Daniels Mark J, Jagielnicki Maciej, Yeager Mark
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA, 22908, USA.
Department of Biochemistry, University of Toronto, Toronto, ON, M5G 1M1, Canada.
Curr Res Struct Biol. 2020 Jun 27;2:144-155. doi: 10.1016/j.crstbi.2020.06.001. eCollection 2020.
The human zinc transporter ZnT8 (SLC30A8) is expressed primarily in pancreatic β-cells and plays a key function in maintaining the concentration of blood glucose through its role in insulin storage, maturation and secretion. ZnT8 is an autoantigen for Type 1 diabetes (T1D) and is associated with Type 2 diabetes (T2D) through its risk allele that encodes a major non-synonymous single nucleotide polymorphism (SNP) at Arg325. Loss of function mutations improve insulin secretion and are protective against diabetes. Despite its role in diabetes and concomitant potential as a drug target, little is known about the structure or mechanism of ZnT8. To this end, we expressed ZnT8 in yeast and 9 insect cells. Guided by a rational screen of 96 detergents, we developed a method to solubilize and purify recombinant ZnT8. An transport assay in and a liposome-based uptake assay for insect-cell derived ZnT8 showed that the protein is functionally active in both systems. No significant difference in activity was observed between full-length ZnT8 (ZnT8A) and the amino-terminally truncated ZnT8B isoform. A fluorescence-based transport assay using proteoliposomes indicated that human ZnT8 functions as a Zn/H antiporter. We also purified -expressed amino- and carboxy-terminal cytoplasmic domains of ZnT8A. Circular dichroism spectrometry suggested that the amino-terminal domain contains predominantly α-helical structure, and indicated that the carboxy-terminal domain has a mixed α/β structure. Negative-stain electron microscopy and single-particle image analysis yielded a density map of ZnT8B at 20 Å resolution, which revealed that ZnT8 forms a dimer in detergent micelles. Two prominent lobes are ascribed to the transmembrane domains, and the molecular envelope recapitulates that of the bacterial zinc transporter YiiP. These results provide a foundation for higher resolution structural studies and screening experiments to identify compounds that modulate ZnT8 activity.
人类锌转运蛋白ZnT8（SLC30A8）主要在胰腺β细胞中表达，通过其在胰岛素储存、成熟和分泌中的作用，在维持血糖浓度方面发挥关键功能。ZnT8是1型糖尿病（T1D）的自身抗原，并且通过其风险等位基因与2型糖尿病（T2D）相关，该风险等位基因在第325位精氨酸处编码一个主要的非同义单核苷酸多态性（SNP）。功能丧失突变可改善胰岛素分泌并预防糖尿病。尽管ZnT8在糖尿病中起作用并具有作为药物靶点的潜在可能性，但对其结构或机制了解甚少。为此，我们在酵母和9种昆虫细胞中表达了ZnT8。在对96种去污剂进行合理筛选的指导下，我们开发了一种溶解和纯化重组ZnT8的方法。在大肠杆菌中的转运测定以及针对昆虫细胞来源的ZnT8的基于脂质体的摄取测定表明，该蛋白在两个系统中均具有功能活性。在全长ZnT8（ZnT8A）和氨基末端截短的ZnT8B异构体之间未观察到活性的显著差异。使用蛋白脂质体的基于荧光的转运测定表明，人类ZnT8作为锌/氢反向转运体发挥作用。我们还纯化了在大肠杆菌中表达的ZnT8A的氨基末端和羧基末端胞质结构域。圆二色光谱表明，氨基末端结构域主要包含α螺旋结构，并表明羧基末端结构域具有混合的α/β结构。负染电子显微镜和单颗粒图像分析产生了分辨率为20 Å的ZnT8B密度图，该图显示ZnT8在去污剂胶束中形成二聚体。两个突出的叶归因于跨膜结构域，并且分子包络概括了细菌锌转运蛋白YiiP的分子包络。这些结果为更高分辨率的结构研究和筛选实验提供了基础，以鉴定调节ZnT8活性的化合物。
