The Role of Mucolipin 1 Block in Transition Metal Toxicity

说明(申请人提供)：过渡金属如铜、铁、钴具有众所周知的毒性作用。很大一部分过渡金属的摄取是通过溶酶体进行的，在过载模型中显示了过渡金属在溶酶体中的积累。过渡金属对溶酶体消化机械的影响还不是很清楚。为该项目获得的初步数据显示，过渡金属阻断离子通道粘蛋白1(TRPML1)。TRPML1是一种溶酶体离子通道突变，可导致溶酶体储存病IV型黏脂沉积症(MLIV)。与MLIV中TRPML1下调的作用类似，过渡金属阻断TRPML1与细胞质储存体的形成、线粒体的碎裂和线粒体对钙的缓冲丧失有关。通过与溶酶体储存疾病中衰老和细胞死亡的溶酶体-线粒体轴模型的类比，这些数据表明，过渡金属阻断TRPML1影响溶酶体功能，从而影响自噬。抑制自噬会导致功能失调的线粒体积累，而线粒体不能吸收细胞质中的钙离子，并保护细胞免受钙离子刺激物的促凋亡作用。这一建议的中心前提是：如果TRPML1阻断对过渡金属的毒性作用，那么所有阻断TRPML1的过渡金属都应该诱导相同的MLIV样表型。这一新的溶酶体对过渡金属毒性的贡献模型将使用几种方法进行测试。首先，将确定阻断TRPML1的过渡金属的范围，以便将金属封闭的TRPML1与这种封闭诱导的溶酶体存储表型相关联。是否所有阻断TRPML1的过渡金属都会诱导自噬、抑制和积累线粒体，这些线粒体的钙缓冲功能受到了损害，这一点将会得到证实。TRPML1被过渡金属阻断的结构决定因素将被建立，并将创建过渡金属抗性突变体。这些突变体将被用来取代天然的TRPML1，并确定TRPML1阻断对过渡金属溶酶体毒性的相对贡献。在本项目过程中获得的结果有望确定过渡金属溶酶体积聚和细胞死亡之间的新联系，并可能为过渡金属毒性的新药理学方法提供理论基础。该项目的基本生物学意义包括更好地了解有毒金属的影响、离子通道的结构和功能以及溶酶体的管家作用。 公共卫生相关性：本项目旨在回答溶酶体离子通道TRPML1的阻断是否有助于过渡金属毒性。阐明溶酶体积累过渡金属与细胞死亡之间的联系将为详细探讨过渡金属毒性的溶酶体方面铺平道路，并可能提出新的药理学方法来补充现有的金属毒性治疗方法。由于溶酶体参与了抗原加工等生物体水平的功能，证明过渡金属影响溶酶体的功能可能会产生更广泛的生物学影响。

DESCRIPTION (provided by applicant): Transition metals such as Cu, Fe, Co have well known toxic effects. A large fraction of the transition metal uptake occurs through lysosomes and accumulation of transition metals in lysosomes was shown in the overload models. The effects of transition metals on lysosomal digestive machinery are not very well established. The preliminary data obtained for this project show that transition metals block ion channel mucolipin 1 (TRPML1). TRPML1 was previously identified as the lysosomal ion channel mutations in which cause lysosomal storage disease mucolipidosis type IV (MLIV). Similar to the effects of TRPML1 downregulation in MLIV, TRPML1 block by transition metals is associated with formation of cytoplasmic storage bodies, mitochondrial fragmentation and the loss of Ca2+ buffering by mitochondria. By analogy with the lysosome-mitochondria axis model of aging and cell death in lysosomal storage diseases, these data suggest that TRPML1 block by transition metals affects lysosomal function and, therefore autophagy. Suppressed autophagy results in accumulation of dysfunctional mitochondria that cannot take up cytoplasmic Ca2+ and guard cells against pro-apoptotic effects of Ca2+ spikes. The central premise of this proposal is: if TRPML1 block contributes to transition metals toxicity, then all transition metals that block TRPML1 should induce the same MLIV-like phenotype. This novel model of the lysosomal contribution to transition metal toxicity will be tested using several approaches. First, the range of transition metals that block TRPML1 will be established in order to correlate TRPML1 block by metals and lysosomal storage phenotype induced by such block. Whether or not all transition metals that block TRPML1 induce autophagy suppression and accumulation of mitochondria that are compromised in their Ca2+ buffering function will be established. Structural determinants of TRPML1 block by transition metals will be established and transition metal resistant mutants will be created. Such mutants will be used to replace native TRPML1 and establish the relative contribution of TRPML1 block into lysosomal toxicity of transition metals. The results obtained in the course of this project are expected to identify a novel link between lysosomal buildup of transition metals and cell death and may provide a rationale for novel pharmacological approaches to transition metals toxicity. The basic biological implications of this project include better understanding of the effect of toxic metals, structure and function of ion channels and housekeeping role of lysosomes. PUBLIC HEALTH RELEVANCE: The present project aims to answer whether the block of the lysosomal ion channel TRPML1 contributes to transition metal toxicity. Delineating the chain of events that connects lysosomal accumulation of transition metals to cell death will pave way for a detailed inquiry into lysosomal aspects of transition metal toxicity and may suggest novel pharmacological approaches to complement the existing treatments for metal toxicity. Since lysosomes are involved in the organism-level functions such as antigen processing, proving that transition metals affect lysosomal function may have even broader biological impact.