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.

这个中期职业发展（MCA）奖调查了甲壳类动物生长过程背后的生理机制，即蜕皮。甲壳类动物（包括螃蟹和虾）的蜕皮过程是一个复杂的调节过程，涉及至少两个内分泌腺（眼柄上的甲壳类窦腺/ x器官和y器官）之间的协调信号。内分泌信号通过基因表达的变化起作用，导致特定组织转化为不同的细胞表型，通过产生的蛋白质补体或蛋白质组表达。本项目将对两种螃蟹蜕皮不同阶段这些组织的蛋白质组进行分析，以了解其中复杂的信号传导和内分泌控制过程。蛋白质组只能通过多步骤分析途径获得，需要先进的质谱分析。该奖项为首席研究员提供了保护时间，以扩展他操作先进质谱仪的技能，并随后教本科生和硕士研究生如何操作它。质谱分析多种生物的蛋白质组学的应用正在推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的认识。例如，这项工作的结果将促进对甲壳类动物和昆虫生长过程的理解，因为这两个生物群体的分子过程相似，这对水产养殖和农业具有重要意义。PI将运用他的专业知识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更广泛的生物体，并建立新的合作关系。甲壳类动物的蜕皮是一个周期性的过程，在甲壳变硬之前，新的外骨骼会膨胀，从而使动物得以生长。脱毛抑制激素（MIH）抑制y器官（YO）的蜕皮甾体形成，这一过程在脱毛期间受到抑制，MIH是由眼柄窦腺/ x器官产生的。MIH水平的降低使脱毛胚从脱毛间转到脱毛前，并增加其合成蜕皮甾体的能力，以启动蜕皮（蜕皮）。研究人员假设，这种转变是通过一种环gmp激活的蛋白激酶（PKG），通过丝氨酸/苏氨酸特异性蛋白激酶雷帕霉素（mTOR）的机制靶标（mTOR）来控制的。利用重组MIH在体外有或没有PKG抑制剂的yo上，研究将测试通过PKG传递的MIH信号是否影响mTOR活性。当不受抑制时，mTOR会影响转录因子的转化生长因子β家族，当YO对MIH不耐受时，mTOR会将YO转向承诺状态。最后，外皮甾体受体和外皮甾体应答基因在YO本身的表达表明，当它转向抑制状态时，它受到外皮甾体发生的自动调节。研究人员还将通过切除窦腺/ x器官（阻止MIH分泌）和多腿自切术（启动蜕皮）来实验性地操纵蜕皮。首席研究员将在质谱分析的帮助下描述操作将如何影响y型器官的蛋白质组。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。