The amidohydrolase superfamily is a functionally diverse group of evolutionarily related proteins which utilize metal cofactors in the activation of a hydrolytic water molecule and in the stabilization of the resulting tetrahedral intermediate. Members of this superfamily have been described which use one or two divalent transition metals. These metal cofactors are located in either or both of two active-site metal binding centers which are labeled as the Ma and MB sites. The goal of this research was to elucidate the nature of the reactions catalyzed by Ma and MB mononuclear members of the amidohydrolase superfamily. This was approached through comprehensive mechanistic evaluations of two enzymes which utilized the different metal sites. Nacetyl- D-glucosamine-6-phosphate deacetylase from E. coli (NagA) and cytosine deaminase from E. coli (CDA) served as models for mononuclear amidohydrolase superfamily enzymes which have evolved to utilize a single B-metal and a single a-metal for hydrolysis, respectively. This research elucidated the different properties imparted by the distinct a and B active sites and the specific interactions utilized by the enzymes for substrate binding and catalysis. These studies led to the eventual proposal of detailed chemical mechanisms and the identification of rate determining steps. Knowledge of sequence-function relationships was applied toward the discovery of function for enzymes related to cytosine deaminase and guanine deaminase. The first group of enzymes investigated was proposed to catalyze the fourth step in riboflavin and coenzyme F420 biosynthesis in Achaea. Three putative deaminases; Mm0823 from Methanosarcina mazei, MmarC7_0625 from Methanococcus maripaludis C7 and Sso0398 from Sulfolobus solfataricus were cloned and expressed. These proteins proved to be intractably insoluble. A second set of enzymes, Pa0142 from Pseudomonas aeruginosa PA01 and SGX-9236e (with crystal structure PDB: 3HPA) were found to catalyze the novel deamination of 8-oxoguanine, a mutagenic product of DNA oxidation. 9236e was cloned from an unidentified environmental sample of the Sargasso Sea. The closest homolog (98% identical) is Bcep18194_A5267 from Burkholderia sp. 383. Additionally, it was discovered that the proteins SGX-9339a (with crystal structure PDB: 2PAJ) and SGX-9236b catalyzed the deamination of isoxanthopterin and pterin-6- carboxylate in a poorly characterized folate degradation pathway. These enzymes were also from unknown environmental samples of the Sargasso Sea. The closest homolog of 9339a (88% identical) is Bxe_A2016 from Burkholderia xenovorans LB400. The closest homolog of 9236b (95% identical) is Bphyt_7136 from Burkholderia phytofirmans PsJN.

Bacteria employ survival strategies to protect themselves against foreign invaders, including bacteriophages. The ‘immune system’ of bacteria relies mostly on restriction-modification (R-M) systems. The primary role of R-M systems is to protect the host from invading foreign DNA molecules. Three major types of R–M system are found in bacteria, viz.Types I, II and III. Type II R–M systems comprise a separate restriction endonuclease (REase) and a methyltransferase (MTase) that act independently of each other. Type II REases generally recognize palindromic sequences in DNA and cleave within or near their recognition sequences and produce DNA fragments of defined sizes. They have become indispensable tools in molecular biology and have been widely exploited for studying site-specific protein–DNA interactions. Surprisingly, these enzymes share little or no sequence homology among them, though the three-dimensional structures determined to date reveal a common-core motif (‘PD...D/EXK’ motif) with a central β-sheet that is flanked by α-helices on both sides. In the motif, two acidic residues (D and D/E) are important for the metal ion binding and catalysis. The work presented in this thesis deals with the determination of active site, elucidation of kinetic mechanism and study of evolution of sequence specificity using the well known, R.KpnI, from Klebsiella pneumoniae. The enzyme is a homodimer, which recognizes a palindromic double stranded DNA sequence, GGTAC↓C, and cleaves as shown. Unlike other REases, R.KpnI shows prolific promiscuous DNA cleavage in presence of Mg2+. Surprisingly, Ca2+ completely suppresses the Mg2+ mediated promiscuous activity and induces high fidelity cleavage at the recognition sequence. These unusual properties of R.KpnI led to the characterization of the active site of the enzyme. This thesis is divided into five chapters. Chapter 1 is a general introduction of R-M systems and an overview of the literature on active sites of Type II REases. It deals with discovery, nomenclature and classification followed by description of the enzymes diversity and general features of Type II REases. The different active site folds of the REases have been discussed in detail. The features of sequence specificity and the efforts undertaken to engineer the new specificity in the REases have been dealt at the end of the chapter. Chapter 2 describes identification and characterization of the R.KpnI active site by bioinformatics analyses, homology modeling and mutational studies. Bioinformatics analyses reveal that R.KpnI contains a ββα-Me-finger fold, which is a characteristic of many HNH-superfamily endonucleases. According to the homology model of R.KpnI, the putative active site residues correspond to the conserved residues present in HNH nucleases. Substitutions of these conserved residues in R.KpnI resulted in loss of the DNA cleavage activity, confirming their importance. This study provides the first experimental evidence for a Type IIP REase that is a member of the HNH superfamily and does not belong to the PD...D/EXK superfamily of nucleases. In Chapter 3 DNA binding and kinetic analysis of R.KpnI is presented. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, it was shown that Ca2+-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. The kinetic constants were obtained through steady-state kinetic analysis of R.KpnI in presence of different metal ions. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg2+- and Mn2+-mediated reactions and was about three times slower with Ca2+. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg2+-mediated reactions unlike any other Type II REases, accounting for its promiscuous behavior. These studies suggest that R.KpnI displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity for DNA recognition and cleavage. In chapter 4, two uncommon roles for Zn2+ in R.KpnI are described. Examination of the sequence revealed the presence of a zinc finger (CCCH) motif rarely found in proteins of prokaryotic origin. Biophysical experiments and subsequent mutational analysis showed that the zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in a REase. Chapter 5 describes generation of highly sequence specific R.KpnI. Towards this end, site-directed mutants were generated at the putative secondary metal binding site. The DNA binding and cleavage analyses of the mutants at putative secondary metal binding site revealed that the secondary site is not important for primary catalysis and have a role in sequence specificity. A single amino acid change at the D163 position abolished the promiscuous activity of the wt enzyme in the presence of Mg2+ and Mn2+. Thus, a single point mutation converts the promiscuous endonuclease to a high fidelity REase. In conclusion, the work described in the thesis reveals new information on the REases in general and R.KpnI in particular. Many of the properties of R.KpnI elucidated in this thesis represent hitherto unknown features amongst REases. The presence of an HNH catalytic motif in the enzyme indicates the diversity of active site fold in REases and their distinct origin. Similarly, the high degree of promiscuity exhibited by the enzyme may hint at the evolutionary link between non-specific and highly sequence specific nucleases. The present studies also provide an example for the role of mutations in the evolution of sequence specificity. The utilization of different metal ions for DNA cleavage and the architectural role for Zn2+ in maintaining the structural integrity are other unusual properties of the enzyme.

SUMMARYThe diversity of cancer molecular origins associated with the genetic variability of patients has encouraged the development of chemotherapeutic treatments adapted not only to the target tumor, but also to a specific patient. This personalized strategy is based on cancer biomarkers allowing a better identification and characterization of each tumor where predictive biomarkers provide the distinction between various factors indicative of the response to the treatment. In this context, several studies highlighted the role of the solute carrier transporter family 22 (solute carriers 22 or SLC22) in the uptake of platinum anticancer drugs. This mechanism being not well understood, our work intends to establish the potential role of SLC22 member A18 (SLC22A18) as predictive biomarker in the aim to help to a better targeted chemotherapeutic strategy for each patient. We optimized a system overexpressing SLC22A18 stably or transiently in HeLa cancer cell line. SLC22A18 expression was confirmed by qRT-PCR, western blotting, microscopy and flow cytometry. The cell lines were treated with taxane, anthracyclin, vinca alkaloid and nitrosoureas anticancer drug families. We showed that doxorubicin, camptothecin, chloroquine, tetracycline and carmustin had no effect on the cell viability assays suggesting that they are not substrates of SLC22A18. Interestingly, the cell line was sensitized in the presence of antimitotic drug with a sensitivity factor of 2.7 in the presence of paclitaxel, 1.4 with docetaxel, 1.8 with vinblastin and 2.2 in the presence of vincristine. To confirm these results, we elaborated a SLC22A18 knockdown cell line in HS683 cells using siRNA technology. The downexpression of SLC22A18 was correlated to a tendency to resist to the accumulation of paclitaxel thereby confirming the previous results. Simultaneously, a knockout cell line was established using the transcription activator-like effectors nuclease (TALEN) technology in U373 cell line. Our studies constitute a robust base of knowledge for further investigation on SLC22A18 transporter as a predictive biomarker promoting antimitotic treatment in tumors where this transporter is detected. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Solute carriers (SLCs) constitute the largest family of membrane-bound transporter proteins in humans, and they convey transport of nutrients, ions, drugs and waste over cellular membranes via facilitative diffusion, co-transport or exchange. Several SLCs are associated with diseases and their location in membranes and specific substrate transport makes them excellent as drug targets. However, as 30 % of the 430 identified SLCs are still orphans, there are yet numerous opportunities to explain diseases and discover potential drug targets. Among the novel proteins are 29 atypical SLCs of major facilitator superfamily (MFS) type. These share evolutionary history with the remaining SLCs, but are orphans regarding expression, structure and/or function. They are not classified into any of the existing 52 SLC families. The overall aim in this thesis was to study the atypical SLCs with a focus on their phylogenetic clustering, evolutionary conservation, structure, protein expression in mouse brains and if and how their gene expressions were affected upon changed food intake. In Papers I-III, the focus was on specific proteins, MFSD5 and MFSD11 (Paper I), MFSD1 and MFSD3 (Paper II), and MFSD4A and MFSD9 (Paper III). They all shared neuronal expression, and their transcription levels were altered in several brain areas after subjecting mice to food deprivation or a high-fat diet. In Paper IV, the 29 atypical SLCs of MFS type were examined. They were divided into 15 families, based on phylogenetic analyses and sequence identities, to facilitate functional studies. Their sequence relationships with other SLCs were also established. Some of the proteins were found to be well conserved with orthologues down to nematodes and insects, whereas others emerged at first in vertebrates. The atypical SLCs of MFS type were predicted to have the common MFS structure, composed of 12 transmembrane segments. With single-cell RNA sequencing and in situ proximity ligation assay, co-expression of atypical SLCs was analysed to get a comprehensive understanding of how membrane-bound transporters interact.   In conclusion, the atypical SLCs of MFS type are suggested to be novel SLC transporters, involved in maintaining nutrient homeostasis through substrate transport.

L'engagement des cellules vers un destin donné, ou détermination cellulaire, est un processus clé du développement. Quels sont les mécanismes qui sous-tendent la détermination cellulaire ? Pour aborder cette question, nous utilisons le lignage des soies mécanosensorielles de la drosophile. Dans ce lignage, la diversité des cellules résulte de l'activation différentielle de la voie Notch ainsi que la ségrégation asymétrique de déterminants cellulaire à chaque division. Cependant, comment la répétition d’un même mécanisme peut-elle être à l’origine des destins cellulaires différents ? D'autres facteurs doivent être impliqués dans ce processus. Afin des les identifier, mon intérêt s’est porté sur les facteurs de transcription de la superfamille Snail, connus pour être impliqués dans la détermination cellulaire au cours du développement de la drosophile.Deux membres de cette superfamille, Escargot (Esg) et Scratch (Scrt) sont exprimés dans le lignage des soies, en particulier dans les cellules neurales et leurs précurseurs. Des analyses de perte et de gain de fonction indiquent qu’Esg et Scrt, agissant de manière redondante, sont nécessaires pour le maintien de l'identité du précurseur secondaire neural. Des tests d’interaction génétique ont montré que ces facteurs agissent en interaction avec la voie Notch, probablement via la répression de l’expression des gènes cibles de la voie. De plus, Esg, mais pas Scrt, a un rôle supplémentaire lors de la formation du lignage des soies. La perte de fonction de ce facteur provoque un défaut de l’arborisation et de la croissance axonales. En outre, l'expression des gènes impliqués dans la différenciation neuronale, tels que Elav et Prospero, est altérée dans ce contexte, suggérant qu’Esg contrôle la différenciation neuronale en régulant l'expression de gènes clés de l’identité neuronale.Ensemble, mes résultats ont montré qu’Esg et Scrt participent à la mise en place de la diversité cellulaire dans le lignage des soies de la drosophile. / The commitment of cells to a given fate, or cell fate determination, is a key process in development. Cell type diversity arises from variations in this process. What are the mechanisms underlying cell determination and how is cell diversity achieved? In order to approach these questions, we use the Drosophila mechanosensory bristle lineage. In this lineage, cell diversity arises from the differential activation of the Notch pathway as well as the asymmetric segregation of cell fate determinants at each division. However, how does the repetition of the same mechanism trigger different cell fates? Other factors might be involved in cell fate commitment. In order to identify such factors, I focused my interest on the transcription factor of the Snail superfamily, known to be involved in cell determination during Drosophila development.Two members of this superfamily, escargot (esg) and scratch (scrt) are expressed in the bristle lineage, specifically in the inner neural cells and their precursor cells. Loss and gain of function analysis indicate that Esg and Scrt, acting redundantly, are necessary for the maintenance of the neural secondary precursor cell identity. A genetics interaction test showed that this role is achieved in interaction with the Notch pathway, probably through the repression of Notch target genes expression. Moreover, Esg, but not Scrt, has an additional role during the inner bristle cell formation. Loss of function of this factor induces a defect in neuronal differentiation, specifically axon growth and patterning. Moreover, the expression of genes involved in neuronal differentiation, such as elav and prospero, is impaired in this context. Altogether, these data suggests that Esg is involved in neuronal differentiation by regulating the expression of key neuronal genes.Together, my results showed that Esg and Scrt participate to the establishment of cell diversity in Drosophila bristle cell lineage.

1. The effects of external H-7, a potent protein kinase inhibitor, on the responses mediated by γ-aminobutyric acid A type (GAGA(A))-, nicotinic acetylcholine (nicotinic ACh)-, ionotropic 5-hydroxytryptamine (5-HT3)-, adenosine 5'-triphosphate (ATP)-, N-methyl-D-aspartate (NMDA)- and kainate (KA)-receptors were studied in freshly dissociated rat dorsal root ganglion neurone by use of whole cell patch-clamp technique. 2. External H-7 (1-1000 μM) produced a reversible, dose-dependent inhibition of whole cell currents activated by GABA, ACh and 5-HT. 3. Whole-cell currents evoked by ATP, 2-methylthio-ATP, NMDA and KA were sensitive to external H-7. 4. External H-7 shifted the dose-response curve of GABA-activated currents downward without changing the EC50 significantly (from 15.0 ± 4.0 μM to 18.0 ± 5.0 μM). The maximum response to GABA was depressed by 34.0 ± 5.3%. This inhibitory action of H-7 was voltage-independent. 5. Intracellular application of H-7 (20 μM), cyclic AMP (1 mM) and BAPTA (10 mM) could not reverse the H-7 inhibition of GABA-activated currents. 6. The results suggest that external H-7 selectively and allosterically modulates the functions of GABA(A)-, nicotine ACh- and 5-HT3 receptors via a common conserved site in the external domain of these receptors.

Myostatin (GDF-8) is the chief chalone in skeletal muscle and negatively controls adult skeletal muscle growth. The role of myostatin during overload-induced hypertrophy of adult muscle is unclear. We tested the hypothesis that overloaded adult rodent skeletal muscle would result in reduced myostatin protein levels. Overload-induced hypertrophy was accomplished by unilateral tenotomy of the gastrocnemius tendon in male adult Sprague-Dawley rats followed by a two-week period of compensatory overload of the plantaris and soleus muscles. Western blot analysis was performed to evaluate changes in active, latent and precursor myostatin protein levels. Significant hypertrophy was noted in the plantaris (494 ± 29 vs. 405 ± 15 mg, p < 0.05) and soleus (289 ± 12 vs. 179 ± 37 mg, p < 0.05) muscles following overload. Overloaded soleus muscle decreased the concentration of active myostatin protein by 32.7 ± 9.4% (p < 0.01) while the myostatin precursor protein was unchanged. Overloaded plantaris muscle decreased the concentration of active myostatin protein by 28.5 ± 8.5% (p < 0.01) while myostatin precursor levels were reduced by 17.5 ± 5.9% (p < 0.05). Myostatin latent complex concentration decreased in the overloaded soleus and plantaris muscle by 15.0 ± 5.9% and 70.0 ± 2.3% (p < 0.05), respectively. These data support the hypothesis that the myostatin signaling pathway in overloaded muscles is generally downregulated and contributes to muscle hypertrophy. Plasma concentrations of total and active myostatin proteins were similar in overloaded and control animals and averaged 8865 ± 526 pg/ml and 569 ± 28 pg/ml, respectively. Tissue levels of BMP-1, an extracellular proteinase that converts myostatin to its active form, also decreased in overloaded soleus and plantaris muscles by 40.4 ± 12.9% and 32.9 ± 6.9% (p < 0.01), respectively. These data support the hypothesis that local, rather than systemic, regulation of myostatin contributes to the growth of individual muscles, and that an association exists between the extracellular matrix proteinase BMP-1 and the amount of active myostatin in overloaded muscles.

No description available.

Scaffolding proteins belonging to the Membrane Associated GUanylate Kinase (MAGUK) superfamily function as adaptors linking cytoplasmic and cell surface proteins to the cytoskeleton to regulate cell-cell adhesion, cell-cell communication and signal transduction. We have identified a novel Drosophila MAGUK member, Varicose (Vari), the homologue of vertebrate scaffolding protein PALS2. Similar to its vertebrate counterpart, Varicose localizes to pleated Septate Junctions (pSJs) of all embryonic, ectodermally derived epithelia and peripheral glia. In vari mutants, essential SJ proteins NeurexinIV and FasciclinIII are mislocalized basally and the cells develop a leaky paracellular seal. Localization of SJ protein Discs Large is not affected, indicating Vari is not involved in cell polarization. In addition, vari mutants display irregular tracheal tube diameters and have reduced lumenal protein accumulation suggesting involvement in tracheal morphogenesis. We found that Vari is distributed in the cytoplasm of optic lobe neuroepithelium and is required for proper ommatidial patterning. As well, Vari is expressed in a subset of neuroblasts and differentiated neurons of the nervous system. We also present a novel MAGUK function in wing hair alignment during adult morphogenesis. We conclude that Varicose is involved in scaffold assembly at the SJ and has a role in patterning adult epithelia and in nervous system development. / Thesis / Doctor of Philosophy (PhD)

No description available.