With the advent of large scale genome sequencing efforts along with more sophisticated methods of genetic mapping, a number of loci have been identified that are associated with human diseases. Intriguingly, many genes identified in these loci remain uncharacterized. Although current annotation can provide a prediction of putative function of some of these proteins at a biochemical level, understanding their cellular roles require analysis at a single gene level. Bioinformatic analysis carried out in the laboratory during studies on cyclic nucleotide metabolism in mycobacteria identified putative Class III cyclic nucleotide phosphodiesterases (Class III cNMP PDEs) from the non-redundant database of proteins. One of the proteins identified was the Rv0805 gene product from Mycobacterium tuberculosis. Detailed biochemical characterization of this protein revealed that Rv0805 is indeed a phosphodiesterase (PDE) and could hydrolyze 3’, 5’-cyclic adenosine monophosphate (cAMP) as well as 3’, 5’-cyclic guanosine monophosphate (cGMP). Structural analysis of Rv0805 revealed a metallophosphoesterase (MPE) like fold and presence of two metal atoms at the binuclear metal centre of the protein. Moreover, overexpression of Rv0805 in E. coli and M. smegmatis reduced intracellular cAMP levels indicating that it possesses cAMP PDE activity in vivo. The majority of proteins identified in this bioinformatic analysis were of bacterial or archaebacterial in origin but it was interesting to find some mammalian proteins, since, till date, no Class III cNMP PDE has been found in higher eukaryotes. Interestingly, two genes were identified in the human genome. These genes, 239FB and 239AB, are expressed in the fetal brain and adult brain, respectively and have been annotated as metallophosphoesterases but there has been no biochemical or functional characterization of these proteins. The 239FB gene is present between the FSHB and PAX6 genes on chromosome 11. This gene locus is present within a deletion interval (11p13-14) that is associated with the mental retardation phenotype of WAGR syndrome (Wilms’ tumor, aniridia, genitourinary anomalies, mental retardation). Inspection of available sequenced mammalian genomes indicated a shared synteny of the genes in the WAGR locus, highlighting it’s evolutionary conservation. Most interestingly, nucleotide sequences within the WAGR locus (which include the 5 genes WT1, PAX6, RCN1, ELP4 and 239FB) are amongst the 481 ultra conserved regions of the human genome. Moreover, 239FB is one of only 24 instances where an ortholog of an ultra-conserved element could be partially traced back by sequence similarity in lower eukaryotes such as Ciona intestinalis, Drosophila melanogaster, or Caenorhabditis elegans. Although the function of the 239FB protein is unknown so far, the distinctive expression of the gene in the fetal brain and the presence of an “ancient conserved region” in this gene suggest that this gene may be vital for the development of the nervous system. The work carried out in this thesis has attempted to understand the physiological functions of the 239FB/AB gene family. Amino acid sequence comparison revealed two amino acids changes between the human and rat proteins indicating the extra-ordinary sequence conservation of these proteins. Therefore, to characterize the biochemical properties of 239FB and 239AB proteins, rat proteins were used as model enzymes. Reverse transcription-PCR analysis of RNA prepared from the fetal and adult rat brains as well as Western blot analysis on cytosolic fractions of rat brains from various developmental stages indicated that 239FB is predominantly expressed in fetal brain. Detailed biochemical analyses of the rat 239FB and 239AB proteins were performed which showed that they possess metallophosphodiesterase activity. 239FB showed activity only in the presence of Mn2+ and Co2+ as the added metal cofactors. Surprisingly, the Km for Mn2+ of 239FB was found to be 1.5 mM, which is nearly 60-fold higher than that of its mycobacterial ortholog, Rv0805. A systematic mutational analysis was performed to characterize the residues that are involved in binding either one or both the metals found in the catalytic site of 239FB. Although 239FB shares some of the residues that have been shown to be essential for metal binding and catalytic activity with other MPEs including Rv0805, there are some differences as well. One histidine residue that has been conserved in other MPEs and has been shown to be important for metal binding is replaced by glycine (Gly-252) in 239FB. To study the consequence of replacing the glycine with a histidine in 239FB, a 239FBGly252His mutant protein was generated and characterized. Interestingly, the single mutation of Gly-252 to a histidine residue not only increased the affinity of the protein for metals but increased catalytic activity as well with various phosphodiesters. Moreover, 239FBGly252His mutant protein showed significant activity with cAMP and cGMP which were not hydrolysed by wild type 239FB. Interestingly, in the 239AB protein, histidine 284 is present at a position equivalent to Gly-252 in the 239FB protein. Biochemical characterization of 239AB showed 2’, 3’-cAMP hydrolyzing activity similar to 239FBGly252His mutant protein. A rat 239FB protein with a mutation (His67Arg) corresponding to a single nucleotide polymorphism seen in human 239FB, led to complete inactivation of the protein. The occurrence of this SNP at a very low frequency and only as a heterozygous condition suggests that a complete loss-of-function mutation of 239FB in human populations cannot be tolerated. To gain insights into the function of 239FB in its physiological milieu, yeast two-hybrid screening was performed with 239FB using human fetal brain cDNA library. Dipeptidyl peptidase III, a zinc dependent metallopeptidase, was found as an interacting partner of 239FB in this analysis and the functional consequences of this interaction would be an interesting area of study in future. While a number of metallophosphoesterases have been characterized biochemically and structurally, their biological role(s) and in vivo substrate(s) remain elusive. In order to elucidate the physiological role of 239FB/AB family, the ortholog of 239FB/AB in D. melanogaster was characterized. Sequence comparison of Drosophila ortholog with both the mammalian proteins indicated that it may be an ortholog of 239AB and hence, it was named as d239AB. Enhancer-promoter analysis with a putative promoter region of the d239AB indicated the expression of d239AB in the mushroom bodies in brain and in enterocytes in mid gut. Characterization of a Drosophila line, BS#16242, with a piggybac element inserted in the intron of d239AB showed disruption of d239AB expression. This suggested that BS#16242 line can serve as a d239AB knockout line and hence, was selected for further phenotypic characterization to unravel the physiological roles of d239AB. Though, BS#16242 flies did not show any developmental defects, a severe reduction in the fecundity of these files was observed. Further analysis revealed defective ovulation as a probable reason for reduced fecundity of these flies. In addition to compromised fecundity, BS#16242 flies showed a significant reduction in the life span of male as well as female flies. Moreover, these flies showed less resistance to thermal stress and desiccation. Most interestingly, all these phenotypes were rescued upon neuronal expression of the d239AB transgene in BS#16242 flies indicating that neuronal function of d239AB is important for diverse physiological processes. The phenotypes observed in BS#16242 flies mimic the physiological state under increased insulin signaling, such as decrease in life span, and susceptibility to various stress conditions suggesting that d239AB could play a role in the insulin signaling pathway. Interestingly, overexpression of d239AB transgene in neurons reduced cAMP levels in the brains of Drosophila, indicating that the protein may have cAMP phosphodiesterase activity in vivo. This is the first analysis of the presence of a Class III phosphodiesterase in eukaryotes. Thus, d239AB mediated regulation of cAMP levels in a particular subsets of cells, such as neurons, could also be one of the molecular mechanisms responsible for reduced fecundity and longevity of BS#16242 flies. Interacting partners of d239AB were inspected in the Drosophila interactome (built on protein-protein interactions identified using a yeast two-hybrid approach). Strikingly, most of the d239AB interacting proteins were involved either in transcriptional or translational regulation indicating that d239AB could be involved in the regulation of expression of genes involved in diverse physiological processes. This could explain why disruption of d239AB led to various physiological defects such as reduced fecundity, decreased life span and compromised fitness. In summary, studies described in this thesis suggest that 239FB and 239AB proteins are the first Class III cyclic nucleotide phosphodiesterases reported in eukaryotes. Results shown here suggest the critical role of their ortholog in the physiology of Drosophila. Further genetic manipulation in D. melanogaster and other organisms which harbor orthologs of the 239FB/AB gene could throw light on the diverse biological roles of these enzymes in humans.

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.