Dissecting the role of TMEM163 protein in cells

黏液脂质沉积症（MLIV）是一种溶酶体沉积紊乱，主要影响大脑、眼睛和胃。该疾病是由TRPML1离子通道的功能突变丧失引起的。我们在MLIV小鼠模型的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，溶酶体中可见明显的离子积累。这些发现影响了我们对MLIV病理学的理解，因为大脑中含有一个锌螯合池，在正常的神经传递或病理事件中与谷氨酸共同释放。我们发现跨膜(TMEM)-163，一种锌结合蛋白和推定的转运蛋白，作为TRPML1的一个新的相互作用伙伴。TMEM163的异源表达表明其定位于质膜（PM），并与TRPML1部分共定位于溶酶体。初步数据显示，暴露于外源锌的tmem163过表达细胞导致氟辛-3荧光和金属硫蛋白- 1a表达显著增加，这是细胞内锌超载的标志。此外，我们的异种共表达TMEM163和锌转运蛋白-4 (ZnT4)，而不是其他ZnT蛋白，显示了氟ozin-3荧光的协同增强。同时，细胞表面生物素化研究表明，野生型（WT）和缺失突变体TMEM163蛋白的PM水平在与WT TRPML1共表达时趋于稳定，而缺失突变体在没有TRPML1共表达时错定位，这表明TRPML1影响了TMEM163的运输。因此，我们假设TMEM163是一种介导细胞内锌稳态的转运蛋白，它独立于其他可能导致MLIV疾病的锌转运蛋白，或者与其他可能导致MLIV疾病的锌转运蛋白结合。本研究的第一个目标是利用从TMEM163- mcherry过表达（OE）细胞的细胞膜中提取的异种表达细胞和脂质体，通过确定TMEM163是内流还是外排，以及ATP依赖还是ph依赖，来研究TMEM163的锌转运蛋白功能。第二个目标是确定TMEM163中负责锌运输的特定氨基酸，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。最后，第三个目标将确定在TRPML1- oe或敲除时，以及在细胞暴露于各种药物抑制剂的运输过程时，细胞中的TMEM163运输是否由TRPML1介导。最终，从该提案中收集的数据将填补目前我们对TMEM163在MLIV病因学方面的生理或潜在有害作用的知识空白，以及导致MLIV病理的细胞内或溶酶体锌积累的机制过程。

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.