Unraveling the role of protein phosphorylation in the regulation of development i

描述(由申请人提供)：本提案的目标是通过对蛋白激酶/磷酸酶网络的初步阐明，开始研究蛋白质丝氨酸/苏氨酸/酪氨酸磷酸化在调节衣原体生理和发病机制中的作用。蛋白质Ser/Thr/Tyr的磷酸化被越来越多地认为是一种广泛使用的机制，通过可逆的蛋白质功能改变来调节对生长和生存(从而致病)至关重要的细菌过程。衣原体(Chlamydia spp.)在环境稳定的EB和复制的细胞内RB之间经历了一个独特的两阶段发育周期，这似乎需要对蛋白质合成和功能进行广泛的调节。令人费解的是，它们5%的基因组被保留给规范的转录调控因子。此外，在DNA被组蛋白样蛋白封闭且仅存在低水平RNA的情况下，EBS早期分化为RBS。这些生物学“问题”提示使用非经典的解决方案来调节衣原体的途径功能。因此，我们推测衣原体属(Chlamydia spp.利用全球蛋白质磷酸化作为应对应激源和调节分化的机制。与这一假说一致，利用2D凝胶电泳、磷蛋白染色和MALDI-TOF-TOF分析对猪囊尾蚴EB/Rb磷蛋白进行定位，在EBS和RBS中发现了42个阶段特异的磷酸化蛋白(98%是泛衣原体蛋白)。此外，还对衣原体进行了一维凝胶电泳磷蛋白染色分析。进一步支持在整个衣原体中存在丰富水平的磷蛋白。这项R15研究中提出的工作将使用一种系统的、自下而上的方法进一步解决我们的假设，该方法侧重于建立和验证专注于沙眼衣原体的激酶/底物和磷酸酶/底物相互作用。在目标1中，我们将使用多种方法来检测蛋白质-蛋白质之间的相互作用，从而描绘出PKN1和PKND激酶/底物网络。将使用体外激酶分析来确认合作关系，并将为选定的底物确定磷酸化位置。我们将在AIM 2中尝试验证CTL0511的功能，CTL0511是一种预测的蛋白质磷酸酶。然后将使用体外磷酸酶检测和各种蛋白质-蛋白质相互作用的方法来鉴定CTL0511底物。最后，在目标3中，我们将进一步定义相互作用的伙伴，包括磷酸化调节的衣原体伙伴转换机制。总之，这些研究将填补我们对衣原体发育的理解空白，提供未来的研究方向，确定治疗和抗感染的新靶点，并阐明蛋白质磷酸化对细菌生理和致病性的更广泛影响。

DESCRIPTION (provided by applicant): The objective of this proposal is to begin addressing the role of protein Ser/Thr/Tyr phosphorylation in regulating chlamydial physiology and pathogenesis through initial elucidation of the protein kinase/phosphatase networks. Protein Ser/Thr/Tyr phosphorylation is increasingly recognized as a widely- employed mechanism for regulating bacterial processes crucial for growth and survival (and therefore pathogenesis) via reversible alteration of protein function. Chlamydia spp. undergo a unique biphasic developmental cycle transitioning between the environmentally stable EB and the replicative, intracellular RB that would appear to require extensive regulation of protein synthesis and function. Perplexingly, <5% of their genomes are reserved for canonical transcriptional regulators. Furthermore, early differentiation of EBs into RBs occurs under conditions where DNA is occluded by histone-like proteins and only low levels of RNA are present. These biological "problems" suggest the use of non-classical solutions for regulating pathway functionality in Chlamydia spp. Consequently, we hypothesize that Chlamydia spp. utilize global protein phosphorylation as a mechanism to respond to stressors and regulate differentiation. Consistent with this hypothesis, mapping of the EB/RB phosphoproteomes of C. caviae using 2D gel electrophoresis, phosphoprotein staining, and MALDI-TOF-TOF analysis identified 42 stage-specific phosphorylated proteins in EBs and RBs (98% were pan-chlamydial proteins). In addition, 1D gel electrophoresis phosphoprotein staining analysis of Chlamydia spp. further supports the presence of abundant levels of phosphoproteins across Chlamydia. The work proposed in this R15 study will further address our hypothesis using a systematic, bottom-up approach that focuses on building and validating kinase/substrate and phosphatase/substrate interactomes focusing on Chlamydia trachomatis. In Aim 1, we will delineate the Pkn1 and PknD kinase/substrate network using multiple methods to detect protein-protein interactions. Partnering will be confirmed using in vitro kinase assays and phosphorylation sites also will be determined for select substrates. We will seek to validate the functionality of CTL0511, a predicted protein phosphatase, in Aim 2. CTL0511 substrates will then be identified using in vitro phosphatase assays and various protein-protein interaction methods. Finally, in Aim 3, we will further define the interacting partners comprising the phosphorylation- regulated chlamydial Partner Switching Mechanism. Collectively, these studies will fill a gap in our understanding of chlamydial development, provide future research directions, identify novel targets for therapeutics and anti-infectives, and illuminate the broader consequences of protein phosphorylation on bacterial physiology and pathogenicity.