De Lazzari, Federica (2019) Exploring the physiological functions of the protein DJ-1 in redox homeostasis. [Ph.D. thesis]
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Abstract (italian or english)
Reactive oxygen and nitrogen species, collectively called RONS, are crucial molecules involved in multiple beneficial functions at the physiological level. However, RONS can assume detrimental roles at higher concentrations.
Among the different cellular sources of RONS, mitochondria play a central role, in particular, when they are not properly functioning. In fact, a defective mitochondrial homeostasis has been recurrently associated with many pathological states, such as neurodegenerative diseases. In this scenario, the anti-oxidant defence exerts a pivotal role in counteracting the damaging effects of RONS. Among the different anti-oxidant molecules, superoxide dismutases (SODs) are often considered as the first line of defence due to their ability to eliminate superoxide anions, from which more harmful species can arise. In recent years, mounting evidence is highlighting the protective role of the Parkinson’s disease-associated protein DJ-1 against redox alterations. In fact, various reports have reported the participation of the protein in the anti-oxidant protection under different oxidative conditions, including exposure to exogenous pro-oxidants and ischemia-reperfusion injury. Nonetheless, the exact mechanism of action of DJ-1 has not been completely clarified yet.
In light of the aforementioned considerations, our project focused on the elucidation of the physiological functions of the protein in vivo. To this aim, we exploited Drosophila melanogaster as a model organism, using fruit flies lacking the expression of the fly DJ-1 homologue.
The loss of DJ-1 does not affect lifespan but results in mild locomotor dysfunctions. Moreover, the absence of the protein appears to influence the cristae morphology, supporting that the protein could play a role at the mitochondrial level. Therefore, we then explored the consequences of DJ-1 loss of function in the mitochondrial homeostasis, under basal and oxidative conditions. Our study showed that the absence of DJ-1 impairs mitochondrial functionality and morphology, especially, under oxidative stimuli. Furthermore, although not affecting the total levels of ATP, DJ-1 null flies are more sensitive to starvation than controls, suggesting a dysregulation in the mobilisation of the energetic storages.
We also set up the experimental conditions to investigate the fly metabolic response to anoxia, through the evaluation of succinate accumulation and ATP depletion, laying the ground for future experiments focused on the fly metabolic response to anoxia and on the role of DJ-1 in this pathway.
A second part of the project was dedicated to the exploration of the involvement of DJ-1 in the SOD1 maturation pathway, which is normally accomplished by a dedicated copper chaperone, named CCS. Nevertheless, a residual activity has been described in the absence of CCS, supporting the existence of a CCS-independent pathway. Our group has found that human DJ-1 can bind and transfer copper to SOD1, rendering the enzyme active in vitro, suggesting a role of DJ-1 in the alternative activation of SOD1. To investigate this pathway in vivo, we overexpressed or silenced the expression of DJ-1, under the absence of the fly CCS homologue.
Our results evidenced that DJ-1 is essential under the absence of CCS and that the ubiquitous overexpression of DJ-1 in CCS null background seems to rescue SOD1 protein levels. Since fly SOD1 is unstable in the absence of CCS, this data may suggest a possible participation of DJ-1 in the maturation of the enzyme, though further confirmation is required.
Overall, with this project, we contributed to add some pieces of information concerning the anti-oxidant role of DJ-1 in vivo. In accordance with the multifaceted nature of the protein, our data indicate that DJ-1 may exert its protective activity acting at different levels, ranging from the maintenance of the mitochondrial homeostasis to the possible activation of the anti-oxidant enzyme SOD1.
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