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

描述（由申请方提供）：本提案的目的是通过初步阐明蛋白激酶/磷酸酶网络，开始阐明蛋白质Ser/Thr/Tyr磷酸化在调节衣原体生理学和发病机制中的作用。蛋白质Ser/Thr/Tyr磷酸化越来越被认为是通过蛋白质功能的可逆改变来调节对生长和存活（以及因此发病机理）至关重要的细菌过程的广泛使用的机制。衣原体属物种经历一个独特的双相发育周期，在环境稳定的EB和复制性的细胞内RB之间过渡，这似乎需要广泛调节蛋白质合成和功能。令人困惑的是，它们的基因组中只有不到5%的基因是为典型的转录调节因子保留的。此外，EB向RB的早期分化发生在DNA被组蛋白样蛋白封闭且仅存在低水平RNA的条件下。这些生物学“问题”表明使用非经典的解决方案来调节衣原体属中的途径功能。因此，我们假设衣原体属。利用整体蛋白质磷酸化作为响应应激源和调节分化的机制。与这一假设相一致，C.使用2D凝胶电泳、磷蛋白染色和MALDI-TOF-TOF分析的细胞周期分析鉴定了EB和RB中的42个阶段特异性磷酸化蛋白（98%是泛衣原体蛋白）。此外，还对衣原体进行了1D凝胶电泳磷蛋白染色分析。进一步支持了跨衣原体存在丰富水平的磷蛋白。在这项R15研究中提出的工作将进一步解决我们的假设，使用系统的，自下而上的方法，重点是建立和验证激酶/底物和磷酸酶/底物相互作用组集中在沙眼衣原体。在目标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Pkn 1和PknD激酶/底物网络。将使用体外激酶测定来确认伙伴关系，并且还将确定所选底物的磷酸化位点。我们将寻求验证目标2中预测的蛋白磷酸酶CTL 0511的功能。然后使用体外磷酸酶测定和各种蛋白质-蛋白质相互作用方法鉴定CTL 0511底物。最后，在目标3中，我们将进一步定义包括磷酸化调节的衣原体伴侣转换机制的相互作用伴侣。总的来说，这些研究将填补我们对衣原体发展的理解的空白，提供未来的研究方向，确定治疗和抗感染的新靶点，并阐明蛋白磷酸化对细菌生理学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.