Adenosine-regulated mechanisms in the pathogenesis of ventilation disorders in patients with pulmonary tuberculosis

Uncovering involvement of the purinergic system in the pathogenesis of ventilation disorders (VD) may provide additional information about the pathophysiological mechanisms leading to the development of VD in pulmonary tuberculosis (PT). The aim was to identify a relationship between the parameters of adenosine metabolism, inflammatory response and altered ventilation metabolism in PT patients. Materials and methods. Obstructive and mixed PT patients were assigned to subgroups with/without VD for assessing adenosine deaminase activity (ADA-1, 2) in serum, mononuclear cells, neutrophils; ecto-5’-nucleotidase (ecto-5’-NT); CD26 (dipeptidyl peptidase-4, DPP-4), phagocyte oxidative burst measured by NO generation. Results. PT patients showed decreased ADA-1 and CD26 (DPP-4), but increased ADA-2. Elevated intracellular adenosine concentration was found in mononuclear cells in patients lacking VD, whereas patients with mixed and obstructive VD — had it in neutrophils. Mononuclear cells of patients with PT lacking VD as well as with obstructive VD type had decreased NO3– concentration. Neutrophil hyperactivity was recorded in all groups of PT patients. Patients with PT lacking VD as well as with mixed VD type showed that the parameters of external respiration were associated with activity of extra-/intracellular ADA, whereas obstructive VD was caused by excessive formation of serum adenosine. Changes in respiratory function in PT were associated with decreased level of serum NO radicals, impaired nitrogen-dependent bactericidal phagocyte activity, and overproduced neutrophil oxygen radicals. Conclusion. Purinergic regulation is involved in regulating inflammatory and compensatory processes in PT patients as well as impaired ventilation efficiency. The most severe respiratory disorders observed in PT patients with mixed VD type are associated with the most prominent changes in nucleotidase activity, particularly ecto-ADA-2 and DPP-4/CD26.

Adenosine turnover in GtoPdb v.2021.3

A multifunctional, ubiquitous molecule, adenosine acts at cell-surface G protein-coupled receptors, as well as numerous enzymes, including protein kinases and adenylyl cyclase. Extracellular adenosine is thought to be produced either by export or by metabolism, predominantly through ecto-5’-nucleotidase activity (also producing inorganic phosphate). It is inactivated either by extracellular metabolism via adenosine deaminase (also producing ammonia) or, following uptake by nucleoside transporters, via adenosine deaminase or adenosine kinase (requiring ATP as co-substrate). Intracellular adenosine may be produced by cytosolic 5’-nucleotidases or through S-adenosylhomocysteine hydrolase (also producing L-homocysteine).

Substrate Specificity of Chimeric Enzymes Formed by Interchange of the Catalytic and Specificity Domains of the 5′-Nucleotidase UshA and the 3′-Nucleotidase CpdB

The 5′-nucleotidase UshA and the 3′-nucleotidase CpdB from Escherichia coli are broad-specificity phosphohydrolases with similar two-domain structures. Their N-terminal domains (UshA_Ndom and CpdB_Ndom) contain the catalytic site, and their C-terminal domains (UshA_Cdom and CpdB_Cdom) contain a substrate-binding site responsible for specificity. Both enzymes show only partial overlap in their substrate specificities. So, it was decided to investigate the catalytic behavior of chimeras bearing the UshA catalytic domain and the CpdB specificity domain, or vice versa. UshA_Ndom–CpdB_Cdom and CpdB_Ndom–UshA_Cdom were constructed and tested on substrates specific to UshA (5′-AMP, CDP-choline, UDP-glucose) or to CpdB (3′-AMP), as well as on 2′,3′-cAMP and on the common phosphodiester substrate bis-4-NPP (bis-4-nitrophenylphosphate). The chimeras did show neither 5′-nucleotidase nor 3′-nucleotidase activity. When compared to UshA, UshA_Ndom–CpdB_Cdom conserved high activity on bis-4-NPP, some on CDP-choline and UDP-glucose, and displayed activity on 2′,3′-cAMP. When compared to CpdB, CpdB_Ndom–UshA_Cdom conserved phosphodiesterase activities on 2′,3′-cAMP and bis-4-NPP, and gained activity on the phosphoanhydride CDP-choline. Therefore, the non-nucleotidase activities of UshA and CpdB are not fully dependent on the interplay between domains. The specificity domains may confer the chimeras some of the phosphodiester or phosphoanhydride selectivity displayed when associated with their native partners. Contrarily, the nucleotidase activity of UshA and CpdB depends strictly on the interplay between their native catalytic and specificity domains.

Anti-5 ′ -Nucleotidases (5 ′ -ND) and Acetylcholinesterase (AChE) Activities of Medicinal Plants to Combat Echis carinatus Venom-Induced Toxicities

Echis carinatus is one of the highly venomous snakes of Pakistan that is responsible for numerous cases of envenomation and deaths. In Pakistan, medicinal plants are commonly used traditionally for snakebite treatment because of their low cost and easy availability in comparison with antivenom. The current research is aimed at evaluating the inhibitory activity of Pakistani medicinal plants against acetylcholinesterase and 5 ′ -nucleotidases present in Echis carinatus venom. Acetylcholinesterase and 5 ′ -nucleotidase enzymatic assays were performed at different venom concentrations to check the activity of these enzymes. Methanolic extracts from different parts of plants were used for in vitro determination of their inhibitory activity against 5 ′ -nucleotidases in snake venom. Active methanolic extracts were subsequently fractioned using different solvents, and these fractions were also assessed for their anti-5 ′ -nucleotidase activity. Results of this study exhibited that Eugenia jambolana Willd. ex O. Berg, Rubia cordifolia L., Trichodesma indicum (L.) R. Br., Calotropis procera (Wild.) R. Br., Curcuma longa L., and Fagonia arabica L. were able to significantly ( p > 0.5 ) neutralize the 5 ′ -nucleotidase activity by 88%, 86%, 86%, 85%, 83.7%, and 83%, respectively, compared with a standard antidote (snake venom antiserum). Thus, this study indicates that these plants possess the potential to neutralize one of the toxic enzymatic components of Echis carinatus venom and hence can help to augment the future efforts of developing alternative therapy for the management of snakebites.

Post-Translational Mechanisms of NT5C2 Activation As Non-Genetic Drivers of Chemotherapy Resistance in Relapsed Acute Lymphoblastic Leukemia

Despite intensive chemotherapy, 20% of pediatric acute lymphoblastic leukemia (ALL) patients fail to achieve a complete remission or relapse after intensified chemotherapy, making relapse and resistance to therapy the most significant challenge in the treatment of this disease. We have shown that NT5C2 mutations resulting in deregulated enzymatic activity are present in 10% of relapsed B-precursor ALL and 20% of relapsed T-ALL cases. These genetic alterations abrogate the activity of an intramolecular switch-off mechanism resulting in increased nucleotidase activity. NT5C2 metabolizes and clears the activated forms of 6-mercaptopurine (6-MP), a critical drug in the treatment of ALL. As a result, activating mutations in the NT5C2 cytosolic nucleotidase gene are characteristically associated with early relapsed leukemia and progression under therapy and confer resistance to 6-mercaptopurine chemotherapy. A saturated mutagenesis positive selection screen for all possible NT5C2 variants (10640 alleles) conferring resistance to 6-MP recovers mutational hotspots involving recurrent relapse-associated mutations and identifies new residues and protein domains involved in NT5C2 regulation whose inactivation drives resistance to 6-MP. Moreover, a genome wide CRISPR screen analysis of 6-MP-gene interactions in NT5C2 wild type REH ALL cells identifies NT5C2 as the most prominent hit whose inactivation increases sensitivity to 6-MP. Taken together, these results point to heretofore uncharacterized mechanisms of NT5C2 regulation and support a role for wild type NT5C2 in 6-MP metabolism and inactivation. Thus post-translational modifications enhancing NT5C2 activity could attenuate the therapeutic activity of 6-MP in NT5C2 wild type cells as non-mutational mechanism of 6-MP resistance. Mass spectrometry analysis of NT5C2 protein revealed the presence of two NT5C2 acetylations and two NT5C2 phosphorylation sites. Functional analyses of these modifications revealed increased nucleotidase activity for the NT5C2 S502D phospho-mimic mutation. Consistently, expression of NT5C2 S502D in leukemia lymphoblasts induced resistance to 6-MP. Importantly, western blot analysis revealed increased NT5C2 S502 phosphorylation in relapsed leukemia xenograft samples compared with matched diagnosis derived ALL xenografts. Crystal structure analysis of NT5C2 recombinant protein supports that S502 engages D229 in the partner protomer of the NT5C2 dimer. Structure-function analysis of S502D, S502A and D229A mutants targeting this interaction supports the disruption of S502-D229 interaction via S502 phosphorylation as mechanism of NT5C2 activation. In all, these results highlight a prominent role for wild type NT5C2 activity in the detoxification of 6-MP in ALL lymphoblasts, identifies NT5C2 S502 phosphorylation as a prevalent non-genetic mechanism of NT5C2 activation at relapse and provides novel insight on the mechanisms of NT5C2 regulation of relevance for the therapeutic targeting of NT5C2 in relapsed ALL. Disclosures No relevant conflicts of interest to declare.

Adenosine turnover (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

A multifunctional, ubiquitous molecule, adenosine acts at cell-surface G protein-coupled receptors, as well as numerous enzymes, including protein kinases and adenylyl cyclase. Extracellular adenosine is thought to be produced either by export or by metabolism, predominantly through ecto-5’-nucleotidase activity (also producing inorganic phosphate). It is inactivated either by extracellular metabolism via adenosine deaminase (also producing ammonia) or, following uptake by nucleoside transporters, via adenosine deaminase or adenosine kinase (requiring ATP as co-substrate). Intracellular adenosine may be produced by cytosolic 5’-nucleotidases or through S-adenosylhomocysteine hydrolase (also producing L-homocysteine).