Dissecting the role of TMEM163 protein in cells

剖析TMEM163蛋白在细胞中的作用

基本信息

批准号：
9305662
负责人：
MATH P CUAJUNGCO
金额：
$ 38.21万
依托单位：
CALIFORNIA STATE UNIVERSITY FULLERTON
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-02-01 至 2020-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9305662
关键词：
Achlorhydria Acute Affect Affinity Chromatography Agonist Alanine Amino Acids Anemia Aspartate Autopsy Binding Binding Proteins Biochemical Biological Assay Biotinylation Brain Brefeldin A Calcium Carrier Proteins Cataract Cell Membrane Permeability Cell membrane Cell surface Cells Chelating Agents Child Chloroquine Clinical Computer Simulation Confocal Microscopy Cultured Cells Cycloheximide Cysteine Data Disease Disease Progression Dyes Etiology Event Exposure to Eye Fibroblasts FluoZin-3 Fluorescence Fluorescence Spectrometry Fluorescent Dyes Future Ganglioside Sialidase Deficiency Disease Gene Expression Glutamates Goals Hereditary Disease Histidine Homeostasis Human Human Biology Image Immobilization Ion Channel Ions Iron Knock-out Knockout Mice Knowledge Liposomes Lysosomes Manganese Mediating Membrane Proteins Metabolism Metallothionein Metals Mutation Nerve Degeneration Neurodegenerative Disorders Pathologic Pathology Patients Permeability Pharmaceutical Preparations Physiological Process Production Proteins Proteomics Rattus Regulation Reporting Retinal Degeneration Role Serum Site-Directed Mutagenesis Stomach Symptoms Techniques Testing Time Trace metal Transmembrane Domain ZIP protein Zinc brain tissue experimental study human disease inhibitor/antagonist knock-down late endosome loss of function mutation mouse model mutant neurotransmission novel overexpression prevent protein degradation protein transport receptor targeted treatment trafficking zinc-binding protein

项目摘要

Mucolipidosis type IV (MLIV) is a lysosomal storage disorder that mainly affects the brain, eyes, and stomach. The disease is caused by a loss of function mutation in the TRPML1 ion channel. We discovered abnormally high zinc levels in MLIV patient fibroblasts and brain tissues of the MLIV mouse model. Indeed, when MLIV fibroblasts are acutely exposed to exogenous zinc, marked accumulation of the ion is seen in lysosomes. These findings impact our understanding of MLIV pathology, because the brain contains a chelatable pool of zinc that is co-released with glutamate during normal neurotransmission, or pathological events. We identified transmembrane (TMEM)-163, a zinc-binding protein and putative transporter, as a novel interaction partner for TRPML1. Heterologous expression of TMEM163 shows that it localizes in the plasma membrane (PM), and that it partially co-localizes with TRPML1 in the lysosomes. Preliminary data show that TMEM163-overexpressing cells exposed to exogenous zinc result in significant increase of Fluozin-3 fluorescence and Metallothionein-1A expression, a marker of intracellular zinc overload. Also, our heterologous co-expression of TMEM163 and zinc transporter-4 (ZnT4), but not other ZnT proteins tested, revealed a synergistic increase of Fluozin-3 fluorescence. Meanwhile, cell surface biotinylation studies showed that the PM levels of wild-type (WT) and deletion mutant TMEM163 proteins become stabilized when co-expressed with WT TRPML1, while deletion mutants mis-localize without TRPML1 co-expression, implying that TRPML1 influences the trafficking of TMEM163. Thus, we hypothesize that TMEM163 is a transporter that mediates intracellular zinc homeostasis independent of, or in conjunction with other zinc transporters that could possibly contribute to MLIV disease. The first goal of this proposal is to investigate the putative zinc transporter function of TMEM163 by determining whether it is influx or efflux, and ATP- or pH-dependent using heterologously expressed cells and liposomes taken from cell membranes of TMEM163-mCherry overexpressing (OE) cells. The second aim is to identity specific amino acids within TMEM163 that are responsible for zinc transport, and determine if the synergistic function between TMEM163 and ZnT4 proteins is due to their physical interaction. Finally, the third goal will determine if TMEM163 trafficking in cells is mediated by TRPML1 upon TRPML1-OE or knock out, and upon exposure of cells with various drug inhibitors of trafficking processes. Ultimately, the data that will be gathered from this proposal would fill current gaps in our knowledge on the physiological or potentially detrimental role of TMEM163 with respect to MLIV etiology, and the mechanistic processes responsible for intracellular or lysosomal zinc accumulation leading to MLIV pathology.

IV型（MLIV）是一种溶酶体储存障碍，主要影响大脑，眼睛和胃。该疾病是由TRPML1离子通道中功能突变丧失引起的。我们在MLIV小鼠模型的MLIV患者成纤维细胞和脑组织中发现异常高的锌水平。实际上，当MLIV成纤维细胞急性暴露于外源锌时，在溶酶体中看到了离子的明显积累。这些发现会影响我们对MLIV病理学的理解，因为大脑包含一个可螯合的锌池，在正常神经传递或病理事件中，该锌与谷氨酸共同发行。我们将锌结合蛋白和推定转运蛋白的跨膜（TMEM）-163确定为TRPML1的新型相互作用伴侣。 TMEM163的异源表达表明，它位于质膜（PM）中，并且它与溶酶体中的TRPML1部分共定位。初步数据表明，暴露于外源锌的TMEM163过表达的细胞导致氟辛-3荧光和金属硫蛋白-1A表达的显着增加，这是细胞内锌过载的标志物。同样，我们对TMEM163和锌转运蛋白4（ZNT4）的异源共表达，但未测试的其他ZNT蛋白显示出荧光素3荧光的协同增加。同时，细胞表面生物素化研究表明，当与WT TRPML1共表达时，野生型（WT）和缺失突变体TMEM163蛋白的PM水平稳定，而缺失突变体失去了trpml1 co-co-Excression，则暗示TRPML1影响TRPML1会影响TMEM163的TRPML1。因此，我们假设TMEM163是一种转运蛋白，它介导了与可能有助于MLIV疾病的其他锌转运蛋白无关或与其他锌转运蛋白共同介导。该提案的第一个目标是通过确定它是涌入还是外排，研究TMEM163的假定锌转运蛋白功能，以及使用从TMEM163-MCHERRY过表达（OE）细胞的细胞膜中采集的异源表达细胞和脂质体的ATP或pH依赖性。第二个目的是识别负责锌转运的TMEM163中的特定氨基酸，并确定TMEM163和Znt4蛋白之间的协同功能是否是由于它们的物理相互作用引起的。最后，第三个目标将确定TMEM163在TRPML1-OE时TRPML1介导的TMEM163运输是在TRPML1-OE时介导的还是敲除，并在使用各种运输过程的各种药物抑制剂接触细胞后。最终，从该提案中收集的数据将填补我们关于TMEM163在MLIV病因方面的生理或潜在有害作用的知识中的当前空白，以及导致MLIV病理学导致MLIV病理学的细胞内或溶酶体锌积累的机械过程。