Molecular evidence of adenosine deaminase linking adenosine A2A receptor and CD26 proteins

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits

Therapeutic Perspectives of Adenosine Deaminase Inhibition in Cardiovascular Diseases

Adenosine deaminase (ADA) is an enzyme of purine metabolism that irreversibly converts adenosine to inosine or 2′deoxyadenosine to 2′deoxyinosine. ADA is active both inside the cell and on the cell surface where it was found to interact with membrane proteins, such as CD26 and adenosine receptors, forming ecto-ADA (eADA). In addition to adenosine uptake, the activity of eADA is an essential mechanism that terminates adenosine signaling. This is particularly important in cardiovascular system, where adenosine protects against endothelial dysfunction, vascular inflammation, or thrombosis. Besides enzymatic function, ADA protein mediates cell-to-cell interactions involved in lymphocyte co-stimulation or endothelial activation. Furthermore, alteration in ADA activity was demonstrated in many cardiovascular pathologies such as atherosclerosis, myocardial ischemia-reperfusion injury, hypertension, thrombosis, or diabetes. Modulation of ADA activity could be an important therapeutic target. This work provides a systematic review of ADA activity and anchoring inhibitors as well as summarizes the perspectives of their therapeutic use in cardiovascular pathologies associated with increased activity of ADA

Changes in cardiac nucleotide metabolism in Huntington’s disease

Huntington’s disease (HD) is a monogenic neurodegenerative disorder with a significant peripheral component to the disease pathology. This includes an HD-related cardiomyopathy, with an unknown pathological mechanism. In this study, we aimed to define changes in the metabolism of cardiac nucleotides using the well-established R6/2 mouse model. In particular, we focused on measuring the activity of enzymes that control ATP and other adenine nucleotides in the cardiac pool, including eNTPD, AMPD, e5'NT, ADA and PNP. We employed HPLC to assay the activities of these enzymes by measuring the concentrations of adenine nucleotide catabolites in the hearts of symptomatic R6/2 mice. We found a reduced activity of AMPD (12.9 ± 1.9 nmol/min/mg protein in control; 7.5 ± 0.5 nmol/min/mg protein in R6/2) and e5'NT (11.9 ± 1.7 nmol/min/mg protein in control; 6.7 ± 0.7 nmol/min/mg protein in R6/2). Moreover, we detected an increased activity of ADA (1.3 ± 0.2 nmol/min/mg protein in control; 5.2 ± 0.5 nmol/min/mg protein in R6/2), while no changes in eNTPD and PNP activities were detected. Analysis of cardiac adenine nucleotide catabolite levels revealed an increased inosine level (0.7 ± 0.01 nmol/mg dry tissue in control; 2.7 ±0.8 nmol/mg dry tissue in R6/2) and a reduced concentration of cardiac adenosine (0.9 ± 0.2 nmol/mg dry tissue in control; 0.2 ± 0.08 nmol/mg dry tissue in R6/2). This study highlights a decreased rate of degradation of cardiac nucleotides in HD mouse model hearts, and an increased capacity for adenosine deamination, that may alter adenosine signaling

The new insight into extracellular NAD+NAD^{+} degradation : the contribution of CD38 and CD73 in calcific aortic valve disease

Nicotinamide adenine dinucleotide (NAD(+)) is crucial for cell energy metabolism and many signalling processes. Recently, we proved the role of ecto‐enzymes in controlling adenine nucleotide–dependent pathways during calcific aortic valve disease (CAVD). This study aimed to investigate extracellular hydrolysis of NAD(+) and mononucleotide nicotinamide (NMN) in aortic valves and aorta fragments of CAVD patients and on the inner aortic surface of ecto‐5′‐nucleotidase knockout mice (CD73−/−). Human non‐stenotic valves (n = 10) actively converted NAD(+) and NMN via both CD73 and NAD(+)‐glycohydrolase (CD38) according to our analysis with RP‐HPLC and immunofluorescence. In stenotic valves (n = 50), due to reduced CD73 activity, NAD(+) was degraded predominantly by CD38 and additionally by ALP and eNPP1. CAVD patients had significantly higher hydrolytic rates of NAD(+) (0.81 ± 0.07 vs 0.56 ± 0.10) and NMN (1.12 ± 0.10 vs 0.71 ± 0.08 nmol/min/cm(2)) compared with controls. CD38 was also primarily engaged in human vascular NAD(+) metabolism. Studies using specific ecto‐enzyme inhibitors and CD73−/− mice confirmed that CD73 is not the only enzyme involved in NAD(+) and NMN hydrolysis and that CD38 had a significant contribution to these pathways. Modifications of extracellular NAD(+) and NMN metabolism in aortic valve cells may be particularly important in valve pathology and could be a potential therapeutic target

CoCl2-Mimicked Endothelial Cell Hypoxia Induces Nucleotide Depletion and Functional Impairment That Is Reversed by Nucleotide Precursors

Chronic hypoxia drives vascular dysfunction by various mechanisms, including changes in mitochondrial respiration. Although endothelial cells (ECs) rely predominantly on glycolysis, hypoxia is known to alter oxidative phosphorylation, promote oxidative stress and induce dysfunction in ECs. Our work aimed to analyze the effects of prolonged treatment with hypoxia-mimetic agent CoCl2 on intracellular nucleotide concentration, extracellular nucleotide breakdown, mitochondrial function, and nitric oxide (NO) production in microvascular ECs. Moreover, we investigated how nucleotide precursor supplementation and adenosine deaminase inhibition protected against CoCl2-mediated disturbances. Mouse (H5V) and human (HMEC-1) microvascular ECs were exposed to CoCl2-mimicked hypoxia for 24 h in the presence of nucleotide precursors: adenine and ribose, and adenosine deaminase inhibitor, 2′deoxycoformycin. CoCl2 treatment decreased NO production by ECs, depleted intracellular ATP concentration, and increased extracellular nucleotide and adenosine catabolism in both H5V and HMEC-1 cell lines. Diminished intracellular ATP level was the effect of disturbed mitochondrial phosphorylation, while nucleotide precursors effectively restored the ATP pool via the salvage pathway and improved endothelial function under CoCl2 treatment. Endothelial protective effects of adenine and ribose were further enhanced by adenosine deaminase inhibition, that increased adenosine concentration. This work points to a novel strategy for protection of hypoxic ECs by replenishing the adenine nucleotide pool and promoting adenosine signaling