The E-NTPDase family of ectonucleotidases: Structure function relationships and pathophysiological significance

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Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that regulates key pathophysiological processes, such as those linked to inflammation. Classically, this reaction has been known to occur in the pericellular milieu catalyzed by membrane bound cellular ecto-nucleotidases, which can be released in the form of both soluble ecto-enzymes as well as being associated with exosomes. Circulating ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1/CD39) and adenylate kinase 1 (AK1) activities have been shown to be present in plasma. However, other ecto-nucleotidases have not been characterized in depth. An in vitro ADPase assay was developed to probe the ecto-enzymes responsible for the ectonucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating exosomes. We noted that microparticle-bound ENTPDases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane expressed E-NTPDases are also found in circulating microparticles in human plasma, inclusive of: CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members and other ecto-nucleotidases are found on distinct microparticle populations. A significant proportion of the microparticle-associated ecto-nucleotidase activity is sensitive to POM6, inferring the presence of NTPDases, either −2 or/and −3. We have refined ADPase assays of human plasma from healthy volunteers and have found that CD39, NTPDases 2, 3, and 8 to be associated with circulating microparticles, whereas soluble AK1 is present in human plasma. These ecto-enzymes constitute the bulk circulating ADPase activity, suggesting a broader implication of CD39 family and other ecto-enzymes in the regulation of extracellular nucleotide metabolism.

Section: Introductionmentioning

confidence: 99%

Characterization of circulating microparticle-associated CD39 family ecto-nucleotidases in human plasma

Jiang

Wu²,

Csizmadia³

et al. 2014

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“…It is confirmed by our study showing that blockade of autophagy achieves efficient cytotoxic effect on hepatoma cells treated with lower concentrations of eATP. Extracellular ATP could be hydrolyzed by CD39/ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1) to AMP, and the latter could further be hydrolyzed by CD73 to adenosine [5]. Several types of cancer cells express CD39, which can hydrolyze eATP to adenosine directly [35].…”

Section: Discussionmentioning

confidence: 99%

“…Effect of eATP on hepatoma cells was further confirmed by non-hydrolysable ATP analogue, BzATP eATP could be degraded to AMP and further adenosine at the presence of ectonucleotidase [5]. To confirm that eATP was responsible for this apoptosis and autophagy switch in anchorage-dependent and anchorage-independent hepatoma cells, BzATP (a non-hydrolysable form of eATP) was used, followed by detection of apoptosis marker caspase 3, Fig.…”

Section: Effect Of Eatp On the Anchorage-independent Hepatoma Cellsmentioning

confidence: 93%

“…Extracellular ATP (eATP) exerts many functions in physiological and pathological conditions, and its effect on cells is mediated by ionotropic (P2X) and metabotropic (P2Y) receptors [2][3][4]. Extracellular ATP could be hydrolyzed by ectonucleotidases; thus, its concentration is maintained at very low level in physiological conditions [5]. However, in some pathological conditions, such as tumor microenvironment and fast-growing tumor center which are less vascularized or have lots of cell death, a large amount of intracellular ATP is released from damaged cells, and the concentration of eATP in pericellular space is maintained at a relatively high level [4,6].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

High dose of extracellular ATP switched autophagy to apoptosis in anchorage-dependent and anchorage-independent hepatoma cells

Wei

Zhang

Xing

et al. 2013

Extracellular adenosine triphosphate (eATP) transduces purinergic signal and plays an important regulatory role in many biological processes, including tumor cell growth and cell death. A large amount of eATP exists in the fast-growing tumor center and inflammatory tumor microenvironment. Tumor cells could acquire anoikis resistance and anchorage independence in tumor microenvironment and further cause metastatic lesion. Whether such a high amount of eATP has any effect on the anchored and nonanchored tumor cells in tumor microenvironment has not been elucidated and is investigated in this study. Our data showed that autophagy helped hepatoma cells to maintain survival under the treatment of no more than 1 mM of eATP. Only when eATP concentration reached a relatively high level (2.5 mM), cell organelle could not be further maintained by autophagy, and apoptosis and cell death occurred. In hepatoma cells under treatment of 2.5 mM of eATP, an AMP-activated protein kinase (AMPK) pathway was dramatically activated while mTOR signaling pathway was suppressed in coordination with apoptosis. Further investigation showed that the AMPK/mTOR axis played a key role in tipping the balance between autophagy-mediated cell survival and apoptosis-induced cell death under the treatment of eATP. This work provides evidence to explain how hepatoma cells escape from eATP-induced cytotoxicity as well as offers an important clue to consider effective manipulation of cancer.

“…The largest families of ectonucleotidases are the ectonucleoside triphosphate diphosphohydrolase (ENTPD) and ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) families that hydrolyze nucleotide di-and triphosphates. The number of phosphate residues removed and the kinetic properties and substrate preferences vary from enzyme to enzyme [34,35]. Alkaline phosphatase, which is highly expressed in neutrophils, can convert ATP directly to adenosine, while CD73 converts AMP to adenosine [33].…”

Section: Breast Cancer Cells Express Ectonucleotidases That Promote Amentioning

confidence: 99%

Adenosine arrests breast cancer cell motility by A3 receptor stimulation

Ledderose

Hefti

Chen

et al. 2016

In neutrophils, adenosine triphosphate (ATP) release and autocrine purinergic signaling regulate coordinated cell motility during chemotaxis. Here, we studied whether similar mechanisms regulate the motility of breast cancer cells. While neutrophils and benign human mammary epithelial cells (HMEC) form a single leading edge, MDA-MB-231 breast cancer cells possess multiple leading edges enriched with A3 adenosine receptors. Compared to HMEC, MDA-MB-231 cells overexpress the ectonucleotidases ENPP1 and CD73, which convert extracellular ATP released by the cells to adenosine that stimulates A3 receptors and promotes cell migration with frequent directional changes. However, exogenous adenosine added to breast cancer cells or the A3 receptor agonist IB-MECA dose-dependently arrested cell motility by simultaneous stimulation of multiple leading edges, doubling cell surface areas and significantly reducing migration velocity by up to 75 %. We conclude that MDA-MB-231 cells, HMEC, and neutrophils differ in the purinergic signaling mechanisms that regulate their motility patterns and that the subcellular distribution of A3 adenosine receptors in MDA-MB-231 breast cancer cells contributes to dysfunctional cell motility. These findings imply that purinergic signaling mechanisms may be potential therapeutic targets to interfere with the motility of breast cancer cells in order to reduce the spread of cancer cells and the risk of metastasis.