Biochemical and functional characterization of the enigmatic MPPED1/MPPED2 metallophosphodiesterases
Evolutionarily conserved genes in nature have been known to perform critical functions inside the cell. Earlier bioinformatic analysis performed in our laboratory identified mammalian orthologs of Rv0805, a novel class III metallophosphoesterase from M. tuberculosis. These have been annotated as MPPED1 and MPPED2 (metallophosphoesterase domain containing protein 1 and 2). It was interesting to note the close relation between Rv0805 and the mammalian proteins in the phylogenetic clustering of metallophosphoesterases. The genes encoding MPPED1 and MPPED2 are highly conserved from Drosophila to Homo sapiens. In vitro biochemical analysis of rat MPPED1 and MPPED2 proteins suggested that these proteins were active metallophosphodiesterases. However, the in vivo function(s) of these proteins remains elusive. In humans, the MPPED2 gene is located on chromosome 11 in a locus associated with WAGR syndrome, and multiple studies suggest that MPPED2 may impair tumorigenesis. The present study aims to identify physiological functions of the MPPED1/MPPED2 family of proteins. In the first part of the study, spatio-temporal characterization of the mouse MPPED1 and MPPED2 was performed during development. Interestingly, it was observed that MPPED1 and MPPED2 expression were not exclusive to adult and fetal brain, respectively, as reported earlier. Additionally, primary glial and neuronal cultures have been established as tools to unravel the brain-specific physiological functions of these proteins. In the second part of the study, Drosophila melanogaster was chosen as a model system to probe into the physiological roles of CG16717, the only ortholog of MPPED1/MPPED2 present. Biochemical analysis suggested that CG16717 is a metallophosphodiesterase.CG16717 transcripts were found to be expressed throughout the life cycle, with maximal expression in the brain. CG16717KO flies exhibited reduced lifespan in comparison to wild-type flies, and lifespan was restored following neuronal expression of CG16717. Neuronal expression of mammalian MPPED2 restored the reduced lifespan of CG16717KO flies as well, suggesting functional conservation during evolution. Interestingly, over-expression of CG16717 in wild-type flies enhanced lifespan. Upon dietary restriction, both CG16717KO and wild-type flies showed an extension in lifespan, and this coupled with similar expression of IIS and TOR pathway transcripts, indicated that CG16717 regulates lifespan independent of Insulin/TOR pathways. RNA-seq analyses using whole flies suggested aberrant regulation of genes involved in innate immunity. Future studies will be directed to elucidate the molecular mechanisms by which CG16717 regulates Drosophila lifespan. In summary, this study characterized the spatio-temporal expression profile of the MPPED1/MPPED2 protein family in rodent model and identified the physiological role of the orthologous, evolutionarily conserved and neuronally-enriched Drosophila metallophosphodiesterase, CG16717, in lifespan regulation.