Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi
Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi
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DOI：
10.1042/bj20060757
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发表时间：
2006-10-15
影响因子：
4.1
通讯作者：
Rascón, Ana
中科院分区：
文献类型：
--
作者：
Diaz-Benjumea, Rocio;Laxman, Sunil;Rascón, Ana
克氏锥虫（Trypanosoma cruzi）是南美锥虫病的病原体，编码许多不同的cAMP特异性PDE（磷酸二酯酶）家族。在这里，我们报告的TcrPDEB 1的鉴定和表征，并与以前确定的TcrPDEB 2（以前称为TcPDE1）的比较。这是TcrPDEB家族的两种不同的PDE酶，根据动质体PDE命名委员会的最近推荐命名[Kunz，Beavo，D'Angelo，Flawia，弗朗西斯，Johner，Laxman，Oberholzer，Rascon，Shakur等人（2006）Mol.生物化学。寄生醇。145，133 - 135]。这两种酶都显示出对许多哺乳动物PDE抑制剂抑制的抗性，并且那些确实抑制的酶对这两种酶的抑制剂谱具有明显的差异。这两种酶都含有两个GAF（c（G）在bar MP特异性和刺激的磷酸二酯酶下，（A）在bar nabaena腺苷酸环化酶下，和大肠杆菌（F）在bar hlA下）结构域和与布氏锥虫TbPDE2/TbrPDEB 2家族高度同源的催化结构域。两种酶的N-末端+GAF-A结构域在它们对环核苷酸结合的亲和力方面显示出显著差异。使用允许精确测量低亲和力结合位点的量热技术，发现TcrPDEB 2N-末端+GAF-A结构域以类似于500 nM的亲和力结合cAMP。TcrPDEB 1 N-末端+GAF-A结构域以略低的亲和力结合cAMP，接近1 μ M。TcrPDEB I的N-末端+GAF-A结构域不结合cGMP，而TcrPDEB 2的N-末端+GAF-A结构域以类似于3 μ M的低亲和力结合cGMP。在相关的锥虫寄生虫中也发现了与这些蛋白质中发现的那些同源的GAF结构域。最后，发现荧光cAMP类似物MANT-cAMP [2'-O-（N-甲基-邻氨基苯甲酰基）腺苷-3'，5'-环一磷酸]是TcPDEB 1催化结构域的底物，从而开启了在非放射性、基于荧光的PDE测定中使用该分子作为底物的可能性，包括筛选锥虫PDE抑制剂。
Trypanosoma cruzi, the causative agent of Chagas disease, encodes a number of different cAMP-specific PDE (phosphodiesterase) families. Here we report the identification and characterization of TcrPDEB1 and its comparison with the previously identified TcrPDEB2 (formerly known as TcPDE1). These are two different PDE enzymes of the TcrPDEB family, named in accordance with the recent recommendations of the Nomenclature Committee for Kinetoplast PDEs [Kunz, Beavo, D'Angelo, Flawia, Francis, Johner, Laxman, Oberholzer, Rascon, Shakur et al. (2006) Mol. Biochem. Parasitol. 145, 133-135]. Both enzymes show resistance to inhibition by many mammalian PDE inhibitors, and those that do inhibit do so with appreciable differences in their inhibitor profiles for the two enzymes. Both enzymes contain two GAF (c (G) under bar MP-specific and -stimulated phosphodiesterases, (A) under bar nabaena adenylate cyclases and Escherichia coli (F) under bar hlA) domains and a catalytic domain highly homologous with that of the T brucei TbPDE2/TbrPDEB2 family. The N-terminus + GAF-A domains of both enzymes showed significant differences in their affinities for cyclic nucleotide binding. Using a calorimetric technique that allows accurate measurements of low-affinity binding sites, the TcrPDEB2N-terminus + GAF-A domain was found to bind cAMP with an affinity of similar to 500 nM. The TcrPDEB1 N-terminus + GAF-A domain bound cAMP with a slightly lower affinity of similar to 1 mu M. The N-terminus + GAF-A domain of TcrPDEB I did not bind cGMP, whereas the N-terminus + GAF-A domain of TcrPDEB2 bound cGMP with a low affinity of similar to 3 mu M. GAF domains homologous with those found in these proteins were also identified in related trypanosomatid parasites. Finally, a fluorescent cAMP analogue, MANT-cAMP [2'-O-(N-methyl-anthraniloyl)adenosine-3',5'-cyclic monophosphate], was found to be a substrate for the TcPDEB1 catalytic domain, opening the possibility of using this molecule as a substrate in non-radioactive, fluorescence-based PDE assays, including screening for trypanosome PDE inhibitors.