Structure and function of ecto-nucleotidases in purinergic signaling

除了具有重要的细胞代谢功能外，ATP还作为细胞外信号物质。细胞外核苷酸影响多种短期（急性）生理过程，包括外分泌和内分泌分泌、免疫反应、炎症、伤害性机械感觉转导、血小板聚集和内皮介导的血管舒张。受影响的长期（营养）过程是细胞增殖、分化、迁移和死亡，例如发育、再生和癌症。细胞外核苷酸酶参与核苷酸的代谢。ntpdase（外核苷三磷酸二磷酸水解酶）通过ADP将ATP脱磷酸为AMP。NTPDase1、-2、-3和-8面向细胞外空间，在嘌呤能信号传递中起作用，而ntpdase 4-7位于细胞内细胞器，如高尔基体。在这里，它们参与蛋白质糖基化并将ndp转化为nmp，以避免糖基转移酶的抑制，并通过反转运体提供nmp与ndp糖的交换。除了ntpase外，核苷酸焦磷酸酶/磷酸二酯酶（NPPs）也参与细胞外核苷酸代谢。该项目的目的是通过x射线晶体学、诱变研究和活性分析来表征ntpases和NPPs的分子结构和催化机制。一个主要的目标是首次确定细胞内ntpase参与蛋白质糖基化的结构。这些酶的活性位点结构和独特的底物特异性不能基于NTPDase1和2的可用结构产生的同源模型来理解。为此，我们将测定人和大鼠ntpases 4-7的晶体结构。与底物和抑制剂的共晶结构将定义底物结合相互作用和底物特异性差异的分子基础（NDP与NTP以及不同的核碱基）。NTPDase1在不释放ADP的情况下将ATP水解成AMP。先前的研究表明，这种处理能力取决于酶的膜锚定。通过诱变研究，我们将验证细胞外ntpases中的长疏水保守环参与膜锚定并介导跨膜部分和外畴之间的偶联的假设。此外，我们将确定尚未表征的外位- ntpases 3和8的晶体结构。项目的另一部分涉及核电的特殊性和机制。我们可以结晶大鼠NPP3。我们将确定酶的x射线结构，并分析不同核苷酸底物和产物的共晶结构，以了解参与嘌呤能信号传导和钙化的不同npp之间底物特异性的差异。在合作中，我们将帮助开发这些酶的特异性抑制剂作为生物和药物工具。

In addition to its important cellular function in metabolism, ATP also serves as an extracellular signaling substance. Extracellular nucleotides influence a wide variety of short-term (acute) physiological processes, including exocrine and endocrine secretion, immune responses, inflammation, nociceptive mechanosensory transduction, platelet aggregation and endothelial-mediated vasodilatation. Long-term (trophic) processes affected are cell proliferation, differentiation, migration and death as for example in development, regeneration and cancer.Extracellular nucleotidases are involved in the metabolism of the nucleotides. The NTPDases (ecto-nucleoside triphosphate diphosphohydrolases) dephosphorylate ATP via ADP to AMP. NTPDase1, -2, -3 and -8 face the extracellular space and function in purinergic signal transmission whereas the NTPDases 4-7 are located in intracellular organelles such as the Golgi. Here they are involved in protein glycosylation and convert NDPs to NMPs to avoid inhibition of glycosyl transferases and to provide NMPs for exchange against NDP-sugars by an antiporter. In addition to the NTPDases, nucleotide pyrophosphatases/phosphodiesterases (NPPs) are involved in extracellular nucleotide metabolism.The aim of the project is to characterize the molecular structures and catalytic mechanisms of NTPDases and NPPs by X-ray crystallography, mutagenesis studies and activity assays. A major goal is the first structure determination of an intracellular NTPDase involved in protein glycosylation. The active site structures and unique substrate specificity of these enzymes cannot be understood based on homology models generated from the available structures of NTPDase1 and 2. To this end, we will determine crystal structures of human and rat NTPDases 4-7. Co-crystal structures with substrates and inhibitors will define the substrate binding interactions and the molecular basis for the differences in substrate specificity (NDP vs. NTP and for different nucleobases). NTPDase1 hydrolyses ATP to AMP without the release of ADP. It was previously shown that this processivity depends on the membrane anchoring of the enzyme. Via mutagenesis studies we will test the hypothesis that a long hydrophobic conserved loop in the extracellular NTPDases is involved in membrane anchoring and mediates the coupling between the transmembrane part and the ectodomain. In addition, we will determine crystal structures of the yet uncharacterized ecto-NTPDases 3 and 8. Another project part concerns the specificity and mechanism of NPPs. We were able to crystallize rat NPP3. We will determine the X-ray structure of the enzyme and analyze co-crystal structures with various nucleotide substrates and products to understand the differences in substrate specificity between various NPPs involved in purinergic signaling and calcification. In collaboration we will aid the development of specific inhibitors of these enzymes as biological and pharmaceutical tools.