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

这一职业中期促进奖(MCA)调查甲壳类动物生长过程背后的生理机制，称为蜕皮。蜕皮过程允许甲壳类动物(包括螃蟹和虾)的生长，是一个复杂的调节过程，涉及至少两个内分泌腺之间的协调信号，即甲壳类鼻窦腺/眼柄中的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表型，通过产生的蛋白质补体或蛋白质组表达。在这个项目中，这些组织在不同的蜕皮阶段的蛋白质组将在两个螃蟹物种中进行表征，以了解涉及的复杂信号和内分泌控制过程。蛋白质组只能通过多步骤分析途径才能获得，需要先进的质谱学。该奖项为首席研究员提供了受保护的时间，让他扩展自己的技能，操作先进的质谱仪，并随后教授本科生和硕士研究生如何操作它。应用质谱仪分析各种生物的蛋白质组，促进了我们对环境变化如何影响多个具有重要经济意义的生物的认识。例如，这项工作的结果将促进对甲壳类和昆虫生长过程的了解，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很重要。PI将应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。甲壳动物的蜕皮是一种周期性事件，它会用新的外骨骼取代甲壳，外骨骼在变硬之前会膨胀，从而使动物得以生长。蜕皮抑制激素(MIH)是由眼柄中的窦腺/X器官产生的，通过抑制Y器官(Yo)中蜕皮类固醇的生成来抑制这一过程。MIH水平的降低将Yo从蜕皮间转移到蜕皮前，并增加了其合成蜕皮类固醇以启动蜕皮(蜕皮)的能力。研究人员假设，这种转变是通过机制靶点雷帕霉素(MTOR)，一种丝氨酸/苏氨酸特异的蛋白激酶，通过循环的GMP激活的蛋白激酶(PKG)来控制的。将重组的MIH在体外有无PKG抑制剂的情况下作用于yos，研究将测试通过PKG的MIH信号是否影响mTOR活性。当mTOR不被抑制时，它会影响转化生长因子β转录因子家族，当YO对MIH无效时，它会将YO转变为承诺状态。最后，蜕皮类固醇受体和蜕皮类固醇反应基因在Yo本身的表达表明，当Yo向抑制状态转变时，它是由蜕皮类固醇合成来自我调节的。研究人员还将使用切除鼻窦腺/X器官来阻止MIH分泌，以及多腿自体切断术来实验性地操纵蜕皮。首席研究人员将借助质谱学来描述操纵将如何影响Y器官的蛋白质组。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.