Towards a system level understanding of non-model organisms sampled from the environment: a network biology approach.

The acquisition and analysis of datasets including multi-level omics and physiology from non-model species, sampled from field populations, is a formidable challenge, which so far has prevented the application of systems biology approaches. If successful, these could contribute enormously to improving our understanding of how populations of living organisms adapt to environmental stressors relating to, for example, pollution and climate. Here we describe the first application of a network inference approach integrating transcriptional, metabolic and phenotypic information representative of wild populations of the European flounder fish, sampled at seven estuarine locations in northern Europe with different degrees and profiles of chemical contaminants. We identified network modules, whose activity was predictive of environmental exposure and represented a link between molecular and morphometric indices. These sub-networks represented both known and candidate novel adverse outcome pathways representative of several aspects of human liver pathophysiology such as liver hyperplasia, fibrosis, and hepatocellular carcinoma. At the molecular level these pathways were linked to TNF alpha, TGF beta, PDGF, AGT and VEGF signalling. More generally, this pioneering study has important implications as it can be applied to model molecular mechanisms of compensatory adaptation to a wide range of scenarios in wild populations.

Attenuation of the activity of the cAMP-specific phosphodiesterase PDE4A5 by interaction with the immunophilin XAP2.

The cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4A5 interacted with the immunophilin XAP2 in a yeast two-hybrid assay. The interaction was confirmed in biochemical pull-down analyses. The interaction was specific, in that PDE4A5 did not interact with the closely related immunophilins AIPL1, FKBP51, or FKBP52. XAP2 also did not interact with other PDE4A isoforms or typical isoforms from the three other PDE4 subfamilies. Functionally, XAP2 reversibly inhibited the enzymatic activity of PDE4A5, increased the sensitivity of PDE4A5 to inhibition by the prototypical PDE4 inhibitor 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram) and attenuated the ability of cAMP-dependent protein kinase to phosphorylate PDE4A5 in intact cells. XAP2 maximally inhibited PDE4A5 by approximately 60%, with an IC50 of 120 nm, and reduced the IC50 for rolipram from 390 nm to 70-90 nm. Co-expression of XAP2 and PDE4A5 in COS7 cells showed that they could be co-immunoprecipitated and also reduced both the enzymatic activity of PDE4A5 and its IC50 for rolipram. Native XAP2 and PDE4A5 could be co-immunoprecipitated from the brain. The isolated COOH-terminal half of XAP2 (amino acids 170-330), containing its tetratricopeptide repeat domain, but not the isolated NH2-terminal half (amino acids 1-169), containing the immunophilin homology region, similarly reduced PDE4A5 activity and its IC50 for rolipram. Mutation of Arg271 to alanine, in the XAP2 tetratricopeptide repeat region, attenuated its ability to both interact with PDE4A5 in two-hybrid assays and to inhibit PDE4A5 activity. Either the deletion of a specific portion of the unique amino-terminal region or specific mutations in the regulatory UCR2 domain of PDE4A5 attenuated its ability be inhibited by XAP2. We suggest that XAP2 functionally interacts with PDE4A5 in cells.

Long PDE4 cAMP specific phosphodiesterases are activated by protein kinase A-mediated phosphorylation of a single serine residue in Upstream Conserved Region 1 (UCR1).

1. Challenge of COS1 cells with the adenylyl cyclase activator forskolin led to the activation of recombinant PDE4A8, PDE4B1, PDE4C2 and PDE4D5 cAMP-specific phosphodiesterase long isoforms. 2. Forskolin challenge did not activate mutant long PDE4 isoforms where the serine target residue (STR) within the protein kinase A (PKA) consensus phosphorylation site in Upstream Conserved Region 1 (UCR1) was mutated to alanine. 3. The PKA inhibitor, H89, ablated forskolin activation of wild-type long PDE4 isoforms. 4. Activated PKA caused the in vitro phosphorylation of recombinant wild-type long PDE4 isoforms, but not those where the STR was mutated to alanine. 5. An antiserum specific for the phosphorylated form of the STR detected a single immunoreactive band for recombinant long PDE4 isoforms expressed in COS1 cells challenged with forskolin. This was not evident in forskolin-challenged cells treated with H89. Neither was it evident in forskolin-challenged cells expressing long isoforms where the STR had been mutated to alanine. 6. In transfected COS cells challenged with forskolin, only the phosphorylated PDE4D3 long form showed a decrease in mobility in Western blotting analysis. This decreased mobility of PDE4D3 was ablated upon mutation of either of the two serine targets for PKA phosphorylation in this isoform, namely Ser54 in UCR1 and Ser13 in the isoform-specific N-terminal region. 7. Activation by forskolin challenge did not markedly alter the sensitivity of PDE4A8, PDE4B1, PDE4C2 and PDE4D5 to inhibition by rolipram. 8. Long PDE4 isoforms from all four sub-families can be phosphorylated by protein kinase A (PKA). This leads to an increase in their activity and may thus contribute to cellular desensitization processes in cells where these isoforms are selectively expressed.

TAPAS-1, a novel microdomain within the unique N-terminal region of the PDE4A1 cAMP-specific phosphodiesterase that allows rapid, Ca2+-triggered membrane association with selectivity for interaction with phosphatidic acid.

Here we identify an 11-residue helical module in the unique N-terminal region of the cyclic AMP-specific phosphodiesterase PDE4A1 that determines association with phospholipid bilayers and shows a profound selectivity for interaction with phosphatidic acid (PA). This module contains a core bilayer insertion unit that is formed by two tryptophan residues, Trp(19) and Trp(20), whose orientation is optimized for bilayer insertion by the Leu(16):Val(17) pairing. Ca(2+), at submicromolar levels, interacts with Asp(21) in this module and serves to gate bilayer insertion, which is completed within 10 ms. Selectivity for interaction with PA is suggested to be achieved primarily through the formation of a charge network of the form (Asp(21-):Ca(2+):PA(2-):Lys(24+)) with overall neutrality at the bilayer surface. This novel phospholipid-binding domain, which we call TAPAS-1 (tryptophan anchoring phosphatidic acid selective-binding domain 1), is here identified as being responsible for membrane association of the PDE4A1 cAMP-specific phosphodiesterase. TAPAS-1 may not only serve as a paradigm for other PA-binding domains but also aid in detecting related phospholipid-binding domains and in generating simple chimeras for conferring membrane association and intracellular targeting on defined proteins.