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：1）支持有罪研究的领域计划目标； 2）使大学生接受研究； 3）加强非研究密集大学的研究环境。这项研究的一般目标是 阐明多羟基烷烃包容生物发生的机制。多羟基烷酸酯（PHAS）是细菌聚合物，当碳水平较高并且另一种必需的营养素（例如氮气）被限制时，它们在包含物中合成。在PHAS中，商业兴趣很大，因为它们包括一个可以形成可生物降解的塑料的聚酯家族。电子显微镜研究无法解析PHA夹杂物的结构，这抑制了朝着包含生物发生的凝聚模型的运动。使用原子力显微镜，我们确定有三层结构，一个 外膜是膜双层的厚度，中间网络层和下面的晶体层状层。遗传研究表明，中间网络至少部分由PHAP组成，并且PHAP可能会易位到周期。因此，似乎通过将蛋白质和/或蛋白质移动到周质和通过细胞质膜萌芽到细胞质中，可能发生包含生物发生，从而促进了细胞质的获取。 膜作为信封。这项研究的目的是证明或反驳这一假设。 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.最终，研究的目的是扩大我们的包容性知识 生物发生以至于可以控制和用于医疗应用。例如，可以预见的是，可以插入生物活性化合物，而不是将聚合物插入包含中，从而将其纳入吸毒工具。莫尔黑德州立大学最近着手强调本科研究的过程，并鼓励教师成为教师/学者。大量资源已分配给该目标。该项目支持该任务，并通过为本科生提供许多学习生物医学研究基础的机会，同时进行研究，从而扩大我们对原核生物过程的了解，从而增强了MSU的研究环境。公共卫生相关性：使用原子力显微镜，我们解决了多羟基烷烃包含物的结构布置，这导致了包容性生物发生的初步模型。这项研究的目的是测试该初步模型，以期适应可以用作药物输送车辆的医疗应用的生物发生策略。