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调节分泌的缺失会导致儿童疾病，如糖尿病、肥胖症以及神经发育、记忆和学习方面的缺陷。因此，阐明分泌囊泡在调节分泌途径(RSP)中的运输机制将有助于开发治疗此类疾病的新方法。候选人发现，高尔基体后将新合成的BDNF/ACTH分泌囊泡从神经元和内分泌细胞的细胞体运输到轴突/树突状终末或质膜的释放部位，需要囊泡羧肽酶E(CPE)细胞质尾巴、管理单元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一起以一种活性依赖的方式参与肽能分泌囊泡的运输。候选假设是Snapin-CPE尾部相互作用蛋白控制着RSP囊泡沿神经突起的顺行运输，并在刺激时参与将这些囊泡从微管运输平台捕获到终末的肌动蛋白网络上。因此，主要的研究目标是在内分泌细胞和神经元中识别CPE-Snapin相互作用蛋白，这些蛋白(A)调节RSP囊泡沿受调节的分泌途径中的突起/轴突的顺行运输；以及(B)在刺激下在末端系住/捕获这些囊泡，并将它们移动到释放位置，以依赖于其货物的活性分泌。在特定目标1中提出的这项研究旨在确定高尔基体后ACTH和BDNF囊泡沿微管运输所需的蛋白质。与Snapin-CPE马达相互作用的蛋白质将通过层析进行纯化，并使用微管下拉和活细胞成像分析其功能。在特定目标2中，他将纯化和鉴定CPE-Snapin相关蛋白，这些蛋白参与将多肽能小泡连接到质膜上的肌动蛋白网络，以实现活性依赖的分泌。CPE-Snapin相互作用蛋白将被检测以增强ACTH囊泡在刺激下与肌动蛋白的连接，使用活细胞成像。这项拟议的研究将揭示ACTH/BDNF囊泡运输在(神经)内分泌系统中调节的分子机制，以及与该机制中断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.