PHYTOCHROME KINASE SUBSTRATE 1 is a phototropin 1 binding protein required for phototropism.

Phototropism, or plant growth in response to unidirectional light, is an adaptive response of crucial importance. Lateral differences in low fluence rates of blue light are detected by phototropin 1 (phot1) in Arabidopsis. Only NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and root phototropism 2, both belonging to the same family of proteins, have been previously identified as phototropin-interacting signal transducers involved in phototropism. PHYTOCHROME KINASE SUBSTRATE (PKS) 1 and PKS2 are two phytochrome signaling components belonging to a small gene family in Arabidopsis (PKS1-PKS4). The strong enhancement of PKS1 expression by blue light and its light induction in the elongation zone of the hypocotyl prompted us to study the function of this gene family during phototropism. Photobiological experiments show that the PKS proteins are critical for hypocotyl phototropism. Furthermore, PKS1 interacts with phot1 and NPH3 in vivo at the plasma membrane and in vitro, indicating that the PKS proteins may function directly with phot1 and NPH3 to mediate phototropism. The phytochromes are known to influence phototropism but the mechanism involved is still unclear. We show that PKS1 induction by a pulse of blue light is phytochrome A-dependent, suggesting that the PKS proteins may provide a molecular link between these two photoreceptor families.

Complex organization of CTF/NF-I, C/EBP, and HNF3 binding sites within the promoter of the liver-specific vitellogenin gene.

Vitellogenin genes are expressed specifically in the liver of female oviparous vertebrates under the strict control of estrogen. To explain this tissue-specific expression, we performed a detailed analysis of the Xenopus laevis vitellogenin gene B1 promoter by DNase I footprinting and gel mobility-shift assays. We characterized five binding sites for the ubiquitous factor CTF/NF-I. Two of these sites are close to the TATA-box, whereas the others are located on both sides of the estrogen responsive unit formed by two imperfect estrogen response elements. Moreover two liver-enriched factors, C/EBP and HNF3, were found to interact with multiple closely spaced proximal promoter elements in the first 100 base pairs upstream of the TATA-box. To confirm the physiological significance of this in vitro analysis, in vivo DNase I footprinting experiments were carried out using the ligation-mediated polymerase chain reaction technique. The various cis-elements characterized in vitro as binding sites for known transcription factors and more particularly for liver-enriched transcription factors are efficiently recognized in vivo as well, suggesting that they play an important role in the control of the liver-specific vitellogenin gene B1 expression.

Point Mutations in the Monocarboxylate Transporter SLC16A12 Lead to Juvenile and Age-Related Cataract

Purpose: Previously we reported on a premature termination mutation in SLC16A12 that leads to dominant juvenile cataract and renal glucosuria. To assess the mutation rate and genotype-phenotype correlations of SLC16A12 in juvenile or age-related forms of cataract, we performed a mutation screen in cataract patients. Methods: Clinical data of approximately 660 patients were collected, genomic DNA was isolated and analyzed. Exons 3 to 8 including flanking intron sequences of SLC16A12 were PCR amplified and DNA sequence was determined. Selected mutations were tested by cell culture assays, in silico analysis and RT-PCR. Results: We found sequence alterations at a rate of approximately 1/75 patients. None of them was found in 360 control alleles. Alterations affect splice site and regulatory region but most mutations caused an amino acid substitution. The majority of the coding region mutations maps to trans-membrane domains. One mutation located to the 5'UTR. It affects translational efficiency of SLC16A12. In addition, we identified a cataract-predisposing SNP in the non-coding region that causes allele-specific splicing of the 5'UTR region. Conclusions: Altered translational efficiency of the solute carrier SLC16A12 and its allele-specific splicing strongly support a model of challenged homeostasis to cause various forms of cataract. In addition, the pathogenic property of the here reported sequence alterations is supported by the lack of known sequence variations within the coding region of SLC16A12. Due to the relatively high mutation rate, we suggest to include SLC16A12 in diagnostic cataract screening. Generally, our data recommend the assessment of regulatory sequences for diagnostic purposes.

Helicase-defective RuvB(D113E) promotes RuvAB-mediated branch migration in vitro.

In Escherichia coli, the RuvA and RuvB proteins interact at Holliday junctions to promote branch migration leading to the formation of heteroduplex DNA. RuvA provides junction-binding specificity and RuvB drives ATP-dependent branch migration. Since RuvB contains sequence motifs characteristic of a DNA helicase and RuvAB exhibit helicase activity in vitro, we have analysed the role of DNA unwinding in relation to branch migration. A mutant RuvB protein, RuvB(D113E), mutated in helicase motif II (the DExx box), has been purified to homogeneity. The mutant protein forms hexameric rings on DNA similar to those formed by wild-type protein and promotes branch migration in the presence of RuvA. However, RuvB(D113E) exhibits reduced ATPase activity and is severely compromised in its DNA helicase activity. Models for RuvAB-mediated branch migration that invoke only limited DNA unwinding activity are proposed.

Use of an in-house approach to study the three-dimensional structures of various outer membrane proteins: structure of the alcaligin outer membrane transporter FauA from Bordetella pertussis.

Bordetella pertussis is the bacterial agent of whooping cough in humans. Under iron-limiting conditions, it produces the siderophore alcaligin. Released to the extracellular environment, alcaligin chelates iron, which is then taken up as a ferric alcaligin complex via the FauA outer membrane transporter. FauA belongs to a family of TonB-dependent outer membrane transporters that function using energy derived from the proton motive force. Using an in-house protocol for membrane-protein expression, purification and crystallization, FauA was crystallized in its apo form together with three other TonB-dependent transporters from different organisms. Here, the protocol used to study FauA is described and its three-dimensional structure determined at 2.3 A resolution is discussed.

IB1, a JIP-1-related nuclear protein present in insulin-secreting cells.

JIP-1 is a cytoplasmic inhibitor of the c-Jun amino-terminal kinase activated pathway recently cloned from a mouse brain cDNA library. We report herein the expression cloning of a rat cDNA encoding a JIP-1-related nuclear protein from a pancreatic beta-cell cDNA library that we named IB1 for Islet-Brain 1. IB1 was isolated by its ability to bind to GTII, a cis-regulatory element of the GLUT2 promoter. The IB1 cDNA encodes a 714-amino acid protein, which differs from JIP-1 by the insertion of 47 amino acids in the carboxyl-terminal part of the protein. The remaining 667 amino acids are 97% identical to JIP-1. The 47-amino acid insertion contains a truncated phosphotyrosine interaction domain and a putative helix-loop-helix motif. Recombinant IB1 (amino acids 1-714 and 280-714) was shown to bind in vitro to GTII. Functionally IB1 transactivated the GLUT2 gene. IB1 was localized within the cytoplasm and the nucleus of insulin-secreting cells or COS-7 cells transfected with an expression vector encoding IB1. Using a heterologous GAL4 system, we localized an activation domain of IB1 within the first 280 amino acids of the protein. These data demonstrate that IB1 is a DNA-binding protein related to JIP-1, which is highly expressed in pancreatic beta-cells where it functions as a transactivator of the GLUT2 gene.

Interaction of antigenic peptides with MHC class I molecules on living cells studied by photoaffinity labeling.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of antigenic peptides with murine MHC class I molecules on living cells. Photoreactive derivatives were prepared by N-terminal amidation with iodo, 4-azido salicylic acid of the Kd restricted Plasmodium berghei circumsporozoite (P.b. CS) peptide 253-260 (YIPSAEKI) and the Db-restricted Adenovirus 5 early region 1A (Ad5 E1A) peptide 234-243 (SGPSNTPPEI). As assessed in functional competition experiments, both peptide derivatives retained the specific binding activity of the parental peptides for Kd or Dd, respectively. The P.b. CS photoprobe specifically labeled Kd molecules on P815 (H-2d) cells, but failed to label RMA (H-2b) cells. Conversely, the Ad5 E1A photoprobe specifically labeled Db molecules on RMA cells, but failed to label P815 cells. When the two photoprobes were tested on a panel of Con A-activated spleen cells expressing 10 different H-2 haplotypes, significant photoaffinity labeling was observed only on H-2d cells with the P.b. CS photoprobe and on H-2b cells with the Ad5 E1A photoprobe. Labeling of cell-associated Kd or Db molecules with the photoprobes was specifically inhibited by antigenic peptides known to be presented by the same class I molecule. Photoaffinity labeling of Kd with the P.b. CS photoprobe was used to study the dynamics of peptide binding on living P815 cells. Binding increased steadily with the incubation period (up to 8 h) at 37 degrees C and at ambient temperature, but was greatly reduced (greater than 95%) at 0 to 4 degrees C or in the presence of ATP synthesis inhibitors. The magnitude of the labeling was twofold higher at room temperature than at 37 degrees C. In contrast, binding to isolated Kd molecules in solution rapidly reached maximal binding, particularly at 37 degrees C. Dissociation of the photoprobe from either cell-associated or soluble Kd molecules was similar, with a half time of approximately 1 h at 37 degrees C, whereas the complexes were long-lived at 4 degrees C in both instances.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Binding of low concentration of peptide to H-2Kd produced in insect cells requires mouse beta 2-microglobulin co-expression.

A recombinant baculovirus expressing the murine class I MHC heavy chain H-2Kd cDNA under the transcriptional control of Autografa californica nuclear polyhedrosis virus (AcNPV) polyhedrin promoter has been isolated and used to infect Sf9 lepidopteran cells either alone or in association with a previously isolated virus expressing mouse beta 2-microglobulina (beta 2-ma). When infected with the heavy chain-encoding virus alone, H-2Kd was produced in a beta 2-m-free conformation detected on the surface of infected cells by conformation-independent antibodies. When Sf9 cells were co-infected with both viruses, approximately 10% of the heavy chain pool was engaged in the formation of native heterodimeric MHC class I molecules, which were glycosylated and transported to the cell surface as demonstrated by radio-binding experiments and flow cytometry. The assembly of the recombinant class I molecule was dependent on peptide, since heterodimer formation was brought about by H-2Kd-specific peptide ligands both in vivo, upon incubation with dually infected cells, and in vitro, in cell-free detergent extracts. In addition, a change in heavy chain conformation was brought about upon incubation with high concentrations (100 microM) of an H-2Kd-restricted octapeptide epitope from Plasmodium berghei. Furthermore, using low concentrations (3 nM) of a photoaffinity label derivative of this peptide, we show direct binding to cells co-expressing class I heavy chain and mouse beta 2-m but not to cells expressing free heavy chain only.

Tasosartan, enoltasosartan, and angiotensin II receptor blockade: the confounding role of protein binding.

Tasosartan is a long-acting angiotensin II (AngII) receptor blocker. Its long duration of action has been attributed to its active metabolite enoltasosartan. In this study we evaluated the relative contribution of tasosartan and enoltasosartan to the overall pharmacological effect of tasosartan. AngII receptor blockade effect of single doses of tasosartan (100 mg p.o. and 50 mg i.v) and enoltasosartan (2.5 mg i.v.) were compared in 12 healthy subjects in a randomized, double blind, three-period crossover study using two approaches: the in vivo blood pressure response to exogenous AngII and an ex vivo AngII radioreceptor assay. Tasosartan induced a rapid and sustained blockade of AngII subtype-1 (AT1) receptors. In vivo, tasosartan (p.o. or i.v.) blocked by 80% AT1 receptors 1 to 2 h after drug administration and still had a 40% effect at 32 h. In vitro, the blockade was estimated to be 90% at 2 h and 20% at 32 h. In contrast, the blockade induced by enoltasosartan was markedly delayed and hardly reached 60 to 70% despite the i.v. administration and high plasma levels. In vitro, the AT1 antagonistic effect of enoltasosartan was markedly influenced by the presence of plasma proteins, leading to a decrease in its affinity for the receptor and a slower receptor association rate. The early effect of tasosartan is due mainly to tasosartan itself with little if any contribution of enoltasosartan. The antagonistic effect of enoltasosartan appears later. The delayed in vivo blockade effect observed for enoltasosartan appears to be due to a high and tight protein binding and a slow dissociation process from the carrier.

O-mannosyl phosphorylation of alpha-dystroglycan is required for laminin binding.

Alpha-dystroglycan (alpha-DG) is a cell-surface glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin-G domains and certain arenaviruses. Receptor binding is thought to be mediated by a posttranslational modification, and defective binding with laminin underlies a subclass of congenital muscular dystrophy. Using mass spectrometry- and nuclear magnetic resonance (NMR)-based structural analyses, we identified a phosphorylated O-mannosyl glycan on the mucin-like domain of recombinant alpha-DG, which was required for laminin binding. We demonstrated that patients with muscle-eye-brain disease and Fukuyama congenital muscular dystrophy, as well as mice with myodystrophy, commonly have defects in a postphosphoryl modification of this phosphorylated O-linked mannose, and that this modification is mediated by the like-acetylglucosaminyltransferase (LARGE) protein. These findings expand our understanding of the mechanisms that underlie congenital muscular dystrophy.

Understanding the two-component sensing System of virulent bacteria

Report for the scientific sojourn carried out at the l’ Institute for Computational Molecular Science of the Temple University, United States, from 2010 to 2012. Two-component systems (TCS) are used by pathogenic bacteria to sense the environment within a host and activate mechanisms related to virulence and antimicrobial resistance. A prototypical example is the PhoQ/PhoP system, which is the major regulator of virulence in Salmonella. Hence, PhoQ is an attractive target for the design of new antibiotics against foodborne diseases. Inhibition of the PhoQ-mediated bacterial virulence does not result in growth inhibition, presenting less selective pressure for the generation of antibiotic resistance. Moreover, PhoQ is a histidine kinase (HK) and it is absent in animals. Nevertheless, the design of satisfactory HK inhibitors has been proven to be a challenge. To compete with the intracellular ATP concentrations, the affinity of a HK inhibidor must be in the micromolar-nanomolar range, whereas the current lead compounds have at best millimolar affinities. Moreover, the drug selectivity depends on the conformation of a highly variable loop, referred to as the “ATP-lid, which is difficult to study by X-Ray crystallography due to its flexibility. I have investigated the binding of different HK inhibitors to PhoQ. In particular, all-atom molecular dynamics simulations have been combined with enhanced sampling techniques in order to provide structural and dynamic information of the conformation of the ATP-lid. Transient interactions between these drugs and the ATP-lid have been identified and the free energy of the different binding modes has been estimated. The results obtained pinpoint the importance of protein flexibility in the HK-inhibitor binding, and constitute a first step in developing more potent and selective drugs. The computational resources of the hosting institution as well as the experience of the members of the group in drug binding and free energy methods have been crucial to carry out this work.

Production of human secretory component with dimeric IgA binding capacity using viral expression systems.

The cDNA encoding the NH2-terminal 589 amino acids of the extracellular domain of the human polymeric immunoglobulin receptor was inserted into transfer vectors to generate recombinant baculo- and vaccinia viruses. Following infection of insect and mammalian cells, respectively, the resulting truncated protein corresponding to human secretory component (hSC) was secreted with high efficiency into serum-free culture medium. The Sf9 insect cell/baculovirus system yielded as much as 50 mg of hSC/liter of culture, while the mammalian cells/vaccinia virus system produced up to 10 mg of protein/liter. The M(r) of recombinant hSC varied depending on the cell line in which it was expressed (70,000 in Sf9 cells and 85-95,000 in CV-1, TK- 143B and HeLa). These variations in M(r) resulted from different glycosylation patterns, as evidenced by endoglycosidase digestion. Efficient single-step purification of the recombinant protein was achieved either by concanavalin A affinity chromatography or by Ni(2+)-chelate affinity chromatography, when a 6xHis tag was engineered to the carboxyl terminus of hSC. Recombinant hSC retained the capacity to specifically reassociate with dimeric IgA purified from hybridoma cells.

Antigen-secretory IgA immune complexes are retro-transported into mouse Peyer's patches: immunotargeting to dendritic cells

RÉSUMÉ Les plaques de Peyer (PP) représentent le site d'entrée majeur des pathogènes au niveau des muqueuses intestinales. Après avoir traversé la cellule M, l'antigène est pris en charge par les cellules dendritiques (DC) de la région sub-épithéliale du dôme des PP. Ces dernières activent une réponse immunitaire qui conduit à la production de l'IgA de sécrétion (SIgA), l'anticorps majeur au niveau muqueux. Des études précédentes dans notre laboratoire ont démontré qu'après administration de SIgA dans des anses intestinales de souris, les SIgA se lient spécifiquement aux cellules M, entrent dans les PP, et sont éventuellement internalisées par les DC. Le but de ce travail est de comprendre la relevance biologique de l'entrée des SIgA dans les PP et leur relevance physiologique dans l'homéostasie mucosale. Dans un premier temps, nous avons montré en utilisant une méthode de purification optimisée basée sur une isolation magnétique, que, en plus des DC myéloïdes (CD11c+/CD11b+) et des DC lymphoïdes (CD11c+/CD8+), les PP de souris contiennent un nouveau sous-type de DC exprimant les marqueurs CD11c et CD19. L'utilisation de la microscopie confocale nous a permis de démontrer que les DC myéloïdes internalisent des SIgA, contrairement aux DC lymphoïdes qui n'interagissent pas avec les SIgA, alors que le nouveau sous-type de DC exprimant CD19 lie les SIgA. En plus, nous avons démontré qu'aucune des DC de rate, de ganglion bronchique ou de ganglion inguinal interagit avec les SIgA. Dans le but d'explorer si les SIgA peuvent délivrer des antigènes aux DC des PP in vivo, nous avons administré des complexes immunitaires formés de Shigella flexneri complexées à des SIgA, dans des anses intestinales de souris. Nous avons observé une entrée dans les PP, suivie d'une migration vers les ganglions mésentériques drainants, contrairement aux Shigella flexneri seules, qui n'infectent pas la souris par la voie intestinale. Shigella flexneri délivrée par SIgA n'induit pas de destruction tissulaire au niveau de l'intestin. En plus de l'exclusion immunitaire, ces résultats suggèrent un nouveau rôle des SIgA, qui consiste à transporter des antigènes à l'intérieur des PP dans un contexte non-inflammatoire. RÉSUMÉ DESTINÉ À UN LARGE PUBLIC L'intestin a pour rôle principal d'absorber les nutriments digérés tout au long du tube digestif, et de les faire passer dans le compartiment intérieur sanguin. Du fait de son exposition chronique avec un monde extérieur constitué d'aliments et de bactéries, l'intestin est un endroit susceptible aux infections et a donc besoin d'empêcher l'entrée de microbes. Pour cela, l'intestin est tapissé de "casernes" appelées les plaques de Peyer, qui appartiennent à un système de défense appelé système immunitaire muqueux. Les plaques de Peyer sont composées de différents types de cellules, ayant pour rôle de contrôler l'entrée de microbes et de développer une réaction immunitaire lors d'infection. Cette réaction immunitaire contre les microbes (antigènes) débute par la prise en charge de l'antigène par des sentinelles, les cellules dendritiques. L'antigène est préparé de façon à être reconnu par d'autres cellules appelées lymphocytes T capables d'activer d'autres cellules, les lymphocytes B. La réaction immunitaire résulte dans la production par les lymphocytes B d'un anticorps spécifique appelé IgA de sécrétion (SIgA) au niveau de la lumière intestinale. De manière classique, le rôle de SIgA au niveau de la lumière intestinale consiste à enrober les microbes et donc exclure leur entrée dans le compartiment intérieur. Dans ce travail, nous avons découvert une nouvelle fonction des SIgA qui consiste à introduire des antigènes dans les plaques de Peyer, et de les diriger vers les cellules dendritiques. Sachant que les SIgA sont des anticorps qui ne déclenchent pas de réactions de défense violentes dites inflammatoires, l'entrée des antigènes via SIgA serait en faveur d'une défense intestinale maîtrisée sans qu'il y ait d'inflammation délétère. Ces résultats nous laissent supposer que l'entrée d'antigènes via SIgA pourrait conduire le système immunitaire muqueux à reconnaître ces antigènes de manière appropriée. Ce mécanisme pourrait expliquer les désordres immunitaires de types allergiques et maladies auto-immunitaires que l'on rencontre chez certaines personnes déficientes en IgA, chez qui cette lecture d'antigènes de manière correcte serait inadéquate. ABSTRACT Peyer's patches (PP) represent the primary site for uptake and presentation of ingested antigens in the intestine. Antigens are sampled by M cells, which pass them to underlying antigen-presenting cells including dendritic cells (DC). This leads to the induction of mucosal T cell response that is important for the production of secretory IgA (SIgA), the chief antibody at mucosal surfaces. Previous studies in the laboratory have shown that exogenous SIgA administrated into mouse intestinal loop binds specifically to M cells, enter into PP, and is eventually internalized by DC. The aim of this work is to understand the biological significance of the SIgA uptake by PP DC and its physiological relevance for mucosal homeostasis. As a first step, we have shown by using an optimized MACS method that, in addition to the CD11c+/CD11b+ (myeloid DC) and CD11c+/CD8+ (lymphoid DC) subtypes, mouse PP contain a novel DC subtype exhibiting both CD11c and CD19 markers. By using a combination of MACS isolation and confocal microscopy, we have demonstrated that in contrast to the lymphoid DC which do not interact with SIgA, the myeloid DC internalize SIgA, while the CD19+ subtype binds SIgA on its surface. Neither spleen DC, nor bronchial-lymph node DC, nor inguinal lymph node DC exhibit such a binding specificity. To test whether SIgA could deliver antigens to PP DC in vivo, we administered SIgA-Shigella flexneri immune complexes into mouse intestinal loop containing a PP. We found that (i) SIgA-Shigella flexneri immune complexes enter the PP and are internalized by sub-epithelial dome PP DC, in contrast to Shigella flexneri alone that does not penetrate the intestinal epithelia in mice, (ii) immune complexes migrate to the draining mesenteric lymph node, (iii) Shigella flexneri carried via SIgA do not induce intestinal tissue destruction. Our results suggest that in addition to immune exclusion, SIgA transports antigens back to the PP under non-inflammatory conditions.