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Molecular basis of purinergic signal metabolism by ectonucleotide pyrophosphatase/phosphodiesterases 4 and 1 and implications in stroke.

ABSTRACT: NPP4 is a type I extracellular membrane protein on brain vascular endothelium inducing platelet aggregation via the hydrolysis of Ap3A, whereas NPP1 is a type II extracellular membrane protein principally present on the surface of chondrocytes that regulates tissue mineralization. To understand the metabolism of purinergic signals resulting in the physiologic activities of the two enzymes, we report the high resolution crystal structure of human NPP4 and explore the molecular basis of its substrate specificity with NPP1. Both enzymes cleave Ap3A, but only NPP1 can hydrolyze ATP. Comparative structural analysis reveals a tripartite lysine claw in NPP1 that stabilizes the terminal phosphate of ATP, whereas the corresponding region of NPP4 contains features that hinder this binding orientation, thereby inhibiting ATP hydrolysis. Furthermore, we show that NPP1 is unable to induce platelet aggregation at physiologic concentrations reported in human blood, but it could stimulate platelet aggregation if localized at low nanomolar concentrations on vascular endothelium. The combined studies expand our understanding of NPP1 and NPP4 substrate specificity and range and provide a rational mechanism by which polymorphisms in NPP1 confer stroke resistance.

SUBMITTER: Albright RA

PROVIDER: S-EPMC3916532 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Molecular basis of purinergic signal metabolism by ectonucleotide pyrophosphatase/phosphodiesterases 4 and 1 and implications in stroke.

Albright Ronald A RA Ornstein Deborah L DL Cao Wenxiang W Chang William C WC Robert Donna D Tehan Martin M Hoyer Denton D Liu Lynn L Stabach Paul P Yang Guangxiao G De La Cruz Enrique M EM Braddock Demetrios T DT

The Journal of biological chemistry 20131212 6

NPP4 is a type I extracellular membrane protein on brain vascular endothelium inducing platelet aggregation via the hydrolysis of Ap3A, whereas NPP1 is a type II extracellular membrane protein principally present on the surface of chondrocytes that regulates tissue mineralization. To understand the metabolism of purinergic signals resulting in the physiologic activities of the two enzymes, we report the high resolution crystal structure of human NPP4 and explore the molecular basis of its substr ...[more]

PMID: 24338010

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Project description:ENPP-1 is a transmembrane enzyme involved in nucleotide metabolism, and its overexpression is associated with various cancers, making it a potential therapeutic target and biomarker for early tumor diagnosis. Current detection methods for ENPP-1 utilize a colorimetric probe, TMP-pNP, which has significant limitations in sensitivity. Here, we present probe CL-ENPP-1, the first nucleic acid-based chemiluminescent probe designed for rapid and highly sensitive detection of ENPP-1 activity. The design of probe CL-ENPP-1 features a phenoxy-adamantyl-1,2-dioxetane luminophore linked to thymidine via a phosphodiesteric bond. Upon cleavage of the enzymatic substrate by ENPP-1, the probe undergoes an efficient chemiexcitation process to emit a green photon. Probe CL-ENPP-1 demonstrates an exceptional signal-to-noise ratio of 15000 and a limit of detection value approximately 4500-fold lower than the widely used colorimetric probe TMP-pNP. A comparison of TMP-pNP activation by ENPP-1 versus alkaline phosphatase (ALP) reveals a complete lack of selectivity. Removal of the self-immolative spacer from probe CL-ENPP-1 resulted in a new chemiluminescent probe, CL-ENPP-2, with an 18.4-fold increase in selectivity for ENPP-1 over ALP. The ability of probe CL-ENPP-2 to detect ENPP-1 activity in mammalian cells was assessed using the human breast cancer cell line MDA-MB-231. This probe demonstrated a 19.5-fold improvement in the signal-to-noise ratio, highlighting its superior ability to detect ENPP-1 activity in a biological sample. As far as we know, to date, CL-ENPP-1 and CL-ENPP-2 are the most sensitive probes for the detection of ENPP-1 catalytic activity. We anticipate that our new chemiluminescent probes will be valuable for various applications requiring ENPP-1 detection, including enzyme inhibitor-based drug discovery assays. The insights gained from our probe design principles could advance the development of more selective probes for ENPP-1 and contribute to future innovations in chemiluminescence research.

Project description:BackgroundIschemic stroke is the combinatorial effect of many pathological processes including the loss of energy supplies, excessive intracellular calcium accumulation, oxidative stress, and inflammatory responses. The brain's ability to maintain energy demand through this process involves metabolism of glycogen, which is critical for release of stored glucose. However, regulation of glycogen metabolism in ischemic stroke remains unknown. In the present study, we investigate the role and regulation of glycogen metabolizing enzymes and their effects on the fate of glycogen during ischemic stroke.ResultsIschemic stroke was induced in rats by peri-vascular application of the vasoconstrictor endothelin-1 and forebrains were collected at 1, 3, 6 and 24 hours post-stroke. Glycogen levels and the expression and activity of enzymes involved in glycogen metabolism were analyzed. We found elevated glycogen levels in the ipsilateral hemispheres compared with contralateral hemispheres at 6 and 24 hours (25% and 39% increase respectively; P<0.05). Glycogen synthase activity and glycogen branching enzyme expression were found to be similar between the ipsilateral, contralateral, and sham control hemispheres. In contrast, the rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 58% lower activity (P<0.01) in the ipsilateral hemisphere (24 hours post-stroke), which corresponded with a 48% reduction in cAMP-dependent protein kinase A (PKA) activity (P<0.01). In addition, glycogen debranching enzyme expression 24 hours post-stroke was 77% (P<0.01) and 72% lower (P<0.01) at the protein and mRNA level, respectively. In cultured rat primary cerebellar astrocytes, hypoxia and inhibition of PKA activity significantly reduced glycogen phosphorylase activity and increased glycogen accumulation but did not alter glycogen synthase activity. Furthermore, elevated glycogen levels provided metabolic support to astrocytes during hypoxia.ConclusionOur study has identified that glycogen breakdown is impaired during ischemic stroke, the molecular basis of which includes reduced glycogen debranching enzyme expression level together with reduced glycogen phosphorylase and PKA activity.

Dataset Information

Molecular basis of purinergic signal metabolism by ectonucleotide pyrophosphatase/phosphodiesterases 4 and 1 and implications in stroke.

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Molecular basis of purinergic signal metabolism by ectonucleotide pyrophosphatase/phosphodiesterases 4 and 1 and implications in stroke.

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