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.

该职业生涯中期进步 (MCA) 奖项研究了甲壳类动物生长过程（称为蜕皮）背后的生理机制。甲壳类动物（包括螃蟹和虾）的蜕皮过程允许生长，并且是一个涉及协调信号的复杂调节过程。至少有两个内分泌腺，即眼柄中的甲壳动物窦腺/X器官和Y器官，内分泌信号通过基因表达的变化发挥作用，导致特定组织转化为不同的细胞。通过所得蛋白质补体或蛋白质组表达的表型在该项目中，将在两个蟹种中表征不同蜕皮阶段的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。多步骤分析途径，需要先进的质谱仪，该奖项为主要研究人员提供了保护时间来扩展他操作先进质谱仪的技能，并随后教授本科生和硕士研究生如何操作质谱仪。用于分析多种生物体蛋白质组的光谱仪正在增进我们对不断变化的环境如何影响多种经济上重要的生物体的了解，例如，这项工作的结果将增进对甲壳类动物和昆虫生长过程的理解，因为分子过程。这两个有机体类群相似，这对水产养殖和农业很重要。首席研究员将运用他的专业知识来培训其他研究人员和新一代学生，将蛋白质组学分析扩展到广泛的生物体并建立新的合作。蜕皮是一种周期性事件，它会用新的外骨骼取代甲壳，新的外骨骼会在变硬之前膨胀，从而使动物能够生长。在蜕皮期间，蜕皮抑制激素 (MIH) 会抑制 Y 器官 (YO) 中的蜕皮类固醇生成，从而抑制这一过程。 )，由眼柄中的窦腺/X 器官产生。MIH 水平的降低使 YO 从蜕皮间转变为蜕皮前，并增加其合成能力。研究人员发现，这种转变是通过重组 MIH 的雷帕霉素靶点（mTOR）（一种丝氨酸/苏氨酸特异性蛋白激酶）来控制的。在体外使用和不使用 PKG 抑制剂的 YO 上，该研究将测试通过 PKG 的 MIH 信号传导是否会影响 mTOR 活性（当 mTOR 未被抑制时）。影响转录因子的转化生长因子β家族，当 YO 对 MIH 难治时，该家族将 YO 转变为定型状态。研究人员还将通过切除窦腺/X器官来阻止 MIH 分泌，并通过多腿自切术来启动蜕皮。首席研究员将在质谱的帮助下描述这些操作将如何影响 Y 器官的蛋白质组。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的评估被认为值得支持。影响审查标准。