FXYD7 is a brain-specific regulator of Na,K-ATPase alpha 1-beta isozymes.

Recently, corticosteroid hormone-induced factor (CHIF) and the gamma-subunit, two members of the FXYD family of small proteins, have been identified as regulators of renal Na,K-ATPase. In this study, we have investigated the tissue distribution and the structural and functional properties of FXYD7, another family member which has not yet been characterized. Expressed exclusively in the brain, FXYD7 is a type I membrane protein bearing N-terminal, post-translationally added modifications on threonine residues, most probably O-glycosylations that are important for protein stabilization. Expressed in Xenopus oocytes, FXYD7 can interact with Na,K-ATPase alpha 1-beta 1, alpha 2-beta 1 and alpha 3-beta 1 but not with alpha-beta 2 isozymes, whereas, in brain, it is only associated with alpha 1-beta isozymes. FXYD7 decreases the apparent K(+) affinity of alpha 1-beta 1 and alpha 2-beta 1, but not of alpha 3-beta1 isozymes. These data suggest that FXYD7 is a novel, tissue- and isoform-specific Na,K-ATPase regulator which could play an important role in neuronal excitability.

Application de la modélisation moléculaire à de nouvelles approches thérapeutiques du cancer [Application of molecular modeling to new therapeutic cancer approaches].

Recent progress in the experimental determination of protein structures allow to understand, at a very detailed level, the molecular recognition mechanisms that are at the basis of the living matter. This level of understanding makes it possible to design rational therapeutic approaches, in which effectors molecules are adapted or created de novo to perform a given function. An example of such an approach is drug design, were small inhibitory molecules are designed using in silico simulations and tested in vitro. In this article, we present a similar approach to rationally optimize the sequence of killer T lymphocytes receptors to make them more efficient against melanoma cells. The architecture of this translational research project is presented together with its implications both at the level of basic research as well as in the clinics.

Epigenetic features of human mesenchymal stem cells determine their permissiveness for induction of relevant transcriptional changes by SYT-SSX1.

BACKGROUND: A characteristic SYT-SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism. METHODOLOGY/PRINCIPAL FINDINGS: We addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression. CONCLUSIONS/SIGNIFICANCE: Our observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects.

Ubiquitylation of voltage-gated sodium channels.

Ion channel proteins are regulated by different types of posttranslational modifications. The focus of this review is the regulation of voltage-gated sodium channels (Navs) upon their ubiquitylation. The amiloride-sensitive epithelial sodium channel (ENaC) was the first ion channel shown to be regulated upon ubiquitylation. This modification results from the binding of ubiquitin ligase from the Nedd4 family to a protein-protein interaction domain, known as the PY motif, in the ENaC subunits. Many of the Navs have similar PY motifs, which have been demonstrated to be targets of Nedd4-dependent ubiquitylation, tagging them for internalization from the cell surface. The role of Nedd4-dependent regulation of the Nav membrane density in physiology and disease remains poorly understood. Two recent studies have provided evidence that Nedd4-2 is downregulated in dorsal root ganglion (DRG) neurons in both rat and mouse models of nerve injury-induced neuropathic pain. Using two different mouse models, one with a specific knockout of Nedd4-2 in sensory neurons and another where Nedd4-2 was overexpressed with the use of viral vectors, it was demonstrated that the neuropathy-linked neuronal hyperexcitability was the result of Nav1.7 and Nav1.8 overexpression due to Nedd4-2 downregulation. These studies provided the first in vivo evidence of the role of Nedd4-2-dependent regulation of Nav channels in a disease state. This ubiquitylation pathway may be involved in the development of symptoms and diseases linked to Nav-dependent hyperexcitability, such as pain, cardiac arrhythmias, epilepsy, migraine, and myotonias.

Characterization and binding affinities of SmLANP: a new Schistosoma mansoni member of the ANP32 family of regulatory proteins.

Members of the leucine-rich repeat protein family are involved in diverse functions including protein phosphatase 2-inhibition, cell cycle regulation, gene regulation and signalling pathways. A novel Schistosoma mansoni gene, called SmLANP, presenting homology to various genes coding for proteins that belong to the super family of leucine-rich repeat proteins, was characterized here. SmLANP was 1184bp in length as determined from cDNA and genomic sequences and encoded a 296 amino acid open reading frame that spanning from 6 to 894bp. The predicted amino acid sequence had a calculated molecular weight of 32kDa. Analysis of the predicted sequence indicated the presence of 3 leucine-rich domains (LRR) located in the N-terminal region and an aspartic acid rich region in the C-terminal end. SmLANP transcript is expressed in all stages of the S. mansoni life cycle analyzed, exhibiting the highest expression level in males. The SmLANP protein was expressed in a GST expression system and antibodies raised in mice against the recombinant protein. By immunolocalization assay, using adult worms, it was shown that the protein is mainly present in the cell nucleus through the whole body and strongly expressed along the tegument cell body nuclei of adult worms. As members of this family are usually involved in protein-protein interaction, a yeast two hybrid assay was conducted to identify putative binding partners for SmLANP. Thirty-six possible partners were identified, and a protein ATP synthase subunit alpha was confirmed by pull down assays, as a binding partner of the SmLANP protein.

New human kallikrein 14: enzymatic characterization and inhibitors development.

RESUME DESTINE A UN LARGE PUBLIC En biologie, si une découverte permet de répondre à quelques questions, en général elle en engendre beaucoup d'autres. C'est ce qui s'est produit récemment dans le monde des kallicréines. De la famille des protéases, protéines ayant la faculté de couper plus ou moins spécifiquement d'autres protéines pour exercer un rôle biologique, la famille des kallicréines humaines n'était composée que de 3 membres lors du siècle dernier. Parmi eux, une kallicréine mondialement utilisée pour détecter le cancer de la prostate, le PSA. En 2000, un chercheur de l'hôpital universitaire Mont Sinaï à Toronto, le Professeur Eleftherios Diamandis, a découvert la présence de 12 nouveaux gènes appartenant à cette famille, situés sur le même chromosome que les 3 premières kallicréines. Cette découverte majeure a placé les spécialistes des kallicréines face à une montagne d'interrogations car les fonctions de ces nouvelles protéases étaient totalement inconnues. La kallicréine humaine 14 (hK14) présente un intérêt particulier, car elle se retrouve associée à différents cancers, notamment les carcinomes ovariens et mammaires. Cette association ne répond cependant pas à la fonction de cette protéase. L'objectif de ce travail de thèse était donc de découvrir, dans un premier temps, la spécificité de cette nouvelle kallicréine, c'est-à-dire le type de coupure qu'elle engendre au niveau des protéines qu'elle cible. Utilisant une technologie de pointe qui exploite la propriété des bactériophages à se répliquer dans les bactéries à l'infini, des dizaines de millions de combinaisons protéiques aléatoires ont été présentées à hK14, qui a pu sélectionner celles qui lui étaient favorables pour la coupure. Cette technique qualitative porte le nom de Phage Display Substrate. Une fois la sélection réalisée, il fallait transférer ces séquences coupées ou substrats dans un système permettant de donner une valeur quantitative à l'efficacité de coupure. Pour cela nous avons développé une technologie qui permet d'évaluer cette efficacité en utilisant des protéines fluorescentes de méduse, modifiées génétiquement, dont l'excitation de la première (CFP : cyan fluorescent protein) par la lumière à une certaine longue d'onde permet le transfert d'énergie à la seconde (YFP : yellow fluorescent protein), via un substrat qui les lie. Pour que ce transfert d'énergie se produise, il faut que les deux protéines fluorescentes soient proches, comme c'est le cas lorsqu'elles sont liées par un substrat. La coupure de ce lien provoque un changement de transfert d'énergie qui est quantifiable en utilisant un spectrofluoromètre. Cette technologie permet donc de suivre la réaction d'hydrolyse (coupure) des protéases. Afin de poursuivre certaines expériences permettant de mieux comprendre la fonction biologique d'hK14 ainsi que son éventuelle implication dans le cancer, nous avons développé des inhibiteurs spécifiques d'hK14. Les séquences qui on été le plus efficacement coupées par hK14 ont été utilisées pour transformer deux types d'inhibiteurs classiques, qui circulent dans notre sang, en inhibiteurs d'hK14 hautement efficaces et spécifiques. Selon les résultats obtenus in vitro, ils pourront être évalués in vivo en tant que traitement potentiel contre le cancer. RESUME Les protéases sont des enzymes impliquées dans des processus physiologiques mais aussi parfois pathologiques. La famille des kallicréines tissulaires humaines représente le plus grand groupe de protéases humaines, dont plusieurs pourraient participer au développement de certaines maladies. D'autre part, ces protéases sont apparues comme des marqueurs de pathogénicité potentiels, notamment dans les cas de cancers hormono-dépendants. La kallicréine humaine 14 a été récemment découverte et son implication dans quelques maladies, particulièrement dans le cas de tumeurs, semble probable. En effet, son expression génique est augmentée au niveau des tissus cancéreux de la prostate et du sein et son expression protéique s'est révélée plus élevée dans le sérum de patientes atteintes d'un cancer du sein ou des ovaires. Cependant, comme c'est le cas pour la plupart des kallicréines, sa fonction est encore inconnue. Afin de mieux connaître son rôle biologique et/ou pathologique, nous avons décidé de caractériser son activité enzymatique. Nous avons tout d'abord mis au point un système de substrats entièrement biologique permettant d'étudier in vitro l'activité des protéases. Ce système est basé sur le phénomène de FRET, à savoir le transfert d'énergie de résonance fluorescente qui intervient entre deux molécules fluorescentes voisines si le spectre d'émission de la protéine donneuse chevauche le spectre d'excitation de la protéine receveuse. Nous avons fusionné de manière covalente une protéine fluorescente bleue (CFP) et une jaune (YFP) en les liant avec diverses séquences. Par clivage de la séquence de liaison, une perte du transfert d'énergie peut être mesurée par un spectrofluoromètre. Cette technologie représente un moyen facile de suivre la réaction d'hydrolyse des protéases. Les conditions optimales de production de ces substrats CFP-YFP ont été déterminées, de même que les paramètres pouvant éventuellement influencer le FRET. Ce système possède une grande résistance à la protéolyse non spécifique et est applicable à un grand nombre de protéase. Contrairement aux substrats fluorogéniques, il permet d'étudier les acides aminés se trouvant des deux côtés du site de clivage. Ce système étant entièrement biologique, il est le reflet des interactions protéine-protéine et représente un outil biologique facile, bon marché et rapide pour caractériser les protéases. Dans un premier temps, hK14 a été mise en présence d' une banque de haute diversité de pentapeptides aléatoires présentée à la surface de phages afin d'identifier des substrats spécifiques. Ensuite, le système CFP-YFP a été employé pour trier les peptides sélectionnés afin d'identifier les séquences de substrats les plus sensibles et spécifiques pour hK14. Nous avons montré, qu'en plus de sa prévisible activité de type trypsine, hK14 possède aussi une très surprenante activité de type chymotrypsine. Les séquences les plus sensibles ont été choisies pour cribler la banque de donnée Swissprot, permettant ainsi l'identification de 6 substrats protéiques humains potentiels pour hK14. Trois d'entre eux, la laminine α-5, le collagène IV et la matriline-4, qui sont des composants de la matrice extracellulaire, ont démontré une grande susceptibilité à l'hydrolyse par hK14. De plus, la séparation éléctrophorétique a montré que la dégradation de la laminine α-5 et de la matriline-4 par hK14 devait se produire aux sites identifiés par la technologie du phage display. Pour terminer, nous avons transformé, par mutagenèse dirigée, deux serpines (inhibiteurs de protéases de type sérine) connues, AAT et ACT (alpha anti-trypsine et alpha anti-chymotrypsine), qui inhibent un vaste éventail d'enzymes humaines en inhibiteurs d'hK14 hautement efficaces et spécifiques. Ces inhibiteurs pourront être utilisés d'une part pour poursuivre certaines expériences permettant de mieux comprendre l'implication d'hK14 dans des voies physiologiques ou dans le cancer et d'autre part pour les évaluer in vivo en tant que traitement potentiel contre le cancer. SUMMARY Proteases consist of enzymes involved in physiological events, but also, in case of dysregulation, in pathogenicity. The human tissue kallikrein family represents the largest human protease cluster and includes several members that either could participate in the course of certain diseases or emerged as potential biological markers, especially in hormone dependent cancers. The human kallikrein 14 has been recently discovered and suggested implications in some disorders, particularly in tumors since its gene expression is up-regulated in prostate and breast cancer tissues and its protein expression increased in the serum of patients with breast and ovarian cancers. However, like most kallikreins, its function remains unknown. To better understand hK14 biological and/or pathological role, we decided to characterize its enzymatic activity. First of all, we developped a biological system suitable for in vitro study of protease activity. This system is based on the so-called FRET phenomenon, that is the Fluorescence Resonance Energy Transfer that occurs between two nearby fluorescent proteins if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. We fused covalently a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) with diverses sequences. Upon cleavage of the linker sequence by protease, the loss of energy transfer can be measured by a spectrofluorometer allowing an easy following of hydrolysis reaction. The optimal conditions to produce in bacterial system these CFP-YFP substrates were determined as well as the parameters that could eventually influence the FRET. This system demonstrated a high degree of resistance to non-specific proteolysis and applicability to various conditions corresponding to a great number of existing proteases. Other avantages are the possibility to study the amino acids located both sides of the cleavage site as well as the interest to work in a full biological system reflecting protein-protein interaction. A phage substrate library with exhaustive diversity was used prior to CFP-substrate-YFP system to isolate specific human kallikrein 14 substrates. After that the CFP-YFP system was used to sort peptides and identify highly sensitive and specific substrate sequences for hK14. We showed that besides its predictable trypsin-like activity, hK14 also possesses a surprising chymotrypsin-like activity. The screening of the Swissprot database was achieved with the most sensitive sequences and allowed the identification of 6 potential human protein substrates for hK14. Three of them, laminin α-5, collagen IV and matrilin-4, which are components of the extracellular matrix were incubated with hK14, by which they were efficiently hydrolyzed. Moreover, electrophoretic separation revealed that degradation of laminin α-5 and matrilin-4 by hK14 generated fragments with identical molecular size than the predicted N-terminal fragments that would result from hK14 specific cleavage, proving the value of phage display substrate to identify potential substrates. Finally, with site-directed mutagenesis, we transformed two well-known serpins (serine protease inhibitors), AAT and ACT (alpha anti-trypsin and alpha anti-chymotrypsin), which inhibit a vast spectrum of human enzymes into highly efficient and specific hK14 inhibitors. These inhibitors will be used to pursue experiments that could help understand hK14 implication in physiological pathways as well as in cancer biology and also to perform their in vivo evalution as potential cancer treatment.

The roles of PKCδ and Src kinases in the glucocorticoid induction and the TGF-β superinduction of the mouse mammary tumor virus transcription in B lymphocytes

Résumé : Le virus tumoral de la glande mammaire de la souris (MMTV) est un rétrovirus provoquant le développement de tumeurs dans les glandes mammaires des souris susceptibles femelles. Au cours de son évolution, le virus s'est adapté et s'exprime dans des cellules spécialisées. Les lymphocytes B sont les premières cellules infectées et elles sont essentielles pour la propagation de l'infection aux glandes mammaires. Dans notre étude, le virus MMTV a été utilisé afin d'examiner les voies de signalisation induites par les glucocorticoïdes (dexaméthasone (dex), une hormone stéroïdienne) et le transforming growth factor-f3 (TGF-P, une cytokine), deux molécules impliquées dans l'activation de la transcription à partir du promoteur du MMTV dans les cellules B. Le TGF-P seul n'influence pas l'activité du promoteur du MMTV. Par contre, en synergie avec dex, le TGF-P provoque une super-induction de l'expression du promoteur par rapport à une stimulation par le glucocorticoïde seul. Cette super-induction est régulée par une famille de protéines, les Smads. Ainsi, dans les lymphocytes B, l'utilisation du MMTV a permis de mettre en évidence une nouvelle synergie entre les glueocortieoïdes et le TGF-p. pans ce travail, l'utilisation d'inhibiteurs pharmacologiques et de mutants « dominant-négatifs » nous a pet mis de démontrer qu'une Protéine Kinase C delta (PKC5) active est impliquée dans la transduction du signal lors de la réponse au dex ainsi que celle au TGF-P. Néanmoins, la PKC5 est régulée différemment dans chaque voie spécifique : la voie du TGF-p nécessitait l'activation du PKC5 par diacylglycerol (DAG) et la phosphorylation de tyrosines spécifiques, alors que la voie impliquant les glucocorticoïdes ne le nécessitait pas. Nous avons aussi démontré qu'une tyrosine kinase de la famille Src est responsable de la phosphorylation des tyrosines sur la PKC5. Les essais de kinase in vitro nous ont permis de découvrir que plusieurs Src kinases peuvent phosphoryler la PKC6 dans les cellules B et qu'elles étaient constitutivement actives. Enfin, nous avons montré qu'il existe une interaction protéine - protéine induite par dex, entre le récepteur aux glucocorticoïdes (GR) et la PKC5 dans les cellules B, une association qui n'a pas été démontrée auparavant. Par ailleurs, nous avons analysé les domaines d'interactions entre PKC5 et GR en utilisant les essais de «GST pull-down». Nos résultats montrent que le domaine régulateur de la PKC5 et celui qui interagit avec l'ADN du GR sont impliqués. En résumé, nous avons trouvé que dans une lignée lymphocytaire B, le virus MMTV utilise des mécanismes pour réguler à la fois la transcription et la voie de signalisation qui sont différents de ceux utilisés dans les cellules mammaires épithéliales et les fibroblastes. Nos découvertes pourraient être utilisées comme modèles pour l'étude de gènes cellulaires impliqués dans des processus tels qu'inflammation, immunité ou cancérogénèse. Summary: Mouse Mammary Tumor Virus (MMTV) is a retrovirus that causes tumors in the mammary glands of susceptible female mice and has adapted evolutionarily to be expressed in specialized cells. The B lymphocytes are the first cells to be infected by the MMTV and are essential for the spread of infection to the mammary glands. Here, we used the MMTV as a model system to investigate the signalling cascade induced by giucocorticoids (dexamethasone, "dex", a steroid hormone), and by Transforming Growth Factor-beta (TGF-P, a cytokine) leading to its transcriptional activation in B lymphocytes. By itself, TGF-I3 does not affect the basal activity of the MMTV promoter. However, TGF-13 significantly increases glucocorticoid-induced expression, through its effectors, the Smad factors. Thus, MMTV in B cells demonstrates a novel synergism between glucocorticoids and TGF-16. In this thesis project, we present evidence, based on the use of pharmacological inhibitors and of dominant-negative mutants, that an active Protein Kinase C delta (PKC6) is required as a signal transducer for the dex response and for the TGF-P superinduction as well. The PKC6 is differentially regulated in each specific pathway: whereas the TGF-13 superinduction required PKC6 to be activated by diacylglycerol (DAG) and to be phosphorylated at specific tyrosine residues, the glueocorticoid-induced pathway did not. We also showed that a protein tyrosine kinase of the Src family is responsible for the phosphorylation of tyrosines on PKC6. By performing in vitro kinase assays, we found that several Src kinases of B cells were able to phosphorylate PKC6 and that they were constitutively active. Finally, we demonstrate a dex-dependent functional protein-protein interaction between the glucocorticoid receptor (GR) and PKC6 in B cells, an association that has not been previously described. We further analysed the interacting domains of PKG6 and GR using in vitro GST pull-down assays, whereby the regulatory domain of PKC6 and the extended DNA-binding domain of the GR were involved. In summary, we found that in B-lymphoid cell lines, MMTV uses novel mechanisms of transcriptional control and signal transduction that are different from those at work in mammary epithelial or fibroblastic cells. These findings will be used as model for cellular genes involved in cellular processes such as immune functions, inflammation, or oncogenic transformation that may have a similar pattern of regulation.

Jasmonate signaling pathway.

Jasmonates in plants are cyclic fatty acid-derived regulators structurally similar to prostaglandins in metazoans. These chemicals mediate many of plants' transcriptional responses to wounding and pathogenesis by acting as potent regulators for the expression of numerous frontline immune response genes, including those for defensins and antifungal proteins. Additionally, the pathway is critical for fertility. Ongoing genetic screens and protein-protein interaction assays are identifying components of the canonical jasmonate signaling pathway. A massive molecular machine, based on two multiprotein complexes, SCF(COI1) and the COP9 signalosome (CNS), plays a central role in jasmonate signaling. This machine functions in vivo as a ubiquitin ligase complex, probably targeting regulatory proteins, some of which are expected to be transcriptional repressors. Some defense-related mediators, notably salicylic acid, antagonize jasmonates in controlling the expression of many genes. In Arabidopsis, NONEXPRESSOR OF PR GENES (NPR1) mediates part of this interaction, with another layer of control provided further downstream by the mitogen-activated protein kinase (MAPK) homolog MPK4. Numerous other interpathway connections influence the jasmonate pathway. Insights from Arabidopsis have shown that an allele of the auxin signaling gene AXR1, for example, reduces the sensitivity of plants to jasmonate. APETALA2 (AP2)-domain transcription factors, such as ETHYLENE RESPONSE FACTOR 1 (ERF1), link the jasmonate pathway to the ethylene signaling pathway. As progress in characterizing several new mutants (some of which are hypersensitive to jasmonic acid) augments our understanding of jasmonate signaling, the Connections Map will be updated to include this new information.

Compartmentalization in membrane rafts defines a pool of N-cadherin associated with catenins and not engaged in cell-cell junctions in melanoma cells.

Melanoma progression is associated with changes in adhesion receptor expression, in particular upregulation of N-cadherin which promotes melanoma cell survival and invasion. Plasma membrane lipid rafts contribute to the compartmentalization of signaling complexes thereby regulating their function, but how they may affect the properties of adhesion molecules remains elusive. In this study, we addressed the question whether lipid rafts in melanoma cells may contribute to the compartmentalization of N-cadherin. We show that a fraction of N-cadherin in a complex with catenins is associated with cholesterol/sphingolipid-rich membrane microdomains in aggressive melanoma cells in vitro and experimental melanomas in vivo. Partitioning of N-cadherin in membrane rafts is not modulated by growth factors and signaling pathways relevant to melanoma progression, is not necessary for cell-cell junctions' establishment or maintenance, and is not affected by cell-cell junctions' and actin cytoskeleton disruption. These results reveal that two independent pools of N-cadherin exist on melanoma cell surface: one pool is independent of lipid rafts and is engaged in cell-cell junctions, while a second pool is localized in membrane rafts and does not participate in cell-cell adhesions. Targeting to membrane rafts may represent a previously unrecognized mechanism regulating N-cadherin function in melanoma cells.

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.

Knowledge management for systems biology a general and visually driven framework applied to translational medicine

Background: To enhance our understanding of complex biological systems like diseases we need to put all of the available data into context and use this to detect relations, pattern and rules which allow predictive hypotheses to be defined. Life science has become a data rich science with information about the behaviour of millions of entities like genes, chemical compounds, diseases, cell types and organs, which are organised in many different databases and/or spread throughout the literature. Existing knowledge such as genotype - phenotype relations or signal transduction pathways must be semantically integrated and dynamically organised into structured networks that are connected with clinical and experimental data. Different approaches to this challenge exist but so far none has proven entirely satisfactory. Results: To address this challenge we previously developed a generic knowledge management framework, BioXM™, which allows the dynamic, graphic generation of domain specific knowledge representation models based on specific objects and their relations supporting annotations and ontologies. Here we demonstrate the utility of BioXM for knowledge management in systems biology as part of the EU FP6 BioBridge project on translational approaches to chronic diseases. From clinical and experimental data, text-mining results and public databases we generate a chronic obstructive pulmonary disease (COPD) knowledge base and demonstrate its use by mining specific molecular networks together with integrated clinical and experimental data. Conclusions: We generate the first semantically integrated COPD specific public knowledge base and find that for the integration of clinical and experimental data with pre-existing knowledge the configuration based set-up enabled by BioXM reduced implementation time and effort for the knowledge base compared to similar systems implemented as classical software development projects. The knowledgebase enables the retrieval of sub-networks including protein-protein interaction, pathway, gene - disease and gene - compound data which are used for subsequent data analysis, modelling and simulation. Pre-structured queries and reports enhance usability; establishing their use in everyday clinical settings requires further simplification with a browser based interface which is currently under development.

A targeted association study of immunity genes and networks suggests novel associations with placental malaria infection

A large proportion of the death toll associated with malaria is a consequence of malaria infection during pregnancy, causing up to 200,000 infant deaths annually. We previously published the first extensive genetic association study of placental malaria infection, and here we extend this analysis considerably, investigating genetic variation in over 9,000 SNPs in more than 1,000 genes involved in immunity and inflammation for their involvement in susceptibility to placental malaria infection. We applied a new approach incorporating results from both single gene analysis as well as gene-gene interactionson a protein-protein interaction network. We found suggestive associations of variants in the gene KLRK1 in the single geneanalysis, as well as evidence for associations of multiple members of the IL-7/IL-7R signalling cascade in the combined analysis. To our knowledge, this is the first large-scale genetic study on placental malaria infection to date, opening the door for follow-up studies trying to elucidate the genetic basis of this neglected form of malaria.

From SNPs to pathways: integration of functional effect of sequence variations on models of cell signalling pathways

Background: Single nucleotide polymorphisms (SNPs) are the most frequent type of sequence variation between individuals, and represent a promising tool for finding genetic determinants of complex diseases and understanding the differences in drug response. In this regard, it is of particular interest to study the effect of non-synonymous SNPs in the context of biological networks such as cell signalling pathways. UniProt provides curated information about the functional and phenotypic effects of sequence variation, including SNPs, as well as on mutations of protein sequences. However, no strategy has been developed to integrate this information with biological networks, with the ultimate goal of studying the impact of the functional effect of SNPs in the structure and dynamics of biological networks. Results: First, we identified the different challenges posed by the integration of the phenotypic effect of sequence variants and mutations with biological networks. Second, we developed a strategy for the combination of data extracted from public resources, such as UniProt, NCBI dbSNP, Reactome and BioModels. We generated attribute files containing phenotypic and genotypic annotations to the nodes of biological networks, which can be imported into network visualization tools such as Cytoscape. These resources allow the mapping and visualization of mutations and natural variations of human proteins and their phenotypic effect on biological networks (e.g. signalling pathways, protein-protein interaction networks, dynamic models). Finally, an example on the use of the sequence variation data in the dynamics of a network model is presented. Conclusion: In this paper we present a general strategy for the integration of pathway and sequence variation data for visualization, analysis and modelling purposes, including the study of the functional impact of protein sequence variations on the dynamics of signalling pathways. This is of particular interest when the SNP or mutation is known to be associated to disease. We expect that this approach will help in the study of the functional impact of disease-associated SNPs on the behaviour of cell signalling pathways, which ultimately will lead to a better understanding of the mechanisms underlying complex diseases.

Topological comparison of methods for predicting transcriptional cooperativity in yeast

Background: The cooperative interaction between transcription factors has a decisive role in the control of the fate of the eukaryotic cell. Computational approaches for characterizing cooperative transcription factors in yeast, however, are based on different rationales and provide a low overlap between their results. Because the wealth of information contained in protein interaction networks and regulatory networks has proven highly effective in elucidating functional relationships between proteins, we compared different sets of cooperative transcription factor pairs (predicted by four different computational methods) within the frame of those networks. Results: Our results show that the overlap between the sets of cooperative transcription factors predicted by the different methods is low yet significant. Cooperative transcription factors predicted by all methods are closer and more clustered in the protein interaction network than expected by chance. On the other hand, members of a cooperative transcription factor pair neither seemed to regulate each other nor shared similar regulatory inputs, although they do regulate similar groups of target genes. Conclusion: Despite the different definitions of transcriptional cooperativity and the different computational approaches used to characterize cooperativity between transcription factors, the analysis of their roles in the framework of the protein interaction network and the regulatory network indicates a common denominator for the predictions under study. The knowledge of the shared topological properties of cooperative transcription factor pairs in both networks can be useful not only for designing better prediction methods but also for better understanding the complexities of transcriptional control in eukaryotes.

Predicting cancer involvement of genes from heterogeneous data

Background: Systematic approaches for identifying proteins involved in different types of cancer are needed. Experimental techniques such as microarrays are being used to characterize cancer, but validating their results can be a laborious task. Computational approaches are used to prioritize between genes putatively involved in cancer, usually based on further analyzing experimental data. Results: We implemented a systematic method using the PIANA software that predicts cancer involvement of genes by integrating heterogeneous datasets. Specifically, we produced lists of genes likely to be involved in cancer by relying on: (i) protein-protein interactions; (ii) differential expression data; and (iii) structural and functional properties of cancer genes. The integrative approach that combines multiple sources of data obtained positive predictive values ranging from 23% (on a list of 811 genes) to 73% (on a list of 22 genes), outperforming the use of any of the data sources alone. We analyze a list of 20 cancer gene predictions, finding that most of them have been recently linked to cancer in literature. Conclusion: Our approach to identifying and prioritizing candidate cancer genes can be used to produce lists of genes likely to be involved in cancer. Our results suggest that differential expression studies yielding high numbers of candidate cancer genes can be filtered using protein interaction networks.