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

揭示蛋白质磷酸化在发育调节中的作用

• 批准号: 8771143
• 负责人: Derek James Fisher
• 金额: $ 41.75万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8771143
• 关键词: 6H,8H-3,4-dihydropyrimido(4,5-c)(1,2)oxazin-7-one; Address; Anti-Infective Agents; Antibodies; Bacteria; Bacterial Physiology; Biological; Biological Assay; Cell Culture Techniques; Chlamydia; Chlamydia trachomatis; Chlamydophila pneumoniae; DNA; Development; Drug Targeting; Gel; Generic Drugs; Genome; Genomics; Glycine decarboxylase; Growth; Growth and Development function; Histones; Human; In Vitro; Infection; Libraries; Maps; Methods; Modeling; Modification; Pathogenesis; Pathogenicity; Pathway interactions; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylated Peptide; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiology; Play; Pneumonia; Process; Production; Protein Biosynthesis; Protein Dephosphorylation; Protein Kinase; Protein phosphatase; Protein-Serine-Threonine Kinases; Proteins; Proteome; Public Health; RNA; Recombinants; Regulation; Reporting; Role; Sexually Transmitted Diseases; Sigma Factor; Site; Solutions; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staging; Staining method; Stains; System; Testing; Time; Trachoma; Two-Dimensional Gel Electrophoresis; United States; Virulence; Western Blotting; Work; acrosome stabilizing factor; base; gel electrophoresis; in vitro Assay; kinase inhibitor; new therapeutic target; novel; pathogen; preference; prevent; protein function; protein protein interaction; public health relevance; sensor; stressor; vector; yeast two hybrid system

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.

描述（由申请人提供）：该建议的目的是开始解决蛋白质Ser/Thr/Tyr磷酸化在调节衣原体生理学和发病机理中的作用，从而初始阐明蛋白激酶/磷酸酶网络。蛋白质Ser/Thr/Tyr磷酸化越来越多地被认为是通过可逆改变蛋白质功能来调节细菌过程对生长和生存至关重要的细菌过程的广泛机制。衣原体。在环境稳定的EB与复制性的细胞内RB之间进行独特的双相发育周期过渡，这似乎需要广泛调节蛋白质合成和功能。令人不安的是，其<5％的基因组保留给规范的转录调节剂。此外，在DNA被组蛋白样蛋白遮住并且仅存在低水平的RNA的条件下，EBS早期分化为RBS。这些生物学“问题”表明使用非古典溶液来调节衣原体中的途径功能。因此，我们假设衣原体属。利用全球蛋白质磷酸化作为响应压力并调节分化的机制。与这一假设一致，使用2D凝胶电泳，磷蛋白染色和MALDI-TOF-TOF分析映射C. caviae的EB/RB磷酸蛋白质组，鉴定了42个EBS和RBS中的42阶段特异性磷酸化蛋白（98％是泛磷脂蛋白）。此外，衣原体属属1D凝胶电泳磷酸蛋白染色分析。进一步支持整个衣原体中大量的磷蛋白水平。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方法，该方法着重于建立和验证针对沙眼衣原体的激酶/底物和磷酸酶/磷酸酶/底物相互作用。在AIM 1中，我们将使用多种方法来检测蛋白质 - 蛋白质相互作用来描述PKN1和PKND激酶/底物网络。将使用体外激酶测定法确认合作伙伴，并且还将确定针对某些底物的磷酸化位点。我们将寻求验证CTL0511（一种预测的蛋白质磷酸酶）的功能。最后，在AIM 3中，我们将进一步定义包括磷酸化调节的衣原体伴侣交换机制的相互作用伙伴。总的来说，这些研究将填补我们对衣原体发展的理解，提供未来的研究方向，确定对治疗和抗感染的新目标，并阐明蛋白质磷酸化对细菌生理和致病性的更广泛后果。