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

这个中期职业发展（MCA）奖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，并且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，甲壳动物窦腺/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起作用，导致特定组织转化为不同的细胞表型，通过所得蛋白质补体或蛋白质组表达。在这个项目中，这些组织在蜕皮的不同阶段的蛋白质组将在两个螃蟹物种的特点，以了解复杂的信号和内分泌控制过程。蛋白质组只能通过多步分析途径获得，需要先进的质谱法。该奖项为首席研究员提供了保护时间，以扩展其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。应用质谱仪分析各种生物体的蛋白质组正在推进我们对不断变化的环境如何影响多种经济重要生物体的认识。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，因为这两种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广泛的生物体，并建立新的合作。甲壳动物的蜕皮是一个周期性的事件，它用一个新的外骨骼取代甲壳，在它变硬之前膨胀，使动物生长。蜕皮抑制激素（MIH）抑制Y器官（YO）中的蜕皮类固醇生成，从而在蜕皮间期抑制该过程，该激素由眼柄中的窦腺/X器官产生。MIH水平的降低使YO从蜕皮间期转变为蜕皮前期，并增加其合成蜕皮类固醇的能力以启动蜕皮（蜕皮）。研究人员假设，这种转变是通过雷帕霉素（mTOR），丝氨酸/苏氨酸特异性蛋白激酶，通过环GMP激活的蛋白激酶（PKG）的机制控制的。在有和没有PKG抑制剂的情况下，在体外对YO使用重组MIH，该研究将测试经由PKG的MIH信号传导是否影响mTOR活性。当它不被抑制时，mTOR影响转录因子的转化生长因子β家族，当它对MIH不敏感时，其将YO向定型状态转变。最后，蜕皮激素受体和蜕皮激素反应基因在YO本身的表达表明，它是自动调节蜕皮激素生成时，它转向抑制状态。研究人员还将使用窦腺/X器官的切除，这将阻止MIH分泌，以及多条腿的自切，这将启动蜕皮，以实验性地操纵蜕皮。主要研究者将在质谱仪的帮助下描述操作如何影响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。

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.