A structural context for the mechanism of Uncoupling protein-1

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Most of the energy that we gain from the breakdown of sugars, fats and other food components is harnessed by mitochondria, the 'power plants' of our cells. However, the process of energy conversion is not 100% efficient. In specialised 'brown fat' cells of mammals, the process is pro-actively 'short circuited' to burn off calories as heat. These cells do this using a particular protein called uncoupling protein 1 (UCP1). Heat production using UCP1 is important for many mammals including livestock to survive cold temperatures, especially newborns. Though it is also beneficial for adult humans. Active brown fat tends to occur in leaner people who are less likely to develop age-related obesity, consistent with the tissue expending excess calories and improving health. UCP1 activity in brown fat helps remove glucose and fat from the blood, which can help combat diabetes. At present, obesity and related poor health conditions are growing as a major national and international problem for which a better understanding of metabolism is urgently needed.There is now significant scientific research worldwide into ways to encourage the development of brown fat as a viable means to increase calorie-expenditure to deliver better health. However, an important feature of brown fat is that UCP1 is not inherently active in the cells and must be specifically 'switched on' to burn calories for heat. This normally occurs through the action of regulator molecules that activate UCP1 in response to physiological stimuli such as cold temperatures. Therapeutic strategies to artificially activate UCP1 in the absence of physiological stimuli have the potential to greatly increase the capacity of the tissue to burn calories. Yet at present we do not know how UCP1 works nor how the regulator molecules interact with the protein to turn it on.UCP1 is a membrane protein, which are generally difficult to study due to their insoluble nature and instability when isolated. Recently, however, there have been some key advances in our understanding of how a membrane protein related to UCP1 works, which provides new opportunities to determine the molecular mechanism of UCP1. My investigations have provided new methods to purify and assess UCP1, which have revealed the basic functional unit of the protein and the associated lipid molecules that stabilise it. The work outlined here aims to capitalize on these advances and clarify the molecular nature of UCP1. The protein will be purified from natural sources (newborn lambs) as well as yeast that have been engineered to make it. Details on how regulator molecules interact with the isolated protein will be investigated using biophysical methods that monitor either the heat released from the binding event or the associated changes in the protein's stability that occurs. UCP1 will also be incorporated into artificial 'liposome' membranes, so that details on how regulators influence its activity can be assessed. Genetic methods will be used to alter key parts of the protein to help determine where activating molecules binds, as well as the function of the protein's other notable structural features. Additionally, experiments to crystallise UCP1 and use x-ray crystallography methods to gain a detailed picture of what the protein looks like will be carried out.These studies will provide valuable insight into the molecular process used by UCP1, which will advance our fundamental understanding of how membrane proteins work but also the way in which energy metabolism is controlled in cells. The details revealed may provide clues to identify other cellular components involved in UCP1's activation, and will also be invaluable to rationalise therapeutic strategies to activate energy expenditure pathways for targeting obesity and metabolic disease.