Search

Oxidative stress plays a pivotal role in pathogenesis of cardiovascular diseases and diabetes; however, the roles of protein kinase A (PKA) and human phosphodiesterase 3A (hPDE3A) remain unknown. Here, we show that yeast expressing wild-type (WT) hPDE3A or K13R hPDE3A (putative ubiquitinylation site mutant) exhibited resistance or sensitivity to exogenous hydrogen peroxide (H 2 O 2 ), respectively. H 2 O 2 -stimulated ROS production was markedly increased in yeast expressing K13R hPDE3A (Oxidative stress Sensitive 1, OxiS1), compared with yeast expressing WT hPDE3A (Oxidative stress Resistant 1, OxiR1). In OxiR1, YAP1 and YAP1-dependent antioxidant genes were up-regulated, accompanied by a reduction in thioredoxin peroxidase. In OxiS1, expression of YAP1 and YAP1-dependent genes was impaired, and the thioredoxin system malfunctioned. H 2 O 2 increased cyclic adenosine monophosphate (cAMP)-hydrolyzing activity of WT hPDE3A, but not K13R hPDE3A, through PKA-dependent phosphorylation of hPDE3A, which was correlated with its ubiquitinylation. The changes in antioxidant gene expression did not directly correlate with differences in cAMP-PKA signaling. Despite differences in their capacities to hydrolyze cAMP, total cAMP levels among OxiR1, OxiS1, and mock were similar; PKA activity, however, was lower in OxiS1 than in OxiR1 or mock. During exposure to H 2 O 2 , however, Sch9p activity, a target of Rapamycin complex 1-regulated Rps6 kinase and negative-regulator of PKA, was rapidly reduced in OxiR1, and Tpk1p, a PKA catalytic subunit, was diffusely spread throughout the cytosol, with PKA activation. In OxiS1, Sch9p activity was unchanged during exposure to H 2 O 2 , consistent with reduced activation of PKA. These results suggest that, during oxidative stress, TOR-Sch9 signaling might regulate PKA activity, and that post-translational modifications of hPDE3A are critical in its regulation of cellular recovery from oxidative stress., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

In adipocytes, PDE3B (phosphodiesterase 3B) is an important regulatory effector in signalling pathways controlled by insulin and cAMP-increasing hormones. Stimulation of 3T3-L1 adipocytes with insulin or the beta3-adrenergic receptor agonist CL316243 (termed CL) indicated that insulin preferentially phosphorylated/activated PDE3B associated with internal membranes (endoplasmic reticulum/Golgi), whereas CL preferentially phosphorylated/activated PDE3B associated with caveolae. siRNA (small interfering RNA)-mediated KD (knockdown) of CAV-1 (caveolin-1) in 3T3-L1 adipocytes resulted in down-regulation of expression of membrane-associated PDE3B. Insulin-induced activation of PDE3B was reduced, whereas CL-mediated activation was almost totally abolished. Similar results were obtained in adipocytes from Cav-1-deficient mice. siRNA-mediated KD of CAV-1 in 3T3-L1 adipocytes also resulted in inhibition of CL-stimulated phosphorylation of HSL (hormone-sensitive lipase) and perilipin A, and of lipolysis. Superose 6 gel-filtration chromatography of solubilized membrane proteins from adipocytes stimulated with insulin or CL demonstrated the reversible assembly of distinct macromolecular complexes that contained 32P-phosphorylated PDE3B and signalling molecules thought to be involved in its activation. Insulin- and CL-induced macromolecular complexes were enriched in cholesterol, and contained certain common signalling proteins [14-3-3, PP2A (protein phosphatase 2A) and cav-1]. The complexes present in insulin-stimulated cells contained tyrosine-phosphorylated IRS-1 (insulin receptor substrate 1) and its downstream signalling proteins, whereas CL-activated complexes contained beta3-adrenergic receptor, PKA-RII [PKA (cAMP-dependent protein kinase)-regulatory subunit] and HSL. Insulin- and CL-mediated macromolecular complex formation was significantly inhibited by CAV-1 KD. These results suggest that cav-1 acts as a molecular chaperone or scaffolding molecule in cholesterol-rich lipid rafts that may be necessary for the proper stabilization and activation of PDE3B in response to CL and insulin.

Fractionation of 3T3-L1 adipocyte membranes revealed that PDE3B (phosphodiesterase 3B) was associated with PM (plasma membrane) and ER (endoplasmic reticulum)/Golgi fractions, that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions, and that there was no significant translocation of PDE3B between membrane fractions. Insulin also induced formation of large macromolecular complexes, separated during gel filtration (Superose 6 columns) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and signalling molecules potentially involved in its activation by insulin, e.g. IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 [p85-subunit of PI3K (phosphoinositide 3-kinase)], PKB (protein kinase B), HSP-90 (heat-shock protein 90) and 14-3-3. Expression of full-length recombinant FLAG-tagged murine (M) PDE3B and M3BDelta604 (MPDE3B lacking N-terminal 604 amino acids) indicated that the N-terminal region of MPDE3B was necessary for insulin-induced activation and recruitment of PDE3B. siRNA (small interfering RNA) knock-down of PDE3B indicated that PDE3B was not required for formation of insulin-induced complexes. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, as well as phosphorylation/activation of PKB and PDE3B, and their co-immunoprecipitation. Another PI3K inhibitor, LY294002, and the tyrosine kinase inhibitor, Genistein, also inhibited insulin-induced activation of PDE3B and its co-immunoprecipitation with PKB. Confocal microscopy indicated co-localization of PDE3B and PKB. Recombinant MPDE3B co-immunoprecipitated, and co-eluted during Superose 12 chromatography, to a greater extent with recombinant pPKB (phosphorylated/activated PKB) than dephospho-PKB or p-DeltaPKB [pPKB lacking its PH domain (pleckstrin homology domain)]. Truncated recombinant MPDE3B proteins and pPKB did not efficiently co-immunoprecipitate, suggesting that structural determinants for their interaction reside in, or are regulated by, the N-terminal portion of MPDE3B. Recruitment of PDE3B in macromolecular complexes may be critical for regulation of specific cAMP pools and signalling pathways by insulin, e.g. lipolysis.

We have identified a new cyclic-nucleotide phosphodiesterase isoform, PDE3A, and cloned its cDNA from cultured aortic myocytes. The nucleotide sequence of its coding region is similar to that of the previously cloned myocardial isoform except for the absence of the initial 300-400 nt that are present in the latter, as confirmed by reverse-transcriptase-mediated PCR, 5' rapid amplification of cDNA ends and a ribonuclease protection assay. Expression in Spodoptera frugiperda (Sf9) cells yields a protein with catalytic activity and inhibitor sensitivity typical of the PDE3 family. The recombinant protein's molecular mass of approx. 131 kDa is compatible with translation from an ATG sequence corresponding to nt 436-438 of the myocardial PDE3A coding region. Antibodies against residues 424-460 (nt 1270-1380) and 1125-1141 (nt 3373-3423) of the myocardial isoform react with an approx. 118 kDa band in Western blots of homogenates of human aortic myocytes, whereas antibodies against residues 29-42 (nt 85-126) do not react with any bands in these homogenates. Our results suggest that a vascular smooth-muscle isoform ('PDE3A2') is a product of the same gene as the longer myocardial ('PDE3A1') and the shorter placental ('PDE3A3') isoforms and is generated pre-translationally in a manner that results in the absence of the 145 N-terminal amino acids of PDE3A1.

To test the validity of thermodynamic parameters from the equilibrium method, we have studied the reversible heat-induced denaturations of lysozyme, ribonuclease A, cytochrome c and myoglobin at various pH values, using absorption spectral measurements. For each protein, if a linear temperature-dependence of the pre- and post-transition baselines is assumed for the analysis of the conformational-transition curve, the estimate of DeltaH (the enthalpy change on denaturation at T(m), the midpoint of denaturation) is significantly less than DeltaH, the value obtained by the calorimetric measurements. If the analysis of thermal-denaturation curves assumes that the temperature-dependence of pre- and post-transition baselines is described by a parabolic function, there exists an excellent agreement between DeltaH(m) values of all proteins obtained from equilibrium and calorimetric methods. The latter analysis is supported by the studies on model compounds, for measurements of absorption properties of tyrosine, tryptophan and haem as a function of temperature suggested that the temperature-dependencies of the optical properties are indeed non-linear. We have observed that for each protein the constant-pressure heat-capacity change on denaturation (DeltaC(p)) determined from the plots of DeltaH versus T(m) is not only independent of the method of analysis of the transition curve, but it is also in excellent agreement with calorimetric DeltaC(p). An important conclusion of this study is that for these proteins that exhibit two-state character, all stability parameters are measured with the same error as that observed with a calorimeter.

This study is a systematic attempt to understand the roles of non-compatible osmolytes, i.e. solutes that have inhibitory effects on enzymes, in the stabilization of proteins against denaturing stress. Thermal denaturation of RNase A, holo-alpha-lactalbumin, apo-alpha-lactalbumin, lysozyme and metmyoglobin in the absence and presence of various concentrations of free basic amino acids was studied by observing changes in the absorption coefficients of these proteins. It has been observed that arginine and histidine destabilize all proteins in terms of the midpoint of the transition curve and Gibbs energy change on denaturation. Study of the heat-induced denaturation of the proteins in the presence of various concentrations of arginine at different pH values demonstrated that arginine binds to the denatured molecules. In contrast with the effect of arginine and histidine on protein stability, it was observed that the effect of lysine on proteins stability is unpredictable, i.e. it may have a stabilizing effect, no effect or a destabilizing effect on proteins during denaturing stress. The results of this study are considered from an evolutionary perspective.

Biotin-binding antibodies were raised in rabbits by injecting biotin-bovine serum albumin conjugate. Neither the protomer nor the polymer of rat mammary-gland acetyl-CoA carboxylase formed precipitin bands with the anti-biotin. By virtue of its ability to bind biotin (apparent binding constant for free biotin about 1mum), the anti-biotin inhibited the carboxylase activity under certain conditions. This property of the antibody was employed to detect the ligand-induced changes affecting the biotinyl group in different conformational states of mammalian carboxylase. Depending on the ligand present, the biotinyl group in the protomeric form was either accessible or inaccessible to the antibody. The biotinyl group of the protomer generated by a relatively high concentration of NaCl (0.5m) reacted with the antibody, and the antibody-carboxylase complex could not be converted into active enzyme by citrate. Further experiments showed that citrate failed to induce polymerization in this protomer-antibody complex and that anti-biotin could be displaced rapidly from this complex with excess of biotin. The resulting protomer was converted into the polymeric state on citrate addition, with parallel regain of enzyme activity. In the presence of ADP+Mg(2+), ATP+Mg(2+) or ATP+Mg(2+)+HCO(3) (-), however, the enzyme remained as a protomer, but its configuration was such that the biotinyl group was essentially inaccessible to the antibody. Likewise, the biotinyl group of the different polymeric forms of the carboxylase (s approximately 30-45S) engendered by phosphate, malonyl-CoA, acetyl-CoA or citrate remained essentially inaccessible, since their activity was minimally affected by the anti-biotin. In the presence of 0.15m-NaCl, the phosphate-induced polymer reverted to a approximately 19S form with concomitant appearance of anti-biotin-sensitivity, whereas the other polymeric forms remained unaffected under similar experimental conditions.