Proteins important for dense-core vesicle function

致密核囊泡是一种释放肽类激素、生长因子和生物胺的细胞器 神经元和神经内分泌细胞对钙增加的反应。致密核囊泡货物 调节多种生物过程，包括神经元存活和发育、痛觉、血液 葡萄糖稳态和突触可塑性。因此，许多疾病，如情绪障碍， 肥胖症和糖尿病是由神经肽和激素分泌缺陷引起的。因此必须 确定的分子机制和机制，协调生物发生和释放这些 囊泡载体然而，与其他囊泡隔室如突触囊泡相比， 已知致密核心囊泡生物发生、运输和释放的分子机制。致密核心 囊泡产生于高尔基体，并在不明确的成熟过程中获得其隔室特性 过程发生后高尔基体，但很少有分子已被确定在这些过程中发挥作用。货物 分选致密核心囊泡仍然是一个难题。本项目的长期目标是了解 致密核心囊泡形成的分子机制，分类货物，并获得其隔室 身份实现这一目标的第一步是确定致密核心囊泡生物发生所需的分子。我们 在线虫C.在致密核心囊泡功能缺陷的突变体 并确定了一些新的蛋白质，这些蛋白质在致密核心囊泡生物发生中起作用， RAB-2和CCCP-1，RAB-2效应物，以及EARP内体运输复合物。本课题 将测试RAB-2及其效应物如何与EARP相互作用，以介导货物分选到致密核心囊泡。在 目的1和2，我们将利用遗传学、生物化学和细胞生物学方法，在C。优雅的，在 哺乳动物胰岛素分泌细胞系INS-1 832/13，以确定RAB-2和EARP如何以及在何处与胰岛素受体结合， 复合物是本地化的，无论它们在物理上和功能上相互作用，并决定它们在 致密核心囊泡货物的分选、加工和分泌。在目标3中，我们将结合使用 体外生物化学方法和体内功能测定来确定golgin样卷曲螺旋蛋白 CCCP-1结合膜并发挥介导致密核心囊泡生物发生的功能。这些研究将 推进我们对致密核心囊泡如何产生和分类货物的理解，并提供一般的 深入了解由多亚基复合物控制的膜运输和货物分选机制， 介导内体区室和高尔基体网络之间的运输。

The dense-core vesicle is an organelle that releases peptide hormones, growth factors, and biogenic amines from neurons and neuroendocrine cells in response to increases in calcium. Dense-core vesicle cargos regulate a variety of biological processes including neuronal survival and development, pain sensation, blood glucose homeostasis, and synaptic plasticity. As a consequence, numerous diseases such as mood disorders, obesity, and diabetes are caused by defects in neuropeptide and hormone secretion. Thus, it is important to determine the molecular machinery and mechanisms that orchestrate the biogenesis and release of these vesicular carriers. However, in comparison to other vesicular compartments such as synaptic vesicles, little is known about the molecular mechanisms of dense-core vesicle biogenesis, trafficking, and release. Dense-core vesicles are generated at the trans-Golgi and gain their compartmental identity in a poorly defined maturation process that occurs post-Golgi, but few molecules have been identified that function in these processes. Cargo sorting to dense-core vesicles remains a puzzle. The long-term goal of this project is to understand the molecular mechanisms by which dense-core vesicles are formed, sort cargos, and gain their compartmental identity. A first step towards this goal is to identify molecules required for dense-core vesicle biogenesis. We performed a genetic screen in the nematode C. elegans for mutants defective in dense-core vesicle function and identified a number of new proteins that act in dense-core vesicle biogenesis, including the small GTPase RAB-2 and CCCP-1, a RAB-2 effector, as well as the EARP endosomal trafficking complex. In this project, we will test how RAB-2 and its effectors interact with EARP to mediate cargo sorting to dense-core vesicles. In Aims 1 and 2, we will use genetic, biochemical and cell biological approaches in C. elegans and in the mammalian insulin-secreting cell line INS-1 832/13 to determine how and where the RAB-2 and EARP complexes are localized, whether they physically and functionally interact, and determine their precise roles in the sorting, processing, and secretion of dense-core vesicle cargos. In Aim 3, we will use a combination of in vitro biochemical approaches and in vivo functional assays to determine how the golgin-like coiled-coil protein CCCP-1 binds membranes and functions to mediate dense-core vesicle biogenesis. These studies will advance our understanding of how dense-core vesicles are generated and sort cargos, and provide general insights into mechanisms of membrane trafficking and cargo sorting controlled by multisubunit complexes that mediate trafficking between endosomal compartments and the trans-Golgi network.