scholarly journals Extracellular vesicles direct migration by synthesizing and releasing chemotactic signals

2018 ◽  
Vol 217 (8) ◽  
pp. 2891-2910 ◽  
Author(s):  
Paul W. Kriebel ◽  
Ritankar Majumdar ◽  
Lisa M. Jenkins ◽  
Hiroshi Senoo ◽  
Weiye Wang ◽  
...  

Chemotactic signals are relayed to neighboring cells through the secretion of additional chemoattractants. We previously showed in Dictyostelium discoideum that the adenylyl cyclase A, which synthesizes the chemoattractant cyclic adenosine monophosphate (cAMP), is present in the intraluminal vesicles of multivesicular bodies (MVBs) that coalesce at the back of cells. Using ultrastructural reconstructions, we now show that ACA-containing MVBs release their contents to attract neighboring cells. We show that the released vesicles are capable of directing migration and streaming and are central to chemotactic signal relay. We demonstrate that the released vesicles not only contain cAMP but also can actively synthesize and release cAMP to promote chemotaxis. Through proteomic, pharmacological, and genetic approaches, we determined that the vesicular cAMP is released via the ABCC8 transporter. Together, our findings show that extracellular vesicles released by D. discoideum cells are functional entities that mediate signal relay during chemotaxis and streaming.

2019 ◽  
Author(s):  
Bella Grigorenko ◽  
Igor Polyakov ◽  
Alexander Nemukhin

<p>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 Mg<sub>A</sub><sup>2+ </sup>are observed in MD simulations of ES complexes. In the lowest energy ES conformation, the coordination shell of Mg<sub>A</sub><sup>2+ </sup>does not include the O<sub>δ1</sub> atom of the conserved Asp440 residue. Starting from this conformation, a one-step reaction mechanism is characterized which includes proton transfer from the ribose O<sup>3'</sup>H<sup>3' </sup>group in ATP to Asp440 via a shuttling water molecule and P<sup>A</sup>-O<sup>3A</sup> bond cleavage and O<sup>3'</sup>-P<sup>A</sup> bond formation. The energy profile of this route is consistent with the observed reaction kinetics. In a higher energy ES conformation, Mg<sub>A</sub><sup>2+</sup> is bound to the O<sub>δ1</sub>(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 O<sup>3'</sup>H<sup>3' </sup>group via shuttling water molecules. </p>


2021 ◽  
Author(s):  
Kaley M. Wilburn ◽  
Christine R. Montague ◽  
Bo Qin ◽  
Ashley K. Woods ◽  
Melissa S. Love ◽  
...  

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.


2017 ◽  
Author(s):  
Kaumudi H Prabhakara ◽  
Albert J Bae ◽  
Eberhard Bodenschatz

AbstractUpon starvation, Dictyostelium discoideum (D.d.) exhibit social behavior mediated by the chemical messenger cyclic adenosine monophosphate (cAMP). Large scale cAMP waves synchronize the population of starving cells and enable them to aggregate and form a multi-cellular organism. Here, we explore the effect of cell-to-cell variability in the production of cAMP on aggregation. We create a mixture of extreme cell-to-cell variability by mixing a few cells that produce cAMP (haves) with a majority of mutants that cannot produce cAMP (have-nots). Surprisingly, such mixtures aggregate, although each population on its own cannot aggregate. We show that (1) a lack of divalent ions kills the haves at low densities and (2) the have-nots supply the cAMP degrading enzyme, phosphodiesterase, which, in the presence of divalent ions, enables the mixture to aggregate. Our results suggest that a range of degradation rates induces optimal aggregation. The haves and the have-nots cooperate by sharing complementary resources.


2020 ◽  
Author(s):  
Alexander Nemukhin ◽  
Maria Khrenova ◽  
Anna M. Kulakova

<p>We report the first computational characterization of an optogenetic system composed of two photosensing BLUF (<u>b</u>lue <u>l</u>ight sensor <u>u</u>sing <u>f</u>lavin 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 <i>Beggiatoa sp.</i> (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. </p>


2019 ◽  
Vol 119 (07) ◽  
pp. 1124-1137 ◽  
Author(s):  
Joanne C. Clark ◽  
Deirdre M. Kavanagh ◽  
Stephanie Watson ◽  
Jeremy A. Pike ◽  
Robert K. Andrews ◽  
...  

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.


2021 ◽  
Vol 11 (2) ◽  
pp. 20200034
Author(s):  
Tom Rossetti ◽  
Stephanie Jackvony ◽  
Jochen Buck ◽  
Lonny R. Levin

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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