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Molecular expression, characterization and mechanism of ALAS2 gain-of-function mutants
Molecular Medicine ( IF 5.7 ) Pub Date : 2019-01-24 , DOI: 10.1186/s10020-019-0070-9
Vassili Tchaikovskii , Robert J. Desnick , David F. Bishop

BackgroundX-linked protoporphyria (XLP) (MIM 300752) is an erythropoietic porphyria due to gain-of-function mutations in the last exon (Ducamp et al., Hum Mol Genet 22:1280-88, 2013) of the erythroid-specific aminolevulinate synthase gene (ALAS2). Five ALAS2 exon 11 variants identified by the NHBLI Exome sequencing project (p.R559H, p.E565D, p.R572C, p.S573F and p.Y586F) were expressed, purified and characterized in order to assess their possible contribution to XLP. To further characterize the XLP gain-of-function region, five novel ALAS2 truncation mutations (p.P561X, p.V562X, p.H563X, p.E569X and p.F575X) were also expressed and studied.MethodsSite-directed mutagenesis was used to generate ALAS2 mutant clones and all were prokaryotically expressed, purified to near homogeneity and characterized by protein and enzyme kinetic assays. Standard deviations were calculated for 3 or more assay replicates.ResultsThe five ALAS2 single nucleotide variants had from 1.3- to 1.9-fold increases in succinyl-CoA Vmax and 2- to 3-fold increases in thermostability suggesting that most could be gain-of-function modifiers of porphyria instead of causes. One SNP (p.R559H) had markedly low purification yield indicating enzyme instability as the likely cause for XLSA in an elderly patient with x-linked sideroblastic anemia. The five novel ALAS2 truncation mutations had increased Vmax values for both succinyl-CoA and glycine substrates (1.4 to 5.6-fold over wild-type), while the Kms for both substrates were only modestly changed. Of interest, the thermostabilities of the truncated ALAS2 mutants were significantly lower than wild-type, with an inverse relationship to Vmax fold-increase.ConclusionsPatients with porphyrias should always be assessed for the presence of the ALAS2 gain-of-function modifier variants identified here. A key region of the ALAS2 carboxyterminal region is identified by the truncation mutations studied here and the correlation of increased thermolability with activity suggests that increased molecular flexibility/active site openness is the mechanism of enhanced function of mutations in this region providing further insights into the role of the carboxyl-terminal region of ALAS2 in the regulation of erythroid heme synthesis.

中文翻译:

ALAS2 功能获得性突变体的分子表达、表征和机制

背景 X 连锁原卟啉症 (XLP) (MIM 300752) 是红细胞生成性卟啉症,这是由于红细胞特异性氨基乙酰丙酸盐的最后一个外显子的功能获得性突变 (Ducamp et al., Hum Mol Genet 22:1280-88, 2013)合酶基因 (ALAS2)。表达、纯化和表征由 NHBLI 外显子组测序项目(p.R559H、p.E565D、p.R572C、p.S573F 和 p.Y586F)鉴定的五个 ALAS2 外显子 11 变体,以评估它们对 XLP 的可能贡献。为了进一步表征 XLP 功能获得区,还表达和研究了五个新的 ALAS2 截断突变(p.P561X、p.V562X、p.H563X、p.E569X 和 p.F575X)。产生 ALAS2 突变体克隆,所有克隆都被原核表达,纯化至接近同质,并通过蛋白质和酶动力学测定进行表征。计算 3 次或更多次重复测定的标准偏差。 结果 5 个 ALAS2 单核苷酸变体的琥珀酰辅酶 A Vmax 增加了 1.3 到 1.9 倍,热稳定性增加了 2 到 3 倍,这表明大多数可能是卟啉症的功能修饰剂而不是原因。一个 SNP (p.R559H) 的纯化产率显着降低,表明酶不稳定性可能是 X 连锁铁粒幼细胞性贫血老年患者 XLSA 的原因。五个新的 ALAS2 截断突变使琥珀酰辅酶 A 和甘氨酸底物的 Vmax 值增加(比野生型高 1.4 至 5.6 倍),而两种底物的 Kms 仅略有变化。有趣的是,截断的 ALAS2 突变体的热稳定性显着低于野生型,与 Vmax 倍数增加呈负相关。结论应始终评估卟啉症患者是否存在此处确定的 ALAS2 功能获得修饰符变体。ALAS2 羧基末端区域的一个关键区域由这里研究的截断突变确定,热稳定性增加与活性的相关性表明分子灵活性/活性位点开放性增加是该区域突变功能增强的机制,提供了对作用的进一步见解ALAS2 羧基末端区域在调节红细胞血红素合成中的作用。
更新日期:2019-01-24
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