Molecular Basis of Transient Neonatal Zinc Deficiency: NOVEL ZnT2 MUTATIONS DISRUPTING ZINC BINDING AND PERMEATION
Molecular Basis of Transient Neonatal Zinc Deficiency: NOVEL ZnT2 MUTATIONS DISRUPTING ZINC BINDING AND PERMEATION
复制标题
DOI：
10.1074/jbc.m116.732693
复制
发表时间：
2016-06-24
影响因子：
4.8
通讯作者：
Assaraf, Yehuda G.
中科院分区：
文献类型：
--
作者：
Golan, Yarden;Itsumura, Naoya;Assaraf, Yehuda G.
近年来，新生儿短暂性锌缺乏症（TNZD）的病例逐渐增多，所有这些病例均与ZnT 2失活突变有关。在这里，我们描述了三种新的杂合ZnT 2突变G280 R，T312 M和E355 Q的影响，这些突变导致日本母亲纯母乳喂养的婴儿TNZD。我们使用的双分子荧光互补（BiFC）测定提供直接的视觉证据，这些ZnT 2突变体的原位二聚化，并探讨其亚细胞定位。此外，使用三种互补的功能测定，使用BiFC-Zinquin和Zinpyr-1荧光的锌积累以及锌毒性测定，我们确定了这些ZnT 2突变对囊泡锌积累的影响。虽然所有三个突变体形成同源二聚体与野生型（WT）ZnT 2和保留大量的囊泡定位，以及囊泡锌积累，他们没有显性负效应超过WT ZnT 2。此外，使用先进的生物信息学，结构建模和定点突变，我们发现这些突变定位在关键残基，这在锌配位（G280 R和E355 Q）和锌渗透（T312 M）中发挥重要的生理作用。总的来说，我们的研究结果表明，一些杂合性功能丧失ZnT 2突变破坏锌结合和锌渗透，从而表明TNZD发病机制中未受影响的WT ZnT 2等位基因的单倍不足状态。这些结果突出了迫切需要开发一种合适的遗传筛查，用于TNZD的早期诊断，以防止发病。
A gradually increasing number of transient neonatal zinc deficiency (TNZD) cases was recently reported, all of which were associated with inactivating ZnT2 mutations. Here we characterized the impact of three novel heterozygous ZnT2 mutations G280R, T312M, and E355Q, which cause TNZD in exclusively breastfed infants of Japanese mothers. We used the bimolecular fluorescence complementation (BiFC) assay to provide direct visual evidence for the in situ dimerization of these ZnT2 mutants, and to explore their subcellular localization. Moreover, using three complementary functional assays, zinc accumulation using BiFC-Zinquin and Zinpyr-1 fluorescence as well as zinc toxicity assay, we determined the impact of these ZnT2 mutations on vesicular zinc accumulation. Although all three mutants formed homodimers with the wild type (WT) ZnT2 and retained substantial vesicular localization, as well as vesicular zinc accumulation, they had no dominant-negative effect over the WT ZnT2. Furthermore, using advanced bioinformatics, structural modeling, and site-directed mutagenesis we found that these mutations localized at key residues, which play an important physiological role in zinc coordination (G280R and E355Q) and zinc permeation (T312M). Collectively, our findings establish that some heterozygous loss of function ZnT2 mutations disrupt zinc binding and zinc permeation, thereby suggesting a haploinsufficiency state for the unaffected WT ZnT2 allele in TNZD pathogenesis. These results highlight the burning need for the development of a suitable genetic screen for the early diagnosis of TNZD to prevent morbidity.