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Characterisation of phosphodiesterase 11 in Drosophila melanogaster

Finlayson, Andrew (2010) Characterisation of phosphodiesterase 11 in Drosophila melanogaster. PhD thesis, University of Glasgow.

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Abstract

The PDE 11 family of dual specificity phosphodiesterases was first identified in 2000, and has not been well characterised, although mutations in the gene have been linked to multiple disorders, including major depressive disorder, and cancer. DmPDE11 is a dual specificity phosphodiesterase, which shows 96% similarity with the catalytic domain of HsPDE11A, and around 40% similarity along the length of the protein. The focus of this project was to characterise this important enzyme using the model organism Drosophila melanogaster. The resources available to Drosophila researchers are unrivalled, and include a sequenced genome, unparalleled transgenic technology, of which stocks are freely available, and Homophila, a database of human disease genes and their Drosophila orthologues. Drosophila is genetically tractable to an extent not seen in any other multicellular organisms. The genetic dissection of gene function in Drosophila has allowed the identification and characterisation of numerous cell signalling genes. For example, mutations to Dunce were shown to affect olfactory learning. This allowed the identification and cloning of the mammalian dnc homologue PDE4. cAMP (and cGMP) were subsequently shown to modulate learning and memory in mammals. The 5.8 kb expressed sequence tag (EST) SD13096 had previously been shown to contain sequence present in the incomplete PDE11 RA ESTs previously released by Flybase, but also incorporating a 5’ UTR, and an in-frame start codon within two novel 5’ exons. A Northern blot of DmPDE11 RA produced one band of approximately 5.8kb; as this matches the size of the DmPDE11 RA ORF, was accepted that SD13096 encodes the entire PDE11 RA ORF (Day, unpublished). Expression of this EST in S2 cells revealed that the construct produced a protein of the accepted size, and the protein localised to the cytoplasm. However, PDE assays of S2 cell lysate revealed that the enzyme did not appear to encode an enzyme with either cA- or cG-PDE activity. DmPDE11 RA was replaced on Flybase by the new isoforms DmPDE11 RB and DmPDE11 RC, which had two key changes to the RA isoform. Both new isoforms had different N termini, sharing a second exon, with distinct first exons. Furthermore, exon 11 of the RA exon is not present in the newly predicted isoforms. These new isoforms were verified by reverse transcriptase- polymerase chain reaction analysis. In the course of this verification, two further novel isoforms were identified, which shared the novel N termini with the RB and RC isoforms, but include a novel exon/exon boundary within the original exon 19, which results in a truncated isoform. As such the four isoforms were named DmPDE11 RB long, DmPDE11 RB short, DmPDE11 RC long, and DmPDE11 RC short. The open reading frames of these isoforms were cloned from Drosophila cDNA using high-fidelity DNA polymerase and sequenced for fidelity. The open reading frames were tagged with YFP, and this tag was used to verify expression of these isoforms. Each isoform expressed a protein of the predicted size when expressed in Drosophila. DmPDE11 B and C proteins show distinct localisation in the Malpighian tubule, where the long and short isoforms of each isoform display indistinguishable localisations. DmPDE11 B localises to the apical and basolateral membranes, and DmPDE11 C localises to an unknown organelle, or to vesicles. All 4 isoforms were verified as dual specificity cA- and cG- PDEs. The previous finding (Day, unpublished) that DmPDE11 co-immunoprecipitates with cGMP dependent protein kinase activity, and that cGMP dependent protein kinases co-immunoprecipitate with cG-PDE activity, and thus that cG-PDE(s) interact with at least one cGMP dependent protein kinase, directly or indirectly, was investigated. DmPDE11 C long and short were co-transfected in Schneider 2 cells with the cGKs DG1, DG2P1 and DG2P2. Co-immunoprecipitation of these showed that both the long and short isoforms of DmPDE11 C interact with every cGK screened. Time did not permit the application of this protocol to screen DmPDE11 B interaction with the cGKs. Whether this interaction is direct or indirect was screened by peptide array. Peptide arrays were generated representing the sequence of DmPDE11, DG1, and DG2, and proteins were generated fusing fragments of these proteins with HIS6 and Glutathione-S-Transferase tags. These were expressed in E. coli, and verified by western blotting. HIS6 tagged protein expression was shown to be of higher quality, and was thus affinity purified, and used to overlay and probe the peptide arrays for putative direct interactions. When the PDE11 array was overlaid with tagged protein representing the C terminal half of DG1, and the N and C terminal halves of DG2, a putative direct interaction was identified between DG1 and PDE11 on two separate regions of the PDE11 array, which both fell within the sequence of PDE11 represented by the Middle-HIS6 fragment. As such, this was used to probe the PDE11 array. A reciprocal putative interaction was identified on three regions of the DG1 array, representing sequence in both DG1N-HIS6 and DG1C-HIS6 fragments. Unfortunately, although DG1-HIS6 was verified by western blotting at the analytical stage, attempts to affinity purify the protein failed. Time did not permit the probing of the array with DG1N-GST fusion protein, and so further putative interaction sites on PDE11 may remain. The generation of alanine substitution arrays, and subsequent mutagenesis analysis with yeast two hybrid or co-immunoprecipitation would be necessary to confirm this direct protein-protein interaction as bona-fide. The investigation into a putative direct interaction between PDE11 and DG2 did not yield conclusive data, and so further investigation is required. The role of DmPDE11 in immunity was investigated by the use of DmPDE11 RNAi and deletion lines. The DmPDE11 deletion line showed a qualitative reduction in survival in individual survival assays, but when these data were merged a significant decrease in survival compared to controls was seen. However, fly numbers did not permit the inclusion of all of the necessary controls, and so these assays should be repeated with these. However, upon immune challenge, progeny from a DmPDE11 RNAi (line 9) x Act5c (a ubiquitous GAL4 driver line) cross did not show a decrease in survival compared to parental lines. Transgenic Drosophila expressing H. sapiens PDE11A3 were generated. The protein localised to the nucleus at low levels of protein; increased expression led to nuclear exclusion, and localisation to the basolateral and especially apical membranes, with cytosolic localisation also. The work has provided the tools needed to further research PDE11. The implication of this gene as a tumour suppressor gene, and its role in other processes, means that it is of the utmost importance that this enzyme is further characterised.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Drosophila melanogaster, cyclic nucleotide signalling, phosphodiesterase 11
Subjects: Q Science > QH Natural history > QH426 Genetics
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology > Molecular Cell and Systems Biology
Supervisor's Name: Davies, Prof. Shireen and Dow, Prof. Julian
Date of Award: 2010
Depositing User: Mr A J Finlayson
Unique ID: glathesis:2010-1954
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 18 Jun 2010
Last Modified: 10 Dec 2012 13:48
URI: http://theses.gla.ac.uk/id/eprint/1954

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