Mast cells are required for the development of renal fibrosis in the rodent unilateral ureteral obstruction model.

Mast cells are associated with inflammation and fibrosis. Whether they protect against or contribute to renal fibrosis is unclear. Based on our previous findings that mast cells can express and secrete active renin, and that angiotensin (ANG II) is profibrotic, we hypothesized that mast cells play a critical role in tubulointerstitial fibrosis.

Expression and biophysical analysis of a triple-transmembrane domain-containing fragment from a yeast G protein-coupled receptor.

Structural characterization of G protein-coupled receptors (GPCRs) is hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Insights into conformational preferences and the three-dimensional (3D) structure of domains of these receptors can be obtained using polypeptide fragments of these proteins. Herein, we report the expression, purification, and biophysical characterization of a three-transmembrane domain-containing 131-residue fragment of the yeast α-factor receptor, Ste2p.

Aldehyde dehydrogenase activation prevents reperfusion arrhythmias by inhibiting local renin release from cardiac mast cells.

Background: Renin released by ischemia/reperfusion from cardiac mast cells activates a local renin-angiotensin system (RAS). This exacerbates norepinephrine release and reperfusion arrhythmias (ventricular tachycardia and fibrillation), making RAS a new therapeutic target in myocardial ischemia.

Methods And Results: We investigated whether ischemic preconditioning (IPC) prevents cardiac RAS activation in guinea pig hearts ex vivo.

Expression and biophysical analysis of two double-transmembrane domain-containing fragments from a yeast G protein-coupled receptor.

Fragments of G protein-coupled receptors (GPCRs) are widely used as models to investigate these polytopic integral-membrane, signal-transducing molecules, but have proven difficult to prepare in quantities necessary for NMR analyses. We report on the biosynthesis of two double transmembrane (TM) containing fragments of Ste2p, the alpha-factor GPCR from the yeast Saccharomyces cerevisiae. Ste2p(G31-T110) [TM1-TM2] and Ste2p(R231-S339) [TM6-TM7-CT40] were expressed as TrpDeltaLE fusion proteins in Escherichia coli and released by CNBr cleavage.

Mast cell renin and a local renin-angiotensin system in the airway: role in bronchoconstriction.

We previously reported that mast cells express renin, the rate-limiting enzyme in the renin-angiotensin cascade. We have now assessed whether mast cell renin release triggers angiotensin formation in the airway. In isolated rat bronchial rings, mast cell degranulation released enzyme with angiotensin I-forming activity blocked by the selective renin inhibitor BILA2157.

Selective labeling of a membrane peptide with 15N-amino acids using cells grown in rich medium.

Nuclear magnetic resonance spectra of membrane proteins containing multiple transmembrane helices have proven difficult to resolve due to the redundancy of aliphatic and Ser/Thr residues in transmembrane domains and the low chemical shift dispersity exhibited by residues in alpha-helical structures. Although (13)C- and (15)N-labeling are useful tools in the biophysical analysis of proteins, selective labeling of individual amino acids has been used to help elucidate more complete structures and to probe ligand-protein interactions. In general, selective labeling has been performed in Escherichia coli expression systems using minimal media supplemented with a single labeled amino acid and nineteen other unlabeled amino acids and/or by using auxotrophs for specific amino acids.

Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor.

The yeast Saccharomyces cerevisiae alpha-factor pheromone receptor (Ste2p) was used as a model G protein-coupled receptor (GPCR). A 73-mer multidomain fragment of Ste2p (residues 267-339) containing the third extracellular loop, the seventh transmembrane domain, and 40 residues of the cytosolic tail (E3-M7-24-T40) was biosynthesized fused to a carrier protein. The multidomain fusion protein (designated M7FP) was purified to near homogeneity as judged by HPLC and characterized by mass spectrometry.

Sexual conjugation in yeast: A paradigm to study G-protein-coupled receptor domain structure.

The yeast Saccharomyces cerevisiae undergoes cell fusion during sexual conjugation to form diploid cells. The haploids participating in this process signal each other through secreted peptide-mating factors (alpha-factor and a-factor) that are recognized by G-protein-coupled receptors. The receptor (Ste2p) recognizing the tridecapeptide alpha-factor is used as a model system in our laboratory to understand various aspects of peptide-receptor interactions and receptor structure.

Biosynthesis and biophysical analysis of domains of a yeast G protein-coupled receptor.

The alpha-factor receptor(Ste2p) is required for the sexual conjugation of the yeast Saccharomyces cerevisiae. Ste2p belongs to the G protein-coupled receptor (GPCR) family sharing a common heptahelical transmembrane structure. Biological synthesis of regions of Ste2p fused to a leader protein in Escherichia coli yielded milligram quantities of polypeptides that corresponded to one or two transmembrane domains.