Endre Kókai

Adenosine and inflammation: what's new on the horizon?

Adenosine contributes to the maintenance of tissue integrity by modulating the immune system. Encouraging results have emerged with adenosine receptor ligands for the management of several inflammatory conditions in preclinical and clinical settings. However, therapeutic applications of these drugs are sometimes complicated by the occurrence of serious adverse effects. The scientific community is making intensive efforts to design novel adenosine receptor ligands endowed with greater selectivity or to develop innovative compounds acting as allosteric receptor modulators. In parallel, research is focusing on novel pharmacological entities (designated as adenosine-regulating agents) that can increase, in a site- and event-specific manner, adenosine concentrations at the inflammatory site, thereby minimizing the adverse systemic effects of adenosine.

Phosphorylation blocks the activity of tubulin polymerization promoting protein (TPPP): Identification of sites targeted by different kinases

Tubulin polymerization-promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Δ3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 ± 0.3, 2.2 ± 0.3, and 0.9 ± 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr14, Ser18, Ser160 for Cdk5; Ser18, Ser160 for ERK2, and Ser32 for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (Kd = 2.5-2.7 μm) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr14 and/or Ser18 in its unfolded N-terminal tail.

Adenosine augments IL‐10‐induced STAT3 signaling in M2c macrophages

The alternatively activated macrophage phenotype induced by IL‐10 is called M2c. Adenosine is an endogenous purine nucleoside that accumulates in the extracellular space in response to metabolic disturbances, hypoxia, inflammation, physical damage, or apoptosis. As adenosine is known to regulate classically activated M1 and IL4‐ and IL‐13‐activated M2a macrophages, the goal of the present study was to explore its effects on M2c macrophages. We found that adenosine augmented the IL‐10‐induced expression of TIMP‐1 and arginase‐1 by the mouse macrophage cell line RAW 264.7 and by mouse BMDMs. The effects of AR stimulation on IL‐10‐induced TIMP‐1 or arginase‐1 expression were lacking in A2BAR KO macrophages. The role of A2BAR on TIMP‐1 production of RAW 264.7 cells was confirmed with specific agonist BAY606583 and antagonist PSB0788. AR stimulation augmented IL‐10‐induced STAT3 phosphorylation in macrophages, and pharmacological inhibition or silencing of STAT3 using siRNA reduced the stimulatory effect of AR stimulation on TIMP‐1 production. In contrast to its stimulatory effect on IL‐10‐induced STAT3 activation, adenosine inhibited IL‐6‐induced STAT3 phosphorylation and SAA3 expression. In conclusion, adenosine enhances IL‐10‐induced STAT3 signaling and M2c macrophage activation.

A2B adenosine receptors prevent insulin resistance by inhibiting adipose tissue inflammation via maintaining alternative macrophage activation

Obesity causes increased classical and decreased alternative macrophage activation, which in turn cause insulin resistance in target organs. Because A2B adenosine receptors (ARs) are important regulators of macrophage activation, we examined the role of A2B ARs in adipose tissue inflammation and insulin resistance. A2B AR deletion impaired glucose and lipid metabolism in mice fed chow but not a high-fat diet, which was paralleled by dysregulation of the adipokine system, and increased classical macrophage activation and inhibited alternative macrophage activation. The expression of alternative macrophage activation–specific transcriptions factors, including CCAAT/enhancer-binding protein-β, interferon regulatory factor 4, and peroxisome proliferator–activated receptor-γ, was decreased in adipose tissue of A2B AR–deficient mice. Furthermore, in in vitro studies, we found that stimulation of A2B ARs suppressed free fatty acid–induced deleterious inflammatory and metabolic activation of macrophages. Moreover, AR activation upregulated the interleukin-4–induced expression of CCAAT/enhancer-binding protein-β, interferon regulatory factor 4, and peroxisome proliferator–activated receptor-γ in macrophages. Altogether, our results indicate that therapeutic strategies targeting A2B ARs hold promise for preventing adipose tissue inflammation and insulin resistance.

CD39 improves survival in microbial sepsis by attenuating systemic inflammation

Sepsis remains the leading cause of morbidity and mortality in critically ill patients. Excessive inflammation is a major cause of organ failure and mortality in sepsis. Ectonucleoside triphosphate diphosphohydrolase 1, ENTPDase1 (CD39) is a cell surface nucleotide‐metabolizing enzyme, which degrades the extracellular purines ATP and ADP, thereby regulating purinergic receptor signaling. Although the role of purinergic receptor signaling in regulating inflammation and sepsis has been addressed previously, the role of CD39 in regulating the hosts response to sepsis is unknown. We found that the CD39 mimic apyrase (250 U/kg) decreased and knockout or pharmacologic blockade with sodium polyoxotungstate (5 mg/kg; IC50 ã 10 μM) of CD39 increased mortality of mice with polymicrobial sepsis induced by cecal ligation and puncture. CD39 decreased inflammation, organ damage, immune cell apoptosis, and bacterial load. Use of bone marrow chimeric mice revealed that CD39 expression on myeloid cells decreases inflammation in septic mice. CD39 expression is upregulated during sepsis in mice, as well as in both murine and human macrophages stimulated with Escherichia coli. Moreover, E. coli increases CD39 promoter activity in macrophages. Altogether, these data indicate CD39 as an evolutionarily conserved inducible protective pathway during sepsis. We propose CD39 as a novel therapeutic target in the management of sepsis—Csóka, B., Németh, Z. H., Törő, G., Koscsó, B., Kókai, E., Robson, S. C., Enjyoji, K., Rolandelli, R. H., Erdélyi, K., Pacher, P., Haskó, G., CD39 improves survival in microbial sepsis by attenuating systemic inflammation. FASEB J. 29, 25–36 (2015). www.fasebj.org

Ribonucleoprotein-masked nicks at 50-kbp intervals in the eukaryotic genomic DNA

By using a microscopic approach, field inversion single-cell gel electrophoresis, we show that preformed single-strand discontinuities are present in the chromatin of resting and proliferating mammalian and yeast cells. These single-strand breaks are primarily nicks positioned at ≈50-kbp intervals throughout the entire genome that could be efficiently labeled in situ by DNA polymerase I holoenzyme but not by Klenow fragment and terminal transferase unless after ribonucleolytic treatments. The RNA molecules involved appear to comprise R-loops, recognized by the S9.6 RNA/DNA hybrid-specific antibody. By using the breakpoint cluster region of the Mixed Lineage Leukemia (MLL) gene as a model, we have found that the number of manifest nicks detected by FISH performed after field inversion single-cell gel electrophoresis depends on epigenetic context, but the difference between germ-line and translocated MLL alleles is abolished by protease treatment. Our data imply that the double-stranded genomic DNA is composed of contiguous rather than continuous single strands and reveal an aspect of higher-order chromatin organization with ribonucleoprotein-associated persistent nicks defining ≈50-kbp domains.

Separation of 1–23-kb complementary DNA strands by urea–agarose gel electrophoresis

Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea–agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of ∼1–20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas.

Protein phosphatase 1

Protein phosphatase 1 (PP1) is one of the first protein phosphatases whose activity was detected and whose catalytic subunit (PP1c) was purified and cloned. It is the representative of the most ancient protein phosphatase family that has a ubiquitous distribution in all eukaryotic organisms. The high level of conservation of the amino acid sequence and protein architecture of the PP1c is remarkable, and the identification of its very similar isoforms was an unexpected result of molecular cloning. The enzyme has a large number of interacting proteins, which tether PP1c to well defined locations and/or regulate its activity. A dynamic exchange of these non-catalytic subunits and the broad substrate specificity of the phosphatase are consistent with a wide range of physiological roles including cell cycle regulation, centrosome separation, interphase chromosome condensation, glycogen metabolism, contractility, morphogenesis, spermatogenesis, learning and memory, as inferred from genetic studies and predicted from biochemical experiments.

Expression of protein phosphatase 1 during the asexual development of Neurospora crassa

We cloned and sequenced the cDNA and the gene encoding the catalytic subunit of protein phosphatase 1 from the filamentous fungus Neurospora crassa. The gene, designated ppp-1 (phosphoprotein phosphatase 1), was mapped by restriction fragment length polymorphism to linkage group III, in the vicinity of con-7 and trp-1. The expression of the gene was monitored by reverse transcriptase and polymerase chain reactions, by Western blotting, and by protein phosphatase activity assays in synchronized cultures. Transcripts of ppp-1 were detected in the dormant conidia. The abundance of ppp-1 mRNA, Ppp-1 protein, and the activity of protein phosphatase 1 increased during germination and subsequent hyphal elongation as well as during the early stages of aerial mycelium formation.

Regulation of calpain B from Drosophila melanogaster by phosphorylation

Calpain B is one of the two catalytically competent calpain (calcium‐activated papain) isoenzymes in Drosophila melanogaster. Because structural predictions hinted at the presence of several potential phosphorylation sites in this enzyme, we investigated the in vitro phosphorylation of the recombinant protein by protein kinase A as well as by the extracellular signal‐regulated protein kinases (ERK) 1 and 2. By MS, we identified Ser845 in the Ca2+ binding region of an EF‐hand motif, and Ser240 close to the autocatalytic activation site of calpain B, as being the residues phosphorylated by protein kinase A. In the transducer region of the protease, Thr747 was shown to be the target of the ERK phosphorylation. Based on the results of three different assays, we concluded that the treatment of calpain B with protein kinase A and ERK1 and ERK2 kinases increases the rate of the autoproteolytic activation of the enzyme, together with the rate of the digestion of external peptide or protein substrates. Phosphorylation also elevates the Ca2+ sensitivity of the protease. The kinetic analysis of phosphorylation mimicking Thr747Glu and Ser845Glu calpain B mutants confirmed the above conclusions. Out of the three phosphorylation events tested in vitro, we verified the in vivo phosphorylation of Thr747 in epidermal growth factor‐stimulated Drosophila S2 cells. The data obtained suggest that the activation of the ERK pathway by extracellular signals results in the phosphorylation and activation of calpain B in fruit flies.