Application of a rank-based genetic association test to age-at-onset data from the Collaborative Study on the Genetics of Alcoholism study.

Association studies of quantitative traits have often relied on methods in which a normal distribution of the trait is assumed. However, quantitative phenotypes from complex human diseases are often censored, highly skewed, or contaminated with outlying values. We recently developed a rank-based association method that takes into account censoring and makes no distributional assumptions about the trait. In this study, we applied our new method to age-at-onset data on ALDX1 and ALDX2. Both traits are highly skewed (skewness > 1.9) and often censored. We performed a whole genome association study of age at onset of the ALDX1 trait using Illumina single-nucleotide polymorphisms. Only slightly more than 5% of markers were significant. However, we identified two regions on chromosomes 14 and 15, which each have at least four significant markers clustering together. These two regions may harbor genes that regulate age at onset of ALDX1 and ALDX2. Future fine mapping of these two regions with densely spaced markers is warranted.

Functional characterization by genetic complementation of aroB-Encoded dehydroquinate synthase from Mycobactetium tuberculosis H37Rv and its heterologous expression and purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.

Functional characterization by genetic complementation of aroB-Encoded dehydroquinate synthase from Mycobactetium tuberculosis H37Rv and its heterologous expression and purification

The recent recrudescence of Mycobacterium tuberculosis infection and the emergence of multidrug-resistant strains have created an urgent need for new therapeutics against tuberculosis. The enzymes of the shikimate pathway are attractive drug targets because this route is absent in mammals and, in M. tuberculosis, it is essential for pathogen viability. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids, and it is found in plants, fungi, bacteria, and apicomplexan parasites. The aroB-encoded enzyme dehydroquinate synthase is the second enzyme of this pathway, and it catalyzes the cyclization of 3-deoxy-D-arabino-heptulosonate-7-phosphate in 3-dehydroquinate. Here we describe the PCR amplification and cloning of the aroB gene and the overexpression and purification of its product, dehydroquinate synthase, to homogeneity. In order to probe where the recombinant dehydroquinate synthase was active, genetic complementation studies were performed. The Escherichia coli AB2847 mutant was used to demonstrate that the plasmid construction was able to repair the mutants, allowing them to grow in minimal medium devoid of aromatic compound supplementation. In addition, homogeneous recombinant M. tuberculosis dehydroquinate synthase was active in the absence of other enzymes, showing that it is homomeric. These results will support the structural studies with M. tuberculosis dehydroquinate synthase that are essential for the rational design of antimycobacterial agents.

Ferrochelatase is a conserved downstream target of the blue light-sensing White collar complex in fungi.

Light is a universal signal perceived by organisms, including fungi, in which light regulates common and unique biological processes depending on the species. Previous research has established that conserved proteins, originally called White collar 1 and 2 from the ascomycete Neurospora crassa, regulate UV/blue light sensing. Homologous proteins function in distant relatives of N. crassa, including the basidiomycetes and zygomycetes, which diverged as long as a billion years ago. Here we conducted microarray experiments on the basidiomycete fungus Cryptococcus neoformans to identify light-regulated genes. Surprisingly, only a single gene was induced by light above the commonly used twofold threshold. This gene, HEM15, is predicted to encode a ferrochelatase that catalyses the final step in haem biosynthesis from highly photoreactive porphyrins. The C. neoformans gene complements a Saccharomyces cerevisiae hem15Delta strain and is essential for viability, and the Hem15 protein localizes to mitochondria, three lines of evidence that the gene encodes ferrochelatase. Regulation of HEM15 by light suggests a mechanism by which bwc1/bwc2 mutants are photosensitive and exhibit reduced virulence. We show that ferrochelatase is also light-regulated in a white collar-dependent fashion in N. crassa and the zygomycete Phycomyces blakesleeanus, indicating that ferrochelatase is an ancient target of photoregulation in the fungal kingdom.

The Bdellovibrio bacteriovorus twin-arginine transport system has roles in predatory and prey-independent growth

Bdellovibrio bacteriovorus grows in one of two ways: either (i) predatorily [in a host-dependent (HD) manner], when it invades the periplasm of another Gram-negative bacterium, exporting into the prey co-ordinated waves of soluble enzymes using the prey cell contents for growth; or (ii) in a host-independent (HI) manner, when it grows (slowly) axenically in rich media. Periplasmic invasion potentially exposes B. bacteriovorus to extremes of pH and exposes the need to scavenge electron donors from prey electron transport components by synthesis of metalloenzymes. The twin-arginine transport system (Tat) in other bacteria transports folded metalloenzymes and the B. bacteriovorus genome encodes 21 potential Tat-transported substrates and Tat transporter proteins TatA1, TatA2 and TatBC. GFP tagging of the Tat signal peptide from Bd1802, a high-potential iron-sulfur protein (HiPIP), revealed it to be exported into the prey bacterium during predatory growth. Mutagenesis showed that the B. bacteriovorus tatA2 and tatC gene products are essential for both HI and HD growth, despite the fact that they partially complement (in SDS resistance assays) the corresponding mutations in Escherichia coli where neither TatA nor TatC are essential for life. The essentiality of B. bacteriovorus TatA2 was surprising given that the B. bacteriovorus genome encodes a second tatA homologue, tatA1. Transcription of tatA1 was found to be induced upon entry to the bdelloplast, and insertional inactivation of tatA1 showed that it significantly slowed the rates of both HI and HD growth. B. bacteriovorus is one of a few bacterial species that are reliant on a functional Tat system and where deletion of a single tatA1 gene causes a significant growth defect(s), despite the presence of its tatA2 homologue.

The protein phosphatase 7 regulates phytochrome signaling in Arabidopsis.

The psi2 mutant of Arabidopsis displays amplification of the responses controlled by the red/far red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) but no apparent defect in blue light perception. We found that loss-of-function alleles of the protein phosphatase 7 (AtPP7) are responsible for the light hypersensitivity in psi2 demonstrating that AtPP7 controls the levels of phytochrome signaling. Plants expressing reduced levels of AtPP7 mRNA display reduced blue-light induced cryptochrome signaling but no noticeable deficiency in phytochrome signaling. Our genetic analysis suggests that phytochrome signaling is enhanced in the AtPP7 loss of function alleles, including in blue light, which masks the reduced cryptochrome signaling. AtPP7 has been found to interact both in yeast and in planta assays with nucleotide-diphosphate kinase 2 (NDPK2), a positive regulator of phytochrome signals. Analysis of ndpk2-psi2 double mutants suggests that NDPK2 plays a critical role in the AtPP7 regulation of the phytochrome pathway and identifies NDPK2 as an upstream element involved in the modulation of the salicylic acid (SA)-dependent defense pathway by light. Thus, cryptochrome- and phytochrome-specific light signals synchronously control their relative contribution to the regulation of plant development. Interestingly, PP7 and NDPK are also components of animal light signaling systems.

Functional differentiation of tbf1 orthologues in fission and budding yeasts.

In Saccharomyces cerevisiae, TBF1, an essential gene, influences telomere function but also has other roles in the global regulation of transcription. We have identified a new member of the tbf1 gene family in the mammalian pathogen Pneumocystis carinii. We demonstrate by transspecies complementation that its ectopic expression can provide the essential functions of Schizosaccharomyces pombe tbf1 but that there is no rescue between fission and budding yeast orthologues. Our findings indicate that an essential function of this family of proteins has diverged in the budding and fission yeasts and suggest that effects on telomere length or structure are not the primary cause of inviability in S. pombe tbf1 null strains.

Prospecção de genes com interesse biotecnológico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

DivIVA is required for polar growth in the MreB-lacking rod-shaped actinomycete Corynebacterium glutamicum.

The actinomycete Corynebacterium glutamicum grows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVA(Cg)) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA from Streptomyces or Mycobacterium localized to the cell poles of DivIVA(Cg)-depleted C. glutamicum and restored polar peptidoglycan synthesis, in contrast to DivIVA proteins from Bacillus subtilis or Streptococcus pneumoniae, which localized at the septum of C. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVA(Cg) localized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that in C. glutamicum DivIVA is not involved in cell division or chromosome segregation.

Hsp110 chaperones control client fate determination in the hsp70-Hsp90 chaperone system.

Heat shock protein 70 (Hsp70) plays a central role in protein homeostasis and quality control in conjunction with other chaperone machines, including Hsp90. The Hsp110 chaperone Sse1 promotes Hsp90 activity in yeast, and functions as a nucleotide exchange factor (NEF) for cytosolic Hsp70, but the precise roles Sse1 plays in client maturation through the Hsp70-Hsp90 chaperone system are not fully understood. We find that upon pharmacological inhibition of Hsp90, a model protein kinase, Ste11DeltaN, is rapidly degraded, whereas heterologously expressed glucocorticoid receptor (GR) remains stable. Hsp70 binding and nucleotide exchange by Sse1 was required for GR maturation and signaling through endogenous Ste11, as well as to promote Ste11DeltaN degradation. Overexpression of another functional NEF partially compensated for loss of Sse1, whereas the paralog Sse2 fully restored GR maturation and Ste11DeltaN degradation. Sse1 was required for ubiquitinylation of Ste11DeltaN upon Hsp90 inhibition, providing a mechanistic explanation for its role in substrate degradation. Sse1/2 copurified with Hsp70 and other proteins comprising the "early-stage" Hsp90 complex, and was absent from "late-stage" Hsp90 complexes characterized by the presence of Sba1/p23. These findings support a model in which Hsp110 chaperones contribute significantly to the decision made by Hsp70 to fold or degrade a client protein.

SnRK1-triggered switch of bZIP63 dimerization mediates the low-energy response in plants

Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites.

Haem uptake is essential for egg production in the haematophagous blood fluke of humans, Schistosoma mansoni

Schistosomes ingest host erythrocytes, liberating large quantities of haem. Despite its toxicity, haem is an essential factor for numerous biological reactions, and may be an important iron source for these helminths. We used a fluorescence haem analogue, palladium mesoporphyrin, to investigate pathways of haem acquisition, and showed that palladium mesoporphyrin accumulates in the vitellaria (eggshell precursor glands) and ovary of female Schistosoma mansoni. Furthermore, incubation of adult females in 10-100 μm cyclosporin A (IC50 = 2.3 μm) inhibits the uptake of palladium mesoporphyrin to these tissues, with tenfold reductions in fluorescence intensity of the ovary. In vitro exposure to cyclosporin A resulted in significant perturbation of egg production, reducing egg output from 34 eggs per female to 5.7 eggs per female over the incubation period, and retardation of egg development. We characterized a S. mansoni homologue of the haem-responsive genes of Caenorhabditis elegans. The gene (Smhrg-1) encodes a protein with a molecular weight of approximately 17 kDa. SmHRG-1 was able to rescue growth in haem transport-deficient HEM1Δ yeast. Transcriptional suppression of Smhrg-1 in adult S. mansoni worms resulted in significant delay in egg maturation, with 47% of eggs from transcriptionally suppressed worms being identified as immature compared with only 27% of eggs laid by control worms treated with firefly luciferase. Our findings indicate the presence of transmembrane haem transporters in schistosomes, with a high abundance of these molecules being present in tissues involved in oogenesis.

Legionella pneumophila secretes a mitochondrial carrier protein during infection

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

An extragenic suppressor of prp24-1 defines genetic interaction between PRP24 and PRP21 gene products of Saccharomyces cerevisiae

The temperature-sensitive prp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperature-sensitive (ts) prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic to prp21-1. This suppressor, prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that of prp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in the prp24-1 strain. Genetic analysis of the suppressor showed that prp21-2 is not a bypass suppressor of prp24-1. The suppression of prp24-1 by prp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2-U6 snRNA interactions.

Characterization of polycystin-1 in ADPKD pathogenetic mechanism : biogenesis and functional implications by genetic approaches in mouse

La polykystose rénale autosomique dominante (ADPKD) est une des maladies génétiques les plus communes. ADPKD se manifeste le plus souvent au stade adulte par la présence de kystes rénaux, et bien souvent de kystes hépatiques, avec une progression très variable. ADPKD mène à une insuffisance rénale: les seuls recours sont la dialyse puis la transplantation rénale. Les mutations dispersées sur les gènes PKD1 (majoritairement; la protéine polycystine-1, PC1) et PKD2 (la protéine polycystine-2, PC2) sont responsables de l’ADPKD. Le mécanisme pathogénétique de perte de fonction (LOF) et donc d’un effet récessif cellulaire est évoqué comme causatif de l’ADPKD. LOF est en effet supporté par les modèles murins d’inactivation de gènes PKD1/PKD2, qui développent de kystes, quoique in utéro et avec une rapidité impressionnante dans les reins mais pas dans le foie. Malgré de nombreuses études in vitro, le rôle de PC1/PC2 membranaire/ciliaire reste plutôt hypothétique et contexte-dépendant. Ces études ont associé PC1/PC2 à une panoplie de voies de signalisation et ont souligné une complexité structurelle et fonctionnelle exceptionnelle, dont l’implication a été testée notamment chez les modèles de LOF. Toutefois, les observations patho-cellulaires chez l’humain dont une expression soutenue, voire augmentée, de PKD1/PC1 et l’absence de phénotypes extrarénaux particuliers remet en question l’exclusivité du mécanisme de LOF. Il était donc primordial 1) d’éclaircir le mécanisme pathogénétique, 2) de générer des outils in vivo authentiques d’ADPKD en terme d’initiation et de progression de la maladie et 3) de mieux connaitre les fonctions des PC1/PC2 indispensables pour une translation clinique adéquate. Cette thèse aborde tous ces points. Tout d’abord, nous avons démontré qu’une augmentation de PKD1 endogène sauvage, tout comme chez l’humain, est pathogénétique en générant et caractérisant en détail un modèle murin transgénique de Pkd1 (Pkd1TAG). Ce modèle reproduit non seulement les caractéristiques humaines rénales, associées aux défauts du cil primaire, mais aussi extrarénales comme les kystes hépatiques. La sévérité du phénotype corrèle avec le niveau d’expression de Pkd1 ce qui supporte fortement un modèle de dosage. Dans un deuxième temps, nous avons démontré par les études de complémentations génétiques que ces deux organes reposent sur une balance du clivage GPS de Pc1, une modification post-traductionelle typique des aGPCR, et dont l’activité et l’abondance semblent strictement contrôlées. De plus, nous avons caractérisé extensivement la biogénèse de Pc1 et de ses dérivés in vivo générés suite au clivage GPS. Nous avons identifié une toute nouvelle forme et prédominante à la membrane, la forme Pc1deN, en plus de confirmer deux fragments N- et C-terminal de Pc1 (NTF et CTF, respectivement) qui eux s’associent de manière non-covalente. Nous avons démontré de façon importante que le trafic de Pc1deN i.e., une forme NTF détachée du CTF, est toutefois dépendant de l’intégrité du fragment CTF in vivo. Par la suite, nous avons généré un premier modèle humanisant une mutation PKD1 non-sens tronquée au niveau du domaine NTF(E3043X) en la reproduisant chez une souris transgénique (Pkd1extra). Structurellement, cette mutation, qui mimique la forme Pc1deN, s’est également avérée causative de PKD. Le modèle Pkd1extra a permis entre autre de postuler l’existence d’une cross-interaction entre différentes formes de Pc1. De plus, nos deux modèles murins sont tous les deux associés à des niveaux altérés de c-Myc et Pc2, et soutiennent une implication réelle de ces derniers dans l’ADPKD tou comme une interaction fonctionnelle entre les polycystines. Finalement, nous avons démontré un chevauchement significatif entre l’ADPKD et le dommage rénal aigüe (ischémie/AKI) dont une expression augmentée de Pc1 et Pc2 mais aussi une stimulation de plusieurs facteurs cystogéniques tel que la tubérine, la β-caténine et l’oncogène c-Myc. Nos études ont donc apporté des évidences cruciales sur la contribution du gène dosage dans l’ADPKD. Nous avons développé deux modèles murins qui serviront d’outil pour l’analyse de la pathologie humaine ainsi que pour la validation préclinique ADPKD. L’identification d’une nouvelle forme de Pc1 ajoute un niveau de complexité supplémentaire expliquant en partie une capacité de régulation de plusieurs voies de signalisation par Pc1. Nos résultats nous amènent à proposer de nouvelles approches thérapeutiques: d’une part, le ciblage de CTF i.e., de style chaperonne, et d’autre part le ciblage de modulateurs intracellulaires (c-Myc, Pc2, Hif1α). Ensemble, nos travaux sont d’une importance primordiale du point de vue informatif et pratique pour un avancement vers une thérapie contre l’ADPKD. Le partage de voies communes entre AKI et ADPKD ouvre la voie aux approches thérapeutiques parallèles pour un traitement assurément beaucoup plus rapide.