Enzymatic activity of the blue light-regulated phosphodiesterase BlrP1 from Klebsiella pneumoniae shows a nonlinear dependence on light intensity.

The blue light-regulated phosphodiesterase BlrP1 from Klebsiella pneumoniae hydrolyzes cyclic dimeric guanosine monophosphate (GMP) in a blue light-dependent manner. It contains a photosensing BLUF domain and a functional EAL domain. Previously, it was reported that conformational changes in the dimer upon light illumination occurred only when both protomers of the dimer were excited. Based on this observation, it was proposed that BlrP1 might be a nonlinear light intensity sensor. To test this, here, the correlation between the turnover number of the hydrolysis reaction (kcat ) and the fraction of the excited protein (fred ) was measured by simultaneously monitoring the reaction rate and fred . Our results show that kcat is proportional to fred2 . Thus, BlrP1 works as a nonlinear light intensity sensor to sense a strong light environment.

Advances, Perspectives and Potential Engineering Strategies of Light-Gated Phosphodiesterases for Optogenetic Applications.

The second messengers, cyclic adenosine 3'-5'-monophosphate (cAMP) and cyclic guanosine 3'-5'-monophosphate (cGMP), play important roles in many animal cells by regulating intracellular signaling pathways and modulating cell physiology. Environmental cues like temperature, light, and chemical compounds can stimulate cell surface receptors and trigger the generation of second messengers and the following regulations. The spread of cAMP and cGMP is further shaped by cyclic nucleotide phosphodiesterases (PDEs) for orchestration of intracellular microdomain signaling. However, localized intracellular cAMP and cGMP signaling requires further investigation. Optogenetic manipulation of cAMP and cGMP offers new opportunities for spatio-temporally precise study of their signaling mechanism. Light-gated nucleotide cyclases are well developed and applied for cAMP/cGMP manipulation. Recently discovered rhodopsin phosphodiesterase genes from protists established a new and direct biological connection between light and PDEs. Light-regulated PDEs are under development, and of demand to complete the toolkit for cAMP/cGMP manipulation. In this review, we summarize the state of the art, pros and cons of artificial and natural light-regulated PDEs, and discuss potential new strategies of developing light-gated PDEs for optogenetic manipulation.

Revisiting and Redesigning Light-Activated Cyclic-Mononucleotide Phosphodiesterases.

As diffusible second messengers, cyclic nucleoside monophosphates (cNMPs) relay and amplify molecular signals in myriad cellular pathways. The triggering of downstream physiological responses often requires defined cNMP gradients in time and space, generated through the concerted action of nucleotidyl cyclases and phosphodiesterases (PDEs). In an approach denoted optogenetics, sensory photoreceptors serve as genetically encoded, light-responsive actuators to enable the noninvasive, reversible, and spatiotemporally precise control of manifold cellular processes, including cNMP metabolism. Although nature provides efficient photoactivated nucleotidyl cyclases, light-responsive PDEs are scarce. Through modular recombination of a bacteriophytochrome photosensor and the effector of human PDE2A, we previously generated the light-activated, cNMP-specific PDE LAPD. By pursuing parallel design strategies, we here report a suite of derivative PDEs with enhanced amplitude and reversibility of photoactivation. Opposite to LAPD, far-red light completely reverts prior activation by red light in several PDEs. These improved PDEs thus complement photoactivated nucleotidyl cyclases and extend the sensitivity of optogenetics to red and far-red light. More generally, our study informs future efforts directed at designing bacteriophytochrome photoreceptors.

Cyclic Nucleotide-Specific Optogenetics Highlights Compartmentalization of the Sperm Flagellum into cAMP Microdomains.

Inside the female genital tract, mammalian sperm undergo a maturation process called capacitation, which primes the sperm to navigate across the oviduct and fertilize the egg. Sperm capacitation and motility are controlled by 3',5'-cyclic adenosine monophosphate (cAMP). Here, we show that optogenetics, the control of cellular signaling by genetically encoded light-activated proteins, allows to manipulate cAMP dynamics in sperm flagella and, thereby, sperm capacitation and motility by light. To this end, we used sperm that express the light-activated phosphodiesterase LAPD or the photo-activated adenylate cyclase bPAC. The control of cAMP by LAPD or bPAC combined with pharmacological interventions provides spatiotemporal precision and allows to probe the physiological function of cAMP compartmentalization in mammalian sperm.

A blue light-inducible phosphodiesterase activity in the cyanobacterium Synechococcus elongatus.

A blue light-inducible phosphodiesterase (PDE) activity, specific for the hydrolysis of cyclic di-GMP (c-di-GMP), has been identified in a recombinant protein from Synechococcus elongatus. Blue light (BL) activation is accomplished by a light, oxygen, voltage (LOV) domain, found in plant phototropins and bacterial BL photoreceptors. The genome of S. elongatus contains two genes coding for proteins with LOV domains fused to EAL domains (SL1 and SL2). In both cases, a GGDEF motif is placed in between the LOV and the EAL motifs. Such arrangement is frequently found with diguanylate-cyclase (DGC) functions that form c-di-GMP. Cyclic di-GMP acts as a second messenger molecule regulating biofilm formation in many microbial species. Both enzyme activities modulate the intracellular level of this second messenger, although in most proteins only one of the two enzyme functions is active. Both S. elongatus LOV-GGDEF-EAL proteins were expressed in full length or as truncated proteins. Only the SL2 protein, expressed as a LOV-GGDEF-EAL construct, showed an increase of PDE activity upon BL irradiation, demonstrating this activity for the first time in a LOV-domain protein. Addition of GTP or c-di-GMP did not affect the observed enzymatic activity. In none of the full-length or truncated proteins was a DGC activity detected.

G-protein deactivation is rate-limiting for shut-off of the phototransduction cascade.

Photoreceptors detect light through a seven-helix receptor (rhodopsin) and heterotrimeric G protein (transducin) coupled to a cyclic GMP phosphodiesterase. Similar pathways are used to amplify responses to hormones, taste and smell. The amplification of phototransduction is reduced by a fall in cytoplasmic Ca2+ , but it is not known how the deactivation of rhodopsin and transducin influence this response and hence the extent and duration of phosphodiesterase activity. Here we investigate this by recording the electrical response to flashes of light in truncated rod photoreceptors. By removing ATP to block the deactivation of rhodopsin by phosphorylation, we show that this reaction limits the amplitude of the response and begins within 3.2 s of a flash in a solution containing 1 microM Ca2+, falling to 0.9 s in a zero-Ca2+ solution. In contrast, the activation and amplitude of the response were unaffected when transducin deactivation by GTP hydrolysis was blocked by replacing GTP with its nonhydrolysable analogue GTP-gammaS, demonstrating that there is little GTP hydrolysis occurring over the period in which photoexcited rhodopsin is quenched. The rapid deactivation of rhodopsin is therefore a Ca2+-sensitive step controlling the amplitude of the light response, whereas transducin deactivation is slower and controls recovery.

[Post-radiation disturbances in the cyclic nucleotide system in rat spleen and thymus lymphocytes]. / Postradiatsionnye narusheniia v sisteme tsiklicheskikh nukeltidov limfotsitov selezenki i timusa krys.

The changes in cAMP and cGMP content in rat spleen and thymus lymphocytes after irradiation in doses of 0.5 and 1 Gy, are determined, indicating to significant disturbances in the system of cyclic nucleotides. Radiation affected the functioning of enzymes both of synthesis and hydrolysis of cAMP and cGMP in spleen lymphocytes. The activity of adenylate cyclase and guanylate cyclase did not change in thymocytes after the exposure, while the activity of phosphodiesterase of cyclic nucleotides slightly increased.

Molecular size of N-acetylglucosaminylphosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase as determined in situ in Golgi membranes by radiation inactivation.

The radiation inactivation method was used to determine the molecular size of the two enzymes that participate in the synthesis of the phosphomannosyl recognition marker of lysosomal proteins. The determinations were carried out in situ, in Golgi membranes isolated from normal human placenta and cultured skin fibroblasts. A molecular size of 228 +/- 29 kDa was found for placental N-acetylglucosaminyl-phosphotransferase, and 129 +/- 11 kDa for placental alpha-N-acetylglucosaminyl phosphodiesterase. The values for the fibroblast enzymes were about 20% higher, 283 +/- 27 kDa and 156 +/- 14 kDa for the transferase and phosphodiesterase respectively. Triton X-100 had no effect on the molecular size of these enzymes.

The substructure of phosphodiesterase as established by radiation inactivation. A reinterpretation of results.

A model for the activation of phosphodiesterase by calmodulin based on a conversion of inactive dimers to active monomers, derived from radiation inactivation studies J. Biol. Chem. (1981) 256, 11351-11355 has been re-examined using a simple probability argument. We conclude that the original model is not supported by the radiation inactivation studies, since our analysis of this model would predict that the rate of radiation inactivation of calmodulin-dependent phosphodiesterase activity be exactly twice that for the decay in total activity in marked contrast with the results obtained.

[Radiation modulation of the activity of the enzymatic systems in isolated plasma membranes in early ontogeny]. / Radiatsionnaia moduliatsiia aktivnosti nekotorykh fermentnykh sistem izolirovannykh plazmaticheskikh membran rannego ontogeneza.

The activity of adenylate cyclase (Ac), cAMP phosphodiesterase (PDE) and 5'-nucleotidase was studied in plasma membranes from the liver of rat embryo of the 20th day of development normally and after exposure to ionizing radiation. Gamma-irradiation of plasma membranes with doses ranging from 0.1 to 100 kR was shown to inhibit the activity of Ac, this effect being more pronounced during stimulation with higher doses of isoproterenol. The activity of 5'-nucleotidase and PDE remained unchanged up to the dose of 100 kR.

Phosphodiesterase and GTPase in rod outer segments. Kinetics in vitro.

The hydrolysis of cyclic guanosine monophosphate (cyclic GMP) and of guanosine triphosphate (GTP) by the broken rods of the frog retina after a flash of light have been studied in vitro with a constant perfusion method. The activation has an onset apparently instantaneous as observed with the existing possible time resolution of 3 s. The activation is followed by a partial inactivation that does not bring the activity back to the pre-flash level. GTP or the non-hydrolysable guanyl-5'-ylimidodiphosphate (GMP-PNP) is required for the normal light-activation of the phosphodiesterase and in its absence both the speed of activation and the sensitivity are greatly reduced. The activation speed, the sensitivity (threshold at approx. 0.00004% bleaching), and the kinetic constants do not exclude a direct role in the process of excitation for the phosphodiesterase and suggest a subsidiary but as yet undefined role for the GTPase.

[Post-radiation disorders in the cyclic AMP system of mouse spleen and thymus lymphoid cells]. / Postradiatsionnye narusheniia v sisteme tsiklicheskogo AMP limfoidnykh kletok selezenki i timusa myshei.

Early alterations in the enzyme activities controlling cyclic adenosine-3',5'-monophosphate (cAMP) metabolism after the irradiation (800 rad) of mice were found in the lymphoid cells of the spleen and thymus. Postradiation disturbances in these activities' ration induced alterations in cAMP steady-state concentrations in the cell. It was also demonstrated that irradiation reduced lymphoid cell ability to accumulate cAMP in response to isoproterenol administration.

[Effect of ionizing radiation on cerebral phosphodiesterase]. / Vliianie na ionizirashtata radiatsiia vurkhu mozuchnata fosfodiesteraza.

The authors examined the influence of x-ray irradiation on phosphodiesterase system in the brain of rats and the sensitivity of the enzyme to inhibitors (caffeine, theophyline, thyopental) and activators (imidozol). The animals (Wistar with weights of 150--200 gm/were irradiated singly with a dose of 737 r. Phosphodiesterase activity (PD) was determined by the method of Filburn and Carn (1972) at two substrate concentrations (1 and 100 microM). X-ray irradiation did not change PD with high Km, while the enzyme with low Km was reduced significantly and transitionaly. Caffeine, theophyline and thyopental inhibited more PD with low Km in the irradiated rats in comparison with the control nonirradiated rats. The activating effect of imidazol on PD with low Km was more marked in irradiated rats. These data could be interpreted in the light of the molecular mechanisms, determining the altered action of some drugs after x-ray irradiation.

[Hepatic adenosine-3',5'-monophosphate metabolism in newborn rats irradiated during the period of organogenesis]. / Metabolizm adenozin-3',5'-monofosfata v pecheni novorozhdennykh krys pri obluchenii v period organogeneza.

The adenylate cylcase (AC) and phosphodiesterase (PDE) activities, as well as the 3'5'-AMP (cAMP) fund were studied in the liver of newborn rats, intact, and after irradiation on the 9th day of the embryonic development. A decrease of AC and PDE activities was noted with a dose of 50 r. The stationary level of cAMP in the tissue remained unchanged. The adrenaline-stimulated AC activity only tended to decrease in case of irradiation. As suggested, during critical periods of development, in the presence of the hormone inductor in the liver of irradiated rats, conditions can be created for an increase of the cAMP pool.

Effect of irradiation on enzyme activities of cyclic AMP system in the neuro- and adenohypophyses.

The enzyme activities of cyclic AMP system in the neuro- and adenohypophyses were studied, immediately after an irradiation by a single whole body exposure of 1600 R, in an attempt to find whether this intervention causes the changes in the responsiveness of the cyclic AMP regulatory system. In the irradiated rats the neurohypophyses revealed a reduced activity of adenylate cyclase, moderately increased activity of phosphodiesterase and slightly decreased activity of protein kinase, including the value stimulated by cyclic AMP. In the adenohypophyses the irradiation did not cause any significant changes in the enzyme activities of the cyclic AMP system, except of slightly decreased adenylate cyclase activity. The possible relationship of the plasma level of antidiuretic hormone immediately after irradiation and the enzyme activities of cyclic AMP system is discussed.