Two-pore channels (TPCs): NAADP-activation mechanisms and function for intracellular transport processes

双孔通道 (TPC)：NAADP 激活机制和细胞内转运过程的功能

• 批准号: 415544668
• 负责人: Professor Dr. Norbert Klugbauer
• 金额: --
• 依托单位: Abteilung I
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/415544668?language=en
• 关键词: Two; pore; channels; TPCs; NAADP

TPCs (two-pore channels) constitute a small family of cation channels that are expressed in membranes of the endolysosomal system. TPCs are activated via the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). However, NAADP does not directly bind to TPCs, instead to other proteins of the channel complex. Recently, in literature two candidate proteins have been suggested as NAADP-binding components, which activate TPCs in various model systems and which induce a Ca2+ current. Within the scope of this application we want to achieve three objectives:(1) By using the CRISPR/Cas-method we will establish cell-based genetic models of the NAADP-binding proteins. We will perform intracellular Ca2+ measurements to investigate the mechanisms of NAADP-activation of TPCs in their natural membrane compartment. NAADP will be packed into liposomes by applying a newly established technique and added to MEF cells (WT, TPC1-, TPC2- and TPC1/2-Double-KO) for Fura2 Ca2+ measurements. We will apply manipulations of the NAADP-binding and the TPC proteins such as knockouts, mutants or overexpressed components and their cellular (co)-localization will be investigated.(2) During the previous funding period we performed detailed RNAseq analyses which allowed new insights into the altered transcriptome of TPC deficient cells. Numerous membrane receptors were differently expressed and their signaling pathways were affected. Now we intend to focus on the mechanisms of endocytosis and intracellular transport processes of selected receptors. We will investigate the consequences of the strongly reduced expression of caveolins in TPC deficient cells for endocytosis, for receptor trafficking and for receptor signaling pathways. Labelled Rab-GTPases will be used for microscopic live-cell-imaging of endolysosomal vesicles.(3) Based on our work for the role of TPCs for the release of cytokines from macrophage cell lines, we will establish in vivo models of autoimmune diseases. We recently started with a model for antigen-induced arthritis (AIA), which will be developed further. Additionally, we will establish a model for chronic inflammatory bowel diseases, the Dextran Sodium Sulfate (DSS) induced colitis.

TPC（双孔通道）构成阳离子通道的一个小家族，其在内溶酶体系统的膜中表达。TPC通过第二信使烟酸腺嘌呤二核苷酸磷酸（NAADP）激活。然而，NAADP不直接结合到TPC，而是结合到通道复合物的其他蛋白质。最近，在文献中，两个候选蛋白质已被建议作为NAADP结合组分，其在各种模型系统中激活TPC并诱导Ca 2+电流。在本申请的范围内，我们希望实现三个目标：（1）通过使用CRISPR/Cas方法，我们将建立NAADP结合蛋白的基于细胞的遗传模型。我们将进行细胞内Ca 2+的测量，以研究其天然膜室中的TPC的NAADP激活的机制。通过应用新建立的技术将NAADP包装到脂质体中，并加入到MEF细胞（WT、TPC 1-、TPC 2-和TPC 1/2-双KO）中，用于Fura 2 Ca 2+测量。我们将应用NAADP结合和TPC蛋白的操作，如敲除、突变体或过表达组分，并研究它们的细胞（共）定位。(2)在之前的资助期间，我们进行了详细的RNAseq分析，这使得对TPC缺陷细胞的转录组改变有了新的见解。许多膜受体表达不同，其信号通路受到影响。现在，我们打算集中在选定的受体的内吞和细胞内转运过程的机制。我们将研究TPC缺陷细胞内吞作用、受体运输和受体信号传导途径中小窝蛋白表达强烈减少的后果。标记的Rab-GTP酶将用于内溶酶体囊泡的显微镜活细胞成像。(3)基于我们对TPC在巨噬细胞系释放细胞因子中的作用的研究，我们将建立自身免疫性疾病的体内模型。我们最近开始了抗原诱导性关节炎（AIA）的模型，这将进一步发展。此外，我们将建立一种慢性炎症性肠病模型，即葡聚糖硫酸钠（DSS）诱导的结肠炎。