Browsing by Subject "Sarcolipin"

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    Multi-omic analysis of hibernator skeletal muscle and calcium handling regulation
    (2016-05) Anderson, Kyle
    Mammalian hibernation is a strategy employed by many species to survive fluctuations in resource availability and environmental conditions. Hibernating mammals endure conditions of dramatically depressed heart rate, body temperature, and oxygen consumption; yet do not show the typical pathological responses. Because of the high abundance and metabolic cost of skeletal muscle, not only must it adjust to the constraints of hibernation, but it is also positioned to play a more active role in the initiation and maintenance of the hibernation phenotype. My M.S. thesis research has primarily focused on the generation and analysis of two high-throughput ‘omics screens in thirteen-lined ground squirrel skeletal muscle. A transcriptomic analysis using Illumina HiSeq2000 technology identified 1,466 differentially expressed genes throughout their circannual cycle. This RNAseq data allowed for greater protein identifications in an iTRAQ based proteogeomic analysis of the same animals. Of the 1,563 proteins identified by this proteogenomic approach, 232 were differentially expressed. These data support previously reported physiological transitions, while also offering new insight into specific mechanisms of how hibernator muscles might be reducing nitrogenous waste, preserving mass and function, and signaling to other tissues. Sarcolipin is a specific gene of interest that shows a 10-fold difference in expression between hibernation and spring collection points. Because of sarcolipin’s interaction with the SERCA pump and their role in muscle-based thermogenesis and calcium homeostasis bioenergetics, I have developed methods to measure the consequences of this differential expression.
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    Structural Characterization of Sarcolipin by Solid State NMR and Investigation of its Role in the Regulation of Sarco(endo)plasmic Reticulum Calcium Adenosine-Triphospatase
    (2014-02) Mote, Kaustubh
    Structural characterization of membrane proteins and their complexes is an important and ever growing challenge to the classical techniques of biomolecular structural characterization. Rapid developments in the field of solid state NMR (ssNMR) have opened up an exciting new alternative to X-ray crystallography, as these studies can now be performed in fully hydrated lipid bilayers that faithfully mimic the physiologically relevant conditions. Nonetheless, routine application of ssNMR on biomolecular systems is hampered by their low sensitivity and spectral resolution. In this work, we have addressed these challenges by developing new strategies to study membrane proteins by ssNMR. With a set of improved pulse sequences for oriented and magic angle spinning techniques in ssNMR, we determined the topology (i.e. the structure and transmembrane orientation) of sarcolipin, a regulator of the Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA), in lipid bilayers. These techniques are further used to study the complex between these two proteins and understand the molecular basis for this regulatory interaction. The methodological developments reported here are transferable to studies on other membrane proteins and they clear several roadblocks in the successful application of ssNMR for these challenging bio-molecular systems. Finally, we present how these studies have furthered our understanding of the regulation of muscle relaxation process by SERCA. These findings represent the first steps in designing new therapeutic approaches for cardiac and skeletal muscle disorders.