A mechanism of lysosomal Calcium entry

溶酶体是高度酸性的细胞器，它整合了所有细胞类型中的重要细胞过程。 与溶酶体功能障碍相关的神经系统疾病的风险基因占优势。 在溶酶体中，腔内钙离子对功能至关重要，而每一种已知的溶酶体钙释放都存在缺陷。 经络会导致一种明显的神经紊乱。然而，溶酶体钙离子调节失调也可能是由于 有缺陷的进口产品。虽然更多的人知道释放溶酶体钙离子的机制，但却很少 关于钙离子输入的病理生理学的信息。值得注意的是，唯一已知的溶酶体钙离子进口体 动物，P型ATPase ATP13A2，是我的实验室最近使用新开发的 溶酶体钙离子成像的报告技术。这个进口商以前被认为是一个主要风险 帕金森氏症的基因。因此，在结构层面上理解ATP13A2如何将钙离子导入到 溶酶体是非常重要的。 这一建议的前提是ATP13A2的功能在机械上类似于SERCA，但 具有较低的亲和力和/或钙离子转运效率。这一前提是基于来自我的 实验室使用同源建模，预测ATP13A2和SERCA之间有很高的相似性。 SERCA是目前研究最多的P2型ATPase之一。在……里面 相反，ATP13A2是一个P5型ATPase，一个尚未被机械描述的ATPase亚类。 该提案中概述的步骤将识别和研究ATP13A2如何 通过在活的哺乳动物细胞中实时绘制溶酶体的钙动力学图来驱动溶酶体的钙输入。 我们计划创造和表征一种光稳定的溶酶体钙离子报告器，并开发一种分析方法来映射 活体细胞中溶酶体钙离子原位输入的动力学。初步数据显示，我们已经确定了 相关分子组分使该细胞器内钙离子具有光稳定性。此外，首字母 生物信息学分析和同源建模揭示了ATP13A2之间显著的相似之处 和SERCA。这将使我们能够通过P5型ATPase精确定位对钙离子输入重要的残基。通过 表达各种ATP13A2突变体，并使用实时钙离子图谱，我们将能够识别 对ATP13A2功能至关重要的残基。 这项研究的成功完成将确定帕金森氏病的一个主要风险基因 引入溶酶体Ca~(2+)，阐明P5型ATPase的第一个构效关系。此外，由 提供了第一个定量绘制活细胞中溶酶体钙通量的实用技术(实时)， 我们将能够研究新的溶酶体钙导入体和现有的溶酶体钙释放通道 与各种神经退行性疾病有关。

Lysosomes are highly acidic organelles that integrate important cellular processes in all cell types. There is a preponderance of risk genes for neurological disorders associated with lysosome dysfunction. In the lysosome, lumenal Ca2+ is critical to function, and defects in every known lysosomal Ca2+ release channel leads to a distinct neurological disorder. Yet, dysregulated lysosomal Ca2+ can also arise due to defective import. While much more known of mechanisms that release lysosomal Ca2+, there is a paucity of information on the pathophysiology of Ca2+ import. Notably, the only known lysosomal Ca2+ importer in animals, the P-type ATPase ATP13A2, was recently discovered by my laboratory using newly developed reporter technology for lysosomal Ca2+ imaging. This importer was previously identified as a major risk gene for Parkinson's disease. Thus, a structural level understanding of how by ATP13A2 imports Ca2+ into the lysosome is highly significant. The premise of this proposal is that ATP13A2 function is mechanistically similar to that of SERCA but with lower affinity and/or efficiency of Ca2+ transport. This premise is based on unpublished data from my laboratory using homology modeling, which predicts very high similarity between ATP13A2 and SERCA. SERCA (Sarco/Endoplasmic Reticulum Ca2+ ATPase) is one of the best studied P2-type ATPases. In contrast, ATP13A2 is a P5-type ATPase, an ATPase sub-class yet to be mechanistically characterized. The steps outlined in this proposal will identify and study the molecular mechanism of how ATP13A2 drives lysosomal Ca2+ import by mapping lysosomal Ca2+ dynamics in real-time in live mammalian cells. We plan to create and characterize a photostable lysosomal Ca2+ reporter and develop an assay to map the kinetics of lysosomal Ca2+ import in situ in live cells. Preliminary data shows that we have identified the relevant molecular components to make this photostable organellar Ca2+ reporter. Further, an initial bioinformatics analysis and homology modeling has revealed a remarkable similarly between ATP13A2 and SERCA. This will allow us to pinpoint residues important to Ca2+ import by a P5-type ATPase. By expressing various ATP13A2 mutants and using real-time Ca2+ mapping, we shall be able to identify residues critical to the function of ATP13A2. Successful completion of this research will identify how a major risk gene for Parkinson's disease imports lysosomal Ca2+ and elucidate the first structure-activity relationship in P5-type ATPases. Also, by providing the first practical technology to quantitatively map lysosomal Ca2+ fluxes in live cells (in real-time), we will be in a position to study new lysosomal Ca2+ importers and existing lysosome Ca2+ release channels connected to various neurodegenerative diseases.