Dissecting the Structure and Function of the PDU Microcompartment in Salmonella

描述（由申请方提供）：细菌微区室是由代谢酶组成的大型亚细胞结构，这些代谢酶封装在由多个亚基构建的蛋白质壳内。它们在细菌中广泛存在，功能多样，与发病机制有关，具有许多重要的潜在生物医学应用，并且似乎包含独特的机械和结构原理。它们的功能是隔离和调节某些代谢途径中发现的有毒或挥发性中间体的产生。然而，很少有人知道这是如何发生在机械水平。该研究的长期目标是阐明沙门氏菌降解1，2-丙二醇的分子原理并建立微区室的三维结构。沙门氏菌系统是无与伦比的知识和工具，可用于微区室的机制研究。拟议的研究结合联合收割机遗传，生物物理和结构的方法来阐明沙门氏菌Pdu微区室的细胞功能的机制水平。提出了三个具体目标：1。确定末端螺旋的作用和其他机制，将蛋白质靶向到Pdu微区室的内腔; 2.确定孔和辅因子再循环在为Pdu微区室的管腔酶提供所需底物和辅因子中的作用;以及3.阐明Pdu微区室的高级结构和组装。结构将通过X射线晶体学，生物物理学和计算方法进行研究和分析。蛋白质-蛋白质结合研究将包括组氨酸标签下拉，生物物理方法和晶体学。功能和机制的见解将来自结构导向诱变结合遗传和生物化学研究。完成拟议的调查将阐明沙门氏菌pdu微区室的机制和结构原则。这将提供对细菌微区室的一般了解。由于细菌微区室在许多微生物（包括几种人类病原体）中起着关键的代谢作用，因此拟议的研究可能最终导致干扰致病过程的新机会。 公共卫生相关性：对在许多人类病原体中发现的细菌微区室的更好理解可能最终导致干扰致病过程的新机会，并且还可能为合理设计用于生产药物或作为药物递送载体的合成蛋白质笼提供基础。

DESCRIPTION (provided by applicant): Bacterial microcompartments are large subcellular structures composed of metabolic enzymes encapsulated within a protein shell built from multiple subunits. They are widespread among bacteria, functionally diverse, linked to pathogenesis, have a number of important potential biomedical applications, and appear to incorporate unique mechanistic and structural principles. Their function is to sequester and regulate the production of toxic or volatile intermediates found in certain metabolic pathways. However, little is known about how this is occurs at the mechanistic level. The long-term goal of the proposed research is to elucidate the molecular principles and to build up a 3-dimensional structure of the microcompartments involved in 1,2-propanediol degradation by Salmonella. The Salmonella system is unmatched with regard to the knowledge and tools available for mechanistic studies of microcompartments. The proposed studies combine genetic, biophysical, and structural methods to elucidate the cellular function of the Salmonella Pdu microcompartment at a mechanistic level. Three specific aims are proposed: 1. Determine the role of terminal helixes and other mechanisms for targeting proteins to the lumen of the Pdu microcompartment; 2. Determine the role of pores and cofactor recycling in supplying the lumen enzymes of the Pdu microcompartment with required substrates and cofactors; and 3. Elucidate the higher order structure and assembly of the Pdu microcompartment. Structures will be investigated and analyzed by x-ray crystallography, biophysical, and computational methods. Protein-protein binding studies will include his-tag pulldowns, biophysical methods, and crystallography. Functional and mechanistic insights will be derived from structure-guided mutagenesis in conjunction with genetic and biochemical studies. Completion of the proposed investigations will elucidate the mechanistic and structural principles of the Salmonella pdu microcompartment. This will provide general insights into bacterial microcompartments. Since bacterial microcompartments play critical metabolic roles in many microbes, including several human pathogens, the proposed studies may ultimately lead to new opportunities for interfering with pathogenic processes. PUBLIC HEALTH RELEVANCE: An improved understanding of bacterial microcompartments, which are found in many human pathogens, may ultimately lead to new opportunities for interfering with pathogenic processes, and may also provide a basis for the rational design of synthetic protein cages for use in the production of pharmaceuticals or as drug delivery vehicles