Polistes spp. it has been proposed that only the European species P. dominulus should be used for VIT. However, this recommendation is not present in any international guideline. Using both laboratory and clinical data, we aimed to evaluate the reliability of this proposal.Venom immunotherapy (VIT) is highly effective in preventing allergic reactions to insect stings, but the appropriate venom must be used to achieve clinical protection. In patients with multiple positive results to venoms, molecular allergy diagnostics or CAP-inhibition may identify the causative venom. Concerning allergy to venom from in vitro study using CAP-inhibition to determine sensitization of 19 patients allergic to Polistes venom. The clinical study included 191 patients with positive tests to Polistes treated with VIT, 102 were treated with P. dominulus and 89 were treated with a mix of American Polistes (mAP).We performed an P. dominulus sIgE by P. dominulus venom (79.8%) compared with inhibition by mAP venom (64.2%) and not significant concerning the inhibition of mAP sIgE by P. dominulus venom (80.1%) and by mAP venom (73.6%). Instead, the clinical protection from stings was not statistically different between the two kinds of venom.The difference in % of inhibition was significant concerning inhibition of P. dominulus venom or mAP venom for VIT is appropriate in patients with CAP inhibition higher than 70%, but the clinical data show the same odds of protection from stings using for VIT P. dominulus or mAP venom.The data from CAP inhibition would suggest that the choice of either  Vespula- and Polistes- IgE in the skin and/or serum were identified as candidates for exclusion of Polistes from immunotherapy because their anti-Polistes IgE was more than 95% cross-inhibitable with Vespula venom [a further advanced, enabling measurement of IgE specific to single venom allergen molecules, thus distinguishing simple cross-reacting components from causative molecules [Vespula and Polistes specific IgE to major allergens such as Ves v 1 and Ves v 5 from Vespula species and Pol d 1 and Pol d 5 from Polistes species, the responsible vespid was identified [P. gallicus, P. dominulus, P. nymphus, P. biglumis, P. associus, P. sulcifer, P. semenowi, and P. atrimandibularis; P. gallicus and P. dominulus are the most medically important. American species include P. annularis, P. fuscatus, P. metricus, P. apaches and P. exclamans. Cross-reactions among venoms from different Polistes spp. are quite extensive [Polistes stings as determined using skin tests, RAST and RAST inhibition (on only 10 patients), Severino et al. reported that RAST inhibition demonstrated only partial cross-reactivity between American and European species and that P. dominulus, and P. gallicus, have exclusive allergens, thus suggesting that such venoms are more suitable than the American mix in Italian patients [P. dominulus and from the mix of American Polistes (mAP) using in vitro CAP-inhibition for diagnosis and retrospectively evaluated the rate of clinical protection from subsequent stings in a group of Italian patients with allergic reactions to Polistes venom previously treated with P. dominulus or mAP, respectively.Venom immunotherapy (VIT) is an effective treatment for preventing anaphylactic reactions to Hymenoptera stings –3. The colecules . Three sentified , 8, 9. Ixtensive . However on only  patientss results to Polistes venom, and who had not previously been treated by VIT (to avoid treatment-induced changes in specific IgE), were included in the in vitro study, which aimed to assess the level of cross-reactivity to P. dominulus and mAP. A result from skin tests or CAP system showing monosensitization to P. dominulus was an exclusion criterion.Nineteen patients  with systemic reactions to Hymenoptera stings of at least Mueller grade II , positivP. dominulus venom from Anallergo  and mAP venom from Stallergenes , by an initial prick test at 100 mcg/ml, followed, if negative, by intradermal testing at 0.1 mcg/ml and 1 mcg/ml.Skin tests were performed by P. dominulus or mAP venom at increasing dilutions . Subsequently, specific IgE values (sIgE) against each of the venoms were determined in the prepared samples [P. dominulus and mAP venoms.(9) The protocol used for CAP inhibition was deposited in protocols.io with the identifier dx.doi-org/10.17504/protocols.io.ht7b6rn.Sera from all patients were analyzed using CAP inhibition to determine sensitization, following the method previously described by Caruso et al. , Savi et samples . The extP. dominulus and mAP were statistically compared using the non-parametric Wilcoxon test.The venom preparations tested were the same as those used for the skin tests. Owing to the limited number of patients, the level of the inhibition obtained with Polistes venom prior to VIT and subsequently restung by an identified insect were retrospectively analyzed to determine the rate of protection from Polistes venom. Monosensitization to P. dominulus was an the exclusion. Of the 191 patients, 89 were treated with mAP and 102 were treated with P. dominulus venom. All patients were instructed to recognize Polistes spp, and distinguish it from Vespula spp., based on anatomical characteristics. In particular, the wasp waist, e.g. the spindle-shaped morphology of the thorax-abdomen joint, is typical of Polistes spp, while in Vespula and Vespa spp this joint is truncated. Only stings from Polistes spp. were considered in the analysis. The difference in the rate of protection from stings were analyzed using the chi square test.For the clinical part of the study, 191 patients  with positive tests results to All participants or parents/tutors for patients of pediatric age gave their written informed consent.in vitro study was to assess the relative importance of allergens from P. dominulus and mAP by measuring their cross-inhibition. P. dominulus before inhibition was 9.85 ± 8.76 kU/L. After inhibition with P. dominulus and mAP the values decreased to 1.86 ± 2.08 and 2.80 ± 2.72 kU/L, respectively. The mean value of sIgE to mAP before inhibition was 6.90 ± 7.71 kU/L. After inhibition with mAP and P. dominulus the values decreased to 1.45 ± 1.60 and 1.35 ± 1.76, respectively. The difference in % of inhibition was significant (p = 0.003) concerning inhibition of P. dominulus sIgE by P. dominulus venom (mean% of inhibiton: 79.8%) compared with inhibition by mAP venom (mean % of inhibiton: 64.2%) and not significant concerning the inhibition of mAP sIgE by P.dominulus venom (mean % of inhibiton: 80.1%) and by mAP venom (mean % of inhibiton: 73.6%). . .P. dominulus with that of mAP. Incomplete protection from stings, with development of mild to moderate systemic reactions (no life-threatening reaction occurred) was found in 4 of 89 patients treated with mAP (4.5%) and 4 of 102 patients treated with P. dominulus (3.9%), this difference was not significant. All patients incompletely protected received a doubled maintenance dose (200 μg) and had no reaction when restung. Tables in vitro inhibition activity of P. dominulus compared to that of mAP did not manifest as clinical protection from stings; rather, the clinical protection between the two was comparable.The rationale of the clinical study was to compare the rate of protection of VIT with ominulus .9%, thisThe clinical study was registered with the Eudract number 2015-003081-92.Polistes spp have a worldwide distribution, with variable presence in different areas. In America the most common species are P. annularis, P. apachus, P. exclamans, P. fuscatus, and P. metricus, while in Europe P. gallicus and P. dominulus prevail. In particular, P. dominulus is the dominant species in Southern Europe [Polistes spp. must be distinguished from the allergological importance. Extensive cross-reactivity is known to occur for Polistes allergens, including the species-specific antigen 5, wish shows an 85% identity the European species P. gallicus and the American species P. annularis [P. dominulus venom for VIT in Polistes allergic patients in Europe.Hymenoptera belonging to n Europe . Howevernnularis . FurtherPolistes stings the CAP-inhibition obtained with P. dominulus was higher than that obtained with mAP. Namely, it was 79.8% for sIgE P. dominulus with the homologous venom vs. 64.2% with the mAP (p <0.01), and 73.6% for the sIgE mAP with the homologous venom vs 80.1% with P. dominulus (n.s.). These differences are lower than those reported by Severino et al, who found a value using RAST inhibition of 80.9% on average with P. dominulus, whereas the inhibition by with mAP was 46.1% on average [P. dominulus for VIT in patients allergic to Polistes venom.In this study we found that in sera from patients with allergic reactions to  average . To estiPolistes  was stronger than that between European and American species, but there was no recommendation to use only P. dominulus venom [has to must be measured against clinical data. Before P. dominulus venom became available for VIT in Europe, all patients were treated using mAP with no report of complete treatment failure, i.e. fatal reactions to stings; therefore, the suggestion to use only P. dominulus venom is flawed. In addition, P. dominulus is present in the United States [Polistes venom available. The rate of incomplete protection, i.e. to have a systemic reaction to a sting following VIT, depends upon the kind of venom. Muller et al reported that honeybee VIT provided complete protection in 77% of patients compared with 91% in patients treated with Vespula spp. VIT [Vespula spp. and Polistes spp. [Polistes venom, distinguishing P. dominulus VIT from mAP VIT. The results showed no statistically significant difference, based on a rate of incomplete protection of 3.9% and 4.5%, respectively, with no life-threatening reaction. This is comparable to the 4% rate of incomplete protection for vespids reported by Rueff et al, though they did not separately analyze Vespula and Polistes stings [P. dominulus and treated by VIT when Polistes was recognized as the stinging insect, 11 (10.6%) had systemic reactions, two of which were severe (one grade III and one grade IV) [The consensus document from the European Academy of Allergy and Clinical Immunology (EAACI) on Hymenoptera venom allergy stated that the cross-reactivity among European species of us venom . Second,d States  and alsospp. VIT . More retes spp. . In the s stings . Again srade IV) .Polistes venom. The first study dates back to 1982,; when studying sera from 62 American patients allergic to Polistes venom, four species' venoms were more closely related to each other than various Vespula spp were related to each other. The authors concluded that the commercial Polistes venom mix appeared to contain all of the relevant allergens [Polistes spp, these allergens are classified as follows: phospholipase A1 is Pol a 1 (from P. annularis) and Pol d 1 (from P. dominulus), hyaluronidase is Pol a 2, and antigen 5 is Pol a 5 and Pol d 5. Hyaluronidase is the main responsible of the cross-reactivity among venom from different species, including not only other vespids but also Apis mellifera, while antigen 5 is considered to be species-specific [P. gallicus and the American species P. annularis showed an 85% identity. The sequence identity increased to 98% within the same subgenus, suggesting that the presence of specific epitopes on antigen 5 should be related to the variations on the superficial loops [Polybia scutellaris, which belongs to the Polistinae subfamily, but does not cause allergic reactions, the amino acid analysis of antigen 5 showed a close similarity with antigen 5 of vespids from different species. However, two forms of antigen 5 were isolated, with the amino acids residues at positions 5 and 11 in P. scutellaris antigen 5 differing from those of the previously known sequences for antigen 5, suggesting that one or the other might be responsible for the lack of allergenicity of the P. scutellaris venom [P. scutellaris induced IgG antibodies which cross-reacted with Pol a 5, while Poly s 5 induced only minimal amounts of IgE and was a poor inducer of basophil-mediator release. This finding led the authors to propose Poly s 5 as a candidate for VIT [Polistinae including P. dominulus. Indeed, a different allergenicity of antigen 5 from Polistes spp. could be a possible factor explaining the different clinical outcome observed with Polistes VIT. This is similar to what is known for food allergy; for example, a divergent allergenicity of apple strains mainly depended on different expression levels of the major allergen Mal d 1, with a proline residue in position 111 causing a drastic reduction of allergenicity [These contrasting data should stimulate discussion regarding the allergens from llergens . In the llergens . Concernspecific . Howeveral loops . Remarkais venom . Vinzon  for VIT , but it genicity .P. dominulus for VIT in patients allergic to Polistes venom, unless the patient is monosensitized to the latter. In patients with positive test results to both P. dominulus and mAP, performing VIT with any of the two venoms has the same odds of clinical success.Currently, there is no scientific report supporting the use of only S1 Fig(DOCX)Click here for additional data file.


Sirt1 inhibition of FcεRI signaling also involved an alternative component, PTP1B, which attenuated the inhibitory AMPK pathway and conversely enhanced the stimulatory Syk pathway, uncovering a novel role of this phosphatase. Moreover, a Sirt1 activator resveratrol stimulated the inhibitory AMPK axis, with reciprocal suppression of the stimulatory PTP1B/Syk axis, thus potently inhibiting anaphylaxis. Overall, our results provide a molecular explanation for the beneficial role of Sirt1 in allergy and underscore a potential application of Sirt1 activators as a new class of anti-allergic agents.Sirt1, a key regulator of metabolism and longevity, has recently been implicated in the regulation of allergic reactions, although the underlying mechanism remains unclear. Here we show that Sirt1 negatively regulates FcεRI-stimulated mast cell activation and anaphylaxis through two mutually regulated pathways involving AMP-activated protein kinase (AMPK) and protein tyrosine phosphatase 1B (PTP1B). Mast cell-specific knockout of Sirt1 dampened AMPK-dependent suppression of FcεRI signaling, thereby augmenting mast cell activation both  Besides these positive signaling pathways, understanding of the negative regulatory mechanisms that turn off positive signals is also of importance to gain comprehensive insights into FcεRI signaling and thereby a novel strategy for treatment of allergic diseases. Examples for such negative regulatory mechanisms involve the tyrosine phosphatases SHP-1 and SHIP in FcεRI- mediated mast cell activation4, the Cbl family ubiquitin ligases, which facilitate degradation or internalization of the activated FcεRI signaling components5, and the inhibitory kinase Csk, which phosphorylates and thereby inactivates the FcεRI-proximal kinases Lyn and Fyn1. Additionally, we have recently shown that AMP-activated protein kinase (AMPK), which is generally known to be activated during energy insufficiency and is essential for metabolic homeostasis7, represents a novel negative regulatory module for FcεRI signaling by altering the subcellular distribution of Fyn and ERK9.Mast cells represent a highly specialized cell population that plays a central role in allergic diseases. Crosslinking of FcεRI-bound IgE with antigen (Ag) on mast cells induces the activation of proximal FcεRI-associated Src kinases (Lyn and Fyn) and Syk, which in turn activate multiple signaling pathways including phospholipase Cγ (PLCγ), mitogen-activated protein kinases, Akt and NF-κB, leading to release of preformed mediators by degranulation and +-dependent type III histone/protein deacetylase, deacetylates several transcriptional and related factors, thereby regulating energy metabolism, aging, senescence and inflammation12. Similar to AMPK, Sirt1 is regulated in response to energy demand and its dysregulation is associated with metabolic syndrome and inflammation16. However, the roles of Sirt1 in allergic diseases are controversial, because Sirt1 reportedly prevents or exacerbates allergic responses in distinct settings21. Some of these studies relied on the pharmacological effects of resveratrol, a red grape-derived polyphenol that has frequently been used as a Sirt1 activator. Indeed, recent studies demonstrated the therapeutic effects of resveratrol on allergic symptoms in both humans and rodents27, supporting the anti-allergic action of Sirt1. However, the roles of Sirt1in allergy have not been firmly established, since it is uncertain whether resveratrol acts on only Sirt1 or some another unknown molecule(s) to exert its actions27.Sirtuin 1 (Sirt1), a ubiquitously expressed NAD30. In hepatocytes, the Sirt1 activator resveratrol activates AMPK, whereas the Sirt1 inhibitor nicotinamide suppresses both Sirt1 and AMPK32. Resveratrol improves insulin sensitivity and mitochondrial function and extends the lifespan of obese mice through activation of Sirt1 and AMPK33. Overexpression of Sirt1 reduces lysine acetylation of LKB1, leading to interaction with and activation of downstream AMPK34. Reciprocally, AMPK functions as a Sirt1 activator by increasing the level of cellular NAD+ or the activity of nicotinamide phosphoribosyltransferase, an NAD+-biosynthetic enzyme35. Together, Sirt1, LKB1 and AMPK are coordinately regulated to form a feed-forward cycle. Given these facts, it can be speculated that Sirt1 may have a negative regulatory role in mast cell activation through interaction with AMPK, although experimental evidence for this hypothesis has currently been lacking.Crosstalk between Sirt1 and AMPK has attracted attention in the fields of energy homeostasis, aging and longevityIn this study, we investigated the roles of Sirt1 in mast cells using pharmacological and genetic approaches. We show that Sirt1 indeed cooperates with AMPK in mast cells, thereby dampening FcεRI signaling. Unexpectedly, the inhibitory action of Sirt1 on FcεRI signaling also relies on an alternative pathway involving protein-tyrosine phosphatase 1B (PTP1B), whose role in mast cells had been controversial. Our results show that PTP1B inhibits AMPK and activates Syk to facilitate FcεRI signaling, and these processes are counteracted by Sirt1.9. Given that Sirt1 lies upstream of AMPK36, we investigated the effect of resveratrol, a Sirt1 activator, on IgE/Ag-stimulated mast cell activation in the context of AMPK signaling. First, to determine the proper concentration of resveratrol for Sirt1 activation, mouse bone marrow-derived mast cells (BMMCs) sensitized with anti-dinitrophenyl (DNP) IgE were treated with various concentrations (1–100 μM) of resveratrol for 1 h prior to stimulation with DNP-human serum albumin (HSA) as an Ag. As reported previously9, IgE/Ag stimulation resulted in a substantial decrease in constitutive phosphorylation of LKB1, AMPK, and their well-known downstream target acyl-CoA carboxylase (ACC) , generation of the lipid mediators LTC4 and PGD2, secretion of the cytokines TNF-α and IL-6, and increase of the intracellular calcium level  Fig. . ResveraIKK Fig. . In addiIKK Fig. , which avel Fig. . These rTo address the role of Sirt1 further, BMMCs were transiently transfected with Sirt1-specific siRNA. Treatment of BMMCs with Sirt1 siRNA, in comparison with that with mock siRNA, greatly if not entirely abrogated the expression of Sirt1, accompanied by a substantial increase in FcεRI-induced Ac-Lys and a reduction in constitutive phosphorylation of LKB1, AMPK, and ACC Fig. , suggest4 and PGD2 were significantly greater in Sirt1-knockdown cells than in control cells  in BMMCs resulted in a marked reduction in IgE/Ag-induced Ac-Lys relative to mock cells . Sirt1 deficiency in BMMCs completely abrogated Sirt1 protein expression and resveratrol-induced deacetylation of FcεRI-driven Ac-Lys  BMMCs , suggesting that the ablation of AMPKα2 decreased the deacetylase activity of Sirt1. This action appeared to be independent of LKB1, because its phosphorylation was barely affected by AMPKα2 deficiency. As reported previously9, FcεRI-dependent phosphorylation of PLCγ1, ERK, JNK, and IKK, but not Akt and p38, was higher in AMPKα2-silenced or -deficient cells than control cells  using mast cell-specific ivo Fig. . Likewisice Fig. , as repoice Fig.  and by oice Fig. . AltogetSirt1−/− cells than in WT cells, and resveratrol reversed it markedly in WT cells and partially in Sirt1−/− cells  other than the Sirt1/LKB1/AMPK axis. Therefore, we examined whether resveratrol could affect the phosphorylation of FcεRI-proximal tyrosine kinases, Lyn, Fyn and Syk. Resveratrol attenuated IgE/Ag-induced phosphorylation of Syk and its target adaptor LAT, without affecting that of Lyn and Fyn Fig. . The efflls Fig. . BecauseNotably, Sirt1 was constitutively associated with LKB1/AMPK in unstimulated BMMCs, whereas it dissociated from LKB1/AMPK and instead interacted with Syk in IgE/Ag-stimulated BMMCs Fig. . Moreove42. While SHP-1 is a well-known protein tyrosine phosphatase that negatively regulates FcεRI signaling43, the role of PTP1B in mast cell activation is currently obscure44. We found that PTP1B phosphorylation was robustly increased following IgE/Ag stimulation, which was reversed by resveratrol  increased Ac-Lys and attenuated the phosphorylation of LKB1, AMPK, and ACC in IgE/Ag-stimulated cells and even in resting cells Fig. , suggestGD2 Fig. . The posGD2 Fig. . AccordiGD2 Fig. .Figure 64 and PGD2 generation in PTP1B-silenced BMMCs  probably due to differences in animal species, anatomical sources, or experimental conditions. Nonetheless, our present study is the first to demonstrate the mechanistic actions of Sirt1 in mast cells.Crosstalk between Sirt1 and AMPK, as evidenced by the findings of their shared common targets and their reciprocal regulation in diverse cellular responsesvice versa. Consistently, overexpression of Sirt1 increased the activation of AMPK. Furthermore, Sirt1 overexpression mimicked the effects of resveratrol, whereas its down-regulation attenuated the sensitivity to resveratrol and increased mast cell activation. Likewise, AMPK knockdown decreased Sirt1 activity and increased FcεRI-mediated signaling. Importantly, both mast cell-specific Sirt1−/− mice and AMPKα2−/− mice displayed increased susceptibility to anaphylaxis, with a diminished anti-allergic effect of resveratrol, implying the physiological relevance of our observations. Although there are contrasting reports of the offensive and protective roles of Sirt1 in mouse asthma models21, our results support the anti-allergic function of Sirt1. Presumably, the pro- or anti-inflammatory roles of Sirt1 may depend on the cell types involved, which may differ among experimental settings.We provide several lines of evidence that Sirt1 and AMPK require each other for their optimal activation, forming a feed-forward cycle. Ablation of Sirt1 by siRNA knockdown or genetic deletion reduced the activation of AMPK, and 42, we herein show, for the first time, that PTP1B has a positive role in FcεRI signaling in two ways. On one hand, PTP1B is activated and associated with Syk toward increased FcεRI signaling. On the other hand, it counteracts the inhibitory AMPK axis by sequestering Sirt1 from the LKB1/AMPK complex. Sirt1 mutually interacts with PTP1B/Syk and LKB1/AMPK toward inhibition and activation of the positive and negative pathways, respectively, thereby dampening mast cell activation. Although it has been reported that Sirt1 downregulates PTP1B expression in insulin signaling42, our results do not agree with this observation, but rather support the idea that Sirt1 inhibits PTP1B activation without affecting its expression. While the activation of LKB1/AMPK by Sirt1 involves deacetylation of LKB1, it remains to be determined whether the deacetylation of PTP1B, Syk, or another unknown component(s) underlies the Sirt1 inhibition of PTP1B/Syk.In light of the finding that Sirt1 activation by resveratrol regulates PTP1B leading to enhanced insulin sensitivity46. Of these sites, phosphorylation of Tyr317 in the linker region of Syk not only suppresses its kinase activity, but also provides a binding site for the ubiquitin ligase Cbl, which promotes the degradation of Syk47. Co-expression of Cbl with Syk decreases the autophosphorylated pool of Syk, eventually hindering Syk signaling5. Reminiscent of this, beyond the contribution of PTP1B to attenuation of insulin signaling49, PTP1B plays a positive role in activation of Src tyrosine kinase, where it dephosphorylates the negative regulatory domain of Src and thereby activates it50. We therefore speculated that PTP1B may be responsible for the dephosphorylation of Tyr317 of Syk, thereby increasing its activity or stability. As opposed to our speculation, however, the phosphorylation of Tyr317 was increased (rather than decreased) by FcεRI-driven mast cell activation and was conterregulated by resveratrol, without a change in the protein level of Syk (data not shown), arguing against the hypothesis that Tyr317 is a dephosphorylation site for PTP1B. Additionally, the other candidate Tyr residues, Tyr352 and Tyr525, of Syk were also phosphorylated, rather than dephosphorylated, following IgE/Ag-stimulation, without being affected by resveratrol (data not shown). These results suggest that PTP1B regulates Syk activation either directly by targeting phosphotyrosine residue(s) other than Tyr317, Tyr352 and Tyr525 of Syk or indirectly by dephosphorylating other signaling molecule(s) involved in Syk activation. Therefore, it should be interesting to identify the target site(s) of PTP1B during mast cell activation in the future study to fully understand the underlying mechanisms for Syk regulation by PTP1B.Syk has multiple tyrosine phosphorylation sites, which are involve in positive or negative regulation of Syk signaling51. In contrast, a study using PTP1B−/− mice has shown that PTP1B plays a role in amelioration of allergen-induced airway inflammation and leukocyte trafficking52. These conflicting outcomes may be because the asthmatic model in the latter study depends on eosinophils and T cells rather than mast cells. In addition, unlike our present results as evaluated by transient by PTP1B knockdown or overexpression, a recent study employing PTP1B knockout has led to the conclusion that PTP1B has a negligible role in mast cell activation44. Although the reason for this discrepancy is unclear, similar situations have also been reported for other FcεRI signaling molecules such as Fyn and Lyn54. This difference could be because permanent knockout might have some developmental changes that ensure compensatory adaptation, while knockdown is an acute effect devoid of such adaptation. Alternatively, PTP1B might affect other signaling pathways which could vary according to experimental conditions or cellular sources.The roles of PTP1B in exacerbation or amelioration of inflammation are controversial. Reminiscent of our present study, PTP1B has been reported to contribute to exacerbation of neuroinflammationSirt1−/− mast cells suggests that the resveratrol effect also relies in part on a Sirt1-independent mechanism, which remains to be elucidated. Considering that mast cell activation can be suppressed by anti-oxidants55 the anti-oxidant moiety of polyphenol might account for the Sirt1-independent action of resveratrol. Alternatively, other sirtuin members may compensate for Sirt1 in mast cells.Overall, our present findings are summarized in Supplementary Figure Understanding of the mechanisms underlying allergic reactions is still incomplete. Besides therapeutics that have been clinically used to date, an alternative approach for the treatment of allergic diseases is desired. In this context, a strategy that activates Sirt1 may have a novel therapeutic potential to treat allergic diseases.AMPKα2−/− mice on the C57BL/6J background were reported previously56.Balb/cJ and C57BL/6J mice were obtained from Samtako, INC. Mast-Cma1-Cre and Sirt1-floxed mice were purchased from Jackson Laboratories . Mast-Cma1-Cre mice were backcrossed for 7 to 9 generations to the C57BL/6J background and then crossed with Sirt1-floxed mice (C57BL/6J background) to produce age-matched Sirt1+/+Mast-Cma1-Cre and Sirt1fl/flMast-Cma1-Cre (termed Sirt1−/− hereafter) mice. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Yeungnam University8.8. Briefly, BMMCs were cultured in RPMI 1640 medium containing 10% (v/v) FBS, 100 U/ml penicillin (Thermo Fisher Scientific), 10 mM HEPES (Sigma-Aldrich), 100 μM MEM non-essential amino acid solution (Invitrogen) and 20% (w/v) PWM-SCM (pokeweed mitogen-spleen cell conditioned medium) as a source of IL-3. For cell stimulation, 1 × 106 cells/ml were sensitized with 500 ng/ml mouse anti-DNP IgE (Sigma-Aldrich) overnight and then stimulated with 100 ng/ml DNP-HAS (Sigma-Aldrich) typically for 15 min at 37 °C. Intracellular Ca2+ levels at 5 min, releases of β-Hex (a marker of mast cell degranulation) and eicosanoids (LTC4 and PGD2) at 15 min, and production of cytokines (IL-6 and TNF-α) at 6 h were evaluated as described previously8. PGD2, LTC4, IL-6 and TNF-α were quantified using respective immunoassay kits for eicosanoids  and for cytokines (R&D Systems). When the effects of resveratrol (Sigma-Aldrich) were examined, it was dissolved in DMSO and added 1 h prior to the addition of Ag, with DMSO at a final concentration of 0.1% (v/v) as a vehicle control in all cases.BMMCs were isolated from 6~7-wk-old male Balb/cJ or C57BL/6 mice, as described previously8. Briefly, cells were washed twice with ice-cold PBS and lysed in SDS-sample buffer containing 1% (v/v) NP-40, 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, and 1 μg/ml pepstatin A for 30 min on ice. Lysates were centrifuged at 14,000 g for 20 min at 4 °C and resulting supernatants were subjected to immunoblotting. For immnoprecipitation, cell lysates were prepared in modified lysis buffer . Total cell lysates  were incubated with various antibodies for 2 h at 4 °C and the immune complexes were precipitated with 20 μl of protein A-Sepharose. The precipitates were then washed three times with ice-cold lysis buffer. The precipitates or total cell lysates were subjected to SDS-PAGE and immunoblotted with corresponding antibodies.Immunoprecipitation and immunoblotting were performed as described previouslyAntibodies against phosphorylated forms of LKB1(Ser428), AMPKα2 (Thr172), ACC (Ser79), PLCγ1 (Tyr783), Akt (Ser473), p38, ERK1/2, JNK, IKKα/β and those against LKB1, AMPKα, ACC, Akt, p38, ERK1/2, JNK and Sirt1 were from Cell Signaling Technology. Antibodies against IKKα/β, PLCγ1, LAT, Syk, Fyn, Lyn, and β-actin were from Santa Cruz Biotechnology. Anti-phosphotyrosine (pY) and –acetyl-lysine (Ac-Lys) antibodies were from Millipore, anti-PTP1B antibody was from ECM Biosciences, and anti-AMPKα2 antibody was from Abcam. Rabbit IgG was from Gen Tex.8. Mouse AMPKα2 siRNA and Sirt1 siRNA in the SMARTpool were obtained from Dharmacon, and non-specific siRNA and mouse PTP1B shRNA were obtained from Santa Cruz Biotechnology. BMMCs were cultured for 16 h in serum-free medium and transfected with a DharmaFECT transfection reagent (Dharmacon) containing siRNA (100 nM per well) or shRNA lentiviral particles (5 × 104 IFU per well) according to the manufacturer’s protocol in 12-well plates. After 24 h, BMMCs were sensitized with IgE in the presence or absence of resveratrol and then stimulated with DNP-HSA as above.Knockdown experiments were carried out as described previouslyBMMCs were infected with adenovirus carrying Sirt1 (Ad-Sirt1) or PTP1B (Ad-PTP1B) (Vector Biolabs) at 100 MOI (multiplicity of infection) according to the manufacturer’s protocol. After 24 h, medium was replaced with fresh RPMI1640 and then the cells were stimulated with IgE/DNP-HSA as described above.8. Briefly, 80 ng of mouse anti-DNP IgE was intradermally injected into ears of 7-wk-old male mice. After 24 h, mice were challenged intravenously with 60 ng of DNP-HSA containing Evans blue. As required for experiments, oral administration of resveratrol was given 1 h before PCA. After 1 h, Evans blue was extracted with formamide at 63 °C overnight and quantified by absorbance at 630 nm. In another set of experiments, 109 PFU of control adenovirus  or 1~2 × 109 PFU of adenovirus bearing shRNA for PTP1B (Ad-PTP1B-shRNA) (Vector Biolabs) was intradermally administered into mice ears. After 6 h, mice were sensitized with anti-DNP IgE for 24 h and then challenged with DNP-HSA as described above. Blood was collected by cardiac puncture at 1 h after Ag challenge to determine serum LTC4 and PGD2 levels as described above. Preparation of immunoblot samples from ear tissues were carried out as described previously57.PCA was carried out as described previouslyt test and multiple comparisons were analyzed using one-way ANOVA. All data are presented as means ± S.E.M. Differences were considered statistically significant at P < 0.05.Data calculation and statistical analysis were performed using GraphPad Prism 3.0 software. The statistical significance of differences between two groups was determined with unpaired Student’s Supplementary information


The Ste20-like kinase, SLK, has diverse cellular functions. SLK mediates organ development, cell cycle progression, cytoskeletal remodeling, cytokinesis, and cell survival. Expression and activity of SLK are enhanced in renal ischemia-reperfusion injury, and overexpression of SLK was shown to induce apoptosis in cultured glomerular epithelial cells (GECs) and renal tubular cells, as well as GEC/podocyte injury in vivo. The SLK protein consists of a N-terminal catalytic domain and an extensive C-terminal domain, which contains coiled-coils. The present study addresses the regulation of SLK activity. Controlled dimerization of the SLK catalytic domain enhanced autophosphorylation of SLK at T183 and S189, which are located in the activation segment. The full-length ectopically- and endogenously-expressed SLK was also autophosphorylated at T183 and S189. Using ezrin as a model SLK substrate (to address exogenous kinase activity), we demonstrate that dimerized SLK 1–373 or full-length SLK can effectively induce activation-specific phosphorylation of ezrin. Mutations in SLK, including T183A, S189A or T193A reduced T183 or S189 autophosphorylation, and showed a greater reduction in ezrin phosphorylation. Mutations in the coiled-coil region of full-length SLK that impair dimerization, in particular I848G, significantly reduced ezrin phosphorylation and tended to reduce autophosphorylation of SLK at T183. In experimental membranous nephropathy in rats, proteinuria and GEC/podocyte injury were associated with increased glomerular SLK activity and ezrin phosphorylation. In conclusion, dimerization via coiled-coils and phosphorylation of T183, S189 and T193 play key roles in the activation and signaling of SLK, and provide targets for novel therapeutic approaches. Glued dynactin subunit . Th. Th24]. In the present study, we examined the effects of coiled-coil domain mutations on SLK phosphorylation and kinase activity. COS-1 cells were transfected with WT or coiled-coil mutants of full-length HA-SLK. Immunoblotting with anti-pT183 antibody detected T183 phosphorylation in HA-SLK WT, as well as coiled-coil domain mutant transfections  . The faiFinally, to monitor kinase activity, cells were transfected with WT or coiled-coil mutants of full-length HA-SLK, plus GFP-ezrin. Transfection of full-length SLK WT stimulated phosphorylation of GFP- and endogenous ezrin, while I848G, L986G and L986G/I989G mutations showed significantly lower ezrin phosphorylation . As expeThe above experiments relied on expression of SLK WT and mutants in cells by transfection. To address the kinase activity of endogenous SLK, we initially employed C2C12 myoblasts, which express a ~3-fold higher level of endogenous SLK, compared with untransfected COS-1 cells . It was We have demonstrated important functional roles for SLK in kidney GECs in culture and in vivo ,8. Next,Erlotinib was originally developed as an inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase, but was later found to have a greater binding affinity for the kinase activity of SLK (and the related lymphocyte-oriented kinase), and to be an effective SLK inhibitor ,37. The To further delineate the specificity of erlotinib, COS-1 cells were transfected with Fv-SLK 1–373 WT. AP20187 was added to induce dimerization of the catalytic domain, in the presence or absence of erlotinib. In keeping with earlier experiments, there was some basal phosphorylation in SLK T183, and this was enhanced by AP20187 . NeitherAs noted above, in vivo, GECs are phenotypically distinct from fibroblasts, but have been viewed as possessing a motile phenotype within the glomerulus, and perhaps becoming hypermotile after injury . To linkTo address SLK activation and phosphorylation in a pathophysiological context, we examined SLK activation in the PHN model of experimental membranous nephropathy, using quantitative immunofluorescence microscopy. In PHN, binding of anti-Fx1A antibody to GEC/podocyte plasma membranes leads to formation of immune deposits, complement activation and podocyte injury ,38. FourThe present study comprehensively addresses the activation and signaling of SLK in the context of intact cells, including a physiologically relevant cell line (GECs). Using phospho-specific antibodies and mutagenesis, the study demonstrates the important role of the SLK T183 and S189 phosphorylation sites in the activation segment, as well as critical amino acid residues in the coiled-coil regions. Controlled dimerization of the SLK catalytic domain enhanced activation segment autophosphorylation at T183 and S189  23,27].,27.23,27Single mutations in T183, S189 or T193 partially reduced phosphorylation in the non-mutated activation segment sites, and showed greater reduction in ezrin phosphorylation Figs . The effPreviously, we employed a protein complementation assay to show that coiled-coil domain mutations reduced the ability of SLK to dimerize . The preThe protein kinase inhibitor, erlotinib, was employed to further characterize the catalytic activity of SLK. Interestingly, erlotinib did not reduce T183 phosphorylation appreciably in full-length SLK, or in monomeric and dimeric SLK 1–373 Figs  and 8. HBased on crystallography studies, phosphorylation in the kinase activation segment, such as pT183 in SLK, is believed to stabilize the kinase in a conformation suitable for substrate binding ,24,26,27Phosphoproteomic analyses have identified S189 and T183 phosphorylation in endogenous SLK in multiple tissues/cells –50. Othe+/H+-exchanger regulatory factor 2 (NHERF2). The ezrin-NHERF2-podocalyxin complex, situated on the apical side of the podocyte foot process, appears to be important in maintaining polarity and architecture [By analogy to other epithelia, in the kidney, SLK and ezrin phosphorylation may be important in maintaining the structure of microvilli on renal tubular epithelial cells . GECs/poitecture ,41,52, aitecture . Previouitecture . Both acitecture ,51,53. Hitecture . The chaitecture ,55, whic


The G protein-coupled bile acid receptor (TGR5) is a cell surface receptor which induces the production of intracellular cAMP and promotes epithelial-mesenchymal transition in gastric cancer cell lines. TGR5 is found in a wide variety of tissues including the kidney. However, the patterns of TGR5 expression have not been well characterized in physiologic kidney or renal neoplasms. We explore the expression of TGR5 in benign renal tissue and renal neoplasms and assess its utility as a diagnostic marker.Sixty-one renal cortical neoplasms from 2000 to 2014 were retrieved. TGR5 protein expression was examined by immunohistochemistry. TGR5 mRNA was also measured by real-time PCR.p < 0.001). TGR5 mRNA was also significantly decreased in clear cell RCCs, suggesting that decreased TGR5 protein expression may be attributable to the downregulation of TGR5 mRNA in these tumors. All 11 papillary RCCs expressed TGR5 with 45% (5/11) exhibiting moderate to strong staining. All chromophobe RCCs and oncocytomas were positive for TGR5 with weak to moderate staining. TGR5 mRNA expression in these tumors was similar to normal kidney. All urothelial carcinomas of the renal pelvis strongly expressed TGR5 including a poorly differentiated urothelial carcinoma with sarcomatoid features.In normal renal tissue, TGR5 was strongly positive in collecting ducts, distal convoluted tubules and thin loop of Henle. Proximal convoluted tubules showed absent or focal weak staining. In clear cell renal cell carcinomas (RCCs), 25 of 27 cases (92%) were negative for TGR5 (TGR5 is strongly expressed in collecting ducts, distal convoluted tubules and thin loop of Henle. TGR5 protein and mRNA expression were notably decreased in clear cell RCCs and may be helpful in differentiating these tumors from other RCCs. Renal malignancies are the 7th most common cancer in men in the US with approximately 14,000 attributable deaths annually . Benign The G protein-coupled bile acid receptor TGR5) is a cell-surface receptor mediating bile acid effects . Both pr is a celWith Institutional Review Board approval at Rhode Island Hospital, sixty-one renal cortical neoplasms were identified from the archives of the Department of Pathology between 2000 and 2014. These included 27 clear cell RCCs and 11 papillary RCCs, 8 of which were type 1 and 3 of which were type 2. The remainder of the cases consisted of 8 papillary urothelial carcinomas including one poorly differentiated case involving the cortex, 6 chromophobe RCCs, 5 oncocytomas, 2 clear cell papillary RCCs, 1 metanephric adenoma, and 1 poorly differentiated RCC with sarcomatoid features. Formalin-fixed paraffin-embedded tissue sections and blocks were retrieved. The corresponding hematoxylin-eosin slides were reviewed for confirmation of diagnosis and adequacy of material by a specialist in genitourinary pathology (AA). Multiple representative sections of each case were examined. Corresponding normal kidney control sections separate from the tumors were also reviewed for all cases. Snap-frozen tissue from normal kidney, clear cell RCCs, papillary RCCs, chromophobe RCC and oncocytomas was obtained from the Rhode Island Hospital tissue bank.Paraffin blocks containing areas of carcinomas were identified on the hematoxylin and eosin-stained sections. Areas of the tumor were identified and marked on the source block. The source block was cored using a 1-mm needle and the tissue was transferred to a recipient “master block” using a Beecher Tissue Microarrayer . Three to six cores of each tumor were arrayed per specimen. Additionally, a core of normal kidney tissue was also sampled when present .Immunohistochemistry for TGR5 was performed on 4-μm paraffin sections of the tissue microarray or whole tissue sections. Slides were stained with TGR5 antibody  using the DAKO Envision + Dual Link System and the DAKO Liquid 3,3′-Diaminobenzidine Substrate Chromogen System . Bile ducts from liver tissue were used as positive controls. Negative controls were included by replacement of the primary antibody with non-reacting antibodies derived from the same species. The specificity of TGR5 antibody has previously been confirmed by Western blot analysis in our lab .Tissue microarray was employed to study normal kidney and 44 cases of renal neoplasms, including all clear cell RCCs, 1 metanephric adenoma, 8 papillary urothelial carcinomas, 4 type 1 papillary RCCs, 3 oncocytomas, and 1 chromophobe RCC.Immunohistochemistry was also performed on whole tissue sections for 17 cases including 4 papillary RCC (4 type 1 papillary RCC and 3 type 2 papillary RCC), 5 chromophobe RCC, 2 oncocytomas, 2 clear cell papillary RCC, and 1 poorly differentiated RCC with some sarcomatoid features.Immunohistochemical results were evaluated semi-quantitatively within neoplastic tissue. Cells displaying strong intensity staining for TGR5 were scored as 3+, moderate staining as 2+, and weak staining as 1+. The extent of staining was scored as follows: 0 (< 5%), 1+ (5–25%), 2+ (26–50%), 3+ (51–100%). A combined score of intensity and extent was calculated and assigned as follows: weak staining 1–2, moderate staining 3–4, and strong staining 5–6. At least three cores were scored per case. Analysis of three cores per case in this fashion has been shown to be comparable to whole tissue sections . All sec’-CGGACAGGATTGACAGATTGATAGC-3’), and 18S antisense (5’-TGCCAGAGTCTCGTTCGTTATCG-3’). Reactions were carried out in an Applied Biosystems StepOnePlus real-time PCR system  for one cycle at 94 °C for 5 min followed by 40 cycles at 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s, 1 cycle at 94 °C for 1 min, and 1 cycle at 55 °C for 30 s. Fluorescence values of SYBR Green I dye, representing the quantity of product amplified at that point in the reaction, were recorded in real time at both the annealing and extension steps of each cycle. The Ct, defined as the point at which the fluorescence signal becomes statistically significant above background, was calculated for each amplicon for each sample using StepOne software . The transcript level of each specific gene was normalized to 18S amplification.TGR5 mRNA was measured using real-time PCR analysis as we have previously described . The prip-value of 0.05 or less was considered statistically significant.A Chi-square test was utilized to compare groups where appropriate. Data were expressed as mean ± SEM. Statistical differences between two groups were determined by Student’s t-test. Differences among multiple groups were tested using analysis of variance (ANOVA) and checked for significance using Fisher’s protected least significant difference test. A In normal kidney, the proximal tubular cells are more columnar and eosinophilic than cells of the distal tubules on H&E slides. The ascending limb has no brush border and the cells are more cuboidal than adjacent proximal tubular cells . Accordip < 0.001, Fig. The expression of TGR5 in renal cell neoplasms is summarized in Table p = 0.152).All papillary RCCs (11/11) demonstrated TGR5 staining. The TGR5 staining in Type 1 papillary RCCs (8 cases) were variable from weak to strong staining Fig. . All 3 cp = 0.082) were identified. The metanephric adenoma  exhibited weak to moderate TGR5 staining Fig. . The oncUrothelial carcinomas all uniformly (8/8) expressed TGR5 Fig.  includinN = 5, p < 0.001, fig. TGR5 mRNA was significantly diminished in clear cell RCC (+/K+-ATPase activity than any other tubular segment [+/K+-ATPase activity in the distal tubular cells. This might account for high TGR5 expression in the distal tubular cells in the kidney.The distal convoluted tubules play a key role in regulating extracellular fluid volume and electrolyte homeostasis. Distal tubular cells are rich in mitochondria and biochemical studies reveal that distal tubular cells have a higher level of Na segment –18. Rece segment . TGR5 ha segment . Our datImmunohistochemical staining demonstrated no expression of TGR5 in the glomeruli with the exception of some parietal cells within Bowman’s capsule. This is consistent with prior studies in the literature . Xiong eStrong expression of TGR5 in distal tubular cells and collecting ducts lends credence to the theory that some renal tumors such as papillary RCCs, chromophobe RCCs, and oncocytomas likely derive from distal convoluted tubules or collecting ducts as suggested by some studies , 21. EleClear cell RCCs comprise 70% to 80% of all RCCs . Clear cPapillary RCCs are the second most common histologic subtype and comprise 7–15% of all RCCs . ImmunohUrothelial carcinomas of the renal pelvis including sarcomatoid type of poorly differentiated urothelial carcinomas strongly expressed TGR5. Although only one case poorly differentiated RCC with sarcomatoid features was included in our study, this case was negative for TGR5. This may suggest that TGR5 could be used to differentiate poorly differentiated urothelial carcinomas from poorly differentiated RCCs. Further studies with a larger cohort are necessary to establish the utility of TGR5 for this application.Our cases suggest that TGR5 would have overall limited diagnostic utility in differentiating among non-clear cell RCCs. All papillary RCCs showed TGR5 positive staining. No significant differences in TGR5 expression between type 1 and type 2 papillary RCCs were detected. Similarly, TGR5 staining may not be useful to differentiate chromophobe RCCs from oncocytomas.In summary, we employed immunohistochemistry to establish that TGR5 expression is robust in the distal convoluted tubules, thin loop of Henle, and collecting ducts. Among renal neoplasms, papillary RCCs, clear cell papillary RCCs, chromophobe RCCs and oncocytomas show various degrees of TGR5 expression. UCCs of the renal pelvis also strongly express TGR5. We demonstrate that the majority of clear cell RCCs, particularly cases without papillary features, are negative attributable to their curtailed expression of TGR5 mRNA. Overall, our data suggest that TGR5 staining may contribute to the diagnostic workup in distinguishing clear cell RCC from other renal tumors.


Innate immune receptors NOD1 and NOD2 are activated by bacterial peptidoglycans leading to recruitment of adaptor kinase RIP2, which, upon phosphorylation and ubiquitination, becomes a scaffold for downstream effectors. The kinase domain (RIP2K) is a pharmaceutical target for inflammatory diseases caused by aberrant NOD2-RIP2 signalling. Although structures of active RIP2K in complex with inhibitors have been reported, the mechanism of RIP2K activation remains to be elucidated. Here we analyse RIP2K activation by combining crystal structures of the active and inactive states with mass spectrometric characterization of their phosphorylation profiles. The active state has Helix αC inwardly displaced and the phosphorylated Activation Segment (AS) disordered, whilst in the inactive state Helix αC is outwardly displaced and packed against the helical, non-phosphorylated AS. Biophysical measurements show that the active state is a stable dimer whilst the inactive kinase is in a monomer-dimer equilibrium, consistent with the observed structural differences at the dimer interface. We conclude that RIP2 kinase auto-phosphorylation is intimately coupled to dimerization, similar to the case of BRAF. Our results will help drug design efforts targeting RIP2 as a potential treatment for NOD2-RIP2 related inflammatory diseases. NOD1 and NOD2 (Nucleotide-binding oligomerization domain-containing proteins 1 and 2) are cytosolic receptors of the innate immune system, which respond to intracellular fragments of the bacterial peptidoglycans, D-glutamyl-meso-diaminopimelic acid (iE-DAP) and muramyl dipeptide (MPD), respectively . Upon liin vivo host responses to MPD . ThThe AS helix encompasses residues 167–178, thus including the phosphorylation sites Ser174, Ser176 and Ser178 while residues 180–184 remain disordered . The rotD311N in complex with staurosporine [K299R/D389N [Interestingly the helical AS from monomer A in the AB dimer projects outwards towards the active site of the asymmetrically related monomer C from the CD dimer, forming a putative substrate-enzyme phosphorylation complex . Despiteosporine , Aurora osporine , and PAK9R/D389N , which aIn the RIP2K-AMPPCP structure, there is an extensive hydrogen-bonding (H-bond) network linking together Helix αC and the ordered part of the AS with their surroundings including the N-terminal anti-parallel β-strands ; S4 Fig.Proper alignment of the R-spine usually depends on the conformation of the AS together with the magnesium binding loop (which comprises the DFG motif) and the HRD (HHD for RIP2K) motif and importantly the correct orientation of the Helix αC , 74 to iFurthermore Glu68 from Helix αC in the active conformation coordinates Trp170 via water molecule . The tryK47R structure, the reorientation of Helix αC and the AS helix creates an alternative hydrogen bonding network between these two elements, the magnesium binding loop and dimer interface at the N-terminus  can be phosphorylated by full length RIP2, indicating that this is trans-phosphorylation rather than cis-phosphorylation. Comparison of the inactive and active states reveals the importance of coupled kinase dimerization and auto-phosphorylation in promoting enzyme activation. The asymmetric unit of RIP2KK47R suggests a possible model of trans-phosphorylation, where the RIP2K phosphorylation occurs between two side-by-side un-phosphorylated RIP2K dimers  [Comparison of our RIP2K structures reveals differences in the conformation of the Lys209 loop. In all structures, Lys209, at the C-terminal end of the Lys209 loop (residues 200–210), is well-ordered with its side-chain engaged in hydrogen bonds with the main-chain carbonyl-oxygens of P277 and R280 thus stabilising a helical turn conformation of residues 277–280. Generally the rest of the Lys209 loop is disordered, notably in the active, AMPPCP bound, structures. However in chain A of the RIP2Krt helix . A similctively) , 39. TheK47R and the RIP2K-AMPPCP dimer, respectively. In particular, their dimeric interfaces are almost identical . Thus, ATP can be bound and trans-phosphorylation can occur between two dimers. Consequently, the phosphorylated AS (green) becomes unstructured and moves closed to the Lys209 loop, which is also disordered. The N-lobe is coloured in dark grey, C-lobe in light grey. Helices are represented as cylinders. Disordered regions are in dashed lines. Phosphorylation sites are represented as red circles, ATP molecules as a brown triangular. Domain colouring is consistent with Figs S1 Fighttp://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) with identical residues highlighted in red, and similar residues written in blue.The secondary structure assignment of PKA is shown on top. Relevant kinase domains and residues are highlighted: Gly-rich loop (residues 50–57 in PKA), the β-strand 3 , Helix αC , the motif HRD , the highly conserved triplets DFG and APE which flank the Activation Segment (Asp184-Phe185-Gly186 and Ala 206-Pro-207-Glu208 in PKA).Residues belonging to the Magnesium binding loop are highlighted with a black dashed line. Residues belonging to the R- and C-spines are highlighted in yellow and light blue respectively  . Figure (TIF)Click here for additional data file.S2 Figm/z 1019.53, and its oxidized form at m/z 1035.52, and two doubly phosphorylated peptides at m/z 1099.65 (oxidized at 1115.55 and 1131.58) and at 1401.77 (oxidized at 1417.48). Two triply phosphorylated peptides were found at m/z 1179.47 (oxidized 1195.62) and 1481.41 (oxidized at 1497.63 and 1513.27). Moreover, a peptide detected at m/z 1561.38 was phosphorylated four times (oxidized at 1577.19 and 1593.19). These signals corresponded to two phospho-peptides differently modified. Those at m/z 1019.53, 1099.65 and 1179.47 corresponded to the modified 172-MMSLSQSR-179 and three phosphorylation sites were assigned to Ser174, Ser176 and Ser178. The phospho-peptides at m/z 1401.77, 1481.41 and 1561.38 corresponded to the phosphorylated 172-MMSLSQSRSSK-182 and the assignment of the three phosphorylation sites  was further confirmed. A fourth phosphorylation site (present in the phospho-peptide at m/z 1561.38) could correspond either to Ser180 or to Ser181. LC-MS/MS analyses on the phosphorylated 172-MMSLSQSR-179 and 172-MMSLSQSRSSK-182 further confirmed the assignment of the Ser174, Ser176 and Ser178 modification sites (data not shown). The presence of additional phosphorylated amino acids within 172-MMS…GQK-203 was suggested by LC-MS/MS experiments, but the exact sites of modification could not be assigned. Overall, the RIP2K AS is phosphorylated on Ser174, Ser176 and Ser178 and on Ser180 or Ser181.A) Sequence of the AS of the RIP2K and its possible phosphorylation sites. B) Phosphopeptides purified by IMAC and analysed by MALDI-TOF MS. We identified a singly phosphorylated peptide at (TIF)Click here for additional data file.S3 FigRibbon diagrams of BRAF kinase domain (A) the active conformation (PDB code:1UWH), (B) the inactive conformation (PDB code: 4WO5) and (C) the side-by-side dimer. N- and C-lobes are represented in dark and light grey respectively. Labelling is consistent with (TIF)Click here for additional data file.S4 FigThe 2Fo-Fc map is shown as counter level at 1.0 σ. Domain colouring is the same as in (TIF)Click here for additional data file.S1 TableAlso shown is the associated experimental molecular weight (MW) obtained by analysing intact proteins by LC/ESI-MS. The MWs shown refer to the un-phosphorylated form.(PDF)Click here for additional data file.S2 TableSecondary structure matching”. PDB codes are shown within parenthesis.The calculations were run with Superpose , using t(PDF)Click here for additional data file.


The cAMP-degrading enzymes, phosphodiesterases (PDEs), localise to specific subcellular domains within which they control local cAMP levels and are key regulators of signal compartmentalisation. Several components of the cAMP/PKA cascade are located to different mitochondrial sub-compartments, suggesting the presence of multiple cAMP/PKA signalling domains within the organelle. The function and regulation of these domains remain largely unknown. Here, we describe a novel cAMP/PKA signalling domain localised at mitochondrial membranes and regulated by PDE2A2. Using pharmacological and genetic approaches combined with real-time FRET imaging and high resolution microscopy, we demonstrate that in rat cardiac myocytes and other cell types mitochondrial PDE2A2 regulates local cAMP levels and PKA-dependent phosphorylation of Drp1. We further demonstrate that inhibition of PDE2A, by enhancing the hormone-dependent cAMP response locally, affects mitochondria dynamics and protects from apoptotic cell death.DOI:http://dx.doi.org/10.7554/eLife.21374.001 Mitochondria produce the majority of ATP required for a cell to function and perform vital functions in the maintenance of cellular metabolism and ion homeostasis. Additionally, mitochondria play an important role in apoptosis and are implicated in the pathogenesis of many diseases . MitochoMitochondria fission involves dynamin-related protein 1 (Drp1), a GTPase of the dynamin superfamily, which resides in the cytosol and translocates to the mitochondria upon activation by calcineurin-dependent dephosphorylation . Drp1 mu2+ and bicarbonate sensitive soluble adenylyl cyclase (sAC) . cAMP is synthesised either by a plasma membrane- associated adenylyl cyclase (pmAC), upon hormonal activation of Gs protein-coupled receptors, or by a Case (sAC) . cAMP/PKse (sAC) . Dysfuncse (sAC) .Phosphodiesterases (PDEs) constitute a superfamily of enzymes, which includes more than 100 isoforms, and are the only enzymes that degrade cAMP. Different PDE isoforms are uniquely regulated and distributed within the cell. Therefore, they differentially determine the local level of cAMP at specific subcellular sites, dictating which PKA targets are phosphorylated and the specificity of the downstream response .A number of components of the cAMP signalling cascade have been located at the mitochondria, including multiple AKAPs  and PDEsPDE2A is a 3',5'-cyclic guanosine monophosphate (cGMP)-activated PDE that degrades both cAMP and cGMP  and is ePrevious evidence suggests localisation of PDE2A2 to the mitochondrial matrix where it regulates ATP production via modulation of cAMP generated locally by sAC . Here, wWhen we expressed PDE2A2-GFP in primary neonatal rat ventricular myocytes (NRVM), we found that it is distinctly located at the mitochondria, unlike PDE2A1-GFP, which is cytosolic, and PDE2A3-GFP, which localises predominantly to the plasmalemma . We alsoDRP1-/-) , rescued the effect of Bay 60–7550 treatment  and found significant mitochondrial elongation compared to DMSO-treated controls . IncreasDRP1-/-) . Expressreatment , confirmPDE2KO). PDE2KO have significantly more elongated mitochondria than the wild type counterpart (MEFswt). The MEFsPDE2KO mitochondrial phenotype could be completely rescued by expression of a red fluorescent protein (RFP)-tagged PDE2A2 (PDE2A2wt-RFP), whereas expression of a catalytically inactive mutant of PDE2A2 (PDE2A2dn-RFP) in MEFsWT resulted in elongated mitochondria  results in a similar increase in cAMP in the bulk cytosol in the two cell types. In contrast, the cAMP increase at the OMM is significantly higher in MEFsPDE2wt than in MEFsPDE2KO  of the IBMX-sensitive PDE activity at the OMM.It has been previously reported that PDE2A2 is found in the mitochondrial matrix, where it hydrolyses cAMP generated locally by sAC . Howevere sensor . We nextFsPDE2KO . As expeTo further investigate the submitochondrial localisation of PDE2A2, we performed western blotting analysis of subcellular fractions from NRVM. PDE2A predominantly segregates with the mitochondrial fraction  and treaTo further assess localisation, we performed super-resolution stimulated emission depletion (STED) microscopy of HeLa cells. Cells expressing PDE2A2-GFP were double labelled with anti-GFP antibodies and antibodies to cytochrome C as a marker for the inter membrane space (IMS). As shown in Mitochondrial localisation of PDE2A in NRVM was further assessed by transmission electron microscopy and immunogold labelling. Analysis of immunogold particle localisation shows predominant particle association with mitochondrial membranes , with miOverall, the above data support localisation of PDE2A2 at the OMM and IMM, whereas the enzyme appears to be largely excluded from the intra-cristae space and from the matrix.PDE2KO and MEFswt to ionomycin (iono), a pro-apoptotic treatment that relies on mitochondrial Ca2+ loading and opening of the mitochondrial permeability transition pore. In addition, we used staurosporin (stau), a compound that triggers apoptosis via non-selective kinase inhibition  and protection from programmed cell death . FCCP aphibition . We founll death . In linell death . Knock de effect  and there effect . Treatmee effect . In addie effect  compared did not . We furtEFsDrp1KO.10.7554/WT) and MEFs derived from sAC knockout mice (MEFssACKO)   to confiFssACKO) . In addiEFssACKO . SimilarEFssACKO , confirmThe cAMP/PKA signalling cascade controls, within the same cell, multiple functions, affecting processes such as gene transcription, hormone secretion, cell metabolism, cell contraction, cell survival and cell death. This complex and multifaceted functional role is executed with accuracy thanks to a tight temporal and spatial control of protein phosphorylation, which is achieved by compartmentalisation of the molecular components of the signalling cascade. Compartmentalisation of cAMP offers the opportunity to target therapeutically individual cAMP pools, rather than global intracellular cAMP levels, in order to achieve greater efficacy and specificity of intervention . To asseNumerous studies have linked PDE2A to pathological conditions, including psychiatric, neurological , cardiovIn this study, we demonstrate that PDE2A2 is part of a novel mitochondrial cAMP/PKA signalling domain. We demonstrate that this domain is fuelled by cAMP generated at the plasma membrane, it drives PKA-dependent phosphorylation of Drp1 and modulates mitochondria dynamics and cell survival. Importantly, we demonstrate that inhibition of PDE2A2 activity results in more elongated mitochondria, increased membrane potential and resistance to pro-apoptotic stimuli. Our STED analysis shows that PDE2A2 localisation at mitochondrial membranes is not homogeneous but shows a dotted appearance , which sA previous study reported that PDE2A2 localises to the mitochondrial matrix in hepatocytes and neurons where it hydrolyses cAMP generated locally by sAC and regulates ATP synthesis . In our Future studies are required to establish the extent to which dysfunction of the cAMP/PKA/PDE2A domain localised at the OMM/IMM underpins PDE2A-related pathological conditions, and whether PDE2A inhibition at this site may be a viable therapeutic avenue. A number of findings warrant these investigations. The positive effect of PDE2A inhibitors on depression/anxiety-like behaviours has been linked to their anti-apoptotic effects . In addiOur discovery of a novel cAMP/PKA/PDE2A2 signalling domain located at mitochondrial membranes provides an original mechanistic framework for understanding the involvement of PDE2A2 in pathology and has potential implications for the treatment of mitochondrial diseases.Forskolin, IBMX, Cilostamide, Ionomycin, Pancreatin and Proteinase K were from Sigma-Aldrich (UK). Bay 60–7550 was from Cambridge Bioscience ((UK). Staurosporin was from Calbiochem . Mitotracker red and Mitotracker green were from Life Technologies . SiGLO red transfection indicator and PDE2 3’UTR siRNA were from Dharmacon . Collagenase was from Roche (Switzerland), Laminin (mouse) from BD Biosciences (UK). Transfectin Lipid Reagent was from BioRAD . FuGene6 was from Promega (UK). Phosphate‐buffered saline (PBS), DMEM high glucose, MEM199, horse serum, new born calf serum, penicillin/streptomycine, trypsin 0.05% and glutamine were from Invitrogen. Polyplus Jetprime transfection reagent was from VWR . pcDNA3.1 (+) Drp1 V2 (plasmid #44599) and pcDNA3.1 (+) Drp1 V2 S600A (plasmid#46344) were from Addgene . The EGFP-tagged versions of DRP1 and DRP1S600A were generated by subcloning EGFP as BamHI fragment at the N-terminus of DRP1 V2 plasmids. The EGFP-tagged version of PDE2A2 was generated by subcloning PDE2A2 as NheI fragment at the N-terminus of EGFP (pEYFP-C1-EGFP). PDE2A1 (RG235036), PDE2A2 (RG226806) and PDE2A3 (RG207219) in pCMV6-AC-GFP were from Origene . HeLa and H9C2 cells were originally purchased from ATCC, which provides authentication by STR profiling, and tested negative in routinely performed mycoplasma tests in the lab. No cell lines from the list of commonly misidentified cells maintained by the International Cell Line Authentication Committee has been used in this study.All procedures were carried out in compliance with the standards for the care and use of animal subjects as stated by the requirements of the UK Home Office (ASPA1986 Amendments Regulations 2012) incorporating the EU directive 2010/63/EU.Primary cultured NRVM were isolated from 1- to 2 day old Sprague Dawley rats as described . Ventrictm1Dgen/H, provided by EMMA, UK), were sacrificed after 13 days of gestation (E.13 dpc) and the embryos collected, then placed into separate dishes containing phosphate buffered saline (PBS). A segment of the tail was kept for genotyping, whereas the head and the internal organs were removed. The remaining tissue was washed with PBS and incubated in trypsin at 37°C for 5–10 min. The solution was pipetted until the tissue was completely homogenised and the isolated cells were plated onto T75 flasks. For transfection the cells were plated onto sterile 24 mm2 coverslips and maintained at 37°C for 24 hr. MEFs were transfected with FuGENE 6 or Polyplus Jetprime according to the manufacturer’s instructions. Immortalized mouse embryo fibroblasts derived from sAC knockout mice (MEFsACKO) (wt) and Drp1 KO (MEFsDrp1KO) were provided by Prof. Naotada Ishihara, Department of Protein Biochemistry, Institute of Life Science, Kurume University, Japan  , and they, Japan .NRVM were washed twice with PBS and lysed for 5 min with RIPA buffer containing complete EDTA-free protease inhibitor cocktail tablets (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche) when required. Pellets were clarified by centrifugation at 10000 rpm for 10 min at 4°C. Protein concentration was determined using the BCA assay  or Bradford assay (Sigma). 30 µg of protein were separated by SDS polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes (Millipore). Primary antibodies used for detection were: PDE2A (Abcam ab125677), DRP1 , phosphoDRP1 , phosphoDRP1 , GAPDH , TOM20 FL-145 , Total OXPHOS rodent , Mitofilin ., Tim23 , Cytochrome C , alpha tubulin , GFP .Cytosolic and mitochondrial fractions from NRVM were generated using the Qproteome Mitochondria Isolation Kit , following the manufacturer’s instructions. Treatment with 10 µM Proteinase K was performed on the mitochondrial fraction for 25 min at 25°C. Mitochondria were then lysed in RIPA buffer containing protease inhibitor provided by the kit. The same volume (15 µl) of cytosol and mitochondria treated and not treated with Proteinase K were loaded onto gels and blotted for cytosol and mitochondrial markers.2, pH 7.4. The supernatant containing the post-mitoplasts fraction was precipitated with 12% TCA and centrifuged at 18,000 g for 15 min at 4°C. The pellet was re-suspended in 200 µL acetone and centrifuged at 18,000 g for 15 min at 4°C. Finally, the pellet was re-suspended in 20 µL of 300 mM Tris-HCl, 10 μM CaCl2, pH 7.4After performing mitochondria fractionation, mitoplasts were prepared as described previously . MitochoFor the protease protection assays, mitoplasts were divided into three tubes: no treatment; proteinase K (PK); PK +1% Triton X-100. Prior to PK treatment, one aliquot of mitoplasts was solubilized with 1% Triton X-100 for 15 min on ice. 10 µM PK treatment was for 20 min at RT, followed by PK inactivation with protease inhibitor.3PO4 1 mmol/L, MgSO41 mmol/L, Hepes 20 mmol/L, Glucose 5.5 mmol/L, CaCl21 mmol/L, pH 7.4), and imaged on an inverted microscope (Olympus IX71) using a PlanApoN, 60X, NA 1.42 oil immersion objective, 0.17/FN 26.5 . The microscope was equipped with a CoolSNAP HQ  for 10 min, washed in TBS, permeabilised with 0.5% (v/v) Triton X-100 (TX) in TBS for 5 min and washed in TBS. Non-specific labelling was blocked with a 10 min incubation with 1% normal goat serum  in Antibody Diluting Solution  and all antibodies were diluted in AbDil 1% NGS. Primary antibodies were incubated for 60 min. After 3 × 5 min washes cells were treated with fluorescent-conjugated species-specific immunoglobulins (1:250 in AbDil 1% NGS) for 60 min. After 3 × 5 min TBS washes, coverslips were mounted in Ibidi mounting media and sealed with nail varnish. Primary antibodies used: mouse anti-GFP , rabbit anti-GFP , rabbit anti-TOM20  and mouse anti-cytochrome-c (ab110325). Species-specific secondary antibodies STAR RED anti-rabbit and STAR 580 anti-mouse were from Abberior.Confocal and STED images were obtained using a Leica TCS SP8 3x inverted microscope with a time-gated stimulated emission depletion (STED) module , equipped with a white light laser (WLL) and an HC PL APO CS2 100x/1.40 oil objective. Excitation wavelength was 561 and 633 nm and depletion was achieved with a 775 nm laser. The pinhole was set to 0.5 Airy units and the pixel size was 25 nm. Images of 1024 × 1024 pixels were acquired at a scan speed of 600 Hz with the number of line average equal to 16 and frame accumulation of 2. Signal was detected with a hybrid detection system (Leica HyD). For confocal imaging the STED depletion beam was turned off and WLL intensity was decreased.Cryo immuno-EM was carried out as previously reported . Brieflymax, where F is MitoTracker fluorescence (background subtracted) averaged within a pixel region of interest (ROI) covering the whole cell area, and Fmax the highest pixel value in the ROI. Before computing E, 20 iterations of a spatial Gaussian filter were performed to the image to improve E sensitivity. Values indicating mitochondria elongation were expressed as 1/E.Quantitative evaluation of mitochondria elongation was performed by deriving a numerical parameter based on the method in . Cells wAlternatively , mitocho6 millions NRVM and MEFs were seeded onto six well plates and incubated with DMSO or Bay 60–7550 for 24 hr. Mitochondrial membrane potential (△Ψm) was measured by loading cells with 50 nM tetramethyl rhodamine methyl ester  for 30 min at 37°C. Cells were then washed and imaged with an inverted microscope (Olympus IX71) using a PlanApoN, 60X, NA 1.42 oil immersion objective, 0.17/FN 26.5 , equipped with a CoolSNAP HQ , according to the manufacturer’s instructions.NRVM and MEFs were seeded on 96 well plates at 2.5 × 106 millions NRVM and MEFs were seeded onto 12 well plates and incubated with DMSO, Bay 60–7550, cilostamide, ionomycin or staurosporin for 24 hr. The TUNEL assay was performed using the in situ Cell Death Detection Kit (ROCHE) according to the manufacturer’s instructions. The cells were then imaged and the nuclei and fluorescent cells were counted in a blind analysis.2.4 × 104 cells were plated onto XF24 plates  and incubated at 37°C, 5% CO2 for 24 hr. The medium was then replaced with 675 μl of unbuffered Dulbecco’s modified Eagle’s medium (Seahorse Bioscience) supplemented with 2 mmol glutamine, 25 mmol glucose and 2% fetal bovine serum (pH was adjusted to 7.4 using sodium hydroxide 0.5 M) and cells were incubated at 37°C in a CO2-free incubator. Basal oxygen consumption rate was then recorded using the XF24 plate reader (Seahorse Bioscience). Oxygen consumption rate was normalized to cell number calculated at the end of the experiments. Homogeneous plating of the cells and cell count was obtained by fixing the cells with trichloroacetic acid 10% for 1 hr at 4°C and then staining the fixes cells with 0.47% solution of Sulforhodamine B (Sigma).Measurements were performed as previously reported . Brieflyt-test was used to compare two groups of experiments. The comparison of 3 or more groups was performed using ANOVA test with Bonferroni correction. The significance level was set at p<0.05 and the following annotation has been used: *0.01≤p≤0.05, **0.001≤p<0.01, ***p<0.001.Data are presented as means ± standard error of the mean. Graph Pad Prism 5 software was used for all statistical analyses. Student Sample sizes, replicate numbers, and p-values are stated in the figure legends. Biological replicates are independent animals or, for cell lines, independent cultures. Technical replicates are number of cells for FRET experiments, mitochondria morphology and TMRM experiments. For TUNEL experiments and cell viability experiments technical replicates are coverslips from the same culture. Outliers, when encountered, where handled using the Dixon’s Q test. For  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Tony Hunter as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission..Summary:Here, the authors showed that the PDE2A2 cAMP phosphodiesterase is localized to the mitochondria, in part at the outer membrane, and, using either knockdown and re-expression of WT and catalytic mutant PDE2A2 or a selective PDE2A2 inhibitor, they used localized FRET-based cAMP sensor analysis to show that PDE2A2 regulates cAMP levels in the vicinity of mitochondria in cardiac myocytes. Functionally, they showed that inhibition or depletion of PDE2A2 caused mitochondrial elongation, whereas overexpression of PDE2A2 but not PDE2A7 caused mitochondrial fragmentation, concomitant with increased or decreased phosphorylation of S637 in the Drp1 fission protein, a PKA site whose phosphorylation inhibits Drp1 mitochondrial fission activity.The compartmentalization of cAMP and PKA signaling is highly topical, and the reviewers found your conclusion that PDE2A2 acts at the periphery of mitochondria to control local cAMP concentrations to be of interest. However, they were not fully convinced that your current data provide unequivocal evidence that cAMP levels in the vicinity of mitochondria are regulated by a pool of PDE2A2 on the surface as opposed to by the intramitochondrial PDE2A2 population.Essential revisions:1) All the experiments on PDE2A2 sub-mitochondrial localization need to be revisited. The data presented in 2) The authors attribute the elongation of mitochondria in PDE2A2 knockout cells to phosphorylation of Drp1 at Ser637, but unequivocal proof is needed to demonstrate that the protective effect of PDE2A on apoptosis is Drp1 mediated. To this end, they need to repeat the experiments in 3) Also in 4) The strongest evidence for a role of PDE2A2 on outer membrane cAMP levels is the data in 5) A major concern is how to reconcile the data in this paper with previous work  that convincingly showed that PDE is localized to the mitochondrial matrix, where it regulates cAMP levels generated by sAC, which is also localized to the mitochondrial matrix. The production of cAMP activated phosphorylation of respiratory chain subunits and increased oxidative phosphorylation. The authors need to provide a convincing explanation.eLife. Your revised article has been favorably evaluated by Tony Hunter (Senior Editor) and three reviewers.Thank you for resubmitting your work entitled "PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling" for further consideration at eLife. The manuscript has been greatly improved by inclusion of new data to address the original reviewers' concerns, but there are a few minor issues raised by the new reviewer 3 and by the original reviewer 1 that need to be addressed before acceptance, as outlined in the reviews below. Please make the small changes that are requested, and resubmit the paper to eLife.I am pleased to say that in principle your paper is now acceptable for publication in Reviewer #1:The revised manuscript has been improved by the addition of some key new experiments. The results in Reviewer #2:The revision greatly improved the paper: the conclusions are now fully supported by the experiments presented and this paper would make a very nice addition to the literature.Reviewer #3:This paper reports the localisation of PDE2A2 at the outer mitochondrial membrane where the authors show that this enzyme contributes to creating a cAMP microdomain that regulates PKA phosphorylation of the mitochondrial fission protein Drp1 (which inhibits its function in fission). Inhibition of PDE2A2 gives mitochondrial elongation, increases membrane potential and protects from apoptosis. The conclusions are supported by pharmacological studies, studies using differentially targeted cAMP FRET reporters and studies in PDE2A2, Drp1 and sAC knockout MEFs. As a new reviewer on the revised version of this manuscript. I have focused on reading the revised paper which I think in general is now a convincing story. I have also gone through the tracked version of the revised manuscript and the reviewers' earlier comments and responses from the authors to these that I in general find have addressed the major comments to the first version.The remaining comment that I have is that I still  think the EM immunolocalization still appears to be on cristae membranes rather than OMM, it would be good if the authors could clarify this. Essential revisions:1) All the experiments on PDE2A2 sub-mitochondrial localization need to be revisited. The data presented in In our original submission we did clearly state that only a fraction (about 40%) of PDE2A2 resides at the OMM and suggested, based also on our EM analysis and STED imaging, that the remaining 60% of PDE2A2 resided inside the mitochondria at the IMM, as it appeared to be excluded from the intra-cristae space and the matrix. As suggested by the reviewers, to further strengthen this point, we have now performed a more detailed biochemical analysis of submitochondrial fractions. The results are presented in 2) The authors attribute the elongation of mitochondria in PDE2A2 knockout cells to phosphorylation of Drp1 at Ser637, but unequivocal proof is needed to demonstrate that the protective effect of PDE2A on apoptosis is Drp1 mediated. To this end, they need to repeat the experiments in wt and MEFDrp1KO. In Drp1KO cells. The ability of PDE2A inhibition to induce elongation of mitochondria is restored in MEFDrp1KO that express wt Drp1 but not a Drp1 mutant that cannot be phosphorylated by PKA. In addition, in Drp1KO, confirming that for PDE2A inhibition to exert a protective effect, Drp1 is required. Expression of a Drp1 phosphomimetic in PDE2AKO cells is not expected to reverse the phenotype (elongated mitochondria) of PDE2AKO cells as lack of PDE2A leads to increased phosphorylation of Drp1 and phosphomimetic Drp1  would also result in elongated mitochondria. We therefore did not perform this experiment.We would like to thank the reviewer for this suggestion as this experiment allows to directly demonstrate the involvement of Drp1 in the effect of PDE2A inhibition. However, we would like to point out that, because inhibition of PDE2A results in an increase of cAMP and PKA-dependent phosphorylation, overexpression of a phospho-null Drp1 is not expected to show additive effect with PDE2A inhibition but is expected to cancel out such effect. Based on this suggestion, to strengthen our conclusion that the effects on mitochondria morphology and cell death that we observe on PDE2 inhibition are dependent on PKA-mediated phosphorylation of Drp1 we performed two completely new sets of experiments using MEFDrp1KO. We believe this depends on the low fraction of Drp1 that is phosphorylated in MEFs. In support of this interpretation is the fact that we could only detect a very weak signal for phospho-Drp1 when the recombinant Drp1 was pulled-down from lysates of MEFs treated with saturating concentrations of Forskolin and IBMX  Also in The presentation of our data on mitochondria membrane potential in the first submission was unclear and has created a misunderstanding. The representative traces shown in our original We thought however that the concern raised by the reviewers about the possibility that a lower membrane potential could result from mitochondria that respire more and, vice versa, that a higher membrane potential may result from mitochondria that respire less, was an important point to address. We therefore performed Seahorse measurement to assess mitochondria respiration in cardiac myocytes treated with the PDE2A inhibitor and found no significant difference compared to untreated myocytes (data are presented in 4) The strongest evidence for a role of PDE2A2 on outer membrane cAMP levels is the data in wt and MEFPDE2KO  A major concern is how to reconcile the data in this paper with previous work  that convincingly showed that PDE is localized to the mitochondrial matrix, where it regulates cAMP levels generated by sAC, which is also localized to the mitochondrial matrix. The production of cAMP activated phosphorylation of respiratory chain subunits and increased oxidative phosphorylation. The authors need to provide a convincing explanation.PDE2KO. By contrast, both IBMX and BAY60 generate a robust response at the OMM in MEFs wt but no response at this site in MEFPDE2KO. Although it is possible that a fraction of PDE2A2 is localised in the matrix in cardiac myocytes and fibroblasts we were not able to detect it with any of the approaches we used.In this study we present multiple lines of evidence that consistently support localisation of PDE2A2 at mitochondrial membranes (both OMM facing the cytosol and IMM facing the IMS). Biochemical fractionation, super resolution microscopy and electron microscopy all confirm predominant localisation of PDE2A2 at these sites. Our FRET imaging experiments also exclude that a major fraction of PDE2A2 localises at the matrix as the response detected at this site on inhibition of PDEs (both with the non-selective inhibitor IBMX and with the PDE2-selective inhibitor BAY60) using a matrix-targeted reporter is the same in MEFs wt and MEFWe would also like to point out that the data presented in Acin-Perez et al., Cell Metab 2009 are not incompatible with localisation of PDE2A at mitochondrial membranes. In that study the evidence that PDE activity is in the matrix is based on an experiment where exogenous cAMP is added to a permeabilised mitoplast preparation with or without addition of the non-selective PDE inhibitor IBMX  the same group addresses directly the submitochondrial localisation of PDE2A2 using a biochemical approach. The key data,  think the EM immunolocalization still appears to be on cristae membranes rather than OMM, it would be good if the authors could clarify this.In mitochondria the surface of the IMM is several times greater than the surface of the OMM. This is reflected in the mitochondria sections captured by EM images, where the length of the IMM largely exceeds that of the OMM. It is therefore to be expected that the number of particles detected at the OMM is significantly smaller than the number found at the IMM. We did perform a more detailed analysis of the number of particles at the OMM and found that this is about 7% of the total. A rough estimation of the particle density per unit of membrane indicates that there is no difference between OMM and IMM. To clarify this point we have now added a sentence to the subsection “Sub-mitochondrial localisation of PDE2A”, including this information.