As a first step towards
As a first step towards this goal, we here set out to investigate aspects of the biology of aRC enzymes in the animal source that we initially selected, C. intestinalis. In particular, in order to obtain clues on their probable physiological functions, we tested how their expression is modified by external conditions, focusing on stressors found in their natural environment. In a parallel set of studies, we investigated how the expression of aRC enzymes, specifically AOX, can modify the physiological responses of model organisms under stressful environmental conditions, focusing initially on temperature and nutrition, and on the already well characterized Drosophila models which are also much more easily manipulated than their mammalian counterparts.
Materials and methods
Results & discussion
Conclusions and perspectives Our global understanding of metabolism and how it impacts cell signaling remains limited. Thus, to predict the effects of introducing a major metabolic modification, such as the introduction of AOX (or NDX) into organisms that lack the aRC, is fraught with difficulties, and we should expect surprises. As summarized in Fig. 7, metazoan AOX, when activated by the accumulation of reduced quinol, plus other, as yet unidentified metabolic trigger(s), accelerates mitochondrial metabolism compared with an inhibited condition that it alleviates. However, it generally decelerates metabolism compared with the fully uninhibited condition, when electrons are able to pass freely to complex III, as inferred from the measured oxygen consumption of cultured AR-13324 . Mitochondrial NADH oxidation and ATP production should follow similar trends, although neither has yet been specifically measured. ATP production should be the more severely affected, given that a much greater proportion of the energy released by AOX-supported respiration is converted to heat rather than being used for ATP synthesis. However, because the net effects on metabolism may be diverse and complex, total cell NAD+ and ATP levels might remain stable or even rise. Similar but opposite considerations apply to ROS. Mitochondrial superoxide production is decreased by AOX in cells where OXPHOS is inhibited, but increased compared with fully uninhibited conditions . How this affects ROS in the rest of the cell is again not clear. Much remains to be documented and tested. Because the metabolic effects of AOX expression are not fully predictable, the many cellular regulatory pathways that respond to ROS, ATP, NAD, TCA cycle intermediates and other metabolites, as well as mitochondrial heat production, are likely to be affected in complex ways, leading to the readouts observed in our studies and elsewhere. These, in turn, are very likely to affect the metabolic triggers that govern AOX activation. About NDX we know even less at this time. For example, when expressed in mammalian cells, AOX does not interact with any of the mitochondrial OXPHOS complexes [54,72], but this has not yet been tested for NDX. A further intriguing question arises as to the functional interactions of AOX and NDX. In principle, if simultaneously active, they would catalyze a completely non proton-motive respiratory chain, although it is not known if this ever happens in a physiological situation, or whether the two enzymes are able to interact physically. An important question is how far they influence each other's activity, and which of them represents the effective control point for the aRC. In Drosophila, they can at least synergize functionally, e.g. in the tko mutant , as already mentioned. Thus, their combined effects on metabolism may differ from that of either alone. If the aRC enzymes are to be used in clinical applications, we will need a much better understanding of all these effects, including how both enzymes are naturally regulated, how they interact, how they access and modify the quinone pool(s), what broader impact they have on metabolism, and the precise mechanisms by which they influence cell signaling.