Structure-function Analysis of the SP85/PsB Spore Coat Protein in Dictyostelium

盘基网柄菌SP85/PsB孢子衣蛋白的结构-功能分析

• 批准号: 0240634
• 负责人: Christopher West
• 金额: --
• 依托单位: University of Florida
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2003-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0240634&HistoricalAwards=false
• 关键词: Structure; function; Analysis; SP85; PsB

Cell walls are essential for the life of many microbial eukaryotes, plants and animals. Polysaccharides including cellulose are a major component of many walls. Studies of protein-rich walls in animals, Chlamydomonas and Volvox have shown that cell-matrix and matrix-matrix interactions are mediated by highly specific contacts involving discrete protein domains. The goal of this project is to study the spore coat of Dictyostelium in order to understand, in the long term, the critical roles played by proteins in the assembly of cellulose-rich walls. Like many cell walls, the spore coat consists of a central cellulose-rich region surrounded on either side by protein-rich layers. One of the major coat proteins, SP85 or PsB, is a prime suspect for coordinating cellulose- protein interactions in the coat. SP85 is located in the inner layer of the coat near the plasma membrane, and can simultaneously bind cellulose and another coat protein, SP65, in vitro . Based on the phenotypes of SP85 knockout strains and strains in which individual domains have been overexpressed, SP85 is thought to contribute to both the timing of cellulose synthesis (early function) and the organization of the outer layer including incorporation of proteins and confinement of cellulose (late function). One construct, a fusion of the N-terminal and cysteine-rich C1-domains, blocks a checkpoint required for cellulose synthesis in 85% of the cells, and disrupts the outer layer in the 15% that break through the block. The C1-domain uniquely binds both cellulose and SP65.To determine the role of binding of SP85 to cellulose and SP65 for function in vivo, the following experiments are planned: 1), the gene for full-length SP85 expressed in growing cells, from which it is secreted away from other coat proteins, will be subjected to random mutagenesis in its C1-domain to specifically block either cellulose-or SP65-binding; 2), the disulfide-bonding and glycosylation patterns of the C1-domain and nearby mucin-like domains will be mapped and used to monitor the structures of normal and mutant SP85s; and 3), the inactivating mutations defined in aim 1 will be introduced into SP85 and NC1 expressed in prespore cells and incorporated into the coat. Phenotype analyses will assess how the early and later SP85-dependent functions are affected. The results are expected to provide information about how SP65 regulates SP85 action in checkpoint inhibition, the hypothesized adaptor role for SP65 in bridging inner-and outer-layer proteins, and whether cellulose-binding is important for localizing SP85 and/or in tethering and organizing cellulose microfibrils. An additional set of experiments, aimed at testing the specificity of mutations that block SP65-binding, will also be carried out if time permits. These approaches will test the in vivo relevance of specific domain activities defined by in vitro studies, therefore avoiding potential artifacts intrinsic to the sole use of partial-length expression constructs. The cellulose-SP85-SP65 trimer is predicted to be a coat assembly core module which can be extended in future studies to understand how discrete protein domains elsewhere in SP85 and in other coat proteins function to organize wall assembly.Broader impacts: The structure-function relationships to be defined for the cysteine-rich domains of SP85 are expected to be generalizable to related domains of other cellulose-rich walls found in plants and eukaryotic microbes of economic and health significance. Ultimately, these studies are likely to establish useful precedents for how cellulose synthase is regulated, how cellulose is crystallized, how cellulose fibrils are constrained within the outer protein layer, and how cell wall layers are formed. The project will continue to engage the participation of graduate,undergraduate and high school students, including women and underrepresented minorities,as research trainees in execution of the aims consistent with the educational commitment of the PI in his academic setting. There will be significant interaction with core laboratories and other research laboratories in both the USA and internationally. These activities will contribute to the overall critical mass of effort in proteomics, glycobiology and cell wall biology, and is expected to constitute a continuing resource for the overall university community. All findings will be shared with the scientific community in timely fashion via meetings and publications.

细胞壁对于许多微生物真核生物，植物和动物的寿命至关重要。包括纤维素在内的多糖是许多壁的主要组成部分。对动物，衣原体和Volvox中蛋白质富含蛋白质壁的研究表明，细胞 - 基质和基质矩阵相互作用是由涉及离散蛋白质结构域的高度特异性接触介导的。该项目的目的是研究Dictyostelium的孢子涂层，以便从长远来看，蛋白质在富含纤维素的壁组装中所扮演的关键作用。像许多细胞壁一样，孢子外套由富含蛋白质的层在两侧包围的中央纤维素富含区域组成。 主要的外套蛋白之一SP85或PSB是用于协调外套中纤维素蛋白相互作用的主要嫌疑犯。 SP85位于质膜附近的外套的内层中，可以同时在体外结合纤维素和另一种涂层蛋白Sp65。 基于SP85敲除菌株的表型和过表达各个结构域的菌株，SP85被认为有助于纤维素合成的时间（早期功能）和外层的组织，包括蛋白质的掺入和纤维素的限制（晚功能）。 一种构建体是N末端和富含半胱氨酸的C1域的融合，阻止了85％细胞中纤维素合成所需的检查点，并破坏了15％的外层破裂。 C1域独特地结合了纤维素和SP65。为了确定SP85与纤维素和SP65在体内功能的结合的作用，计划了以下实验：1），从生长细胞中表达的全长SP85基因，从它被分泌在其他外套蛋白中，将在其C1域中随机诱变，以特异性地阻断任何一种纤维素或SP65结合； 2），将绘制C1域和附近粘蛋白样结构域的二硫键键合和糖基化模式，并用于监测正常和突变体SP85S的结构；和3），在AIM 1中定义的灭活突变将引入在预孔细胞中表达并掺入外套中的SP85和NC1中。 表型分析将评估如何影响早期和较晚的SP85依赖性功能。预期结果将提供有关SP65如何调节SP85在检查点抑制中如何调节SP85作用的信息纤维素微纤维。 如果时间允许，还将进行另一组实验，旨在测试阻断SP65结合的突变的特异性。这些方法将测试由体外研究定义的特定域活动的体内相关性，因此避免了唯一使用部分长度表达构建体的潜在伪像。预计纤维素SP85-SP65三聚体是外套组件核心模块，可以在以后的研究中扩展，以了解SP85和其他外套蛋白质中其他位置的离散蛋白质结构域如何组织墙壁组装。预计为SP85的富含半胱氨酸的域定义的关系可推广到在经济和健康意义的植物和真核微生物中发现的其他富含纤维素的壁的相关域。 最终，这些研究可能为如何调节纤维素合酶，纤维素结晶，纤维素原纤维的约束以及如何形成细胞壁层的方式建立有用的先例。 该项目将继续吸引研究生，本科和高中生，包括妇女和代表性不足的少数民族，作为研究受训者执行与PI在其学术环境中的教育承诺一致的目标。在美国和国际上，将与核心实验室和其他研究实验室进行重大互动。 这些活动将有助于蛋白质组学，糖生物学和细胞壁生物学的整体关键量，并有望构成整个大学社区的持续资源。所有发现将通过会议和出版物及时与科学界分享。