Phenylketonuria:: Genotype-phenotype correlations based on expression analysis of structural and functional mutations in PAH
Phenylketonuria:: Genotype-phenotype correlations based on expression analysis of structural and functional mutations in PAH
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DOI：
10.1002/humu.10198
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发表时间：
2003-04-01
期刊：
影响因子：
3.9
通讯作者：
Pérez, B
中科院分区：
文献类型：
--
作者：
Pey, AL;Desviat, LR;Pérez, B
当在苯丙氨酸羟化酶（PAH）三维结构的背景下进行分析时，世界范围内描述的PKU突变中只有少数影响催化残基。与这些观察结果一致，最近的数据指出，折叠缺陷和随后的聚集/降解是几种突变的主要疾病机制。在这项工作中，我们使用了在真核细胞中不同温度下的表达和伴侣蛋白共表达的原核系统的组合方法来阐明和确认18个PKU突变的结构后果。3个突变位于氨基末端调节结构域，15个突变位于催化结构域。发现四个突变在所有条件下都破坏了特异性活性。两个是催化突变（Y277D和E280K），两个是严重的结构缺陷（IVS10-11G>A和L311P）。其余突变（D59Y、165T、E76G、P122Q、R158Q、G218V、R243Q、P244L、R252W、R261Q、A309V、R408Q、r408w和Y414C）均为折叠缺陷，导致稳定性降低和降解加速，尽管其中一些可能影响参与调控的残基。在这些情况下，我们已经证明PAH突变蛋白的数量和残留活性可以通过体外实验条件来调节，因此观察到的体内代谢变化可以通过质量控制系统的个体间差异来解释。所得结果提供了一个实验框架来定义基因型与表型相关的突变严重程度。他们还解释了在基因型相似而表型不同的患者中观察到的一些突变的不一致性。(C) 2003 Wiley-Liss, Inc。
When analyzed in the context of the phenylalanine hydroxylase (PAH) three-dimensional structure, only a minority of the PKU mutations described world-wide affect catalytic residues. Consistent with these observations, recent data point to defective folding and subsequent aggregation/degradation as a predominant disease mechanism for several mutations. In this work, we use a combined approach of expression in eukaryotic cells at different temperatures and a prokaryotic system with co-expression of chaperonins to elucidate and confirm structural consequences for 18 PKU mutations. Three mutations are located in the amino terminal regulatory domain and 15 in the catalytic domain. Four mutations were found to abolish the specific activity in all conditions. Two are catalytic mutations (Y277D and E280K) and two are severe structural defects (IVS10-11G>A and L311P). All the remaining mutations (D59Y, 165T, E76G, P122Q, R158Q, G218V, R243Q, P244L, R252W, R261Q, A309V, R408Q, R408W. and Y414C) are folding defects causing reduced stability and accelerated degradation, although some of them probably affect residues involved in regulation. In these cases, we have demonstrated that the amount of mutant PAH protein and residual activity could be modulated by in vitro experimental conditions, and therefore the observed in vivo metabolic variation may be explained by interindividual variation in the quality control systems. The results derived provide an experimental framework to define the mutation severity relating genotype to phenotype. They also explain the observed inconsistencies for some mutations in patients with similar genotype and different phenotypes. (C) 2003 Wiley-Liss, Inc.