Bicarbonate, carbon dioxide and pH sensing via mammalian bicarbonate-regulated soluble adenylyl cyclase

Soluble adenylyl cyclase (sAC; ADCY10) is a bicarbonate (HCO 3 − )-regulated enzyme responsible for the generation of cyclic adenosine monophosphate (cAMP). sAC is distributed throughout the cell and within organelles and, as such, plays a role in numerous cellular signalling pathways. Carbonic anhydrases (CAs) nearly instantaneously equilibrate HCO 3 − , protons and carbon dioxide (CO 2 ); because of the ubiquitous presence of CAs within cells, HCO 3 − -regulated sAC can respond to changes in any of these factors. Thus, sAC can function as a physiological HCO 3 − /CO 2 /pH sensor. Here, we outline examples where we have shown that sAC responds to changes in HCO 3 − , CO 2 or pH to regulate diverse physiological functions.

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Expression of Soluble Adenylyl Cyclase in Lentigo Maligna: Use of Immunohistochemistry With Anti–Soluble Adenylyl Cyclase Antibody (R21) in Diagnosis of Lentigo Maligna and Assessment of Margins

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2011-0617-oa ◽

2012 ◽

Vol 136 (12) ◽

pp. 1558-1564 ◽

Cited By ~ 19

Author(s):

Cynthia M. Magro ◽

Sung-Eun Yang ◽

Jonathan H. Zippin ◽

Artur Zembowicz

Keyword(s):

Adenylyl Cyclase ◽

Cyclic Adenosine Monophosphate ◽

Positive Margin ◽

Adenosine Monophosphate ◽

Resection Margins ◽

Nuclear Staining ◽

Margin Assessment ◽

Lentigo Maligna ◽

High Power Field ◽

Soluble Adenylyl Cyclase

Context.—Soluble adenylyl cyclase (sAC) is an enzyme that generates cyclic adenosine monophosphate, a signaling molecule involved in regulating melanocyte functions. R21, a mouse monoclonal antibody against sAC, shows a striking pan-nuclear staining in lentigo maligna, indicating possible utility for diagnosis and margin assessment. Objective.—To evaluate R21 in the diagnosis and evaluation of margins in lentigo maligna. Design.—Thirty one re-excision specimens for lentigo maligna were evaluated for R21 expression using previously published protocol. In addition, 153 cases including 41 lentigo malignas, 30 non–lentigo maligna-type melanomas, 38 lentigos, and 44 nevi were evaluated using a modified stringent protocol to eliminate all nonmelanocyte staining. Results.—The sensitivity of nuclear staining with R21 in lentigo maligna was 87.8%. Nuclear expression of sAC was observed in 40% of other melanomas and 2.3% of benign nevi. R21 did not stain nuclei of resting melanocytes but was observed in 28.9% of melanocytic hyperplasias. These cases were easily distinguished from lentigo maligna in routine sections. R21 staining facilitated extent of the lesion in resection margins. In cases examined under the less stringent conditions, interpretation was facilitated by comparing R21 and Mart1/Melan A staining. Greater than 9 pan-nuclear staining melanocytes within one high-power field along with a pan-nuclear sAC/Melan A ratio greater than 0.5 was consistent with a positive margin whereas 5 or less pan-nuclear staining melanocytes along with a sAC/Melan A ratio of less than 0.3 constituted a negative margin. Conclusion.—R21 is a useful diagnostic adjunct in the diagnosis and evaluation of margins in re-excision specimens in lentigo maligna.

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“Soluble” adenylyl cyclase-generated cyclic adenosine monophosphate promotes fast migration in PC12 cells

Journal of Neuroscience Research ◽

10.1002/jnr.21458 ◽

2007 ◽

Vol 86 (1) ◽

pp. 118-124 ◽

Cited By ~ 11

Author(s):

Jennifer J. Young ◽

Amna Mehdi ◽

Lori L. Stohl ◽

Lonny R. Levin ◽

Jochen Buck ◽

...

Keyword(s):

Adenylyl Cyclase ◽

Pc12 Cells ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Soluble Adenylyl Cyclase

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Increased Levels of cAMP by the Calcium-Dependent Activation of Soluble Adenylyl Cyclase in Parkin-Mutant Fibroblasts

Cells ◽

10.3390/cells8030250 ◽

2019 ◽

Vol 8 (3) ◽

pp. 250 ◽

Cited By ~ 2

Author(s):

Paola Tanzarella ◽

Anna Ferretta ◽

Simona Barile ◽

Mariella Ancona ◽

Domenico De Rasmo ◽

...

Keyword(s):

Adenylyl Cyclase ◽

Calcium Homeostasis ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Soluble Adenylyl Cyclase ◽

Calcium Dependent ◽

Level Of Activity ◽

Fluorimetric Analysis ◽

Polymerase Chain ◽

Low Activity

Almost half of autosomal recessive early-onset parkinsonism has been associated with mutations in PARK2, coding for parkin, which plays an important role in mitochondria function and calcium homeostasis. Cyclic adenosine monophosphate (cAMP) is a major second messenger regulating mitochondrial metabolism, and it is strictly interlocked with calcium homeostasis. Parkin-mutant (Pt) fibroblasts, exhibiting defective mitochondrial respiratory/OxPhos activity, showed a significant higher value of basal intracellular level of cAMP, as compared with normal fibroblasts (CTRL). Specific pharmacological inhibition/activation of members of the adenylyl cyclase- and of the phosphodiesterase-families, respectively, as well as quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, indicate that the higher level of cAMP observed in Pt fibroblasts can contribute to a higher level of activity/expression by soluble adenylyl cyclase (sAC) and to low activity/expression of the phosphodiesterase isoform 4 (PDE4). As Ca2+ regulates sAC, we performed quantitative calcium-fluorimetric analysis, showing a higher level of Ca2+ in the both cytosol and mitochondria of Pt fibroblasts as compared with CTRL. Most notably, inhibition of the mitochondrial Ca2+ uniporter decreased, specifically the cAMP level in PD fibroblasts. All together, these findings support the occurrence of an altered mitochondrial Ca2+-mediated cAMP homeostasis in fibroblasts with the parkin mutation.

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Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires

10.26434/chemrxiv.9209180 ◽

2019 ◽

Author(s):

Bella Grigorenko ◽

Igor Polyakov ◽

Alexander Nemukhin

Keyword(s):

Adenylyl Cyclase ◽

Bond Cleavage ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Md Simulations ◽

Coordination Shell ◽

Energy Profile ◽

One Step ◽

Type V

We report a mechanism of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) conversion by the mammalian type V adenylyl cyclase revealed in molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) simulations. We characterize a set of computationally derived enzyme-substrate (ES) structures showing an important role of coordination shells of magnesium ions in the solvent accessible active site. Several stable six-fold coordination shells of MgA2+ are observed in MD simulations of ES complexes. In the lowest energy ES conformation, the coordination shell of MgA2+ does not include the Oδ1 atom of the conserved Asp440 residue. Starting from this conformation, a one-step reaction mechanism is characterized which includes proton transfer from the ribose O3'H3' group in ATP to Asp440 via a shuttling water molecule and PA-O3A bond cleavage and O3'-PA bond formation. The energy profile of this route is consistent with the observed reaction kinetics. In a higher energy ES conformation, MgA2+ is bound to the Oδ1(Asp440) atom as suggested in the relevant crystal structure of the protein with a substrate analog. The computed energy profile initiated by this ES is characterized by higher energy expenses to complete the reaction. Consistently with experimental data, we show that the Asp440Ala mutant of the enzyme should exhibit a reduced but retained activity. All considered reaction pathways include proton wires from the O3'H3' group via shuttling water molecules.

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Light-Induced Change of Arginine Conformation Modulates the Rate of Adenosine Triphosphate to Cyclic Adenosine Monophosphate Conversion in the Optogenetic System Containing Photoactivated Adenylyl Cyclase

Journal of Chemical Information and Modeling ◽

10.1021/acs.jcim.0c01308 ◽

2021 ◽

Author(s):

Maria G. Khrenova ◽

Anna M. Kulakova ◽

Alexander V. Nemukhin

Keyword(s):

Adenylyl Cyclase ◽

Adenosine Triphosphate ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Photoactivated Adenylyl Cyclase

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Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis

10.1101/2021.08.03.454881 ◽

2021 ◽

Author(s):

Kaley M. Wilburn ◽

Christine R. Montague ◽

Bo Qin ◽

Ashley K. Woods ◽

Melissa S. Love ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Adenylyl Cyclase ◽

Cholesterol Metabolism ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Camp Signaling ◽

Camp Synthesis ◽

Pharmacokinetic Properties ◽

Bacterial Utilization

There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3’, 5’-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required for cholesterol metabolism. Finally, in this work the pharmacokinetic properties of Rv1625c agonists are optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a novel role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.

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Molecular Mechanism of Light-Induced Acceleration of the Adenosine Triphosphate Conversion to the Cyclic Adenosine Monophosphate Catalyzed by the Photoactivated Adenylyl Cyclase bPAC

10.26434/chemrxiv.13139723.v1 ◽

2020 ◽

Author(s):

Alexander Nemukhin ◽

Maria Khrenova ◽

Anna M. Kulakova

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Adenylyl Cyclase ◽

Adenosine Triphosphate ◽

Catalytic Domain ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Bluf Domain ◽

Dynamics Simulations ◽

Photoactivated Adenylyl Cyclase

We report the first computational characterization of an optogenetic system composed of two photosensing BLUF (blue light sensor using flavin adenine dinucleotide) domains and two catalytic adenylyl cyclase (AC) domains. Conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) and pyrophosphate (PPi) catalyzed by ACs coupled with excitation in photosensing domains has emerged in the focus of modern optogenetic applications because of the request in photoregulated enzymes to modulate cellular concentrations of signaling messengers. The photoactivated adenylyl cyclase from the soil bacterium Beggiatoa sp. (bPAC) is an important model showing considerable increase of the ATP to cAMP conversion rate in the catalytic domain after the illumination of the BLUF domain. The 1 μs classical molecular dynamics simulations reveal that the activation of the BLUF domain leading to tautomerization of Gln49 in the chromophore binding pocket results in switching of position of the side chain of Arg278 in the active site of AC. Allosteric signal transmission pathways between Gln49 from BLUF and Arg278 from AC were revealed by the dynamical network analysis. The Gibbs energy profiles of the ATP → cAMP + PPi reaction computed using QM(DFT(ωB97X-D3/6-31G**))/MM(CHARMM) molecular dynamics simulations for both Arg278 conformations in AC clarify the reaction mechanism. In the light-activated system, the corresponding arginine conformation stabilizes the pentacoordinated phosphorus of the α-phosphate group in the transition state, thus lowering the activation energy. Simulations of the bPAC system with the Tyr7Phe replacement in BLUF demonstrate occurrence of both arginine conformations in an equal ratio, explaining the experimentally observed intermediate catalytic activity of the bPAC-Y7F variant as compared with the dark and light states of the wild type bPAC.

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Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but not the Proximal Signalling Events

Thrombosis and Haemostasis ◽

10.1055/s-0039-1688788 ◽

2019 ◽

Vol 119 (07) ◽

pp. 1124-1137 ◽

Cited By ~ 3

Author(s):

Joanne C. Clark ◽

Deirdre M. Kavanagh ◽

Stephanie Watson ◽

Jeremy A. Pike ◽

Robert K. Andrews ◽

...

Keyword(s):

Adenylyl Cyclase ◽

G Protein ◽

Platelet Activation ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Adenosine Diphosphate ◽

Receptor Activation ◽

Inhibitory Effect ◽

Human Platelets ◽

Adenosine Receptor Agonist

Background The G protein-coupled receptor, adenosine A2A, signals through the stimulatory G protein, Gs, in platelets leading to activation of adenylyl cyclase and elevation of cyclic adenosine monophosphate (cAMP) and inhibition of platelet activation. Objective This article investigates the effect of A2A receptor activation on signalling by the collagen receptor glycoprotein (GP) VI in platelets. Methods Washed human platelets were stimulated by collagen or the GPVI-specific agonist collagen-related peptide (CRP) in the presence of the adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA) or the adenylyl cyclase activator, forskolin and analysed for aggregation, adenosine triphosphate secretion, protein phosphorylation, spreading, Ca2+ mobilisation, GPVI receptor clustering, cAMP, thromboxane B2 (TxB2) and P-selectin exposure. Results NECA, a bioactive adenosine analogue, partially inhibits aggregation and secretion to collagen or CRP in the absence or presence of the P2Y12 receptor antagonist, cangrelor and the cyclooxygenase inhibitor, indomethacin. The inhibitory effect in the presence of the three inhibitors is largely overcome at higher concentrations of collagen but not CRP. Neither NECA nor forskolin altered clustering of GPVI, elevation of Ca2+ or spreading of platelets on a collagen surface. Further, neither NECA nor forskolin, altered collagen-induced tyrosine phosphorylation of Syk, LAT nor PLCγ2. However, NECA and forskolin inhibited platelet activation by the TxA2 mimetic, U46619, but not the combination of adenosine diphosphate and collagen. Conclusion NECA and forskolin have no effect on the proximal signalling events by collagen. They inhibit platelet activation in a response-specific manner in part through inhibition of the feedback action of TxA2.

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