Lysosome remodeling mediates high zinc homeostasis

溶酶体重塑介导高锌稳态

• 批准号: 10531021
• 负责人: Kerry Kornfeld
• 金额: $ 36.95万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-06-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10531021
• 关键词: Address; Affect; Animals; Binding; Biogenesis; Biological; Biological Models; Biology; Caenorhabditis elegans; Cell Culture Techniques; Cell Nucleus; Cells; Contracts; Data; Dietary Zinc; Disease; Drug Metabolic Detoxication; Family; Fluorescence Microscopy; Genes; Genetic; Genetic Transcription; Goals; Health; Homeostasis; Human; Intestines; Kinetics; Laboratories; Lead; Lysosomal Storage Diseases; Lysosomes; Mammalian Cell; Mammals; Mediating; Medical Research; Membrane; Messenger RNA; Metabolism; Methods; Microscopy; Modeling; Molecular; Monitor; Nature; Nutrient; Organelles; Pathway interactions; Pattern; Play; Population; Process; Proteins; Proteome; Public Health; Reagent; Regulation; Research; Resistance; Resolution; Roentgen Rays; Role; Site; Structure; Surface; Techniques; Testing; Time; ZIP protein; Zinc; Zinc deficiency; base; cofactor; experimental study; flexibility; forward genetics; innovation; insight; lysosomal proteins; lysosome membrane; macromolecule; novel; novel therapeutic intervention; response; transcription factor; zinc-binding protein

Abstract Zinc is an essential nutrient that profoundly affects human health, since ~10% of the proteome binds zinc. A key to zinc homeostasis is storage during times of excess and release during periods of deficiency. Lysosomes, which have a well-established role in the degradation of macromolecules, are emerging as a conserved site of zinc storage. How lysosomes integrate the dual functions of zinc storage and degradation is not well defined. Our data indicate that lysosomes mediate these dual functions in separate compartments. We used C. elegans to demonstrate excess zinc is stored in lysosomes of intestinal cells; CDF-2 (ZnT2 in mammals) is the SLC30 family transporter that loads zinc into lysosomes, and ZIPT-2.3 is the SLC39 family transporter that releases zinc. Reciprocal regulation of CDF-2 and ZIPT-2.3 regulates the direction of zinc flow; in excess zinc, CDF-2 is upregulated to increase storage and ZIPT-2.3 is downregulated to decrease release. How do lysosomes rapidly change the composition of transporters on their surface? Using super-resolution microscopy, we discovered that lysosomes have an expansion compartment connected to the acidified central compartment. The expansion compartment is contracted in zinc-replete conditions, but grows dramatically in response to high zinc. Our overall hypothesis is that the expansion compartment allows the rapid delivery of CDF-2 to lysosomes to promote zinc homeostasis –without disturbing degradative processes in the acidified compartment. This hypothesis is innovative, since we only observed the expansion compartment with the recent availability of super-resolution microscopy. To determine if this mechanism is conserved, we examined lysosome dynamics in mammalian cells; the ZnT2 zinc transporter alters its localization on lysosomes in response to high zinc, consistent with lysosome remodeling. To test the predictions of our model, we will characterize the molecular nature of the compartment, identify transcriptional changes during assembly and disassembly, and validate our findings in mammalian cells. Specific Aim 1, First, we will use TEM to determine whether lysosomes have a second membrane-bound compartment that contains CDF-2. Second, we will use X-ray fluorescence microscopy to test whether zinc is sequestered in this structure. Specific Aim 2, we will extend these findings to mammalian cells. We will test whether the ZnT2 transporter is required to store zinc, identify the ZIP protein that releases zinc, and test whether these transporters are reciprocally regulated to remodel lysosomes. Specific Aim 3, we will analyze the regulation of lysosome remodeling. We demonstrated that the lysosome biogenesis regulator HLH-30 (TFEB in mammals) is required for remodeling. We will determine how the transcriptional response to high zinc mediates lysosome remodeling. These studies will have a major impact by defining a new aspect of lysosome biology that is critical for zinc homeostasis.

抽象的 锌是一种重要的营养素，对人类健康产生深远影响，因为约有10％的蛋白质组结合锌。 锌稳态的关键是在不足期间过度和释放的时间存储。 溶酶体在大分子降解中具有良好的作用，正在作为一个 锌存储的保守地点。溶酶体如何整合锌存储和降解的双重功能是 定义不当。我们的数据表明，溶酶体在单独的隔室中介导了这些双重功能。 我们使用秀丽隐杆线虫来证明超过锌，存储在肠细胞的溶酶体中。 CDF-2（Znt2 in 哺乳动物）是将锌加载到溶酶体的SLC30家族转运蛋白，ZIPT-2.3是SLC39家族 释放锌的转运蛋白。 CDF-2和ZIPT-2.3的相互调节调节锌流的方向； 在多余的锌中，CDF-2已更新以增加存储空间，并且ZIPT-2.3被下调以减少释放。 溶酶体如何迅速改变其表面转运蛋白的组成？使用超分辨率 显微镜，我们发现溶酶体具有连接到酸化中央的膨胀室 车厢。扩展室在锌恢复条件下收缩，但在 对高锌的反应。我们的总体假设是扩展室允许快速交付 CDF-2至溶酶体以促进锌稳态 - 而不会干扰酸化的降解过程 车厢。该假设具有创新性，因为我们仅观察到与 超分辨率显微镜的最新可用性。为了确定这种机制是否保存，我们检查了 哺乳动物细胞中的溶酶体动力学； Znt2锌转运蛋白改变了其在溶酶体上的定位 对高锌的反应，与溶酶体重塑一致。为了测试我们的模型的预测，我们将 表征隔室的分子性质，确定组装过程中的转录变化 拆卸并验证我们在哺乳动物细胞中的发现。 特定目标1，首先，我们将使用TEM确定溶酶体是否具有第二个膜结合 包含CDF-2的车厢。其次，我们将使用X射线荧光显微镜测试锌是否为 在这种结构中隔离。 具体目标2，我们将将这些发现扩展到哺乳动物细胞。我们将测试ZNT2运输蛋白是 需要储存锌，确定释放锌的拉链蛋白，并测试这些转运蛋白是否是 相互调节对重塑溶酶体。 具体目标3，我们将分析溶酶体重塑的调节。我们证明了溶酶体 重塑需要生物发生调节剂HLH-30（哺乳动物中的TFEB）。我们将确定如何 对高锌的转录反应介导溶酶体重塑。这些研究将对 定义溶酶体生物学的新方面，这对于锌稳态至关重要。