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:
  • 金额:
    $ 38.21万
  • 依托单位:
  • 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

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.
粘脂沉积症IV型(MLIV)是一种溶酶体储存疾病,主要影响大脑,眼睛和胃。这种疾病是由TRPML 1离子通道功能缺失突变引起的。我们在MLIV小鼠模型的MLIV患者成纤维细胞和脑组织中发现了异常高的锌水平。事实上,当MLIV成纤维细胞急性暴露于外源性锌时,在溶酶体中观察到离子的显著积累。这些发现影响了我们对MLIV病理学的理解,因为大脑含有一个可螯合的锌池,在正常神经传递或病理事件期间与谷氨酸共同释放。我们确定了跨膜(TMEM)-163,锌结合蛋白和推定的转运蛋白,作为TRPML 1的一种新的相互作用伙伴。TMEM 163的异源表达显示其定位于质膜(PM)中,并且其与TRPML 1部分共定位于溶酶体中。初步数据显示,TMEM 163过表达细胞暴露于外源性锌导致Fluozin-3荧光和金属硫蛋白-1A表达显著增加,金属硫蛋白-1A是细胞内锌过载的标志物。此外,我们的TMEM 163和锌转运蛋白-4(ZnT 4)的异源共表达,而不是测试的其他ZnT蛋白,揭示了Fluozin-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 TRPML 1共表达时,野生型(WT)和缺失突变体TMEM 163蛋白的PM水平变得稳定,而缺失突变体在没有TRPML 1共表达的情况下错误定位,这意味着TRPML 1影响TMEM 163的运输。因此,我们假设TMEM 163是一种转运蛋白,其介导细胞内锌稳态,独立于或与可能导致MLIV疾病的其他锌转运蛋白结合。本提案的第一个目标是通过使用从TMEM 163-mCherry过表达(OE)细胞的细胞膜获取的异源表达细胞和脂质体确定TMEM 163是否为流入或流出以及ATP或pH依赖性来研究TMEM 163的推定锌转运蛋白功能。第二个目的是鉴定TMEM 163中负责锌转运的特定氨基酸,并确定TMEM 163和ZnT 4蛋白之间的协同功能是否是由于它们的物理相互作用。最后,第三个目标将确定在TRPML 1-OE或敲除后,以及在细胞暴露于运输过程的各种药物抑制剂后,TMEM 163在细胞中的运输是否由TRPML 1介导。最终,将从该提案中收集的数据将填补我们对TMEM 163在MLIV病因学方面的生理或潜在有害作用以及负责导致MLIV病理的细胞内或溶酶体锌积累的机制过程的知识中的现有空白。

项目成果

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MATH P CUAJUNGCO其他文献

MATH P CUAJUNGCO的其他文献

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{{ truncateString('MATH P CUAJUNGCO', 18)}}的其他基金

U-RISE at Cal State Fullerton
加州州立大学富勒顿分校的 U-RISE
  • 批准号:
    10629662
  • 财政年份:
    2023
  • 资助金额:
    $ 38.21万
  • 项目类别:
THE ROLE OF TMEM163 IN ZINC HOMEOSTASIS
TMEM163 在锌稳态中的作用
  • 批准号:
    10287961
  • 财政年份:
    2021
  • 资助金额:
    $ 38.21万
  • 项目类别:
Analysis of the Transcriptional Regulation and Expression of TRPML2
TRPML2转录调控及表达分析
  • 批准号:
    8101779
  • 财政年份:
    2011
  • 资助金额:
    $ 38.21万
  • 项目类别:

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