Browsing by Subject "Uncoupling protein 3"
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Item Investigation of the proteomic interaction profile of uncoupling protein 3 and its effect on epigenetics(2014-08) Yan, Xiwei; Mills, Edward MichaelUncoupling proteins (UCPs) are localized on the inner mitochondrial membrane (IMM) and “uncouple” the electrochemical proton gradient formed by the electron transport chain (ETC) from ATP production. Though the prototypical uncoupling protein 1 (UCP1) is known to mediate the cold-induced thermogenesis in rodents and human neonates, the physiological and biochemical functions of the homologs UCP2-5 are still under debate. Our research focuses on UCP3, the homolog prevalently expressed in skeletal muscle (SKM), the most important metabolic organs. UCP3 has long been speculated to have a pivotal role in maintaining the mitochondrial metabolism. Several biochemical roles have been attributed to UCP3, including the regulation of fatty-acid transport and oxidation, reactive oxygen species (ROS) scavenging and calcium uptake. And several proteins have been identified to directly bind with UCP3 and facilitate its function. But to further understand how UCP3 relates to different aspects of mitochondrial functions, a more comprehensive profile of the UCP3 interaction partners is needed. We performed a mass spectrometry-based experiment and successfully identified a list of over 170 potential proteins that may directly or indirectly interact with UCP3, and several novel functions of UCP3 are implied by these protein-protein interactions. Additionally, researches have shown that the metabolic defects are important contributing factors to the epigenetic changes. Considering the roles of UCP3 in sustaining the normal mitochondrial metabolism, we hypothesized that UCP3 has a novel function in regulating the genomic DNA methylation processes. The data we obtained from the pilot study confirms that loss of UCP3 will lead to aberrant DNA methylation changes. But further experiment is still needed to investigate the regulatory pathway between UCP3 and DNA methylation. The physiological role of UCP3 in defending against cancer, diabetes and obesity has been investigated, but the mechanisms how UCP3 protect the organism from these diseases have not been elucidated. Our research sheds light on the understanding of UCP3 functions and may be of significant therapeutic benefit in the prevention and treatment of these diseases.Item Mitochondrial uncoupling protein 3 blocks skin carcinogenesis and drives bulge stem cell differentiation and epidermal turnover(2009-05) Lago, Cory Ungles; Mills, Edward MichaelMalignant cells increase glycolysis and down regulate mitochondrial respiration for ATP production. Mechanisms for respiratory impairment in cancerous cells and their importance for carcinogenesis are not well defined. We found that expression of the respiration-inducing uncoupling protein 3 (UCP3) was normally expressed in murine skin and was greatly decreased in cutaneous malignancies. To better understand the significance of UCP3 in epidermal biology and to test the importance of respiratory changes in cancer development, we generated hemizygous mice expressing a keratin-5 promoter-UCP3 transgene (K5-UCP3). Compared to wild type, K5-UCP3 mice exhibited increased cutaneous mitochondrial respiration, had decreased mitochondrial membrane potential in isolated keratinocytes, and were completely resistant to chemically-induced skin carcinogenesis. We showed that the mechanism of UCP3-dependent cancer protection is most likely not due to increased intracellular heat production or ATP depletion in pre-cancerous cells. Therefore, because hair follicle "bulge" stem cells (bSC) are K5⁺ and progenitors of cutaneous carcinomas, we hypothesized that K5-UCP3 animals were protected from skin carcinogenesis due to alterations in their bSC population. Unlike WT, most (85%) hair follicle bulge regions in K5-UCP3 mice lost biochemical markers of quiescent bSC, but bSC functions were fully intact. Supporting our hypothesis that increased skin turnover protected K5-UCP3 mice from skin cancer; we showed that basal keratinocyte cell cycling was increased 3% in K5-UCP3 skin compared to WT. Moreover, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced similar proliferative responses in both WT and K5-UCP3 skin, but the magnitude of TPA-induced skin thickening was greatly decreased in K5-UCP3 versus WT mice. Together with microarray, histochemical and in vitro morphologic analyses showing that keratinocyte differentiation was sharply increased in K5-UCP3 skin, this implies that UCP3 may increase keratinocyte transit from stem to differentiated daughter cells. Thus, the cancer resistance mechanism in K5-UCP3 mice likely stems from UCP3-induced mitochondrial respiration, which promotes the differentiation and abrogates the tumorigenicity of progenitor keratinocytes. This is the first demonstration in any context that UCP3 blocks carcinogenesis and promotes cellular differentiation. These observations support Warburg's contention that respiratory dysfunction promotes cancer development, and suggest that mitochondrial uncoupling may be a novel target for cancer prevention and treatment.Item The underlying mechanisms of UCP3-dependent thermogenesis in skeletal muscle(2015-12) Dao, Christine Ky Linh; Mills, Edward Michael; Wright, Casey; Bratton, Shawn; Mukhopadhyay, Somshuvra; Ivy, JohnMitochondrial uncoupling proteins (UCPs) are anion / solute transporters that dissipate the proton gradient used to drive ATP generation. By allowing protons to flow down their electrochemical gradient, UCP activation releases the energy generated from mitochondrial substrate oxidation as heat. This thermogenic process is important in normal thermoregulation (i.e. non-shivering thermogenesis), and also serves as an attractive target in the treatment of obesity by lowering metabolic efficiency. The skeletal muscle (SKM) enriched UCP homologue, UCP3, is associated with increased energy expenditure, fatty acid metabolism, and insulin sensitivity. Unlike the cold-induced prototypical pathway of UCP1-mediated non-shivering thermogenesis in brown adipose tissue (BAT), the mechanisms underlying the thermogenic actions of UCP3 in SKM are not well characterized. Although global UCP3 knockout mice exhibit normal thermoregulatory responses to cold under fed conditions, they exhibit an attenuated hyperthermic response when administered amphetamine-type drugs. In our initial investigation, we show that selective overexpression of UCP3 in SKM by the human α-skeletal actin promoter restored methamphetamine (Meth)-induced hyperthermia in the UCP3⁻/⁻ background (TgSKM UCP3⁻/⁻), but not in the UCP1/UCP3 double knockout background (TgSKM UCP1⁻/⁻+UCP3⁻/⁻). Taken together, these findings further bolster the role of UCP3 as a thermogenic mediator in SKM, and suggest a novel mechanism of crosstalk between BAT UCP1 and SKM UCP3 in Meth-induced hyperthermia. In the second aspect of my project, we characterized the underlying mechanisms of UCP3-dependent thermogenesis within SKM by utilizing an immunoprecipitation- based mass spectrometry approach to identify interacting partners of UCP3. These analyses corroborated previous work performed by our lab, and demonstrated that UCP3 interacts with a subset of fatty acid metabolizing enzymes. Interestingly, one such enzyme, enoyl-CoA hydratase-1 (ECH1), is involved in the metabolism of oleic acid, a known ligand activator of UCP3. This work reveals that ECH1:UCP3 complex formation enhances uncoupled-respiration and fatty acid metabolism, and that genetic mouse models in vivo show that UCP3 and ECH1 participate in a common pathway of thermogenesis. These findings support a new model by which UCP3-dependent thermogenesis in SKM is mediated in part through its cooperation with ECH1, and suggest new approaches for treatment of obesity and related metabolic diseases.