Analysis of Polyhydroxyalkanoate Inclusion Biogenesis

聚羟基脂肪酸酯包合物生物发生分析

• 批准号: 7515485
• 负责人: Douglas Dennis
• 金额: $ 18.43万
• 依托单位: MOREHEAD STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7515485
• 关键词: Atomic Force Microscopy; Bacteria; Biogenesis; Biomedical Research; Carbon; Cell membrane; Cytoplasm; Data; Drug Delivery Systems; Electron Microscopy; Environment; Faculty; Family; Fluorescence; Genetic; Goals; Knowledge; Learning; Ligands; Medical; Membrane; Mission; Modeling; Molecular; Molecular Genetics; Movement; Nitrogen; Nutrient; Plastics; Polyesters; Polymers; Process; Protein Binding; Proteins; Proteomics; Public Health; Purpose; Research; Resources; Scaffolding Protein; Solid; Sorting - Cell Movement; Structure; Students; Subcellular Fractions; Surface; Testing; Thick; Tissues; Universities; Western Blotting; interest; member; periplasm; programs; receptor; teacher

DESCRIPTION (provided by applicant): This proposal accomplishes the AREA program objectives of: 1) supporting meritorious research; 2) exposing undergraduates to research; and 3) strengthening the research environment in non-research intensive universities. The general goal of this research is to elucidate the mechanism of polyhydroxyalkanoate inclusion biogenesis. Polyhydroxyalkanoates (PHAs) are bacterial polymers that are synthesized in inclusions when carbon levels are high and another essential nutrient, such as nitrogen is limited. There is considerable commercial interest in PHAs because they comprise a family of polyesters that can be formed into plastics that are biodegradable. Electron microscopy studies have been unable to resolve the structure of PHA inclusions and this has inhibited movement toward a cohesive model of inclusion biogenesis. Employing atomic force microscopy, we have determined that there are three layers of structure, an outer envelope that is the thickness of a membrane bilayer, a middle network layer, and an underlying crystalline lamellar layer. Genetic studies have indicated that the middle network is comprised at least partially of PhaP and that PhaP is likely to be translocated to the periplasm. Thus, it would appear that inclusion biogenesis may occur by movement of protein and/or proteins to the periplasm and budding through the cytoplasmic membrane into the cytoplasm, facilitating the acquisition of the cytoplasmic membrane as an envelope. The goal of this research is to prove or disprove this supposition. The specific aims of the research are: 1) definitively prove periplasmic localization of PhaP via fluorescence localization and Western blot analyses of subcellular fractions, 2) demonstrate that the inclusion envelope is derived from the cytoplasmic membrane by proteomic analysis, and 3) characterize proteins that bind transiently and permanently to PhaP in hopes of elucidating the mechanism of inclusion biogenesis. Ultimately, the goal of the research is to enlarge our knowledge of inclusion biogenesis to the point that this process can be controlled and utilized for medical applications. For instance, it could be envisioned that instead of polymer being inserted into the inclusion, bioactive compounds could be inserted, making the inclusion into a drug delivery vehicle. Morehead State University has recently embarked upon a process whereby undergraduate research is emphasized and faculty members are encouraged to become teacher/scholars. Significant resources have been allocated to this goal. This project supports this mission and will enhance the research environment at MSU by providing undergraduate students with numerous opportunities to learn the fundamentals of biomedical research while conducting research that will enlarge our knowledge of prokaryotic processes. PUBLIC HEALTH RELEVANCE: Using atomic force microscopy we have resolved the structural arrangement of polyhydroxyalkanoate inclusions and this has led to a preliminary model for inclusion biogenesis. The purpose of this research is to test this preliminary model in hopes of adapting the strategy of biogenesis for medical applications in which inclusions could be used as drug delivery vehicles.

描述（由申请人提供）：该提案实现了 AREA 计划的目标：1) 支持有价值的研究； 2）让本科生进行研究； 3）加强非研究密集型大学的研究环境。本研究的总体目标是 阐明聚羟基脂肪酸酯包合物生物发生机制。聚羟基脂肪酸酯 (PHA) 是一种细菌聚合物，当碳含量较高且另一种必需营养素（例如氮）有限时，内含物中会合成这种聚合物。 PHA 具有相当大的商业兴趣，因为它们包含一系列聚酯，可以制成可生物降解的塑料。电子显微镜研究无法解析 PHA 内含物的结构，这抑制了内含物生物发生的内聚模型的发展。利用原子力显微镜，我们确定了三层结构，一层 外壳是双层膜、中间网络层和下面的结晶层状层的厚度。遗传学研究表明，中间网络至少部分由 PhaP 组成，并且 PhaP 很可能易位至周质。因此，似乎包涵体生物发生可能是通过蛋白质和/或多种蛋白质移动到周质并通过细胞质膜出芽进入细胞质而发生的，从而促进细胞质的获得 膜作为信封。这项研究的目的是证明或反驳这一假设。该研究的具体目标是：1) 通过荧光定位和亚细胞组分的蛋白质印迹分析明确证明 PhaP 的周质定位，2) 通过蛋白质组分析证明包涵膜源自细胞质膜，3) 表征蛋白质短暂且永久地与 PhaP 结合，希望能够阐明包涵体生物发生的机制。最终，研究的目标是扩大我们对包容性的了解 生物发生使得该过程可以被控制并用于医疗应用。例如，可以设想，不是将聚合物插入到内含物中，而是可以插入生物活性化合物，从而使内含物成为药物输送载体。莫尔黑德州立大学最近开始了一项强调本科生研究并鼓励教职员工成为教师/学者的进程。为此目标分配了大量资源。该项目支持这一使命，并将通过为本科生提供大量学习生物医学研究基础知识的机会，同时开展扩大我们对原核过程知识的研究，来改善密歇根州立大学的研究环境。公共健康相关性：使用原子力显微镜，我们已经解决了聚羟基脂肪酸酯包裹体的结构排列，这导致了包裹体生物发生的初步模型。这项研究的目的是测试这个初步模型，希望将生物发生策略应用于医学应用，其中内含物可以用作药物输送载体。