A thesis submitted to the Faculty of Medicine, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 1988

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Thesis (Ph. D.)--University of Lund, 1992. / Published dissertation. Includes bibliographical references.

Thesis (Ph. D.)--University of Lund, 1992. / Published dissertation. Includes bibliographical references.

Dissertation (Ph.D.)--Auburn University,2009. / Abstract. Vita. Includes bibliographic references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted. Errata slip inserted. Includes bibliographical references.

A large number of evolutionarily conserved genes have been identified by comparative genomics approaches. However, a considerable fraction of these genes lack functional characterization despite the availability of several bioinformatics approaches for prediction of protein function. Moreover, with the advent of genome sequencing efforts, numerous disease associated genes have been identified. While high throughput approaches aid in identification of genes, studying individual genes is important to understand their cellular roles. During studies on cyclic AMP metabolism in mycobacteria conducted in the laboratory, a Class III cyclic nucleotide phosphodiesterase, Rv0805 was identified from Mycobacterium tuberculosis. Interestingly, additional bioinformatics analysis identified orthologs were in higher eukaryotes. These were members of the metallophosphoesterase-domain-containing protein 1 (MPPED1) and metallophosphoesterase-domain-containing protein 2 (MPPED2) family. Class III cyclic nucleotide phosphodiesterases were previously reported only in prokaryotes and are distinct from Class I cyclic nucleotide phosphodiesterases generally found in eukaryotes. Thus MPPED1 and MPPED2 proteins were the first identified eukaryotic Class III cyclic nucleotide phosphodiesterases. In humans, MPPED2 is located on chromosome 11 in the region p13-14 that has been associated with WAGR (Wilms’ tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome. Inspection of this region across sequenced mammalian genomes has revealed a shared synteny. Most interestingly, a stretch of 200 bp within the coding sequence of MPPED2 is identified to be one of 481 ultra conserved regions within the human genome. Furthermore, orthologs of MPPED2 can be traced all the way back to Drosophila melanogaster and Caenorhabditis elegans. All of these observations indicate that MPPED2 is highly conserved and hints at its likely importance in many organisms. MPPED1 and MPPED2 have been reported to be expressed in adult and fetal brain respectively and have been annotated as metallophosphoesterases. Metallophosphoesterases are a superfamily of proteins that show wide phyletic distribution and exhibit diversity in their substrate utilization and function. Previous studies from the laboratory have shown that MPPED1 and MPPED2 are indeed metallophosphoesterases and demonstrate cyclic nucleotide phosphodiesterase activity. The crystal structure of MPPED2 was obtained in collaboration with Dr. Marjetka Podobnik (National Institute of Chemistry, Slovenia). Interestingly, the crystal structure of MPPED2 revealed the presence of bound 5’GMP molecule at the active site, and this finding was investigated further in this thesis. MPPED2 bound 5’GMP and 5’AMP with high affinity (IC50 of ~70 nM) which inhibited the activity of MPPED2. Key residues involved in stabilising the 5’ nucleotide have been identified by structure guided mutational analysis. The MPPED2-G252H mutant, generated to mimic the active site of MPPED1, also bound 5’GMP or 5’AMP but with much lower affinity. Given the high affinity of MPPED2 towards 5’GMP/5’AMP, it can be speculated that MPPED2 may show poor phosphodiesterase activity in the cell, and could function in a catalytically-independent manner, perhaps as a scaffolding protein. MPPED1 on the other hand may have a catalytic role that could be regulated by intracellular levels of 5’AMP, 5’GMP and their respective cyclic nucleotides. In order to investigate the biological role of the MPPED1/MPPED2 family of proteins, Drosophila melanogaster was chosen as a model organism owing to the presence of a single ortholog, CG16717, in its genome. Biochemical characterization of CG16717 revealed that the protein was in fact a metallophosphodiesterase capable of hydrolysing cyclic AMP and cyclic GMP, albeit poorly. CG16717 could be inhibited by 5’ nucleotides at high concentrations that may seldom be achieved in-vivo, suggesting that CG16717 may have roles in the organism that depend on its catalytic activity. CG16717 has not been functionally characterized previously. In this thesis, a detailed analysis of CG16717 expression pattern has been performed. CG16717 was found to be expressed in all stages of the fly lifecycle. In adult female flies, levels of CG16717 increased across age. Moreover, CG16717 was not differentially regulated under conditions of starvation, paraquat-induced oxidative stress or in the presence of heavy metals. Spatial expression analysis revealed that CG16717 was expressed in all adult tissues tested, with maximal expression in the brain, suggesting that neuronal expression of CG16717 may be important for its function. Attempts to identify specific cells expressing CG16717 using an enhancer-promoter analysis were not successful. In order to elucidate the physiological role of CG16717, and after having ruled out options of using a P-element insertion mutant and RNA interference approaches, a targeted knock-out of CG16717 was generated using homologous recombination based genomic engineering. CG16717KO flies generated were homozygous viable suggesting that CG16717 was dispensable for fly survival at least under normal laboratory conditions. In line with high expression of CG16717 in the brain and in-vitro ability of CG16717 to hydrolyse cAMP and cGMP, CG16717KO flies showed two to three-fold higher levels of cyclic nucleotides in the head fraction than wild-type flies. C25E10.12, one of the three C. elegans orthologs of CG16717 has been identified to be a target of the transcription factor daf-16 (FOXO) that is inhibited by active insulin signalling. Moreover, knock-down of C25E10.12 reduced the lifespan of age-1 (PI3K) mutant worms. In contrast to this, CG16717 was not found to be differentially regulated in dFOXO null flies. CG16717KO flies however, showed median lifespan that was shorter than control wild-type flies even in the presence of functional PI3K. Various genetic approaches were employed to verify if reduced lifespan was indeed a consequence of loss of CG16717. In the first approach, a wild-type copy of CG16717 was re-introduced at the genomic locus of CG16717 in the CG16717KO flies using attP-attB recombination. However, this approach could not rescue the reduced lifespan of CG16717KO flies, probably due to very low expression of CG16717. In the second approach, CG16717 was reconstituted using genomic constructs containing a copy of CG16717. Finally, CG16717 was expressed ubiquitously using the bipartite Gal4/UAS system. Both the genomic construct and the expression of CG16717 using the Gal4/UAS approach were able to restore the lifespan of CG16717KO flies. More importantly, overexpression of CG16717 in an otherwise wild-type fly led to enhanced lifespan over and above that of control flies. All of these together suggested that CG16717 plays a critical role in regulating lifespan. Mutants of the insulin and target of rapamycin (TOR) signalling pathways have previously been reported to show lifespan extension. Moreover, these mutants have also been associated with reduced growth, increased stress resistance and reduced fecundity. Given the reduction in lifespan of CG16717KO flies, the other insulin/TOR signalling associated phenotypes were tested. While CG16717KO flies showed no difference in terms of developmental growth, and resistance to starvation or paraquat induced oxidative stress, CG16717KO flies were less fecund compared to wild-type controls. Multiple approaches were adopted even in the case of reduced fecundity to verify if the observed phenotype was a consequence of loss of CG16717. However, neither reconstitution of CG16717 using the genomic construct nor ubiquitous expression of CG16717 using the bipartite Gal4/UAS system were able to rescue the reduced fecundity phenotype of CG16717KO flies. This suggested that reduced fecundity in CG16717KO flies was probably not linked to CG16717 and was a consequence of a second mutation at a site distinct from CG16717. Two other approaches were employed to confirm these observations. When CG16717KO/Deficiency lines were tested, these showed fecundity comparable to wild-type control flies despite the lack of CG16717. CG16717KO flies were extensively out-crossed in an attempt to segregate the second site mutation from the CG16717 locus and their fecundity was tested. However, these flies which retained the deletion of CG16717, showed fecundity comparable to wild-type control flies, reiterating that reduced fecundity was not linked to loss of CG16717. In an attempt to find possible links between reduced longevity of CG16717KO flies and the well-established insulin/TOR pathways, transcript levels of key players of these pathways were measured by qRT-PCR. The translational repressor 4EBP was found to be upregulated in CG16717KO flies compared to wild-type control flies. Interestingly, increased 4EBP levels have been associated with enhanced lifespan but in this case despite higher levels of 4EBP, CG16717KO flies showed reduced lifespan. Phosphorylation status of 4EBP and other players involved in the insulin/TOR phosphokinase signalling cascade would shed light on the activity of these pathways. In summary, this thesis has attempted to understand the biochemistry and physiological functions of an evolutionarily conserved metallophosphoesterase. Its apparent role in regulating life span in the fly suggests that the functions of this protein are likely to impinge on a number of diverse and important pathways involved in basic physiological processes in the organism. Further investigation would shed light on the molecular basis by which CG16717 affects lifespan, and opens up new avenues to understanding the contributions of CG16717 in regulating lifespan and diverse neurological functions.

Pathogenic bacteria such as M.tuberculosis have evolved several mechanisms to aid their intracellular survival and subvert host defenses. One of the contributing factors is thought to be the production and secretion of large amount of cAMP, Mycobacterial genomes encode a large number of adenylyl cyclases distinct in their structure and regulatory mechanisms. The roles of these enzymes in the physiology and pathogenesis of virulent mycobacteria are only now being elucidated. The roles of phosphodiesterases (PDEs), which serve to lower cAMP levels through degradation are, however, relatively unexplored. The Rv0805 gene was previously shown to code for an active phosphodiesterase from Mycobacterium tuberculosis. Bioinformatics analysis revealed that orthologs of Rv0805 were found even in eukaryotes. Biochemical and structural characterization of Rv0805 revealed that it was a class III cAMP phosphodiesterase. Comparative genomics identified a close ortholog of Rv0805 in M. leprae (ML2210). The genome of M. leprae Encodes only 1,604 predicted proteins and possesses the highest number of pseudogenes, 1,116. The retention of a functional PDE, the ortholog of Rv0805, in the minimal genome of M. leprae is indicative of its importance in cellular physiology. Biochemical characterization of proteins from M. leprae and use of heterologous hosts will help understand this human pathogen better, since there are no tools currently available to genetically manipulate this bacterium. Sequence analysis of ML2210 revealed the presence of conserved motifs and residues known to be critical for catalysis and unique to class III phosphodiesterases. ML2210 shares 83% sequence identity with Rv0805 and 24% sequence identity with the phosphodiesterase from E. coli (cpdA). In vitro biochemical characterization of ML2210 using non-nucleotide colorigenic and cyclic nucleotide substrates revealed that it was an enzymatically active phosphodiesterase. Kinetic parameters of ML2210 with respect ot colorigenic substrates revealed that its catalytic properties were similar to that of Rv0805. However, with respect to hydrolysis of 3’, 5’-cAMP, ML2210 was catalytically more efficient than Rv0805, suggesting that in spite of being orthologs, these enzymes have evolved distinct specificities at their active site. A parallel of monoclonal antibodies raised to Rv0805 was also used understand the differences in the biochemical properties of Rv0805 and ML2210 better. It was observed that only one monoclonal antibody was able to recognize ML2210 by ELISA and not by Western blot analysis. These results revealed that conformational differences between ML2210 and Rv0805 exist. Over-expression of ML2210 in M. smegmatis resulted in a modest decrease in intracellular cAMP levels. Despite the absence of a predicted transmembrane region or a membrane-targeting signal, ML2210 localized to the cell envelop fraction upon over expression in M. smegmatis. Moreover, like Rv0805, over-expression of ML2210 also resulted in perturbation of the cell wall of M. smegmatis, arguing for additional cellular roles of this protein. Orthologs of Rv0805 or ML2210 are found only in slow growing mycobacteria suggesting that other cyclic nucleotide phosphodiesterases could regulate cAMP levels in fast growing mycobacteria like M. smegmatis. Since BLAST results did not retrieve an ortholog of Rv0805 or ML2210, COG1409 (COG database) containing Rv0805 was examined for the presence of other mycobacterial phosphodiesterases. Bioinformatics analysis identified Rv2795c as another PDE from M. tuberculosis. Sequence analysis of Rv2795c revealed the presence of all the motifs conserved in the class III PDEs but Rv2795c shared only 22% sequence identity with Rv0805 and 19% sequence identity with CpdA. Importantly, an ortholog of Rv2795c was identified in M. leprae. Interestingly. Rv2795c and its orthologs branched away from Rv0805, making it phylogenetically distinct and hence warranting further characterization. Recombinant, purified MSMEG_2647 (the Rv2795c ortholog from M. smegmatis) was able to hydrolyze cyclic nucleotides and other phosphodiester substrates in vitro. The Km for colorigenic substrates was higher when compared to the Km of ML2210 or Rv0805 for these substrates. However, the kinetic parameters of MSMEG_2647 for cyclic nucleotides were comparable to those of ML2210 or Rv0805. MSMEG_2647 was a metal dependent enzyme and among the panel of metals tested, Mn2+ supported the highest in vitro catalytic activity of MSMEG_2647. Zn2+ inhibited the catalytic activity of MSMEG_2647. In order to gain insight into the catalysis of MSMEG_2647, the end products of cAMP hydrolysis by MSMEG_2647 were analysed using reverse phase HPLC. The assay revealed that the end products of cyclic nucleotide hydrolysis by MSMEG_2647 were different when compared to the end products of hydrolysis of the same substrates by Rv0805 or ML2210. This suggests differences in the architecture of the active site residues of the mycobacterial MPEs. A mutational anlaysis of the active site residues in MSMEG_2647 was carried out to identify residues involved in substrate recognition and metal coordination. Although Rv0805 and MSMEG_2647 shared only a 22% sequence identity, MSMEG_2647 displayed strict conservation in the core MPE motifs. Mutation of the active residues N97 and H98 in Rv0805 had led to an abrogation of its catalytic activity. However, corresponding mutations of N76A and H77A in MSMEG_2647, did not lead to a loss in its catalytic activity. A third mutation known to be important for the catalytic activity of Rv0805 (D19) was incorporated. The corresponding residue at D19 position was mutated to an alanine. The catalytic activity of MSMEG_2647D19AN76AH77A mutant was abrogated, suggesting that while the core MPE motifs are conserved between mycobacterial PDEs, differences in the ensemble of the active site residues contributing to their catalytic activity exist. Thus, at least two biochemically diverse PDE clades are found in mycobacterial species. In order to decipher the function of MSMEG_2647, its expression was monitored during the growth of M. Smegmatis. The promoter of MSMEG_2647 displayed maximum activity during the logarithmic phase of M. smegmatis growth after which its activity declined as M. smegmatis entered the stationary phase. However in contrast to this, the transcript corresponding to msmeg_2647 mRNA was found at both logarithmic and stationary phases. The MSMEG_2647 protein was also detected at both logarithmic and stationary phases of M. smegmatis. These results suggest that additional factors may contribute to the stability of msmeg_2647 mRNA and protein levels. Localization studies of MSMEG_2647 revealed that MSMEG_2647 was present in the cytosol as well as in the cell envelope fractions. Interestingly, over-expression of MSMEG_2647 did not result in a significant increase in PDE activity in various subcellular fractions, suggesting tight regulation on the in vivo activity in various subcellular fractions, suggesting tight regulation on the in vivo activity of MSMEG_2647. In addition, over-expression of MSMEG_2647 in M. smegmatis led to only a modest decrease in cAMP levels in M. smegmatis. These results suggested additional roles of MSMEG_2647 in the biology of mycobacteria. Overexpression of MSMEG_2647 peturbed the integrity of cell wall as assessed by the use of lipophillic indicators of cell growth, crystal violet and malachite green, and a cell wall targeting antibiotic, isoniazid. Analyzing the gene neighborhood of MSMEG_2647 provided an insight into its putative function. It was observed that the stop codon of msmeg_2647 overlapped with the start codon of msmeg_2648 and stop codon of msmeg-2648 overlapped with the start codon of msmeg_2649. RT PCR was carried out at logarhtimic and stationary phases of M. smegmatis growth, which revealed that a polycistronic mRNA was being transcribed. These results confirmed that msmeg_2647, msmeg_2648 and msmeg_2649 were a part of an operon. Interestingly, these three genes as a gene cluster were confined to only those actinobacteria that produced mycolic acids. An operon often encodes products that form multiprotein complexes and operate in a common pathway. Since there were a part of an operon, a GST pull-down approach was employed to test if MSMEG_2647, MSMEG_2648 and MSMEG_2649 could physically interact. It was observed that MSMEG_2647 interacted with MSMEG_2648 and MSMEG_2649. MSMEG_2648 in turn interacted with MSMEG_2649. A role for MSMEG_2647 as a scaffold recruiting MSMEG_2648 and MSMEG_2649 is therefore proposed. In turn, a complex formation with these proteins may regulate the activity of MSMEG_2647. Attempts to generate a knock out of msmeg_2647 in M. smegmatis by homologous recombination were not successful suggesting either the gene was essential or a polar effect on msmeg_2648(an essential gene for the viability of M. smegmatis) may not allow msmeg_2647 to be deleted from the genome of M. smegmatis. In summary, this study has identified and characterized two new phosphodiesterases from mycobacteria, one from the pathogenic mycobacterium, M. leprae and the other, a PDE from M. smegmatis that is conserved in all species of mycobacteria. Several, key biochemical differences were observed using biochemical and biological approaches. It appears that the cellular roles of mycobacterial phsophodiesterases may extend beyond cAMP hydrolysis, with these proteins not only regulating cell wall properties but also acting as scaffolding proteins in the cell.

Orientador: Gilberto de Nucci / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T11:10:20Z (GMT). No. of bitstreams: 1 Arruda_AndreMoreiraMartins_M.pdf: 11172054 bytes, checksum: db7fb87cc200cb091d3be4733e8d8af5 (MD5) Previous issue date: 2013 / Resumo: A presença de um par de órgãos copuladores, os hemipênis, é a característica mais singular do grupo Squamata, que reúne as serpentes e os lagartos. Para que ocorra a ereção, o hemipênis sofre ingurgitamento dos corpos cavernosos por sangue e linfa, além de contar com o auxílio da contração do músculo propulsor do pênis e o relaxamento do músculo retrator. O coito nestes animais pode durar até 28 horas, porém, os mecanismos envolvidos, as estruturas e sua base farmacológica de funcionamento são ainda pouco conhecidas. O hemipênis consiste de dois corpos cavernosos funcionalmente concêntricos, um deles contendo feixes de fibras musculares lisas. Em mamíferos, sintases de NO neuronais e endoteliais estão presentes em estruturas neurais e no endotélio, respectivamente, enquanto a guanilato ciclase solúvel e PDE5 (fosfodiesterase tipo 5) estão expressas no músculo liso trabecular. Partindo disto, para investigar as vias presentes no tecido das cobras, foram construídas curvas concentração-resposta cumulativas de relaxamento para a acetilcolina (ACh), nitroprussiato de sódio (SNP), BAY41-2272 e tadalafil em corpos cavernosos de Crotalus (CCC) pré-contraídos com fenilefrina. Relaxamentos induzidos por estímulo elétrico (EFS) também foram feitos na ausência e presença de L-NAME (100 mm), ODQ (10 mM) e tetrodotoxina (TTX, 1 mM). Em CCC pré-contraídos, o relaxamento dependente de frequência, gerado por EFS, durou três vezes mais do que aqueles em CC mamíferos. Embora estes relaxamentos sejam praticamente abolidos por L-NAME ou ODQ, eles não foram afetados pela TTX. Em contraste, o EFS promoveu relaxamento em corpos cavernosos de sagui que haviam sido incubados com TTX / Abstract: The presence of a pair of copulatory organs, the hemipenes, is the most unique feature of the group Squamata, which includes snakes and lizards. For an erection to occur, the hemipenes suffer engorgement of the corpora cavernosa with blood and lymph, besides counting with the aid of contraction of the propellant muscle and relaxation of penis retractor muscle. Coitus in these animals can last up to 28 hours, however, the mechanisms involved, the structures and their pharmacological basis are still little known. The hemipenis consists of two concentric functionally cavernous bodies, one containing bundles of smooth muscle fibers. In mammals, neuronal NO synthases and endothelial cells are present in the endothelium and neuronal structures, respectively, whereas the soluble guanylate cyclase and PDE5 (phosphodiesterase type 5) are expressed in trabecular smooth muscle. To investigas the tissue were constructed cumulative concentration-response curves for relaxation to acetylcholine (Ach), sodium nitroprusside (SNP), BAY41-2272 and tadalafil in the corpora cavernosa of Crotalus (CCC) pre contracted with phenylephrine. Relaxations induced by electrical stimulation (EFS) was also tested in the presence and absence of L-NAME (100 mm), ODQ (10 mM) and tetrodotoxin (TTX, 1 mM). In precontracted CCC, dependent relaxation frequency generated by EFS last three-times more than those in DC mammals. Although these relaxations are virtually abolished by L-NAME or ODQ, they were not affected by TTX. In contrast, EFS caused a relaxation of the corpus cavernosum in marmosets that had been incubated with TTX / Mestrado / Farmacologia / Mestra em Farmacologia