THE ROLE OF TMEM163 IN ZINC HOMEOSTASIS

项目总结 在包括人类在内的许多生物中，锌的动态平衡是通过组织特异性的和高度的 保守的低分子量蛋白质，称为锌转运蛋白(ZNT)和类Zrt和IRT蛋白 (Zip)家庭。在提供锌的组织中发现了几个多余的ZNT和ZIP转运蛋白 流出属性和流入属性。细胞内锌水平的严格调控存在于许多组织中， 尤其是在大脑中，因为这个组织含有相当数量的可螯合锌库，这种锌是... 在正常的神经元通讯过程中与谷氨酸一起释放。事实上，znt3锌喷射器已经 表明锌在隔离和运送锌进入谷氨酸能小泡中很重要，并敲除了 这种蛋白质消除了大脑中锌泡的区隔。我们确定了跨膜163 (TMEM163)是一种锌结合蛋白和转运蛋白，作为TRPML1离子通道的蛋白质相互作用因子。 TRPML1功能丧失会导致粘脂病IV。与此同时，TMEM163最近被报道 通过与神经元型P2X3受体离子通道的相互作用来调节痛觉。TMEM163 又称突触小泡31(Synaptic Vesicle31，SV31)蛋白，最早在大鼠脑突触体内发现并存在 作为一个调光体。TMEM163定位于质膜和囊泡室内，如 突触小泡和溶酶体。我们有初步证据表明TMEM163与ZnT3相互作用 锌外流转运体。功能锌通量分析表明，TMEM163-ZnT3的外排活性 异二聚体与它们各自的同源二聚体的异构体相似。这些结果不仅证实了 TMEM163是一种锌转运体，但它与相关的锌转运蛋白的异二聚化增加了 细胞内锌水平的动态平衡控制系列。因此，TMEM163似乎很重要 用于维持独立于或与另一种锌结合的大脑锌稳态 外排转运体。这个项目的首要目标是调查人类的生物学意义。 TMEM163使用其对应的鼠标TMem163。为此，我们将使用Tem163淘汰赛(KO) 用组织化学、组织化学和组织化学方法测定小鼠脑内可螯合锌分布模式的变化。 荧光和生化技术。我们还将通过以下方式检查Tem163 KO的表型 使用RNA测序分析大脑转录组以确定是否存在遗传补偿(a 众所周知的生物学现象)由其他锌转运蛋白基因引起的 功能。总体而言，该项目可以填补目前关于TMEM163生物学功能的知识空白， 可以为缺乏TMEM163的细胞或组织如何影响人类健康或人类提供深入的见解 涉及锌代谢紊乱的疾病过程。

PROJECT SUMMARY Zinc homeostasis in many organisms, including humans, is achieved by tissue-specific and highly conserved low molecular weight proteins known as the zinc transporter (ZnT) and Zrt- and Irt-like protein (ZIP) families. Several redundant ZnT and ZIP transporters have been identified in tissues that confer zinc efflux and influx property, respectively. A strict regulation of intracellular zinc levels exists in many tissues, especially in the brain, because this tissue contains a sizeable amount of chelatable zinc pool that is co- released with glutamate during normal neuronal communication. Indeed, the ZnT3 zinc effluxer has been shown to be important in sequestering and shuttling zinc into glutamatergic vesicles and that knocking out this protein obliterates vesicular zinc compartmentalization in the brain. We identified Transmembrane 163 (TMEM163), a zinc-binding protein and transporter, as a protein interactor of the TRPML1 ion channel. Loss of TRPML1 function causes Mucolipidosis IV disease. Meanwhile, TMEM163 was recently reported to modulate pain perception via its interaction with the neuronal P2X3 receptor ion channel. TMEM163 also known as synaptic vesicle 31 (SV31) protein was first identified in rat brain synaptosomes and exists as a dimer. TMEM163 localizes within the plasma membrane and vesicular compartments such as synaptic vesicles and lysosomes. We have preliminary evidence that TMEM163 interacts with the ZnT3 zinc efflux transporters. Functional zinc flux assays show that the efflux activity of TMEM163-ZnT3 heterodimers parallels that of their respective homodimer isoforms. These results not only confirm that TMEM163 is a zinc effluxer, but that its heterodimerization with a related zinc transporter adds to a repertoire of homeostatic control for intracellular zinc levels. Thus, it appears that TMEM163 is important for the maintenance of brain zinc homeostasis that is independent of, or in conjunction with, another zinc efflux transporter. The overarching goal of this project is to investigate the biological significance of human TMEM163 using its mouse Tmem163 counterpart. To this end, we will use a Tmem163 knockout (KO) mouse to determine changes in chelatable zinc distribution patterns in the brain using histochemical, histo- fluorescence, and biochemical techniques. We will also examine the Tmem163 KO phenotype by analyzing the brain transcriptome using RNA sequencing to establish whether genetic compensation (a well-known biological phenomenon) by other zinc transporter genes results from the loss of Tmem163 function. Overall, this project could fill current gaps in knowledge on the biological function of TMEM163, and could provide insights on how cells or tissues devoid of TMEM163 impact human health or human disease processes where zinc dyshomeostasis has been implicated.