Functional modulation of phosphodiesterase-6 by calcium in mouse rod photoreceptors

Phosphodiesterase-6 (PDE6) is a key protein in the G-protein cascade converting photon information to bioelectrical signals in vertebrate photoreceptor cells. Here, we demonstrate that PDE6 is regulated by calcium, contrary to the common view that PDE1 is the unique PDE class whose activity is modulated by intracellular Ca2+. To broaden the operating range of photoreceptors, mammalian rod photoresponse recovery is accelerated mainly by two calcium sensor proteins: recoverin, modulating the lifetime of activated rhodopsin, and guanylate cyclase-activating proteins (GCAPs), regulating the cGMP synthesis. We found that decreasing rod intracellular Ca2+concentration accelerates the flash response recovery and increases the basal PDE6 activity (βdark) maximally by ~ 30% when recording local electroretinography across the rod outer segment layer from GCAPs−/−recoverin−/−mice. Our modeling shows that a similar elevation in βdarkcan fully explain the observed acceleration of flash response recovery in low Ca2+. Additionally, a reduction of the free Ca2+in GCAPs−/−recoverin−/−rods shifted the inhibition constants of competitive PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) against the thermally activated and light-activated forms of PDE6 to opposite directions, indicating a complex interaction between IBMX, PDE6, and calcium. The discovered regulation of PDE6 is a previously unknown mechanism in the Ca2+-mediated modulation of rod light sensitivity.

Plasmodium falciparum Guanylyl Cyclase-Alpha and the Activity of Its Appended P4-ATPase Domain Are Essential for cGMP Synthesis and Blood-Stage Egress

ABSTRACT Guanylyl cyclases (GCs) synthesize cyclic GMP (cGMP) and, together with cyclic nucleotide phosphodiesterases, are responsible for regulating levels of this intracellular messenger which mediates myriad functions across eukaryotes. In malaria parasites (Plasmodium spp), as well as their apicomplexan and ciliate relatives, GCs are associated with a P4-ATPase-like domain in a unique bifunctional configuration. P4-ATPases generate membrane bilayer lipid asymmetry by translocating phospholipids from the outer to the inner leaflet. Here, we investigate the role of Plasmodium falciparum guanylyl cyclase alpha (GCα) and its associated P4-ATPase module, showing that asexual blood-stage parasites lacking both the cyclase and P4-ATPase domains are unable to egress from host erythrocytes. GCα-null parasites cannot synthesize cGMP or mobilize calcium, a cGMP-dependent protein kinase (PKG)-driven requirement for egress. Using chemical complementation with a cGMP analogue and point mutagenesis of a crucial conserved residue within the P4-ATPase domain, we show that P4-ATPase activity is upstream of and linked to cGMP synthesis. Collectively, our results demonstrate that GCα is a critical regulator of PKG and that its associated P4-ATPase domain plays a primary role in generating cGMP for merozoite egress. IMPORTANCE The clinical manifestations of malaria arise due to successive rounds of replication of Plasmodium parasites within red blood cells. Once mature, daughter merozoites are released from infected erythrocytes to invade new cells in a tightly regulated process termed egress. Previous studies have shown that the activation of cyclic GMP (cGMP) signaling is critical for initiating egress. Here, we demonstrate that GCα, a unique bifunctional enzyme, is the sole enzyme responsible for cGMP production during the asexual blood stages of Plasmodium falciparum and is required for the cellular events leading up to merozoite egress. We further demonstrate that in addition to the GC domain, the appended ATPase-like domain of GCα is also involved in cGMP production. Our results highlight the critical role of GCα in cGMP signaling required for orchestrating malaria parasite egress.

Plasmodium falciparum guanylyl cyclase-alpha and the activity of its appended P4-ATPase domain are essential for cGMP synthesis and blood stage egress

In malaria parasites, guanylyl cyclases (GCs), which synthesise cyclic GMP (cGMP), are associated with a P4-ATPase-like domain in a unique bifunctional configuration. P4-ATPases generate membrane bilayer lipid asymmetry by translocating phospholipids from the outer to the inner leaflet. Here we investigate the role of Plasmodium falciparum guanylyl cyclase alpha (GCα) and its associated P4-ATPase module, showing that asexual blood stage parasites lacking both the cyclase and P4-ATPase domains are unable to egress from host erythrocytes. GCα-null parasites cannot synthesise cGMP, or mobilise calcium, a cGMP-dependent protein kinase (PKG)-driven requirement for egress. Using chemical complementation with a cGMP analogue and point mutagenesis of a crucial conserved residue within the P4-ATPase domain, we show that ATPase activity is up stream of and linked to cGMP synthesis. Collectively, our results demonstrate that GCα is a critical regulator of PKG and that its associated P4-ATPase domain plays a primary role in generating cGMP for merozoite egress.

195 Lipid profiling of bovine oocytes matured with different stimulation of cyclic GMP synthesis by multiple reaction monitoring profiling

Intrafollicular lipid metabolism is very important for production species such as cattle. Lipids are essential substrates to produce energy during growth, maturation, and acquisition of high competence for the development of oocytes. However, the quantity and distribution of these lipids has been identified as responsible for hindering the process of cryopreservation of oocytes and embryos produced invitro. Previous studies have indicated that the cyclic (c) GMP pathway may be involved in the lipid metabolism of bovine cumulus-oocyte complexes (COC). The synthesis of this nucleotide can be activated through guanylate cyclases (soluble, sGC; or membrane, mGC). Therefore, the objective of this study was to investigate the lipid profile of bovine oocytes matured invitro (IVM) when stimulated by specific stimulators of sGC (protoporphyrin IX) and mGC (NPPB: peptide natriuretic type B). Pools of ovum pickup (OPU) oocytes were matured invitro for 24h in TCM-199 medium, with 15% bovine serum (BS), 0.5µgmL−1 of FSH, 5µgmL−1 of LH, 0.8mM L-glutamine, and 50µgmL−1 of gentamicin at 38.5°C and 10−5 M protoporphyrin IX or 10−6 M NPPB. The control group was matured without NPs or protoporphyrin IX. After IVM, cumulus cells (CC) were removed and oocytes (OO) collected, washed in 1:3 methanol:water (v/v) and frozen at −80°C. The lipid extraction of the samples was performed based on a standard protocol (Bligh and Dyer 1959 Can. J. Biochem. Physiol. 37, 911-917) but adapted for small samples. The samples were diluted and analysed on an Agilent 6410 QQQ (Agilent Technologies) mass spectrometer and analysed according to the multiple reaction monitoring (MRM) method described (de Lima et al. 2018 J. Mass. Spectrom. 53, 1247-52). Data for 3 replicates/group were normalized and then submitted to ANOVA statistical analysis, followed by Tukey test and principal components analysis, by Metaboanalyst 4.0, with an α-level of 5%. The results, representing the analysis of 164 lipids, showed that the lipid profile was not affected when we used the cGMP synthesis stimulators protoporphyrin IX and NPPB, maintaining the same profile of lipid classes in control and treatments. In addition, the quantitative values of the major lipid classes, sphingomyelin, triglycerides, and phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol), were not altered in the treated groups. The values for each class (ng/oocyte) for control, NPPB, and protoporphyrin, respectively, were 0.89, 0.86, and 1.12 for sphingomyelin, 5.63, 5.66, and 6.90 for phosphatidylcholine, 7.34, 6.48, and 7.89 for triglycerides, 209.0, 244.0, and 207.4 for phosphatidylserine, 3.05, 3.0, and 2.35 for phosphatidylethanolamine, 3.40, 3.34, and 3.29 for phosphatidylglycerol, and 3.47, 3.52, and 3.51 for phosphatidylinositol (P<0.05). Further, the amount of these lipids per class was not affected by cGMP synthesis when stimulated by protoporphyrin IX and NPPB, showing that the relationship of this pathway with lipid metabolism needs additional study.

66 Lipid profiling of bovine blastocysts produced invitro with and without a stimulator of cyclic guanosine monophosphate synthesis by multiple reaction monitoring profiling

Cryopreservation of oocytes and embryos is an essential technique for invitro-produced cattle worldwide. One of the great difficulties of cryopreservation of oocytes and blastocysts is the accumulation of lipids in the cytoplasm when produced invitro. The lipid metabolism of oocytes and embryos is classically regulated by the cAMP pathway. Furthermore, previous studies have suggested that the cyclic guanosine monophosphate (cGMP) pathway may also be involved in modulating lipid metabolism through protein kinase G activation. The objective of this study was to investigate the lipid profile of bovine blastocysts produced invitro when stimulated by specific stimulator of cGMP synthesis (NPPB). Pools of oocytes were matured invitro for 24h in tissue culture medium 199, with 15% bovine serum, 0.5µgmL−1 FSH, 5µgmL−1 LH, 0.8mM L-glutamine, and 50µgmL−1 gentamicin at 38.5°C and 10−6 M NPPB. The control group was matured without NPPB. After 22h, the oocytes were fertilised invitro with frozen sperm. The IVM oocytes were fertilised and cultured according standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347-1355). After the 7 days (Day 7), the blastocysts (from early blastocyst to expanded blastocyst) were collected, washed in methanol:water (vol/vol) 1:3, and frozen at −80°C. The lipid extraction of the samples was performed based on the standard protocol (Bligh and Dyer 1959 Can. J. Biochem. Physiol. 37, 911-917) but adapted for small samples. The samples were diluted and analysed in the Agilent 6410 QQQ (Agilent Technologies) mass spectrometer and analysed according to the multiple reaction monitoring method described by de Lima et al. (2018 J. Mass Spectrom. 53, 1247-1252). Data for 3 replicates/group were normalized and then submitted to t-test statistical analysis and principal component analysis, by Metaboanalyst 4.0, with a significance level of 5%. The rates of cleavage and blastocysts were not affected when we used the mGC stimulator presenting a 61% rate of cleavage for both groups, and 24.4% and 25% of blastocyst rate for control and NPPB, respectively (P<0.05). The results, regarding 164 lipids analysed, showed that the lipid profile was not affected when we used NPPB, maintaining the same profile of lipid classes. When we observe the quantitative values, we see a nonsignificant decrease in the lipid classes sphingomyelin, phosphatidylcholine, and triacylglycerol. The values for each class for control and NPPB, respectively, were 0.70 and 0.64 ng/blastocyst for sphingomyelin, 6.45 and 6.07 ng/blastocyst for phosphatidylcholine, and 11.82 and 10.51 ng/blastocyst for triacylglycerol (P<0.05). For the other classes of phospholipids (PE, PG, and PI), we observed a small increase when treated with NPPB, also not significant. We conclude that although we do not have significant differences between the control and the treatment, each class of lipid can respond differently when stimulated with cGMP synthesis.

cGMP dynamics underlie thermosensation in C. elegans

Animals sense ambient temperature so that they can adjust their behavior to the environment; they avoid noxious heat and coldness and stay within a survivable temperature range. C. elegans can sense temperature, memorize past cultivation temperature and navigate towards preferable temperature, for which a thermosensory neuron, AFD, is essential. AFD responds to temperature increase from the past cultivation temperature by increasing intracellular Ca2+ level. We aimed to reveal how AFD encodes and memorizes the information of temperature. Although cGMP synthesis is crucial for thermosensation by AFD, whether and how cGMP level temporally fluctuates in AFD remained elusive. We therefore monitored cGMP level in AFD and found that cGMP dynamically responded to temperature change in a manner dependent on past cultivation temperature. Given that cGMP dynamics is supposed to be upstream of Ca2+ dynamics, our results suggest that AFD’s memory is formed by simpler molecular mechanisms than previously expected from the Ca2+ dynamics. Moreover, we analyzed how guanylyl cyclases and phosphodiesterases, which synthesize and degrade cGMP, respectively, contributed to cGMP and Ca2+ dynamics and thermotaxis behavior.