THE ROLE OF TMEM163 IN ZINC HOMEOSTASIS

项目摘要 锌稳态在许多生物体中，包括人类，是通过组织特异性和高度依赖性来实现的。 保守的低分子量蛋白质，称为锌转运蛋白（ZnT）和Zrt和Irt样蛋白质 (ZIP)家庭已经在组织中鉴定了几种冗余的ZnT和ZIP转运蛋白， 外排和内排特性。细胞内锌水平的严格调节存在于许多组织中， 特别是在大脑中，因为这种组织含有大量的可螯合锌库， 在正常的神经元交流过程中释放谷氨酸。事实上，ZnT 3锌流出物已经被广泛应用于各种场合。 显示出在螯合和穿梭锌进入谷氨酸能囊泡和敲除 这种蛋白质消除了脑中的囊泡锌区室化。我们鉴定了跨膜163 （TMEM 163），锌结合蛋白和转运蛋白，作为TRPML 1离子通道的蛋白质相互作用物。 TRPML 1功能丧失导致粘脂沉积症IV。与此同时，TMEM 163最近被报道 通过其与神经元P2 X3受体离子通道的相互作用来调节疼痛感知。TMEM163 也被称为突触囊泡31（SV 31）蛋白首先在大鼠脑突触体中被鉴定并且存在于 作为二聚体。TMEM 163定位于质膜和囊泡隔室内，例如 突触囊泡和溶酶体。我们有初步证据表明TMEM 163与ZnT 3相互作用， 锌外排转运蛋白。功能性锌通量测定显示TMEM 163-ZnT 3的外排活性 异二聚体的构象与它们各自的同二聚体同种型的构象相似。这些结果不仅证实， TMEM 163是一种锌外排剂，但其与相关锌转运蛋白的异二聚化增加了锌转运蛋白的功能。 细胞内锌水平的稳态控制库。因此，TMEM 163似乎很重要， 用于维持独立于或与另一种锌结合的脑锌稳态 外排转运蛋白该项目的总体目标是研究人类的生物学意义， TMEM 163使用其小鼠Tmem 163对应物。为此，我们将使用Tmem 163敲除（KO） 小鼠，以确定脑中螯合锌分布模式的变化，使用组织化学，组织- 荧光和生物化学技术。我们还将通过以下方法检查Tmem 163 KO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 Tmem 163缺失导致其他锌转运蛋白基因的作用 功能总的来说，该项目可以填补目前对TMEM 163生物功能的知识空白， 并且可以提供关于缺乏TMEM 163的细胞或组织如何影响人类健康或人类免疫的见解。 涉及锌稳态异常的疾病过程。

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.