RUI: Investigation of Copine Function in Dictyostelium

RUI：盘基网柄菌的 Copine 功能研究

• 批准号: 0110555
• 负责人: Cynthia Damer
• 金额: $ 15.29万
• 依托单位: Vassar College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2005-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0110555&HistoricalAwards=false
• 关键词: RUI; Investigation; Copine; Function; Dictyostelium

In the past 10 years, great strides have been made in the identification of proteins that mediate and regulate membrane vesicle trafficking in eukaryotic cells. Different proteins function in distinct steps of vesicular trafficking such as budding, transport, targeting, and fusion of vesicles. Most of these proteins are well conserved with homologs found in organisms from yeast to mammals. However, the exact roles of many of these proteins remain unclear. Furthermore, it is likely that not all proteins involved in these processes have been identified. One new candidate for a role in vesicle membrane trafficking is a family of recently discovered proteins called copines. Copines were first isolated from Paramecium in 1997 by their ability to bind phospholipids in a calcium-dependent manner. Paramecium have two closely related copine genes and analysis of current sequence databases indicates that multiple copine homologs exist not only in ciliates, but also in slime molds, green plants, nematodes, mice, and humans. The high degree of conservation of copines among diverse organisms suggests they play a fundamental role in eukaryotic cells. Structurally, copines have two C2 domains at the N-terminus and a region similar to the A domain found in integrins at the C-terminus. The C2 domain is a calcium-/phospholipid- binding motif originally identified in protein kinase C. The A domain in an intracellular soluble protein is a unique characteristic of copines because this domain is typically found in extracellular proteins or extracellular portions of membrane proteins. Following the A domain, copines have a variable length C-terminal domain that is relatively rich in prolines. This domain may confer unique characteristics to the different copine family members and a site for protein-protein interactions. Several lines of evidence suggest that copines may function in vesicular trafficking. First, antibodies raised against a human copine recognize a protein that binds to chromaffin granules indicating that copines bind secretory vesicles. In addition, several proteins thought to be involved in membrane trafficking, such as synaptotagmin, rabphilin, DOC2, and munc13, contain multiple C2 domains that confer calcium/phospholipid binding properties. The long-term research objective is to define in molecular terms the mechanisms underlying membrane vesicle trafficking. In the short term, the research goal is to determine the general role of copines in eukaryotic cells using the model genetic organism, Dictyostelium discoideum. Genetic studies in yeast have been fundamental to the identification and characterization of proteins involved in vesicular membrane trafficking. However, no copine homologs exist in yeast. Dictyostelium provides the same genetic advantages as yeast and preliminary research demonstrates the existence of two copine homologs in Dictyostelium. The two main objectives of the research are: 1. Determine the localization of copines in live cells by expressing green fluorescent protein tagged copines in Dictyostelium and examining the cells by fluorescence microscopy. This experimental approach will be used to determine transient changes in localization during different cellular behaviors. 2. Create copine gene knockout mutants in Dictyostelium with gene replacement by homologous recombination and analyze the mutant phenotypes. This experimental approach will be used to determine loss of function in membrane trafficking pathways due to loss of copine gene function. Once these two objectives are met, it will be possible to correlate the intracellular location of copines with loss-of-function phenotypes in copine mutants and produce a more specific hypothesis about the function of copines in eukaryotic cells.

在过去的10年里，在鉴定真核细胞中介导和调节膜囊转运的蛋白质方面取得了很大的进展。不同的蛋白质在囊泡运输的不同步骤中起作用，如囊泡的出芽、运输、靶向和融合。从酵母菌到哺乳动物，这些蛋白质中的大多数都与生物中的同源物保持良好的保守性。然而，其中许多蛋白质的确切作用仍不清楚。此外，可能并非所有参与这些过程的蛋白质都已被确定。一个在囊泡膜运输中起作用的新候选蛋白是最近发现的一种叫做copines的蛋白质家族。Copines于1997年首次从草履虫中分离出来，因为它们能够以钙依赖的方式结合磷脂。草履虫有两个密切相关的copine基因，对现有序列数据库的分析表明，不仅在纤毛虫中，在黏菌、绿色植物、线虫、小鼠和人类中也存在多个copine同源基因。复制体在不同生物中的高度保守性表明它们在真核细胞中起着重要作用。在结构上，复制体在n端有两个C2结构域，在c端有一个类似于整合素的a结构域。C2结构域是最初在蛋白激酶c中发现的钙/磷脂结合基元。胞内可溶性蛋白中的a结构域是copines的独特特征，因为该结构域通常存在于细胞外蛋白或膜蛋白的细胞外部分。在A结构域之后，复制体有一个可变长度的c端结构域，其中脯氨酸相对丰富。这个结构域可能赋予不同的copine家族成员独特的特征和蛋白质-蛋白质相互作用的位点。一些证据表明，复制蛋白可能在囊泡运输中起作用。首先，针对人类copines产生的抗体识别与嗜铬蛋白颗粒结合的蛋白质，表明copines与分泌囊泡结合。此外，一些被认为参与细胞膜运输的蛋白质，如synaptotagmin、rabphilin、DOC2和munc13，含有多个C2结构域，赋予钙/磷脂结合特性。长期的研究目标是在分子术语中定义膜泡运输的机制。在短期内，研究目标是利用模式遗传生物盘状盘齿骨确定仿铜在真核细胞中的一般作用。酵母的遗传研究是鉴定和表征参与囊泡膜运输的蛋白质的基础。然而，在酵母中不存在copine同源物。盘形网柱体具有与酵母相同的遗传优势，初步研究表明盘形网柱体中存在两个同源物。研究的两个主要目标是：1。通过在Dictyostelium中表达绿色荧光蛋白标记的copines，并通过荧光显微镜检查细胞，确定copines在活细胞中的定位。这种实验方法将用于确定不同细胞行为期间定位的瞬态变化。2. 同源重组基因置换在盘基骨菌中产生copine基因敲除突变体，并分析突变体表型。这种实验方法将用于确定由于copine基因功能丧失而导致的膜运输途径的功能丧失。一旦这两个目标得到满足，就有可能将copines的细胞内定位与copines突变体的功能丧失表型联系起来，并产生关于copines在真核细胞中的功能的更具体的假设。