Protein-bound 3,4-dihydroxyphenylalanine (PB-DOPA),a long-lived, redox-active product of protein oxidation, is capable of functioning as both a pro- and anti-oxidant. A number of in vitro and in vivo studies have demonstrated a toxic, non-toxic or even beneficial effect of free DOPA, however little investigation has examined the physiological activity of PB-DOPA. Furthermore, as free DOPA is currently the major treatment available for Parkinson's disease, most studies have focused on the effect of DOPA within neurological cells or tissues, although the presence of PB-DOPA in other locations, for example within atherosclerotic plaques, suggests that broader research is needed to fully understand the physiological effects of both free and PB-DOPA. The hypothesis presented in this thesis is that under physiological conditions, when little redox active transition metal is available, PB-DOPA can function as a redox signalling molecule, triggering an enhancement of cellular antioxidant defences, with a potentially specific role in the regulation of defences targeted against protein oxidation. Physiological levels of PB-DOPA are very low, however the level on individual proteins can change to a proportionally large degree during oxidative stress, an appropriate property for a signalling molecule. In addition, remarkably elevated levels occur in some pathologies, including atherosclerosis. As an initial and commonly formed product of protein oxidation, PB-DOPA is well placed for a signalling role, promoting a significant up-regulation of antioxidant defences in the early stages of oxidative stress, before extensive damage has occurred. As an initiator of antioxidant defences, PB-DOPA would be potentially useful as a therapeutic for the treatment of diseases involving oxidative stress or the accumulation of oxidative damage. The main objective of this thesis was, therefore, to examine the effect of PB-DOPA on the cellular antioxidant defence system using monocytic and macrophage-like cells, key cells involved in the formation of atherosclerotic plaques. The incorporation of free DOPA into protein during protein synthesis, a process previously shown to occur both in vitro and in vivo, was used to generate PB-DOPA. Neither free nor PB-DOPA were found to be toxic to monocytic or macrophage-like cells in culture, but rather were both capable of protecting these cells from oxidative stress. Free DOPA was shown to be capable of directly scavenging radicals, a process that was thought to be in part responsible for the protection induced during oxidative stress. The presence of free and PB-DOPA up-regulated the activity of catalase and NAD(P)H: quinone oxidoreductase, two enzymatic antioxidants, however the activity of superoxide dismutase and the concentration of oxidised and reduced glutathione were not affected. Whilst it was thought that PB-DOPA would have a specific effect on the activity of antioxidant defences targeted against protein oxidation, proteolysis and bulk chaperone activity were not affected by a combination of free and PB-DOPA. Oxidatively-induced protein aggregation, however, was inhibited by the presence of free and PB-DOPA, suggesting that a more specific chaperone regulation may be taking place. The regulation of gene and protein expression was thought to be one possible mechanism by which PB-DOPA could function as a signalling molecule. To test this hypothesis, the effect of free and PB-DOPA on transcription factor activation and protein expression were investigated. Free and PB-DOPA did not induce the expression or activation of Nrf2,AP-1 or NFJB, three transcription factors thought to be involved in the expressional regulation of genes involved in the antioxidant defence system. However, the expression of a number of proteins, including antioxidants, chaperones and proteins involved in cell cycle progression, were regulated in monocytic and macrophage-like cells following the administration of free DOPA under conditions that resulted in either a high or low level of PB-DOPA generation. The regulated proteins differed between the two conditions, suggesting that the level of PB-DOPA may be a key factor in determining the specific defences targeted. The results presented in this thesis support the hypothesis that PB-DOPA can function as a signalling molecule, triggering an enhancement of cellular antioxidant defences, with a specific role in the regulation of the chaperone system, a key defence targeted against protein oxidation. This thesis may provide the basis for the potential use of free or PB-DOPA as a therapeutic for diseases known to involve oxidative stress or oxidative damage, however more research will be required to determine if the effects demonstrated in this thesis are also capable of occurring in vivo.
|Date of Award||2008|
|Supervisor||Ruth Foxwell (Supervisor), Roger DEAN (Supervisor) & Jenelle Kyd (Supervisor)|