Dysfunction of cGMP signalling in photoreceptors by a macular dystrophy-related mutation in the calcium sensor GCAP1
Dysfunction of cGMP signalling in photoreceptors by a macular dystrophy-related mutation in the calcium sensor GCAP1
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DOI：
10.1093/hmg/ddw374
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发表时间：
2016-12
影响因子：
3.5
通讯作者：
Farina Vocke;N. Weisschuh;V. Marino;Silvia Malfatti;S. Jacobson;Charlotte M. Reiff;D. Dell’Orco;K. Koch
中科院分区：
文献类型：
--
作者：
Farina Vocke;N. Weisschuh;V. Marino;Silvia Malfatti;S. Jacobson;Charlotte M. Reiff;D. Dell’Orco;K. Koch
黄斑营养不良导致中心视力的进行性丧失，并表现出与年龄相关性黄斑变性相似的症状。诊断患有黄斑营养不良的患者的遗传筛查揭示了GUCA 1A基因中的新突变，即c.526C > T取代导致鸟苷酸环化酶激活蛋白1（GCAP 1）中的氨基酸取代p.L176F。在三个常染色体显性遗传的家系中发现了相同的变异。对于L176 F突变体的完整功能表征，我们表达并纯化了突变体蛋白，并测量了其活化特性、其Ca 2 +/Mg 2+结合以及其与野生型蛋白相比的Ca 2+诱导的构象变化的关键参数。该突变体是不太敏感的变化，游离钙，导致在生理钙浓度下的组成型活性形式，显示出显着较高的激活率比野生型（90倍与20倍），并与其目标鸟苷酸环化酶的更高的表观亲和力相互作用。然而，突变体的直接Ca 2+结合几乎与野生型相似; Mg 2+的结合以更高的亲和力发生。我们进行了分子动力学模拟比较的Ca 2 +-饱和抑制状态的GCAP 1与Mg 2+结合的激活状态。L176 F突变体表现出显着较低的灵活性，当三个Ca 2+或两个Mg 2+结合形成可能的结构基础的修改GCAP 1功能。
Macular dystrophy leads to progressive loss of central vision and shows symptoms similar to age-related macular degeneration. Genetic screening of patients diagnosed with macular dystrophy disclosed a novel mutation in the GUCA1A gene, namely a c.526C > T substitution leading to the amino acid substitution p.L176F in the guanylate cyclase-activating protein 1 (GCAP1). The same variant was found in three families showing an autosomal dominant mode of inheritance. For a full functional characterization of the L176F mutant we expressed and purified the mutant protein and measured key parameters of its activating properties, its Ca2+/Mg2+-binding, and its Ca2+-induced conformational changes in comparison to the wildtype protein. The mutant was less sensitive to changes in free Ca2+, resulting in a constitutively active form under physiological Ca2+-concentration, showed significantly higher activation rates than the wildtype (90-fold versus 20-fold) and interacted with an higher apparent affinity with its target guanylate cyclase. However, direct Ca2+-binding of the mutant was nearly similar to the wildtype; binding of Mg2+ occurred with higher affinity. We performed molecular dynamics simulations for comparing the Ca2+-saturated inhibiting state of GCAP1 with the Mg2+-bound activating states. The L176F mutant exhibited significantly lower flexibility, when three Ca2+ or two Mg2+ were bound forming probably the structural basis for the modified GCAP1 function.