituitary prolactin is synthesized by a wide variety of cells but is not stored in secretory granules. Extrapituitary prolactin displays immunomodulatory activities and acts as a growth factor for cancers of the breast, prostate and tissues of the female reproductive system. We have determined the tertiary structure of human prolactin using three-dimensional (3D) and four-dimensional (4D) heteronuclear NMR spectroscopy. As expected, prolactin adopts an "up-up-down-down" four-helical bundle topology and resembles other members of the family of hematopoietic cytokines. Prolactin displays three discrete structural differences from growth hormone: (1) a structured N-terminal loop in contact with the first helix, (2) a missing mini-helix in the loop between the first and second helices, and (3) a shorter loop between the second and third helices lacking the perpendicular mini-helix observed in growth hormone. Residues necessary for functional binding to the prolactin receptor are clustered on the prolactin surface in a position similar to growth hormone. The backbone dynamics of prolactin were investigated by analysis of 15N NMR relaxation phenomena and demonstrated a rigid four-helical bundle with relatively mobile interconnecting loops. Comparison of global macromolecular tumbling at 0.1mM and 1.0mM prolactin revealed reversible oligomerization, which was correlated to dynamic light scattering experiments. The existence of a reversible oligomerization reaction in solution provides insight into previous results describing the in vitro and in vivo aggregation properties of human prolactin.     

Lipomannan and lipoarabinomannan from a clinical isolate of Mycobacterium kansasii: novel structural features and apoptosis-inducing properties

Although Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M. kansasii pathogenicity. Lipoarabinomannan (LAM), a major mycobacterial cell wall lipoglycan, is an important virulence factor for many mycobacteria, as it modulates the host immune response. Therefore, the detailed structures of the of M. kansasii LAM (KanLAM), as well as of its biosynthetic precursor lipomannan (KanLM), were determined in a clinical strain isolated from a human immunodeficiency virus-positive patient. Structural analyses revealed that these lipoglycans possess important differences as compared with those from other mycobacterial species. KanLAM carries a mannooligosaccharide cap but is devoid of the inositol phosphate cap present in Mycobacterium smegmatis. Characterization of the mannan core of KanLM and KanLAM demonstrated the following occurrences: 1) alpha1,2-oligo-mannopyranosyl side chains, contrasting with the single mannopyranosyl residues substituting the mannan core in all the other structures reported so far; and 2) 5-methylthiopentose residues that were described to substitute the arabinan moiety from Mycobacterium tuberculosis LAM. With respect to the arabinan domain of KanLAM, succinyl groups were found to substitute the C-3 position on 5-arabinofuranosyl residues, reported to be linked to the C-2 of the 3,5-arabinofuranose in Mycobacterium bovis bacillus calmette-guerin LAM. Because M. kansasii has been reported to induce apoptosis, we examined the possibility of the M. kansasii lipoglycans to induce apoptosis of THP-1 cells. Our results indicate that, in contrast to KanLAM, KanLM was a potent apoptosis-inducing factor. This work underlines the diversity of LAM structures among various pathogenic mycobacterial species and also provides evidence of LM being a potential virulence factor in M. kansasii infections by inducing apoptosis.     

Inhibition of InhA activity,but not KasA activity,induces formation of a KasA-containing complex in mycobacteria

Isoniazid (INH) remains one of the key drugs used to control tuberculosis, with the enoyl-AcpM reductase InhA being the primary target. However, based on the observation that INH-treated Mycobacterium tuberculosis overproduces KasA, an enzyme involved in the biosynthesis of mycolic acids, and induces the formation of a covalent complex consisting of AcpM, KasA, and INH, it has been proposed that KasA represents the primary target of INH. However, the relevance of this complex to INH action remains obscure. This study was aimed at clarifying the role of InhA and KasA in relation to INH activity. By using anti-KasA antibodies we detected the KasA-containing complex in INH-treated Mycobacterium smegmatis. In addition, INH-treated cells also produced constant levels of KasA that were not sequestered in the complex and presumably were sufficient to ensure mycolic acid biosynthesis. Interestingly, a furA-lacking strain induced the complex at lower concentrations of INH compared with the control strain, whereas higher INH concentrations were necessary to induce the complex in a strain that lacks katG, suggesting that INH needs to be activated by KatG to induce the KasA-containing complex. The InhA inhibitors ethionamide and diazaborine also induced the complex; thus, its formation was not specifically relevant to INH action but was because of InhA inhibition. In addition, in vitro assays using purified InhA and KasA demonstrated that KatG-activated INH, triclosan, and diazaborine inhibited InhA but not KasA activity. Moreover, several thermosensitive InhA mutant strains of M. smegmatis constitutively expressed the KasA-containing complex. This study provides the biochemical and genetic evidence. 1) Only inhibition of InhA, but not KasA, induces the KasA-containing complex. 2) INH is not part of the complex. 3) INH does not target KasA, consistent with InhA being the primary target of INH.     

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500-A-long secreted protein that is rich in beta-structure and contains two regions, R1 and R2, of tandem 19-residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel beta-sheets; or (ii) a beta-helix in which the polypeptide chain is coiled to form three long parallel beta-sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional "covert" copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the beta-helix model that the shaft domain--a 350 A rod--should consist of a continuous run of these motifs, apart from loop inserts. The N-terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin-like structure. Drawing on crystal structures of known beta-helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins--all large secreted proteins of bacterial provenance--that have similar repeats and probably also similar structures.     

New mutations of CIAS1 that are responsible for Muckle-Wells syndrome and familial cold urticaria: a novel mutation underlies both syndromes

Mutations of CIAS1 have recently been shown to underlie familial cold urticaria (FCU) and Muckle-Wells syndrome (MWS), in three families and one family, respectively. These rare autosomal dominant diseases are both characterized by recurrent inflammatory crises that start in childhood and that are generally associated with fever, arthralgia, and urticaria. The presence of sensorineural deafness that occurs later in life is characteristic of MWS. Amyloidosis of the amyloidosis-associated type is the main complication of MWS and is sometimes associated with FCU. In FCU, cold exposure is the triggering factor of the inflammatory crisis. We identified CIAS1 mutations, all located in exon 3, in nine unrelated families with MWS and in three unrelated families with FCU, originating from France, England, and Algeria. Five mutations--namely, R260W, D303N, T348M, A439T, and G569R--were novel. The R260W mutation was identified in two families with MWS and in two families with FCU, of different ethnic origins, thereby demonstrating that a single CIAS1 mutation may cause both syndromes. This result indicates that modifier genes are involved in determining either a MWS or a FCU phenotype. The finding of the G569R mutation in an asymptomatic individual further emphasizes the importance of such modifier a gene (or genes) in determining the disease phenotype. Identification of this gene (or these genes) is likely to have significant therapeutic implications for these severe diseases.     

Enhanced bacterial virulence through exploitation of host glycosaminoglycans

Present in the extracellular matrix and membranes of virtually all animal cells, proteoglycans (PGs) are among the first host macromolecules encountered by infectious agents. Because of their wide distribution and direct accessibility, it is not surprising that pathogenic bacteria have evolved mechanisms to exploit PGs for their own purposes, including mediating attachment to target cells. This is achieved through the expression of adhesins that recognize glycosaminoglycans (GAGs) linked to the core protein of PGs. Some pathogens, such as Bordetella pertussis and Chlamydia trachomatis, may express more than one GAG-binding adhesin. Bacterial interactions with PGs may also facilitate cell invasion or systemic dissemination, as observed for Neisseria gonorrhoeae and Mycobacterium tuberculosis respectively. More-over, pathogenic bacteria can use PGs to enhance their virulence via a shedding of PGs that leads to there lease of effectors that weaken the host defences.The exploitation of PGs by pathogenic bacteria is thus a multifaceted mechanistic process directly related to the potential virulence of a number of microorganisms.     

An european pupil project linked to the scientific aims of the experiment AQUARIUS-XENOPUS on the taxi Soyuz flight Andromede to ISS

The German-French biological experiment AQUARIUS-XENOPUS which flew on the Soyuz flight Andromede to the International Space Station ISS (launched October 21, 2001 in Baikonour/Kazakhstan) was extended by an outreach project. Pupils of class 10 to 12 from Ulm/D and Nancy-Tomblaine/F studied swimming behavior of Xenopus tadpoles on ground. They were instructed to perform all experimental steps following the protocol of similar video recordings on ISS. After the flight, they evaluated the kinetics of swimming of both ground controls and space animals. The pupil project included theoretical components to introduce them to the field of gravitational biology. One feature of the project was the exchange of ideas between pupils by meetings which took place in Ulm (June 2001), Nancy (February 2002) and Paris (May 2002). We consider our approach as a successful way to include young people in space experiments on a cheap cost level and to bring ideas of gravitational biology into the curricula of European schools.     

A novel strategy for the functional cloning of enzymes using filamentous phage display: the case of nucleotidyl transferases

In vitro selections for catalytic activity have been designed for the isolation of genes encoding enzymes from libraries of proteins displayed on filamentous phages. The proteins are generally expressed as C-terminal fusions with the N-terminus of the minor coat protein p3 for display on phages. As full-length cDNAs generally contain several stop codons near their 3′ end, this approach cannot be used for their expression on the surface of phages. Here we show that in vitro selection for catalytic activity is compatible with a system for expression of proteins as N-terminal fusions on the surface of bacteriophages. It is highlighted for the Stoffel fragment of Taq DNA polymerase I and makes use of (p3–Jun/Fos–Stoffel fragment) fusions. The efficiency of the selection is measured by an enrichment factor found to be about 55 for a phage polymerase versus a phage not expressing a polymerase. This approach could provide a method for the functional cloning of nucleotidyl transferases from cDNA libraries using filamentous phage display.