Mechanism of transport of secretory vesicles in regulated secretory pathway

描述（由申请人提供）：激素、神经肽和神经营养因子的调节分泌缺失导致儿童疾病，如糖尿病、肥胖、神经发育、记忆和学习障碍。因此，阐明调节分泌通路（RSP）中分泌囊泡运输的机制将有助于开发治疗此类疾病的新方法。候选人发现，新合成的BDNF/ACTH分泌囊泡从神经元和内分泌细胞的细胞体到轴突/树突末端或质膜的释放位点的高尔基转运需要囊泡羧基肽酶E （CPE）细胞质尾部、snapin和微管马达之间的相互作用。研究还发现，在分泌的终末阶段，snapin与肌动蛋白相互作用，肌动蛋白与肌球蛋白V一起以活性依赖的方式参与肽能分泌囊泡的运输。候选人假设snapin-CPE尾部相互作用蛋白控制RSP囊泡沿神经突的顺行运输，并且还参与在刺激时将这些囊泡从微管运输平台捕获到末端的肌动蛋白网络上。因此，主要的研究目标是在内分泌细胞和神经元中鉴定cpe -snapin相互作用蛋白(a)，该蛋白在受调节的分泌途径中调节RSP囊泡沿过程/神经突的顺行运输；(b)在受到刺激时，在末端拴住/捕获这些囊泡，并将它们移动到释放点，以活性依赖性地分泌它们的货物。在Specific Aim 1中提出的研究旨在鉴定ACTH和BDNF囊泡沿微管的高尔基转运所需的蛋白质。与snapin- cpe马达相互作用的蛋白质将通过色谱纯化，并使用微管下拉和活细胞成像分析其功能。在Specific Aim 2中，他将纯化和鉴定CPE-snapin相关蛋白，这些蛋白参与将肽能囊泡系在质膜上的肌动蛋白网络上，以进行活性依赖性分泌。cpe -snapin相互作用蛋白将通过活细胞成像检测刺激增强ACTH囊泡与肌动蛋白的捆绑。这些研究将揭示神经内分泌系统中ACTH/BDNF囊泡运输调控的分子机制，以及与该机制破坏相关的疾病的潜在治疗方法。

DESCRIPTION (provided by applicant): Obliteration of regulated secretion of hormones, neuropeptides, and neurotrophins leads to disorders in children, such as diabetes, obesity, and deficits in neural development, memory, as well as learning. Thus, elucidation of the mechanism for secretory vesicle transport in the regulated secretory pathway (RSP) will facilitate development of new approaches to treat such diseases. The candidate found that post-Golgi transport of newly synthesized BDNF/ACTH secretory vesicles from the cell body of neurons and endocrine cells to the release site at axonal/dendritic terminals or plasma membrane requires interaction among vesicular carboxypeptidase E (CPE) cytoplasmic tail, snapin, and microtubule motors. It was also found that snapin interacted with actins, which are known to be involved along with myosin V in the transport of peptidergic secretory vesicles in an activity-dependent manner, in the terminal stages of secretion. The candidate hypothesizes that snapin-CPE tail interacting proteins govern the anterograde transport of RSP vesicles along neurites, and are also involved in capturing these vesicles from the microtubule transport platform onto the actin network at the terminals upon stimulation. Thus, the primary research goal is to identify, in endocrine cells and neurons, CPE-snapin-interacting proteins (a) that regulate the anterograde transport of RSP vesicles along processes/neurites in the regulated secretory pathway; and (b) that tether/capture these vesicles at the terminals upon stimulation and moves them to the release site for activity-dependent secretion of their cargo. The research proposed in Specific Aim 1 is designed to identify proteins necessary for post-Golgi transport of ACTH and BDNF vesicles along microtubules. Proteins that interact with snapin-CPE-motor will be purified by chromatography and analyzed for their function using microtubule pulldown and live cell imaging assays. In Specific Aim 2, he will purify and identify CPE-snapin associated proteins involved in tethering peptidergic vesicles to the actin network at the plasma membrane for activity-dependent secretion. CPE-snapin-interacting proteins will be assayed for enhancement of tethering of ACTH vesicles to actin with stimulation using live cell imaging. The proposed studies will uncover the molecular mechanism for regulation of ACTH/BDNF vesicle transport in (neuro)endocrine systems and potential treatments of diseases associated with the disruption of the mechanism.