Proteomics as a tool for Cloning Leptin from fence lizard Sceloporus undulatus Peter H. Niewiarowski1, Richard L. Londraville1, Michael Kinter2 1The University of Akron and 2The Cleveland Clinic Foundation Fat 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.
