Analysis of Polyhydroxyalkanoate Inclusion Biogenesis

描述(由申请者提供)：这项建议实现了以下领域的计划目标：1)支持有价值的研究；2)让本科生接触研究；3)加强非研究密集型大学的研究环境。这项研究的总体目标是 目的：阐明聚羟基烷酸包合物的生物形成机制。聚羟基烷酸酯(PHAs)是一种细菌聚合物，在碳水平高而另一种基本营养物质(如氮)有限的情况下，在包裹体中合成。PHA有相当大的商业利益，因为它们由一系列聚酯组成，可以形成可生物降解的塑料。电子显微镜研究一直无法解析PHA包裹体的结构，这阻碍了包裹体生物发生的内聚模型的发展。利用原子力显微镜，我们已经确定有三层结构，一层 外包层，即膜双层、中间网状层和下面的晶片层的厚度。遗传学研究表明，中间网络至少部分由Phap组成，Phap很可能被移位到周质。因此，似乎可以通过蛋白质和/或蛋白质移动到周质并通过细胞质膜发芽进入细胞质，促进细胞质的获得来发生包涵体生物发生。 薄膜作为一个信封。本研究的目的是证明或反驳这一假设。本研究的具体目的是：1)通过亚细胞组分的荧光定位和Western印迹分析，明确Phap的周质定位；2)通过蛋白质组学分析，证明包涵体包膜来源于细胞质膜；3)鉴定与Phap瞬时和永久结合的蛋白质，以期阐明包涵体生物发生的机制。归根结底，这项研究的目标是扩大我们对包容性的了解 生物发生，以使这一过程可以被控制和用于医疗应用。例如，可以设想，不是将聚合物插入包合物，而是可以插入生物活性化合物，使包合物进入药物输送载体。莫尔黑德州立大学最近开始了一个过程，强调本科生研究，鼓励教职员工成为教师/学者。已经为这一目标分配了大量资源。该项目支持这一使命，并将改善密歇根州立大学的研究环境，为本科生提供大量学习生物医学研究基础知识的机会，同时进行将扩大我们对原核过程的知识的研究。公共卫生相关性：利用原子力显微镜，我们已经解决了聚羟基烷酸盐包裹体的结构排列，这导致了包裹体生物发生的初步模型。本研究的目的是测试这一初步模型，希望将生物发生策略应用于医疗应用，其中包合物可用作药物输送载体。

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.