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The mechanism of activation and inhibition of proton transport mediated by the adenine nucleotide translocase
Jürgen Kreiter
Elena E. Pohl
Lars Gille
Dissertation - Veterinärmedizinische Universität Wien - 2021
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Dissertation - University of Veterinary Medicine Vienna - 2021
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Mitochondrial uncoupling is the dissipation of the proton gradient that bypasses the production of ATP. For a long time, it was only associated with the uncoupling protein 1 (UCP1) in brown-adipose tissue, where it mediates non-shivering thermogenesis. The role of uncoupling was challenged by the discovery of further UCP homologs, which are expressed in diferent tissues. The finding that further mitochondrial carriers with diferent primary function, such as the adenine nucleotide translocase (ANT), also possess similar uncoupling function, raised the physiological relevance of mitochondrial uncoupling to a new level of complexity. Thus, it seems to be an inherent function of several carriers and is present in all mitochondria of all tissues. However, diferent experimental approaches lead to assorted results regarding the regulation of UCP- and ANT-mediated uncoupling. Despite the known crystallographic structures of ANT, the structure-function relationship for the ANT-mediated uncoupling remains elusive. In this work, we implemented the production of recombinant murine ANT1 to our research and showed its proper ADP/ATP transporting function in liposomes using a novel fluorescence-based approach. Furthermore, we established a protocol which allows a more precise and robust investigation of the proton turnover number using versatile salt-bridge electrodes and showed that the proton transfer rate is similar for UCP1, UCP3 and ANT1. Our results showed that the activation and inhibition of ANT1-mediated proton transport is well described by the fatty acid cycling mechanism, similar to UCP1 and UCP2. We postulate that UCP1-3 and ANT1 have a similar regulation mechanism and that fatty acid anion transport is an inherent function of UCPs and ANT1. Based on our results, we concluded that the fatty acid anion transport by UCPs and ANT follows a conserved pathway. Thus, UCPs and ANT have dual transport function: (i) substrate transport in the absence and (ii) fatty acid anion transport in the presence of free fatty acids. Our results significantly contribute to understand how mitochondrial carriers are involved in the interdependence of mitochondrial coupling and uncoupling. Since the imbalance of the mitochondrial coupling and uncoupling is involved in diseases such as cancer, diabetes and obesity, our findings will be crucial for the design of drugs and therapies against mitochondria associated diseases.
Englisch
2021
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