Bioreactor operation shows haloplasticity of a synthetic nitrifying community, enabling urine treatment in space

Additional file 4: Figure S2. Detection of aabgl1 copy numbers in the transformants by qPCR analysis. Tef1Îą gene was used as a single copy control

Rapid, Facile Detection of Heterodimer Partners for Target Human G-Protein-Coupled Receptors Using a Modified Split-Ubiquitin Membrane Yeast Two-Hybrid System

<div><p>Potentially immeasurable heterodimer combinations of human G-protein-coupled receptors (GPCRs) result in a great deal of physiological diversity and provide a new opportunity for drug discovery. However, due to the existence of numerous combinations, the sets of GPCR dimers are almost entirely unknown and thus their dominant roles are still poorly understood. Thus, the identification of GPCR dimer pairs has been a major challenge. Here, we established a specialized method to screen potential heterodimer partners of human GPCRs based on the split-ubiquitin membrane yeast two-hybrid system. We demonstrate that the mitogen-activated protein kinase (MAPK) signal-independent method can detect ligand-induced conformational changes and rapidly identify heterodimer partners for target GPCRs. Our data present the abilities to apply for the intermolecular mapping of interactions among GPCRs and to uncover potential GPCR targets for the development of new therapeutic agents.</p></div

Activation of human somatostatin receptor subtype-5 (hSSTR5) produced in yeast by exogenously-added somatostatin.

<p>Yeast strains IMFD-70 (a, b); IMFD-72 (c, d); IMFD-70Zs (e, f); IMFD-72Zs (g, h); IMFD-70ZsD (i, j) and IMFD-72ZsD (k, l) were transformed with either pGK421 (empty vector) (a, c, e, g, i, k) or pGK421-SSTR5 (b, d, f, h, j, l). All transformants were grown in SD selective medium for 18 h. The cells then were incubated for another 4 h in pH-adjusted SD selective medium with or without 10 μM somatostatin (SST, 14 aa peptide). (<i>A</i>) The GFP fluorescence of 10,000 cells was measured by flow cytometry. Mean values of the green fluorescence signal of 10,000 cells. Error bars represent the standard deviation from three separate runs (<i>n</i> = 3); ***, <i>p</i> < 0.001, by one-way ANOVA, Tukey’s post test. (<i>B</i>, <i>C</i>) Visualization of the green fluorescence. (<i>B</i>) Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 1 s. The left photographs are fluorescence micrographs, and the right photographs are bright-field micrographs. (<i>C</i>) Cells were examined using the 100× objective lens of a fluorescence microscope. Exposure time was 0.2 s.</p

Bright Fluorescence Monitoring System Utilizing<i>Zoanthus</i> sp. Green Fluorescent Protein (<i>ZsGreen</i>) for Human G-Protein-Coupled Receptor Signaling in Microbial Yeast Cells

<div><p>G-protein-coupled receptors (GPCRs) are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N) ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer’s disease and Parkinson’s disease, respectively) were chosen as human GPCR(s). The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s).</p> </div

Screening of candidate heterodimer partners of AT₁ angiotensin receptor (AGTR1).

<p>(A) Workflow of a yeast two-hybrid screen. Prey library was transformed into the NMY63 yeast strains harboring AGTR1 bait vector, and the selection with growth reporter genes was performed. Following isolation of prey plasmids from each colony, the obtained GPCR clones were determined by sequencing analysis. (B) Quantitative β-galactosidase assays for homo- and hetero-dimerization of AGTR1 in NMY63 strain. NMY63 yeast strain was transformed with GPCR-Nub<i>G</i> indicated at the left and AGTR1-Cub-LexA-VP16. The control prey plasmid was pPR3-C mock vector. Error bars represent the standard deviations (<i>n = </i>3).</p

Activation of human neurotensin receptor subtype-1 (hNTSR1) by membrane-tethered neurotensin.

<p>(<i>A</i>) Amino acid sequences of membrane-tethered peptides. (<i>B</i>, <i>C</i>) Yeast strain IMFD-72ZsD, which coexpresses pGK421-NTSR1 and either pGK426-NTS42 (NTS), pGK426-NTS(8-13)42 (NTS(8-13)), pGK426-NMN42 (NMN) or pGK426-alpha42 (α-factor), was incubated in pH-adjusted SD selective medium. (<i>B</i>) The GFP fluorescence of 10,000 cells was measured by flow cytometry. Mean values of the green fluorescence signal of 10,000 cells. Error bars represent the standard deviations (<i>n</i> = 3); *, <i>p</i> < 0.05, and ***, <i>p</i> < 0.001, by one-way ANOVA, Tukey’s post test. (<i>C</i>) Fluorescence microscopy analysis of the cells incubated for 24 h. Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 0.67 s. The left photographs are fluorescence micrographs, and the right photographs are bright-field micrographs. </p

Activation of human somatostatin receptor subtype-2 (hSSTR2) by exogenously-added somatostatin.

<p>Yeast strains IMFD-70, IMFD-72, IMFD-70ZsD and IMFD-72ZsD were transformed with pGK421-SSTR2. All transformants were grown in SD selective medium for 18 h. The cells then were incubated for another 4 h in pH-adjusted SD selective medium with or without 10 μM somatostatin (SST, 14 aa peptide). (<i>A</i>) The GFP fluorescence of 10,000 cells was measured by flow cytometry. Mean values of the green fluorescence signal of 10,000 cells. Error bars represent the standard deviations (<i>n</i> = 3); ***, <i>p</i> < 0.001, by one-way ANOVA, Tukey’s post test. (<i>B</i>, <i>C</i>) Visualization of the green fluorescence. (<i>B</i>) Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 1 s. The left photographs are fluorescence micrographs, and the right photographs are bright-field micrographs. (<i>C</i>) Cells were examined using the 100× objective lens of a fluorescence microscope. Exposure time was 0.33 s. </p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Schematic illustration of signal activation of human GPCRs by membrane-tethered peptide ligands.

<p>(<i>A</i>) Overview of this study. The membrane-tethered peptide activates human GPCR, which is heterologously produced in yeast, thereby activating the chimeric Gα proteins. This promotes the mitogen-activated protein kinase (MAPK) cascade and transcription factor Ste12p. Phosphorylated Ste12p induces transcription of the GFP reporter gene by binding to a pheromone response element in the <i>FIG1</i> promoter. (<i>B</i>) Functional domains encoded by the membrane-tethered peptide plasmids. After processing by the secretory pathway, the signal sequence and glycosyl-phosphatidylinositol (GPI) targeting sequence are cleaved and the peptide sequence, which contains a free N-terminus, is tethered on the plasma membrane by GPI covalently linked to the C-terminus.</p

MOESM2 of Designing intracellular metabolism for production of target compounds by introducing a heterologous metabolic reaction based on a Synechosystis sp. 6803 genome-scale model

Additional file: Table S2. Detailed intracellular metabolic reactions that need to be activated, if the yield of succinic acid production according to the SyHyMeP is 155