Dissecting the role of TMEM163 protein in cells

IV型粘脂沉积症(MLIV)是一种溶酶体储存障碍，主要影响大脑、眼睛和胃。这种疾病是由TRPML1离子通道功能突变丧失引起的。我们发现MLIV患者成纤维细胞和MLIV小鼠模型的脑组织中锌水平异常高。事实上，当MLIV成纤维细胞急性暴露于外源性锌时，溶酶体中可以看到离子的显著积累。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，在正常的神经传递或病理事件中与谷氨酸共同释放。我们确定跨膜蛋白(TMEM)-163是一种锌结合蛋白，也是一种可能的转运蛋白，是TRPML1的新的相互作用伙伴。TMEM163的异源表达表明它定位于质膜(PM)，并与溶酶体内的TRPML1部分共定位。初步数据显示，TMEM163过表达细胞暴露于外源性锌后，可导致细胞内锌超载的标志物--荧光蛋白3和金属硫蛋白-1A的表达显著增加。此外，我们的异源共表达TMEM163和锌转运蛋白4(ZnT4)，而不是其他被测试的ZNT蛋白，显示了Fluozin-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蛋白在与野生型TRPML1共表达时PM水平趋于稳定，而缺失突变体在没有TRPML1共表达的情况下错位定位，这表明TRPML1影响TMEM163的运输。因此，我们假设TMEM163是一种转运蛋白，它独立于其他可能导致MLIV疾病的锌转运蛋白，或与其他可能导致MLIV疾病的锌转运蛋白一起介导细胞内锌的稳态。该方案的第一个目标是通过使用异源表达的细胞和从TMEM163-mCherry过度表达(OE)细胞的细胞膜上提取的脂质体来确定TMEM163的锌转运蛋白的功能，确定它是内流还是外流，以及ATP或pH依赖的。第二个目的是确定TMEM163中负责锌转运的特定氨基酸，并确定TMEM163和ZnT4蛋白之间的协同功能是否源于它们的物理相互作用。最后，第三个目标将确定TMEM163在细胞内的贩运是由TRPML1-OE上的TRPML1介导的还是由敲除的TRPML1介导的，以及在细胞暴露于各种贩运过程的药物抑制剂时。最终，从这一提案中收集的数据将填补我们目前关于TMEM163在MLIV病原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聚的机制过程。

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.