Analysis of aldosterone-induced differential receptor-independent protein patterns using 2D-electrophoresis and mass spectrometry

Abstract

In the human body the mineralocorticoid aldosterone is responsible for maintaining water and electrolyte homeostasis and therefore controlling blood pressure. In addition, aldosterone has recently been associated with severe heart failure. Besides receptor-dependent action, the damaging effects of aldosterone may also be partly mediated through non-genomic mechanisms. The present study focuses on the mineralocorticoid receptor-independent action of aldosterone at the protein level. We chose the fission yeast Schizosaccharomyces pombe as a model organism, since this yeast does not contain nuclear steroid receptors, but many genes and regulatory mechanisms that are close to those of mammals. Using 2D-electrophoresis we identified for the first time protein spots affected by aldosterone in a nuclear receptor-free system. Mass spectrometry analysis using MALDI-TOF MS and nanoLC-MS/MS approaches allowed the unambiguous identification of 11 proteins that showed increased or decreased levels, which may represent newly identified players and pathways of aldosterone-induced action. Two proteins with a connection to osmotic regulation (NAD-dependent malic enzyme and glycerol-3-phosphate-dehydrogenase), as well as two proteins involved in the overall organization of the cytoskeleton, vip1 and glyceraldehyde-3-phosphate dehydrogenase, which was also found to be specifically affected by aldosterone in human HCT116 cells, are discussed.

Aldosterone Stimulates Elastogenesis in Cardiac Fibroblasts via Mineralocorticoid Receptor-independent Action Involving the Consecutive Activation of Gα13, c-Src, the Insulin-like Growth Factor-I Receptor, and Phosphatidylinositol 3-Kinase/Akt

“…However, it has been proposed that some of these nongenomic effects of aldosterone also require the presence of MR or a closely related protein (14). In contrast, other studies have shown that the nongenomic aldosterone effects still occur in cell lines lacking the classical MR and in yeast devoid of MR or in normal cells treated with MR antagonists (2,11,15). Such results strongly suggest the involvement of other receptor(s), distinct from the classic MR, that may interact with aldosterone and trigger the nongenomic effects of this hormone.…”

Shotgun proteomic analysis of the microsomal fraction of eukaryotic cells using a two‐dimensional reversed‐phase×ion‐pair reversed‐phase HPLC setup

“…Cultivation of S. pombe was done as previously described [1,18]. Cells were harvested at 30006g for 20 min.…”

“…The applicability of the protocol for eukaryotic proteomes was proven using a simple eukaryotic organism, the fission yeast S. pombe. This selection furthermore allowed us to complement the proteomic studies on this model organism, previously reported by us [1,18,19]. After proving the general applicability to eukaryotic proteomes, we set the focus of the study onto human hepatocytes.…”

Cloning and heterologous expression in Escherichia coli of the fission yeast vip1 gene, showing differential expression after aldosterone treatment

“…The differential protein patterns in S. pombe upon mineralocorticoid administration were studied using 2D-gel electrophoresis and mass spectrometry. Eleven proteins that were differentially regulated by aldosterone [29] and 19 proteins that were specifically affected by the aldosterone-precursor 11-deoxycorticosterone (DOC) [30] have been identified. Four of these proteins showed altered levels in both cases [31] and therefore may be specifically associated with non-genomic actions, which makes them potential targets for the development of new drugs (Table 1).…”