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

描述（由申请人提供）：本提案的目的是通过对蛋白激酶/磷酸酶网络的初步阐明，开始解决蛋白Ser/Thr/Tyr磷酸化在调节衣原体生理和发病机制中的作用。蛋白质丝氨酸/苏氨酸/酪氨酸磷酸化越来越被认为是一种广泛应用的机制，通过可逆的蛋白质功能改变来调节细菌生长和生存的关键过程（因此是发病机制）。衣原体经历了一个独特的双相发育周期，在环境稳定的EB和复制的细胞内RB之间过渡，这似乎需要广泛的蛋白质合成和功能调节。令人困惑的是，它们的基因组中只有不到5%是为典型转录调控因子保留的。此外，EBs向RBs的早期分化发生在DNA被组蛋白样蛋白阻断且仅存在低水平RNA的条件下。这些生物学“问题”提示衣原体使用非经典解决方案来调节途径功能。因此，我们假设衣原体利用全局蛋白磷酸化作为一种机制来应对应激源并调节分化。与这一假设相一致，利用2D凝胶电泳、磷酸化蛋白染色和MALDI-TOF-TOF分析对C. caviae的EB/RB磷酸化蛋白质组进行了定位，在EBs和RBs中发现了42个阶段特异性磷酸化蛋白（98%为泛衣原体蛋白）。此外，衣原体的1D凝胶电泳磷酸化蛋白染色分析进一步支持衣原体中存在丰富水平的磷酸化蛋白。在这项R15研究中提出的工作将使用系统的、自下而上的方法进一步解决我们的假设，该方法侧重于构建和验证针对沙眼衣原体的激酶/底物和磷酸酶/底物相互作用组。在Aim 1中，我们将使用多种方法来检测蛋白质-蛋白质相互作用，描绘Pkn1和PknD激酶/底物网络。合作将通过体外激酶测定来确认，磷酸化位点也将针对选定的底物进行确定。我们将在Aim 2中验证CTL0511（一种预测的蛋白磷酸酶）的功能。然后使用体外磷酸酶测定和各种蛋白质相互作用方法鉴定CTL0511底物。最后，在Aim 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。总的来说，这些研究将填补我们对衣原体发育的理解空白，为未来的研究方向提供指导，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.