Characterization of the neuronal microtubule regulator SCG10 in transfected cells and " in vivo "

SUMMARY The ability of neuronal processes to find their way along complex paths and to establish appropriate connections depends on continual rearrangements of the cytoskeletal components. The regulation of microtubules plays an important role for morphological changes underlying nevrite outgrowth, axonal elongation, and growth cone steering. SCG10 (superior cervical ganglion clone 10) is a neuronal growthassociated protein developmentally regulated and highly enriched in the neuronal growth cones. SCG10 presents a microtubule destabilizing activity that could participate to the regulation of microtubule dynamics and thus explain microtubule behaviors in the growth cone during axonal elongation and turning. It is here suggested that a tight control of the opposite effects on microtubules of SCG10 and the stabilizing microtubule-associated protein MAP1B allows a fine tuning of cytoskeletal rearrangement and may provide the required microtubule dynamic instability to promote axonal growth. Moreover, antibodyblockade of SCG10 function, that leads to growth cone pauses similar as those triggered by the guidance molecule EphB, and the modulation of SCG10 activity by the Rho GTPase Rnd1 suggest a potential role for SCG10 in the signal transduction pathways of extracellular guidance cues. The identification of the active zone protein Bassoon as a potential interaction partner for the SCG10-related protein NPC2, using atomic force microscopy as well as COS-7 and neuronal cell cultures, also gives new insights for a role of this protein family into the processes of synapse genesis or plasticity. Finally, SCG10 mutant mice generated by gene targeting and expressing a soluble form of the protein have been characterized during early postnatal development and in the adulthood. Due to the deletion of its membrane binding domain, SCG10 specific subcellular targeting to growth cones is compromised and results in impairments of motor and coordination development. Further histological analysis in the sciatic nerve reveal that these symptoms are associated with neurodegenerative signs. RESUME Une navigation correcte des prolongements cellulaires neuronaux leur permettant de former des connections appropriées repose sur de continuels réarrangements des constituants de leur cytosquelette. La régulation des microtubules joue notamment un rôle important dans les changements morphologiques qui accompagnent la croissance axonale et les réorientations du cône de croissance. SCG10 (superior cervical ganglion clone 10) est une protéine étroitement associée à la croissance neuronale, hautement régulée durant le développement et abondante au niveau du cône de croissance. SCG10 présente une activité déstabilisatrice sur les microtubules qui pourrait permettre une régulation des paramètres dynamiques propres aux microtubules et ainsi expliquer leur comportement durant la navigation du cône de croissance. Il est ici proposé qu'un contrôle précis des effets opposés de SCG10 et d'une autre protéine stabilisante associée aux microtubules (MAP1 B) permette un réglage fin des réarrangements du cytosquelette et puisse ainsi produire l'instabilité dynamique nécessaire à la croissance anale. Par ailleurs, le blocage de la fonction de SCG10 par un anticorps spécifique, conduisant à des pauses du cônes de croissance similaires à celles provoquées par la molécule de guidage EphB, ainsi que la modulation de l'activité de SCG10 par la Rho GTPase Rnd1 suggèrent une potentielle implication de SCG10 dans les voies de transduction des signaux provenant de molécules de guidage extracellulaires. L'identification d'une interaction de la protéine synaptique Bassoon avec la protéine NPC2 apparentée à SCG10, au moyen de la microscopie à force atomique et dans des cultures de cellules neuronales et COS-7, ouvre des perspectives concernant ces protéines dans la formation et la plasticité synaptiques. Finalement, des souris mutantes pour SCG10 produites par ciblage de gène et exprimant une forme soluble de la protéine ont été caractérisées durant la phase précoce du développement et à l'âge adulte. La délétion du domaine permettant l'ancrage de SCG10 aux membranes compromet sa sub-localisation au niveau du cône de croissance et résulte en l'apparition de troubles moteurs et de la coordination. Des analyses histologiques complémentaires au niveau du nerf sciatique montrent que ces symptômes sont associés avec des signes neurodégénératifs.

The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis.

Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.

RDM1, a novel RNA recognition motif (RRM)-containing protein involved in the cell response to cisplatin in vertebrates.

A variety of cellular proteins has the ability to recognize DNA lesions induced by the anti-cancer drug cisplatin, with diverse consequences on their repair and on the therapeutic effectiveness of this drug. We report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searching databases for sequences showing similarities to RAD52, a protein involved in homologous recombination and DNA double-strand break repair. Ablation of RDM1 in the chicken B cell line DT40 led to a more than 3-fold increase in sensitivity to cisplatin. However, RDM1-/- cells were not hypersensitive to DNA damages caused by ionizing radiation, UV irradiation, or the alkylating agent methylmethane sulfonate. The RDM1 protein displays a nucleic acid binding domain of the RNA recognition motif (RRM) type. By using gel-shift assays and electron microscopy, we show that purified, recombinant chicken RDM1 protein interacts with single-stranded DNA as well as double-stranded DNA, on which it assembles filament-like structures. Notably, RDM1 recognizes DNA distortions induced by cisplatin-DNA adducts in vitro. Finally, human RDM1 transcripts are abundant in the testis, suggesting a possible role during spermatogenesis.

In vitro characterization of PlySK1249, a novel phage lysin, and assessment of its antibacterial activity in a mouse model of Streptococcus agalactiae bacteremia.

Beta-hemolytic Streptococcus agalactiae is the leading cause of bacteremia and invasive infections. These diseases are treated with β-lactams or macrolides, but the emergence of less susceptible and even fully resistant strains is a cause for concern. New bacteriophage lysins could be promising alternatives against such organisms. They hydrolyze the bacterial peptidoglycan at the end of the phage cycle, in order to release the phage progeny. By using a bioinformatic approach to screen several beta-hemolytic streptococci, a gene coding for a lysin was identified on a prophage carried by Streptococcus dysgalactiae subsp. equisimilis SK1249. The gene product, named PlySK1249, harbored an original three-domain structure with a central cell wall-binding domain surrounded by an N-terminal amidase and a C-terminal CHAP domain. Purified PlySK1249 was highly lytic and bactericidal for S. dysgalactiae (2-log10 CFU/ml decrease within 15 min). Moreover, it also efficiently killed S. agalactiae (1.5-log10 CFU/ml decrease within 15 min) but not several streptococcal commensal species. We further investigated the activity of PlySK1249 in a mouse model of S. agalactiae bacteremia. Eighty percent of the animals (n = 10) challenged intraperitoneally with 10(6) CFU of S. agalactiae died within 72 h, whereas repeated injections of PlySK1249 (45 mg/kg 3 times within 24 h) significantly protected the mice (P < 0.01). Thus, PlySK1249, which was isolated from S. dysgalactiae, demonstrated high cross-lytic activity against S. agalactiae both in vitro and in vivo. These encouraging results indicated that PlySK1249 might represent a good candidate to be developed as a new enzybiotic for the treatment of systemic S. agalactiae infections.

Characterization and functional identification of a novel plant extradiol 4,5-dioxygenase involved in betalain pigment biosynthesis in Portulaca Grandiflora

RESUME Les bétalaïnes sont des pigments chromo-alcaloïdes violets et jaunes présents dans les plantes appartenant à l'ordre des Caryophyllales et dans les champignons des genres Amanita et Hygrocybe. Leur courte voie de biosynthèse est élucidée chimiquement depuis de nombreuses années, mais les enzymes impliquées dans cette biosynthèse chez les plantes ne sont toujours pas caractérisées. L'enzyme de la DOPA-dioxygénase d' Amanita muscaria a été identifiée (Girod et Zryd, 1991a), mais de nombreuses tentatives d'isolation d'un homologue chez les plantes ont échoué. Afin d'isoler les gènes spécifiques des bétalaïnes chez les plantes, nous avons construit des banques soustraites d'ADNc à partir d'ARN total de pétales immatures de Portulaca grandiflora (Pg) de génotypes jaunes et blancs, respectivement violets et blancs. Les clones couleur- spécifiques ont été détectés en premier par analyse Northem du RNA de pétales blancs et colorés. Les candidats positifs ont alors été soumis à une analyse de transcription au niveau des tiges colorées, vertes et des feuilles, afin d'établir leur expression spécifique. Deux ARNs messagers complets ont une expression corrélée avec l'accumulation des bétalaïnes dans les tissus. Le premier de ces clones, A.16, code pour une oxydase de l'acyl-Coenzyme A (ACX) putative, mais le domaine de liaison du FAD essentiel pour l'activité d'ACX est absent. Toutes nos tentatives pour démontrer sa fonction ont échoué. Le rôle de cette protéine dans la voie de synthèse des bétalaïnes reste inconnu. Le deuxième de ces clones spécifique aux bétalaïnes, L.6 (isolé par Zaiko, 2000), a été renommé DODA en raison de son homologie avec le domaine LigB (pfam02900) d'une 4,5-dioxygénase extradiol bactérienne. DODA a été identifié in silico comme une dioxygénase extradiol en raison de la conservation stricte, au niveau de sa séquence peptidique, des résidus catalytiques de LigB et de ceux liant le cofacteur fer. Une analyse de transfert Southem a montré que ce gène est unique dans Pg. L'expression transitoire de DODA par transformation biolistique dans des pétales blancs de Pg a produit des taches violettes ou jaunes dans des cellules transformées. Une analyse HPLC de ces taches a démontré leur identité avec les bétalaïnes présentes naturellement dans les pétales violets et jaunes de Pg, confirmant ainsi la complémentation par le gène Pg DODA de l'allèle récessif cc présent dans les pétales blancs de Pg. Des homologues de DODA (DOPA-dioxygénase) ont été identifiés dans de nombreuses espèces de plantes, y compris dans celles sans bétalaïne. L'alignement de ces homologues a permis l'identification d'un motif spécifique aux bétalaïnes à côté d'une histidine catalytique conservée. Ce motif [H-P-(S,A)-(N,D)-x-T-P] remplace le motif [H-N-L-R] conservé dans les plantes sans bétalaïne et le motif [H-N-L-x] présent dans tous les homologues bactériens et archaebactériens. Une modélisation tridimensionnelle préliminaire du site actif de Pg DODA et de son homologue dans la mousse Physcomitrella patens a montré l'importance de ce motif spécifique aux bétalaïnes pour l'accessibilité du substrat au site actif. L'analyse phylogénétique de DODA a confirmé l'évolution séparée de cette protéine chez les plantes à bétalaïnes par comparaison avec celle des plantes sans bétalaïne. Nous avons donc conclu que les bétalaïnes sont apparues par modification de l'affinité pour un substrat d'enzymes similaires à DODA, chez un ancêtre unique des Caryophyllales qui a perdu toute capacité de biosynthèse des anthocyanes. Finalement, Pg DODA n'a aucune similarité avec la protéine DODA d' Amanita muscaria, bien que celle-ci complémente aussi la pigmentation des pétales blancs de Pg. La biosynthèse des bétalaïnes est un exemple remarquable de convergence évolutive biochimique indépendante entre espèces de règnes différents. ABSTRACT Betalains are violet and yellow chromo-alkaloid pigments present in plants belonging to the order Caryophyllales and also in the fungal genera Amanita and Hygrocybe. Their short biosynthetic pathway is chemically well understood since many years, but enzymes involved in the plant pathway are still uncharacterized. The DOPA-dioxygenase from Amanita muscaria was identified (Girod and Zryd, 1991a), but numerous attempts to identify a plant homologue to the corresponding gene, failed. In order to isolate betalain-specific genes in plants, subtractive cDNA libraries were built with total RNA from white and yellow and respectively, violet immature petals from Portulaca grandiflora (Pg) genotypes. Colour-specific clones were first detected by Northern blot analysis using RNA from white and coloured petals. Positive candidates were submitted to further transcription analysis in coloured, green stems and leaves in order to assess their specific expression. Two full-length mRNAs showed a correlated expression with betalain accumulation in tissues. One of them, A.16, encodes a putative acyl-Coenzyme A oxidase (ACX), but missing the FAD binding domain essential for the ACX activity. Thus, all attempts to demonstrate its function failed. The role of this protein in the betalain biosynthesis pathway, if any, is still unknown. The second betalain-specific mRNA, L.6 (isolated by Zaiko, 2000) shows a homology with a LigB domain (pfam02900) from a bacterial extradiol 4,5-dioxygenase. It was then renamed DODA (DOPA-dioxygenase). DODA was identified in silico as a highly conserved extradiol dioxygenase due to the strict conservation of its peptidic sequence with LigB catalytic residues and iron-binding cofactor residues. Southern blot analysis showed that this gene is a single copy-gene in Pg. Transient expression of DODA protein through biolistic transformation of Pg white petals produced violet or yellow spots in individual cells. HPLC analysis of these spots showed an identity with betalain pigments present naturally in yellow and violet Pg petals, thus confirming the complementation of the recessive cc allele present in Pg white petals by Pg DODA gene. DODA homologues were identified in numerous plant species including those without betalain. Alignment of these homologues allowed the identification of a betalain-specific pattern beside a highly conserved catalytic histidine. This [H-P-(S,A)-(N,D)-x-T-P] pattern replaces a [H-N-L-R] pattern strictly conserved in non-betalain plants and a [H-N-L-x] pattern present in all bacterial and archaebacterial homologues. Preliminary three-dimensional modeling of the active site of Pg DODA and its Physcomitrella patens moss homologue revealed the importance of this betalain-specific pattern for the substrate accessibility to the DODA active site. DODA phylogenetic analysis confirmed the separate evolution of this protein in betalain-producing plants. We conclude that betalain pigments appeared in a unique ancestor of the Caryophyllales order in which anthocyanin biosynthetic pathway was impaired, by a modification of enzymes of the DODA family for substrate affinity. The Pg DODA protein has no sequence similarity with Amanita muscaria DODA, despite the fact that they both complement Pg white petals for their pigmentation. Betalain biosynthesis is an interesting example of independent biochemical evolutionary convergence between species from different kingdoms.

Ewing sarcoma cancer stem cells : from mechanisms to therapeutic targeting

Le sarcome d'Ewing (SE) est la 2ème tumeur des os la plus fréquente chez les enfants, et le pronostic est sombre au stade métastatique. La pathogenèse du SE repose sur une translocation, provocant la fusion du domaine activateur du facteur de transcription EWS, avec la partie liant l'ADN de la protéine FLI-1. Les cellules souches cancéreuses (CSC) sont supposées être les moteurs de la croissance tumorale, et représente de ce fait des cibles thérapeutiques préférentielles. Dans ce travail nous nous sommes efforcés de comprendre, ainsi que de cibler les mécanismes liés à l'émergence des CSC dans le sarcome d'Ewing. La formation des CSC du ES est liée à un défaut de maturation des miRNAs provoqué par une sous-expression d'un gène, TARBP2, dans les CSC. Ce défaut de maturation peut être corrigé par un traitement des cellules avec de l'enoxacine, une fluoroquinolone utilisée pour traiter les infections urinaires. L'enoxacine seule n'étant pas suffisante pour éradiquer les tumeurs in vivo, nous avons testé la combinaison d'une thérapie ciblée sur les CSC avec une chimiothérapie classique, la doxorubicine, ciblant les cellules différentiées. In vitro l'enoxacine induit l'apoptose dans les CCS sans affecter les cellules différentiées, alors que à l'inverse, la doxorubicine n'affecte que les cellules de la « masse » tumorale. In vivo la combinaison de ces deux drogues inhibe la croissance de tumeurs provenant de cellules primaires xenotranplantées et éradique les CSCs. Nos résultats mettent en lumière une nouvelle approche thérapeutique directement applicable pour le sarcome d'Ewing, et pourraient ainsi rapidement déboucher sur des essais cliniques. Dans la deuxième partie de ce travail nous avons essayé de comprendre comment EWS-FLI1, la protéine de fusion issue de la translocation chromosomique du sarcome d'Ewing conduit à la génération des CSC. Pour cela nous avons effectué des ChIPseq (immunoprecipitation de la chromatine suivi de séquençage) pour EWS-FLI1 ainsi que pour certaines modifications histoniques. -- Ewing sarcoma family tumors (ESFT) are the second most frequent bone tumors in children and have a high rate of recurrence when metastatic at presentation. The pathogenesis of Ewing sarcoma is underlayed by a translocation, leading to the fusion of the trans-activating domain of EWS with the FLU DNA binding domain. Cancer stem cells (CSCs) are thought to be the driving force of tumor growth. In this work we focused on understanding the mechanisms underlying ESFT CSC emergence as well as defining targeted therapeutic strategies. Emergence of CSCs in ESFT has been shown to arise from a defect in TARBP2-dependent microRNA maturation, which can be corrected by exposure to the fluoroquinolone enoxacin. As enoxacin alone is not sufficient to reverse tumor growth in vivo, we assessed the effect of combining a drug that abrogates CSC properties with doxorubicin, a standard-of-care therapy in ESFT. Primary ESFT CSCs and bulk tumor cells were treated with different concentration of drugs and displayed divergent responses to doxorubicin and enoxacin. Doxorubicin, which targets the tumor bulk, displayed toxicity toward primary adherent ESFT cells in culture but not to CSC-enriched ESFT spheres. Conversely, enoxacin induced apoptosis but only in ESFT spheres and specifically on the CD133+ population. In combination, the two drugs markedly depleted CSC and strongly reduced primary growth in xenograft assays of two primary ESFT. Our results identify a potentially attractive therapeutic strategy for ESFT that combines mechanism-based targeting of CSC using a low toxicity antibiotic with a standard-of-care cytotoxic drug, offering immediate applications for clinical evaluation. In the second part of this work we performed chromatin immunopercipitation on CSCs and bulk cells for EWS-FLI1 binding as well as some chromatin modifications, and concluded that EWS-FLI1 shows cell context dependent binding.

Congenital hypogonadotropic hypogonadism with split hand/foot malformation: a clinical entity with a high frequency of FGFR1 mutations.

PURPOSE: Congenital hypogonadotropic hypogonadism (CHH) and split hand/foot malformation (SHFM) are two rare genetic conditions. Here we report a clinical entity comprising the two. METHODS: We identified patients with CHH and SHFM through international collaboration. Probands and available family members underwent phenotyping and screening for FGFR1 mutations. The impact of identified mutations was assessed by sequence- and structure-based predictions and/or functional assays. RESULTS: We identified eight probands with CHH with (n = 3; Kallmann syndrome) or without anosmia (n = 5) and SHFM, seven of whom (88%) harbor FGFR1 mutations. Of these seven, one individual is homozygous for p.V429E and six individuals are heterozygous for p.G348R, p.G485R, p.Q594*, p.E670A, p.V688L, or p.L712P. All mutations were predicted by in silico analysis to cause loss of function. Probands with FGFR1 mutations have severe gonadotropin-releasing hormone deficiency (absent puberty and/or cryptorchidism and/or micropenis). SHFM in both hands and feet was observed only in the patient with the homozygous p.V429E mutation; V429 maps to the fibroblast growth factor receptor substrate 2α binding domain of FGFR1, and functional studies of the p.V429E mutation demonstrated that it decreased recruitment and phosphorylation of fibroblast growth factor receptor substrate 2α to FGFR1, thereby resulting in reduced mitogen-activated protein kinase signaling. CONCLUSION: FGFR1 should be prioritized for genetic testing in patients with CHH and SHFM because the likelihood of a mutation increases from 10% in the general CHH population to 88% in these patients.Genet Med 17 8, 651-659.

Synergism between a foldase and an unfoldase: reciprocal dependence between the thioredoxin-like activity of DnaJ and the polypeptide-unfolding activity of DnaK.

The role of bacterial Hsp40, DnaJ, is to co-chaperone the binding of misfolded or alternatively folded proteins to bacterial Hsp70, DnaK, which is an ATP-fuelled unfolding chaperone. In addition to its DnaK targeting activity, DnaJ has a weak thiol-reductase activity. In between the substrate-binding domain and the J-domain anchor to DnaK, DnaJ has a unique domain with four conserved CXXC motives that bind two Zn(2+) and partly contribute to polypeptide binding. Here, we deleted in DnaJ this Zn-binding domain, which is characteristic to type I but not of type II or III J-proteins. This caused a loss of the thiol-reductase activity and strongly reduced the ability of DnaJ to mediate the ATP- and DnaK-dependent unfolding/refolding of mildly oxidized misfolded polypeptides, an inhibition that was alleviated in the presence of thioredoxin or DTT. We suggest that in addition to their general ability to target misfolded polypeptide substrates to the Hsp70/Hsp110 chaperone machinery, Type I J-proteins carry an ancillary protein dithiol-isomerase function that can synergize the unfolding action of the chaperone, in the particular case of substrates that are further stabilized by non-native disulfide bonds. Whereas the unfoldase can remain ineffective without the transient untying of disulfide bonds by the foldase, the foldase can remain ineffective without the transient ATP-fuelled unfolding of wrong local structures by the unfoldase.

PPARα is Required for PPARδ action in regulation of body weight and hepatic steatosis in mice.

NlmCategory="UNASSIGNED">Peroxisome proliferator activated receptors alpha (PPARα) and delta (PPARδ) belong to the nuclear receptor superfamily. PPARα is a target of well established lipid-lowering drugs. PPARδ (also known as PPARβ/δ) has been investigated as a promising antidiabetic drug target; however, the evidence in the literature on PPARδ effect on hepatic lipid metabolism is inconsistent. Mice conditionally expressing human PPARδ demonstrated pronounced weight loss and promoted hepatic steatosis when treated with GW501516 (PPARδ-agonist) when compared to wild type mice. This effect was completely absent in mice with either a dominant negative form of PPARδ or deletion of the DNA binding domain of PPARδ. This confirmed the absolute requirement for PPARδ in the physiological actions of GW501516 and confirmed the potential utility against the human form of this receptor. Surprisingly the genetic deletion of PPARα also abrogated the effect of GW501516 in terms of both weight loss and hepatic lipid accumulation. Also the levels of the PPARα endogenous agonist 16:0/18:1-GPC were shown to be modulated by PPARδ in wild type mice. Our results show that both PPARδ and PPARα receptors are essential for GW501516-driven adipose tissue reduction and subsequently hepatic steatosis, with PPARα working downstream of PPARδ.

Novel CO2 Regulated Proteins in Synechocystis PCC 6803

The large biodiversity of cyanobacteria together with the increasing genomics and proteomics metadata provide novel information for finding new commercially valuable metabolites. With the advent of global warming, there is growing interest in the processes that results in efficient CO2 capture through the use of photosynthetic microorganisms such as cyanobacteria. This requires a detailed knowledge of how cyanobacteria respond to the ambient CO2. My study was aimed at understanding the changes in the protein profile of the model organism, Synechocystis PCC 6803 towards the varying CO₂ level. In order to achieve this goal I have employed modern proteomics tools such as iTRAQ and DIGE, recombinant DNA techniques to construct different mutants in cyanobacteria and biophysical methods to study the photosynthetic properties. The proteomics study revealed several novel proteins, apart from the well characterized proteins involved in carbon concentrating mechanisms (CCMs), that were upregulated upon shift of the cells from high CO₂ concentration (3%) to that in air level (0.039%). The unknown proteins, Slr0006 and flavodiiron proteins (FDPs) Sll0217-Flv4 and Sll0219-Flv2, were selected for further characterization. Although slr0006 was substantially upregulated under C_i limiting conditions, inactivation of the gene did not result in any visual phenotype under various environmental conditions indicating that this protein is not essential for cell survival. However, quantitative proteomics showed the induction of novel plasmid and chromosome encoded proteins in deltaslr0006 under air level CO₂ conditions. The expression of the slr0006 gene was found to be strictly dependent on active photosynthetic electron transfer. Slr0006 contains conserved dsRNA binding domain that belongs to the Sua5/YrdC/YciO protein family. Structural modelling of Slr0006 showed an alpha/beta twisted open-sheet structure and a positively charged cavity, indicating a possible binding site for RNA. The 3D model and the co-localization of Slr0006 with ribosomal subunits suggest that it might play a role in translation or ribosome biogenesis. On the other hand, deletions in the sll0217-sll218- sll0219 operon resulted in enhanced photodamage of PSII and distorted energy transfer from phycobilisome (PBS) to PSII, suggesting a dynamic photoprotection role of the operon. Constructed homology models also suggest efficient electron transfer in heterodimeric Flv2/Flv4, apparently involved in PSII photoprotection. Both Slr0006 and FDPs exhibited several common features, including negative regulation by NdhR and ambiguous cellular localization when subjected to different concentrations of divalent ions. This strong association with the membranes remained undisturbed even in the presence of detergent or high salt. My finding brings ample information on three novel proteins and their functions towards carbon limitation. Nevertheless, many pathways and related proteins remain unexplored. The comprehensive understanding of the acclimation processes in cyanobacteria towards varying environmental CO₂ levels will help to uncover adaptive mechanisms in other organisms, including higher plants.

P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships

Resistance to chemotherapy in cancer cells is mainly mediated by overexpression of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. Pgp consists of two homologous halves each containing a transmembrane domain and a cytosolic nucleotide-binding domain (NBD) which contains two consensus Walker motifs, A and B, involved in ATP binding and hydrolysis. The protein also contains an S signature characteristic of ABC transporters. The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellular function has been described as a "hydrophobic vacuum cleaner". The limited knowledge about the mechanism of Pgp, partly due to the lack of a high-resolution structure, is well reflected in the failure to efficiently inhibit its activity in cancer cells and thus to reverse multidrug resistance (MDR). In contrast to the difficulties encountered when studying the full-length Pgp, the recombinant NBDs can be obtained in large amounts as soluble proteins. The biochemical and biophysical characterization of recombinant NBDs is shown here to provide a suitable alternative route to establish structure-function relationships. NBDs were shown to bind ATP and analogues as well as potent modulators of MDR, such as hydrophobic steroids, at a region close to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new promising class of bifunctional modulators of Pgp.

A calcineurin inhibitory protein overexpressed in Down's syndrome interacts with the product of a ubiquitously expressed transcript

The Down's syndrome candidate region 1 (DSCR1) protein, encoded by a gene located in the human chromosome 21, interacts with calcineurin and is overexpressed in Down's syndrome patients. As an approach to clarifying a putative function for this protein, in the present study we used the yeast two-hybrid system to identify DSCR1 partners. The two-hybrid system is a method that allows the identification of protein-protein interactions through reconstitution of the activity of the yeast GAL 4 transcriptional activator. The gene DSCR1 fused to the GAL 4 binding domain (BD) was used to screen a human fetal brain cDNA library cloned in fusion with the GAL 4 activation domain (AD). Three positive clones were found and sequence analysis revealed that all the plasmids coded for the ubiquitously expressed transcript (UXT). UXT, which is encoded in human Xp11, is a 157-amino acid protein present in both cytosol and nucleus of the cells. This positive interaction of DSCR1 and UXT was confirmed in vivo by mating the yeast strain AH109 (MATa)expressing AD-UXT with the strain Y187 (MATalpha) expressing BD-DSCR1, and in vitro by co-immunoprecipitation experiments. These results may help elucidate a new function for DSCR1 and its participation in Down's syndrome pathogenesis.

Glutamate receptors as seen by light: spectroscopic studies of structure-function relationships

Ionotropic glutamate receptors are major excitatory receptors in the central nervous system and also have a far reaching influence in other areas of the body. Their modular nature has allowed for the isolation of the ligand-binding domain and for subsequent structural studies using a variety of spectroscopic techniques. This review will discuss the role of specific ligand:protein interactions in mediating activation in the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors as established by various spectroscopic investigations of the GluR2 and GluR4 subunits of this receptor. Specifically, this review will provide an introduction to the insight gained from X-ray crystallography and nuclear magnetic resonance investigations and then go on to focus on studies utilizing vibrational spectroscopy and fluorescence resonance energy transfer to study the behavior of the isolated ligand-binding domain in solution and discuss the importance of specific ligand:protein interactions in the mechanism of receptor activation.

Synechococcus sp. PCC 7002 CpcB lyase null mutations alter phycocyanin chromophore function and, consequently, affect the redistribution of excitation energy via the light state transition /

Phycobilisomes are the major light harvesting complexes for cyanobacteria and phycocyanin is the primary phycobiliprotein of the phycobilisome rod. The phycocyanobilin lyases responsible for chromophorylating the phycocyanin p subunit (CpcB) have been recently identified in the cyanobacterium Synechococcus sp. PCC 7002. Surprisingly, mutants missing the CpcB lyases were nevertheless capable of producing pigmented phycocyanin. 10K absorbance measurements revealed that the energy states of the p phycocyanin chromophores were only subtly shifted; however, 77K steady state fluorescence emission spectroscopy showed excitation energy transfer involving the targeted chromophores to be highly disrupted. Such evidence suggests that phycobilin orientation within the binding domain is specifically modified. We hypothesized that alternate, less specific lyases are able to act on the p binding sites. A phycocyanin linker-polypeptide deficient mutant was similarly characterized. The light state transition, a short term adaptation of the photosynthetic light harvesting apparatus resulting in the redistribution of excitation energy among the photo systems, was shown to be dominated by the reallocation of phycocyanin-absorbed excitation energy. Treatment with a high M phosphate buffer effectively prevented the redistribution of both chlorophyll a- and phycobilisome- absorbed excitation energy, suggesting that the two effects are not strictly independent. The mutant strains required a larger redistribution of excitation energy between light states, perhaps to compensate for their loss in phycobilisome antenna function.

Rapid [Gamma]-amino acid synthesis and the inhibition of the growth and development of oblique banded leaf-roller larvae

The hypothesis that rapid y-aminobutyric acid (GABA) accumulation is a plant defense against phytophagous insects was investigated. Simulation of mechanical damage resulting from phytophagous insect activity increased soybean (Glycine max L.) leaf GABA 10- to 25-fold within 1 to 4 min. Pulverizing leaf tissue resulted in a value of 2. 15 (±O. 11 SE) ~mol GABA per gram fresh weight. Increasing the GABA levels in a synthetic diet from 1.6 to 2.6 Jlffiol GABA per gram fresh weight reduced the growth rates, developmental rates, total biomass (50% reduction), and survival rates (30% reduction) of cultured Oblique banded leaf-roller (OBLR) (Choristonellra rosacealla Harris) larvae. In field experiments OBLR larvae were found predominantly on young terminal leaves which have a reduced capacity to produce GABA in response to mechanical damage. Glutamate decarboxylase (GAD) is a cytosolic enzyme which catalyses the decarboxylation of L-Glu to GABA. GAD is a calmodulin binding enzyme whose activity is stimulated dramatically by increased cytosolic H+ or Ca2 + ion concentrations. Phytophagous insect activity will disrupt the cellular compartmentation of H+ and Ca2 +, activate GAD and subsequent GABA accumulation. In animals GABA is a major inhibitory neurotransmitter. The possible mechanisms resulting in GABA inhibited growth and development of insects are discussed.