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的阻断导致儿童疾病，如糖尿病、肥胖症和神经发育、记忆和学习缺陷。 因此，阐明调节分泌途径（RSP）中分泌囊泡转运的机制将有助于开发治疗此类疾病的新方法。 该候选人发现，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体后运输需要囊泡羧肽酶E（CPE）胞质尾、snapin和微管马达之间的相互作用。 人们还发现，snapin相互作用与肌动蛋白，这是已知的参与沿着与肌球蛋白V在肽能分泌囊泡的运输活动依赖性的方式，在分泌的终端阶段。 该候选人假设，snapin-CPE尾部相互作用蛋白质管理的顺行运输的RSP囊泡沿着轴突，也参与捕获这些囊泡从微管运输平台上的肌动蛋白网络在终端刺激后。 因此，主要的研究目标是确定，在内分泌细胞和神经元，CPE-snapin相互作用的蛋白质（a）调节顺行运输的RSP囊泡沿着过程/神经突在调节分泌途径;和（B），拴系/捕获这些囊泡在刺激后的终端，并将它们移动到释放位点，其货物的活性依赖性分泌。 具体目标1中提出的研究旨在鉴定ACTH和BDNF囊泡沿沿着微管在高尔基体后转运所必需的蛋白质。 与snapin-CPE-motor相互作用的蛋白质将通过色谱法纯化，并使用微管下拉和活细胞成像测定分析其功能。 在具体目标2中，他将纯化和鉴定CPE-snapin相关蛋白质，这些蛋白质参与将肽能囊泡束缚到质膜上的肌动蛋白网络，以进行活性依赖性分泌。 CPE-snapin-相互作用蛋白将被测定用于使用活细胞成像的刺激增强ACTH囊泡与肌动蛋白的束缚。 拟议的研究将揭示（神经）内分泌系统中ACTH/BDNF囊泡转运调节的分子机制，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.