SGER: Proteomics as a Tool for Cloning Leptin from Fence Lizard Sceloperus Undulatus

蛋白质组学作为从波状蜥蜴克隆瘦素的工具peter H. Niewiarowski1, Richard L. Londraville1， Michael kinter2阿克伦大学和克利夫兰诊所基金会脂肪代谢主要影响生物体对其获得的卡路里做出的生理选择。无论目标是发现人类肥胖的分子基础，还是了解冬眠动物如何以及何时积累脂肪以度过冬季，研究影响脂肪代谢的蛋白质都能让我们深入了解这些过程。瘦素是最近发现的一种蛋白质激素，影响脂肪代谢的合成代谢（脂肪储存）和分解代谢（脂肪燃烧）方面。由于瘦素有可能解释脂肪代谢的许多方面，人们对它进行了深入研究（自1994年以来有6000项研究）。几乎所有这些研究都集中在哺乳动物身上，对变温动物的关注相对较少。研究非哺乳动物体内的瘦素（以及其他影响脂肪代谢的蛋白质），为脂肪代谢研究带来了历史上强有力的比较生物学方法。尽管瘦素已经在几十种哺乳动物中被克隆和测序，但尽管付出了相当大的努力，却没有公布任何异温动物的序列。该奖项资助了一种新的方法，蛋白质组学，克隆和表征瘦素从栅栏蜥蜴（Sceloporus波动）。蛋白质组学是一门研究特定时间特定组织中所有蛋白质的学科。在先前的资助（IBN-0099303给PHN和RLL）下，在蜥蜴血液和大脑中发现了一种与瘦素抗体结合的蛋白质，其大小与哺乳动物的瘦素相同。在栅栏蜥蜴的自然种群中测量了这种假定的蜥蜴瘦素的浓度；蜥蜴的瘦素在春季和夏季达到峰值，然后在秋季显著下降，因为蜥蜴会积累脂肪，为冬季冬眠做准备。这与一些哺乳动物冬眠时瘦素浓度的模式是一致的。现在有可能利用这种季节变化的模式来比较蜥蜴在瘦素浓度的高峰和低谷时的蛋白质组。从肝脏、大脑、脂肪体和血清中提取蛋白质，并根据其电荷（第一维）和大小（第二维）通过凝胶电泳进行分离。这一过程产生了一个二维的蛋白质“地图”，这是每个组织的特征。这些二维地图将被扫描，并通过数字成像对同一种群的秋季和夏季蜥蜴进行比较。所有随季节相对量变化的蛋白质将提交质谱分析进行鉴定。质谱实验可以通过在已知氨基酸（蛋白质消化）处切割蛋白质，然后测量所得片段的质量来确定蛋白质的身份；这些质量的集合与所有已知的蛋白质序列进行比较，所有这些序列都是以相同的方式切割的（实际上，是由计算机切割的）。如果没有匹配，未知的可以进一步分离，以确定其氨基酸序列。该氨基酸序列随后可作为传统分子克隆实验和其他实验确定新蛋白功能的基础。拟议研究的更广泛影响是，通过将蛋白质组学纳入阿克伦大学（UA）本科生的实验室练习，它将促进教学、培训和学习。PHN和RLL都持有当前的教学奖学金（GK-12和CCLI），其中研究和教学的整合是一个重要目标。因此，这项研究将无缝地用于促进本科生和研究生的学习。该提案还将扩大代表性不足的群体的参与。亚利桑那大学生物学院有大量的少数民族学生，他们将接触到拟议的研究。此外，PN和RL在过去已经成功，并将继续招募代表性不足的群体参与拟议的研究。通过在UA和克利夫兰诊所基金会之间建立新的伙伴关系，我们将加强研究和教育的基础设施。拟议中的活动对社会的好处是显著的。这项研究是研究一种激素（瘦素）的新方法，这种激素在肥胖的全球健康流行中起着重要作用。

Proteomics as a tool for Cloning Leptin from fence lizard Sceloporus undulatusPeter H. Niewiarowski1, Richard L. Londraville1, Michael Kinter21The University of Akron and 2The Cleveland Clinic FoundationFat metabolism centrally influences physiological choices that organisms make with the calories they acquire. Whether the goal is to discover the molecular basis of obesity in humans, or to understand how and when a hibernator accumulates fat to survive the winter, studying proteins that influence fat metabolism gives insight into those processes. Leptin is a recently discovered protein hormone that affects both the anabolic (fat storage) and catabolic (fat burning) aspects of fat metabolism. Because leptin has the potential to explain so many aspects of fat metabolism, it has been studied intensely (6000 studies since 1994). Nearly all of these studies have been focused on mammals, with relatively little attention paid to ectotherms. Studying leptin (and other proteins that influence fat metabolism) in non-mammals brings the historically strong approach of comparative biology to studies of fat metabolism. Although leptin has been cloned and sequenced in dozens of mammals, no sequence is published for any ectotherms despite considerable effort. This award funds a new approach, proteomics, to cloning and characterizing leptin from fence lizard (Sceloporus undulatus). Proteomics is the study of all proteins present in a given tissue at a given time. Under previous funding (IBN-0099303 to PHN and RLL), a protein in lizard blood and brain that binds to leptin antibodies and is the same size as mammalian leptins was identified. Concentrations of this putative lizard leptin were measured in natural populations of fence lizards; lizard leptin peaks in the spring and summer and then declines significantly in the fall as the lizards accumulate fat and prepare for winter hibernation. This is consistent with the pattern of leptin concentrations described for some mammalian hibernators. It is possible now to use this pattern of seasonal variation to compare the proteomes of lizards at their peak and nadir of leptin concentrations. Proteins from liver, brain, fat bodies, and serum will be extracted and separated according to their charge (first dimension) and size (second dimension) by gel electrophoresis. This procedure results in a two-dimensional 'map' of proteins that is characteristic for each tissue. The two-dimensional maps will be scanned and compared (via digital imaging) between fall and summer lizards from the same population. All proteins that change in relative amount with season will be submitted to mass-spectrometry analyses for identification. The mass-spectrometry experiments can determine the identity of the protein by cutting it at known amino acids (protein digest) and then measuring the mass of the resulting pieces; this collection of masses is compared to all known protein sequences, all of which have been cut the same way (virtually, by a computer). If there is no match, the unknown can be further fractionated to determine its amino acid sequence. This amino acid sequence can subsequently be used as the basis for traditional molecular cloning experiments and other experiments to determine the function of the novel protein.The broader impacts of the proposed research are that it will promote teaching, training, and learning via incorporation of proteomics into laboratory exercises for undergraduates at the University of Akron (UA). PHN and RLL both hold current teaching scholarship grants (GK-12 and CCLI) in which integration of research and teaching is an essential goal. Therefore, this research will be used seamlessly to enhance learning for undergraduate and graduate students. The proposal will also broaden the participation of underrepresented groups. UA Biology has a significant minority student population, and they will be exposed to the proposed research. In addition, PN and RL have successfully in the past, and will continue to recruit underrepresented groups to work on the proposed research. By establishing a new partnership between UA and The Cleveland Clinic Foundation, we will enhance infrastructure for research and education. The benefits of the proposed activity to society are significant. This research is a new approach to studying a hormone (leptin) that is established to play a role in the global health epidemic of obesity.