Tyrosine hydroxylase, an enzyme involved in hypertension development, is shortterm regulated by the ubiquitin-proteasome system

LONGO CARBAJOSA NADIA ANDREA; GIRONACCI MM; GERARDO CORRADI

Buenos Aires

Conferencia; Ubiquitin and ubiquitin-like proteins: At the crossroads from chromatin to protein; 2014

European Molecular Biology Organization (EMBO)

Cellular protein degradation is a highly complex, temporally controlled, and tightly regulated process that plays a fundamental role in cell life and death as well as in health and disease. The ubiquitin-proteasome system (UPS) is the major pathway for intracellular protein degradation in eukaryotic cells. Aberrations in the UPS are implicated in the pathogenesis of many diseases including cancer, neurodegenerative, immune and cardiovascular diseases. Tyrosine hydroxylase (TH), the enzyme that catalyses the first and rate-limiting step in catecholamines biosynthesis, is involved in the development and maintenance of hypertension. TH half-life has been reported to be around 20 h. Up to now no UPS short-term regulation was shown for TH. Our aim was to investigate whether TH protein expression and activity are short-term modulated by the UPS. Given that aberrations in the UPS are implicated in the pathogenesis of many diseases, and that TH expression and activity are augmented in spontaneously hypertensive rats (SHR), which may be a consequence of impairment in the UPS activity, we investigated proteasome activity and expression centrally in SHR. PC12 cells were treated with proteasome or lysosomes inhibitors and TH expression was evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86+15% in TH expression after 30 min of incubation, then began to decrease up to 6 h to reach equal levels to control group and finally it rose up to 45.2+8.5% after 24 h. The decrease in TH protein expression observed after 6 h proteasome inhibition in PC12 cells may be due to another degradating pathway acting on TH content, such as lysosomes. Bafilomycin, a lysosome inhibitor, did not alter TH expression during short times while it induced an increase of 92+22% above basal after 6 h treatment. TH was found to be monoubiquitinated prior to be recognized by the proteasome and efficacy of proteasome inhibition on TH turnover in PC12 cells was evidenced by accumulation of ubiquitinylated TH after 30 min treatment with lactacystin. TH activity can be short-term regulated by phosphorylation at Ser residues. Phosphorylation of TH at Ser-19 and Ser-40 was increased by 5.6+1.7 and 2.7+0.3 fold above basal, respectively, in cells treated with the proteasome inhibitor. In addition, phospho-Ser19-TH or phospho-Ser40-TH colocalized with either Ub or the proteasome 26S. Altogether these results suggest that TH expression and activity are short-term regulated by the UPS. Given that TH expression and activity are augmented in SHR, which may be a consequence of impairment in the UPS activity, we investigated proteasome activity and expression centrally in SHR. We first further confirmed the involvement of the UPS on TH turnover centrally in SHR. Neuronal cultures from hypothalami and brainstem of SHR were treated with the proteasome inhibitor during 30 min and TH expression was evaluated. Under this condition, an increase in TH expression was observed, demonstrating that TH expression is effectively regulated centrally by the UPS in SHR. Since that the proteasome regulates TH expression, consequently it may also be involved in blood pressure regulation in SHR. We tested the hypothesis whether the blockade of the proteasome may influence blood pressure in SHR. Intracerebroventricular injection of the proteasome inhibitor into SHR induced a significant increase in blood pressure. We investigated whether activity and expression of the proteasome is altered in neurons from SHR. Proteasome expression was not modified in neuronal cultures from SHR compared to its normotensive control. However, proteasome activity was significantly reduced 67±4% in neurons from hypothalami and brainstem from SHR. These results demonstrate a centrally proteasome dysfunction in SHR that may explain the increased expression and activity of TH reported in hypertension.