Validating the GTP-cyclohydrolase 1-feedback regulatory complex as a therapeutic target using biophysical and in vivo approaches.
Validating the GTP-cyclohydrolase 1-feedback regulatory complex as a therapeutic target using biophysical and in vivo approaches.
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DOI：
10.1111/bph.13202
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发表时间：
2015-08
影响因子：
7.3
通讯作者：
Nandi M
中科院分区：
文献类型：
--
作者：
Hussein D;Starr A;Heikal L;McNeill E;Channon KM;Brown PR;Sutton BJ;McDonnell JM;Nandi M
6 R-L-胆-5，6，7，8-四氢生物蝶呤（6 R-L-tetrahydrobiopterin，BH 4）是一氧化氮生物合成的重要辅助因子。大量临床证据表明，静脉注射BH 4可恢复患者的血管功能。不幸的是，口服BH 4的功效有限。因此，内源性BH 4生物合成的口服生物可利用的药理学激活剂具有显著的治疗潜力。GTP-环化水解酶1（GCH 1）是BH 4合成的限速酶，与GCH 1反馈调节蛋白（GFRP）形成蛋白质复合物。该复合物受到L-苯丙氨酸（L-phe）的变构前馈激活。我们研究了L-phe对GCH 1和GFRP的生物物理相互作用及其改变体内BH 4水平的潜力的影响。GCH 1-GFRP蛋白质-蛋白质相互作用的详细表征使用表面等离子体共振（SPR）与或不与L-phe。研究了L-phe处理（100 mg·kg-1，p.o.）后对体内全身和血管BH 4生物合成的影响。GCH 1和GFRP蛋白相互作用的情况下，已知的配体或基板，但L-phe的存在下，最大的结合和增强的结合亲和力的8倍。此外，复合物显示出非常缓慢的缔合和解离速率。在体内，L-phe激发诱导了主动脉BH 4的持续升高，这在GCH 1（fl/fl）-Tie 2Cre小鼠中不存在。生物物理数据表明，GCH 1和GFRP是组成性结合的。在体内，数据表明，L-phe升高血管BH 4的内皮GCH 1依赖性的方式。模拟L-phe对GCH 1-GFRP复合物的变构作用的药理学药物有可能提高许多心血管疾病的内皮BH 4生物合成。
6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide biosynthesis. Substantial clinical evidence indicates that intravenous BH4 restores vascular function in patients. Unfortunately, oral BH4 has limited efficacy. Therefore, orally bioavailable pharmacological activators of endogenous BH4 biosynthesis hold significant therapeutic potential. GTP-cyclohydrolase 1 (GCH1), the rate limiting enzyme in BH4 synthesis, forms a protein complex with GCH1 feedback regulatory protein (GFRP). This complex is subject to allosteric feed-forward activation by L-phenylalanine (L-phe). We investigated the effects of L-phe on the biophysical interactions of GCH1 and GFRP and its potential to alter BH4 levels in vivo. Detailed characterization of GCH1–GFRP protein–protein interactions were performed using surface plasmon resonance (SPR) with or without L-phe. Effects on systemic and vascular BH4 biosynthesis in vivo were investigated following L-phe treatment (100 mg·kg−1, p.o.). GCH1 and GFRP proteins interacted in the absence of known ligands or substrate but the presence of L-phe doubled maximal binding and enhanced binding affinity eightfold. Furthermore, the complex displayed very slow association and dissociation rates. In vivo, L-phe challenge induced a sustained elevation of aortic BH4, an effect absent in GCH1(fl/fl)-Tie2Cre mice. Biophysical data indicate that GCH1 and GFRP are constitutively bound. In vivo, data demonstrated that L-phe elevated vascular BH4 in an endothelial GCH1 dependent manner. Pharmacological agents which mimic the allosteric effects of L-phe on the GCH1–GFRP complex have the potential to elevate endothelial BH4 biosynthesis for numerous cardiovascular disorders.