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

SGER:蛋白质组学作为克隆篱笆蜥蜴瘦素的工具

基本信息

  • 批准号:
    0328554
  • 负责人:
  • 金额:
    $ 8万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
    Standard Grant
  • 财政年份:
    2003
  • 资助国家:
    美国
  • 起止时间:
    2003-08-01 至 2005-07-31
  • 项目状态:
    已结题

项目摘要

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.
蛋白质组学作为从篱笆蜥蜴 Sceloporus undulatus 克隆瘦素的工具Peter H. Niewiarowski1、Richard L. Londraville1、Michael Kinter21阿克伦大学和 2克利夫兰诊所基金会脂肪代谢集中影响生物体利用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,还是了解冬眠者如何以及何时积累脂肪以度过冬天,研究影响脂肪代谢的蛋白质都可以深入了解这些过程。 瘦素是一种最近发现的蛋白质激素,它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,因此对其进行了深入的研究(自 1994 年以来已进行了 6000 项研究)。 几乎所有这些研究都集中在哺乳动物身上,而对变温动物的关注相对较少。 研究非哺乳动物的瘦素(和其他影响脂肪代谢的蛋白质)将传统的比较生物学方法引入到脂肪代谢的研究中。 尽管已经在数十种哺乳动物中克隆并测序了瘦素,但尽管付出了相当大的努力,但尚未公布任何变温动物的序列。 该奖项资助一种新的蛋白质组学方法,用于克隆和表征栅栏蜥蜴(Sceloporus undulatus)的瘦素。 蛋白质组学是对给定时间给定组织中存在的所有蛋白质的研究。 在之前的资助(IBN-0099303 给 PHN 和 RLL)的资助下,蜥蜴血液和大脑中的一种蛋白质被发现,它与瘦素抗体结合,并且与哺乳动物瘦素的大小相同。 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;蜥蜴瘦素在春季和夏季达到峰值,然后在秋季显着下降,因为蜥蜴会积累脂肪并为冬季冬眠做准备。 这与一些哺乳动物冬眠者的瘦素浓度模式一致。 现在可以利用这种季节性变化模式来比较瘦素浓度峰值和最低点时蜥蜴的蛋白质组。 来自肝脏、大脑、脂肪体和血清的蛋白质将通过凝胶电泳根据其电荷(第一维)和大小(第二维)被提取和分离。 该过程产生每个组织特有的二维蛋白质“图”。 二维地图将被扫描并比较(通过数字成像)同一种群的秋季和夏季蜥蜴。所有随季节相对量发生变化的蛋白质都将进行质谱分析以进行鉴定。 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,然后测量所得碎片的质量来确定蛋白质的身份;将这些质量集合与所有已知的蛋白质序列进行比较,所有这些序列都以相同的方式(实际上是由计算机)切割。 如果不匹配,则可以进一步分级未知物以确定其氨基酸序列。 该氨基酸序列随后可用作传统分子克隆实验和其他实验的基础,以确定新型蛋白质的功能。该研究的更广泛影响是,它将通过蛋白质组学的结合促进教学、培训和学习为阿克伦大学 (UA) 本科生进行实验室练习。 PHN 和 RLL 均持有当前的教学奖学金(GK-12 和 CCLI),其中研究与教学的整合是一个基本目标。因此,这项研究将无缝地用于加强本科生和研究生的学习。该提案还将扩大代表性不足群体的参与。 UA 生物学拥有大量少数族裔学生,他们将接触到拟议的研究。此外,PN 和 RL 过去已经取得了成功,并将继续招募代表性不足的群体来开展拟议的研究。通过建立 UA 和克利夫兰诊所基金会之间的新合作伙伴关系,我们将加强研究和教育基础设施。拟议活动对社会的好处是显着的。这项研究是研究一种激素(瘦素)的新方法,该激素在全球肥胖健康流行中发挥着作用。

项目成果

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Peter Niewiarowski其他文献

Peter Niewiarowski的其他文献

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{{ truncateString('Peter Niewiarowski', 18)}}的其他基金

Workshop: Designing a Network for Undergraduate Biomimicry Research and Education; October 6-8, 2017, Cleveland/Akron, Ohio
研讨会:设计本科生仿生学研究和教育网络;
  • 批准号:
    1747598
  • 财政年份:
    2017
  • 资助金额:
    $ 8万
  • 项目类别:
    Standard Grant
GK-12 Formal Proposal
GK-12 正式提案
  • 批准号:
    0086378
  • 财政年份:
    2001
  • 资助金额:
    $ 8万
  • 项目类别:
    Continuing Grant
Isolating and Characterizing the Fat-Regulating Hormone Leptin in the Fence Lizard
栅栏蜥蜴中脂肪调节激素瘦素的分离和表征
  • 批准号:
    0099303
  • 财政年份:
    2001
  • 资助金额:
    $ 8万
  • 项目类别:
    Standard Grant

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