Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension.

Nadia A Longo Carbajosa,María J Guil,Mariela M Gironacci,Gerardo Corradi,Marcelo S Vatta,María A Lopez Verrilli

doi:10.1371/journal.pone.0116597

Abstract

Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86±15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2±8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92±22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7±0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasme activity. Proteasome activity was significantly reduced by 67±4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.

Highlights

Cellular protein degradation is a highly complex, temporally controlled, and tightly regulated process that plays a critical role in a variety of basic pathways during cell life and death and in health and disease
Given that aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of many diseases, and that Tyrosine hydroxylase (TH) protein levels 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 protein levels centrally in SHR
As the increase in TH protein content reported by others and us in neurons from SHR [12, 13] may result from impaired UPS activity, we investigated proteasome activity and protein content in SHR brain areas related to blood pressure regulation

Summary

Introduction

Cellular protein degradation is a highly complex, temporally controlled, and tightly regulated process that plays a critical role in a variety of basic pathways during cell life and death and in health and disease. The ubiquitin-proteasome system (UPS) is the major pathway for intracellular protein degradation in eukaryotic cells [1,2]. Degradation of proteins by the UPS occurs in two successive steps: 1) conjugation of multiple ubiquitin (Ub) moieties to the substrate and 2) degradation of the tagged protein by the downstream 26S proteasome complex. This complex is composed of a 20S core particle which embodies the catalytic activity and two 19S regulatory particles [1,2]. Different E3 ligase target specific substrates for degradation and its activity could be under local control depending on the presence of regulatory cofactors [1,3,4,5]

Objectives

Methods

Results

Conclusion