Mitochondria are a major site of reactive oxygen species (ROS) production in cells. While ROS can cause oxidative damage, they are vital in many signaling processes. Recently, mitochondrial superoxide flashes (mSOF) were defined through sensitive measurements of temporal and spatial differences in superoxide production. mSOF are stochastic events of quantal bursts in superoxide production, which are temporally linked to transient mitochondrial inner membrane depolarizations. The aims of the present study were to characterize a hydrogen peroxide sensitive biosensor to monitor these events and elucidate a role for uncoupling protein 3 (UCP3) and the mechanistic details of mSOF. While pHyPer- dmito was sensitive enough to monitor these dynamic changes its kinetics were insufficient to detect these ~20s long flashes. Additionally, analyses showed a prolonged duration of flashes in the absence of UCP3. Furthermore, we unearthed a novel relationship between flash amplitude and mitochondrial depolarization. Finally, investigations of mSOF in muscles of various fiber type compositions showed no differences, though additional investigations are warranted.

Uncoupling 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. / text

Myocardial and skeletal muscle high energy phosphate metabolism is abnormal in heart failure, but the pathophysiology is not understood. Plasma non-esterified fatty acids (NEFA) increase in heart failure due to increased sympathetic drive, and regulate the transcription of mitochondrial uncoupling protein-3 (UCP3), through peroxisome proliferator-activated receptor-α. The aim of the work in this thesis was to determine whether cardiac PCr/ATP ratios and skeletal muscle PCr kinetics during exercise were related to cardiac and skeletal muscle UCP3 levels respectively, thus providing a mechanism for the apparent mitochondrial dysfunction observed in heart failure. Patients having cardiac surgery underwent pre-operative testing, including cardiac and gastrocnemius 31P magnetic resonance spectroscopy. Intra-operatively, ventricular, atrial and skeletal muscle biopsies were taken for measurement of mitochondrial protein levels by immunoblotting, along with mitochondrial function by tissue respiration rates. Fasting plasma NEFA concentrations increased in patients with ventricular dysfunction and with New York Heart Association (NYHA) class. Ventricular UCP3 levels increased and cardiac PCr/ATP decreased with NYHA class, however, demonstrated no relationship to each other. In skeletal muscle, maximal rates of oxidative ATP synthesis (Qmax) related to functional capacity. Skeletal muscle UCP3 levels increased with NYHA class but were unrelated to skeletal muscle Qmax. Tissue respiration experiments revealed no relationship between ventricular function and indices of mitochondrial coupling, furthermore, indices of mitochondrial coupling were unrelated to tissue UCP3 levels. No evidence was found to support mitochondrial uncoupling, mediated through UCP3, as a cause of the abnormalities in cardiac and skeletal muscle high energy phosphate metabolism.

Malignant 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. / text

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