Analysis of Polyhydroxyalkanoate Inclusion Biogenesis

描述（由申请人提供）：该提案实现了区域计划的目标：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。本研究的总体目标是 阐明聚羟基烷酸酯包合物的生物成因机制。聚羟基链烷酸酯（PHA）是细菌聚合物，当碳水平高而另一种必需营养素（如氮）有限时，它们在内含物中合成。PHA具有相当大的商业利益，因为它们包含可形成为可生物降解的塑料的聚酯家族。电子显微镜的研究一直无法解决PHA包涵体的结构，这抑制了运动的包容物生物成因的凝聚力模型。采用原子力显微镜，我们已经确定有三层结构， 外层，其为膜双层、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可能易位到周质。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出芽进入细胞质，促进细胞质的获得，从而发生包涵体生物发生。 膜作为包膜。这项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印迹分析明确证明PhaP的周质定位，2）通过蛋白质组学分析证明包涵体包膜来自细胞质膜，3）表征瞬时和永久结合PhaP的蛋白质，以期阐明包涵体生物发生的机制。最终，这项研究的目标是扩大我们对包容性的认识 生物发生到可以控制该过程并用于医学应用的程度。例如，可以设想，代替聚合物插入到包合物中，可以插入生物活性化合物，使包合物成为药物递送载体。莫尔黑德州立大学最近开始了一个过程，即强调本科生的研究，并鼓励教师成为教师/学者。为实现这一目标已划拨了大量资源。该项目支持这一使命，并将通过为本科生提供大量机会来学习生物医学研究的基础知识，同时进行研究，扩大我们对原核生物过程的知识，从而增强MSU的研究环境。公共卫生相关性：使用原子力显微镜，我们已经解决了聚羟基烷酸酯夹杂物的结构排列，这导致了一个初步的模型，为夹杂物的生物成因。本研究的目的是测试这个初步的模型，希望适应的战略，生物成因的医疗应用中，夹杂物可用作药物输送车辆。

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.