Unique Utilization of a Phosphoprotein Phosphatase Fold by a Mammalian Phosphodiesterase Associated with WAGR Syndrome

Metallophosphoesterase-domain-containing protein 2 (MPPED2) is a highly evolutionarily conserved protein with orthologs found from worms to humans. The human MPPED2 gene is found in a region of chromosome 11 that is deleted in patients with WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome, and MPPED2 may function as a tumor suppressor. However, the precise cellular roles of MPPED2 are unknown, and its low phosphodiesterase activity suggests that substrate hydrolysis may not be its prime function. We present here the structures of MPPED2 and two mutants, which show that the poor activity of MPPED2 is not only a consequence of the substitution of an active-site histidine residue by glycine but also due to binding of AMP or GMP to the active site. This feature, enhanced by structural elements of the protein, allows MPPED2 to utilize the conserved phosphoprotein-phosphatase-like fold in a unique manner, ensuring that its enzymatic activity can be combined with a possible role as a scaffolding or adaptor protein. (C) 2011 Elsevier Ltd. All rights reserved

Biochemical Characterization of dMPPED.

Evolutionarily conserved genes often play critical roles in organismal physiology. Here, we describe multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. We observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism.</div

Expression of dMPPED.

Evolutionarily conserved genes often play critical roles in organismal physiology. Here, we describe multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. We observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism.</div

Generation of <i>dMPPED</i>^<i>KO</i> flies.

Evolutionarily conserved genes often play critical roles in organismal physiology. Here, we describe multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. We observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism.</div

Neuronal expression of dMpped determines fly lifespan, AMP expression and cyclic nucleotide levels in the brain.

(A) Western blot to confirm that dMppedKO is a protein null allele, and expression of dMpped in the brain of flies where dMpped expression is driven in the neurons. The blot was re-probed with an anti-tubulin antibody to normalize protein loading. (B) Survival curves of backcrossed flies of indicated genotypes. Flies (w1118, n = 66; dMppedKO, n = 66; elav-Gal4>dMpped; dMppedKO, n = 30) were from at least three independent experiments. Log-rank test was used to compare across genotypes. Values shown at each time point are the mean ± SD, and the line represents the average across all experiments. p values are shown and compare the average life span across all three replicates of each genotype. (C) RT-qPCR to confirm upregulation of AMPs in dMppedKO flies. Levels were restored following neuronal expression of dMpped in dMppedKO flies. Each data point represents RNA prepared from 10 female flies, and histograms correspond to the mean value ± SD of three independent experiments. Data were analyzed by ANOVA and corrected for multiple comparisons using Tukey’s test. p values are indicated across genotypes. (D) Cyclic AMP and Cyclic GMP levels were measured in the heads of flies of the indicated genotypes. Levels of cAMP and cGMP were significantly higher in dMppedKO flies, correlated with reduced phosphodiesterase activity in flies deleted for dMpped. Extracts were prepared from 10 female fly heads, and histograms correspond to the mean value ± SD of three independent experiments. Data were analyzed by ANOVA and corrected for multiple comparisons using Tukey’s test. p values are indicated across genotypes.</p

dMpped is a metallophosphoesterase and its expression is enriched in neurons.

(A) Sequence alignment of dMpped with human MPPED1, human MPPED2 and rat MPPED2. Red boxes highlight the conserved residues characteristic of metallophosphoesterases. * indicates residues D49 and H52 mentioned in the text. Highlighted in the blue box is the histidine residue that distinguishes MPPED1 from MPPED2. (B) A Coomassie R stained gel showing purified dMpped protein used for biochemical assays. (C) The catalytic activity of dMpped with the indicated colorigenic substrates (10 mM), in the presence of Mn2+ (5 mM) with dMpped (500 ng protein). Values represent the mean ± S.E. of duplicate determinations of experiments performed using two independent protein preparations. BispNPP, bis(p-nitrophenyl) phosphate; pNPPP, p-nitrophenyl phenylphosphonate; pNPP, p-nitrophenyl phosphate; TmPP, thymidine 5’-monophosphate-p-nitrophenyl ester; pNPPC, p-nitrophenylphosphoryl-choline. (D) Expression of CG16717 (dMpped) obtained from an analysis of RNAseq data from the modEncode data hosted in Flybase (www.flybase.org). Transcript levels are seen at low levels in many tissues, including the CNS and larvae (E) Expression pattern of dMpped across developmental stages of the fly. Embryos (2 h after egg laying; 50), third instar larvae (wandering; 10), pupae (21 h after pupae formation; 10), male and female flies (3 days old; 10 each) were collected and RT-qPCR was performed. dMpped transcript levels have been normalized to RpL32 transcript levels. The graph represents mean ± S.D. from 2 sets of samples collected independently. (F) Expression pattern of dMpped in different adult tissues. Testis and ovaries were collected from 50 male and female flies, respectively. The intestine (gut) was dissected from ∼ 50 flies in total, and 100 flies were used for muscles and brains. The graph represents mean ± S.D. from 2 sets of samples collected independently. (G) Confocal images of the ovary, testis, posterior midgut (R3-R4) and brain in pBAC(IT.GAL4)CG16717/UAS-mCD8GFP. pBac(IT.GAL4) enhancer trap element is positioned within the CG16717 gene. Ovaries show expression in the oviduct, the testis in secondary cells, and scattered enterocytes are GFP-positive in the posterior midgut. In the brain, strong expression is seen in the antennal lobe, marked with white arrowheads and in the optic lobe. The scale bar indicates 100 μm. 3D renderings of the brain sections of female brains are shown in S1 Movie, and enlarged images are shown in S1 Fig.</p

List of flies used in this study.

Evolutionarily conserved genes often play critical roles in organismal physiology. Here, we describe multiple roles of a previously uncharacterized Class III metallophosphodiesterase in Drosophila, an ortholog of the MPPED1 and MPPED2 proteins expressed in the mammalian brain. dMpped, the product of CG16717, hydrolyzed phosphodiester substrates including cAMP and cGMP in a metal-dependent manner. dMpped is expressed during development and in the adult fly. RNA-seq analysis of dMppedKO flies revealed misregulation of innate immune pathways. dMppedKO flies showed a reduced lifespan, which could be restored in Dredd hypomorphs, indicating that excessive production of antimicrobial peptides contributed to reduced longevity. Elevated levels of cAMP and cGMP in the brain of dMppedKO flies was restored on neuronal expression of dMpped, with a concomitant reduction in levels of antimicrobial peptides and restoration of normal life span. We observed that dMpped is expressed in the antennal lobe in the fly brain. dMppedKO flies showed defective specific attractant perception and desiccation sensitivity, correlated with the overexpression of Obp28 and Obp59 in knock-out flies. Importantly, neuronal expression of mammalian MPPED2 restored lifespan in dMppedKO flies. This is the first description of the pleiotropic roles of an evolutionarily conserved metallophosphodiesterase that may moonlight in diverse signaling pathways in an organism.</div

Schematic showing pleiotropic roles of dMPPED.

Class III metallophosphoesterases are evolutionarily conserved and members of the MPPED family are neuronally expressed in the brain of mammals and flies. dMPPEDKO flies show misregulation of Obps, elevated levels of cAMP and cGMP and higher expression of AMPs. All these changes can influence longevity, with dMPPEDKO flies showing a decreased life span. The Figure was created with BioRender.com.</p