MCA Pilot PUI: The role of proteomic changes during crustacean molting: from discovery to testing hypotheses

MCA Pilot PUI：甲壳类动物蜕皮过程中蛋白质组变化的作用：从发现到检验假设

• 批准号: 2321487
• 负责人: Lars Tomanek
• 金额: $ 25.69万
• 依托单位: California Polytechnic State University Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2321487&HistoricalAwards=false
• 关键词: MCA; Pilot; PUI; role; proteomic

This Mid-Career Advancement (MCA) award investigates the physiological mechanisms behind the crustacean growth process referred to as molting. The molting process allows growth in crustaceans (a group that includes crabs and shrimp), and is an intricately regulated process involving coordinated signals between at least two endocrine glands, the crustacean sinus gland/X-organ in the eyestalk and the Y-organ. The endocrine signals work through changes in gene expression, resulting in specific tissues being transformed into different cellular phenotypes, expressed through the resulting protein complement or proteome. In this project, the proteome of these tissues at different stages of molting will be characterized in two crab species to understand the complex signaling and endocrine control processes involved. The proteome is only accessible through a multi-step analytical pathway, requiring advanced mass spectrometry. The award provides protected time for the principal investigator to expand his skills to operate an advanced mass spectrometer and subsequently teach undergraduate and master’s level graduate students how to operate it. The application of mass spectrometers to analyze the proteomes of a wide range of organisms is advancing our knowledge of how the changing environment affects multiple economically important organisms. For example, the results from this work will advance understanding of the growth process of crustaceans and insects, because the molecular processes are similar in these two organismal groups, which are important for aquaculture and agriculture. The PI will apply his expertise to train other investigators and a new generation of students to extend proteomic analyses to a wide range of organisms and to build new collaborations. Crustacean molting is a cyclical event which replaces the carapace with a new exoskeleton that expands before it hardens, allowing the animal to grow. This process is suppressed during intermolt by the inhibition of ecdysteroidogenesis in the Y-organ (YO) by molt inhibiting hormone (MIH), which is produced by the sinus gland/X-organ in the eyestalk. Decreasing levels of MIH shift the YO from intermolt to premolt and increase its capacity to synthesize ecdysteroids to initiate the molt (ecdysis). The investigators hypothesize that this shift is controlled through mechanistic Target Of Rapamycin (mTOR), a Serine/Threonine-specific protein kinase, via a cyclic GMP-activated protein kinase (PKG). Using recombinant MIH on YOs in vitro with and without a PKG inhibitor, the research will test whether MIH signaling via PKG affects mTOR activity. When it is not inhibited, mTOR affects the transforming growth factor beta family of transcription factors, which shift the YO towards the committed state when it is refractory to MIH. Finally, the expression of ecdysteroid receptors and ecdysteroid responsive genes in the YO itself suggests that it is autoregulated by ecdysteroidogenesis when it shifts towards the repressed state. The investigators will also use the removal of the sinus gland/X-organ, which stops MIH secretion, and multiple leg autotomy, which initiates ecdysis, to experimentally manipulate molting. The principal investigator will characterize how the manipulations will affect the proteome of the Y-organ with the help of mass spectrometry.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

这项职业中期进步（MCA）奖调查了甲壳类增长过程背后的物理机制，称为摩擦。摩擦过程允许甲壳类动物（包括蟹和虾）的生长，并且是一个复杂的调节过程，涉及至少两个内分泌网格之间的协调信号，眼镜中的甲壳类窦/X-Organ和Y-Organ。内分泌信号通过基因表达的变化起作用，导致特定的组织被转化为不同的细胞表型，这些表型通过所得的蛋白质完成或保护小体表示。在这个项目中，将在两个螃蟹物种中表征这些组织的蛋白质组，以了解所涉及的复杂信号传导和内分泌控制过程。只有通过多步分析途径才能访问Protectome，需要高级质谱。该奖项为主要研究人员提供了受保护的时间，以扩大其技能以操作高级质谱仪，然后教授本科生和硕士学位的研究生如何操作。质谱仪在分析广泛生物的蛋白质组中的应用正在促进我们对不断变化环境如何影响多种经济上重要的生物的了解。例如，这项工作的结果将提高人们对甲壳类动物和昆虫的生长过程的理解，因为这两个有机群中的分子过程相似，这对于水产养殖和农业很重要。 PI将把他的专业知识应用于培训其他调查人员和新一代学生，以将蛋白质组学分析扩展到广泛的生物体并建立新的合作。甲壳类动物是一个周期性的事件，它用新的外骨骼取代了甲壳，该外骨骼在变硬之前膨胀，从而使动物生长。通过抑制Y-ORGAN（YO）在摩尔特（MIH）中抑制e骨（MIH）的霉菌生成在摩尔特间内抑制，这是由眼神中的鼻窦腺/X-Organ产生的。 MIH的水平降低了YO从木间转移到预抛出量，并增加了其合成ecd固醇以启动蜕皮的能力（递归）。研究人员假设这种转移是通过雷帕霉素（MTOR）（一种丝氨酸/苏氨酸特异性蛋白激酶的机械靶标）通过环状GMP激活的蛋白激酶（PKG）来控制的。在具有和不使用PKG抑制剂的情况下，使用重组MIH在YOS的体外使用，该研究将测试MIH信号通过PKG是否会影响MTOR活性。当不抑制它时，mTOR会影响转化因子的转录因子家族的转化因子家族，在对MIH的难治之下时，将YO转移到了所承诺的状态。最后，在YO本身中，ecdy-甾体受体和ecdysteroid的反应基因的表达表明，当ecdylysteroidepenopen朝向反射状态时，它会自动调节。研究人员还将使用停止MIH分泌的鼻窦腺/X-轨道的去除，并使用多个腿部自动切开术（启动逐渐分析）来实验操纵摩擦。首席研究人员将在质谱法的帮助下表征操作将如何影响Y-Organ的蛋白质组。该奖项反映了NSF的法定任务，并通过使用基金会的知识分子优点和更广泛的影响来审查标准，被视为通过评估而被视为珍贵的支持。