Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae.

Background: Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital.

Results: We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies.

G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody.

G-protein-coupled receptors are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states. Conformational changes induced by extracellular ligands binding to G-protein-coupled receptors result in a cellular response through the activation of G proteins. The A(2A) adenosine receptor (A(2A)AR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain.

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist.

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases.

Cloning, expression and purification of the anion exchanger 1 homologue from the basidiomycete Phanerochaete chrysosporium.

Anion exchangers are membrane proteins that have been identified in a wide variety of species, where they transport Cl(-) and HCO3(-)across the cell membrane. In this study, we cloned an anion-exchange protein from the genome of the basidiomycete Phanerochaete chrysosporium (PcAEP). PcAEP is a 618-amino acid protein that is homologous to the human anion exchanger (AE1) with 22.

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis.

Background: Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P.

Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein.

Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system.

Bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin.

Background: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown.

Methods: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships.

Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae.

Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure-function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.

Comparison of functional non-glycosylated GPCRs expression in Pichia pastoris.

N-linked glycosylation is the most common post-translational modification of G-protein-coupled receptors (GPCRs) and is correlated to the localization and function of the receptors depending on each receptor. However, heterogeneity of glycosylation can interfere with protein crystallization. The removal of N-linked glycosylation from membrane proteins improves the ability to crystallize these proteins.

Advanced method for high-throughput expression of mutated eukaryotic membrane proteins in Saccharomyces cerevisiae.

Crystallization of eukaryotic membrane proteins is a challenging, iterative process. The protein of interest is often modified in an attempt to improve crystallization and diffraction results. To accelerate this process, we took advantage of a GFP-fusion yeast expression system that uses PCR to direct homologous recombination and gene cloning.

Augmentation of neovascularization [corrected] in hindlimb ischemia by combined transplantation of human embryonic stem cells-derived endothelial and mural cells.

Background: We demonstrated that mouse embryonic stem (ES) cells-derived vascular endothelial growth factor receptor-2 (VEGF-R2) positive cells could differentiate into both endothelial cells (EC) and mural cells (MC), and termed them as vascular progenitor cells (VPC). Recently, we have established a method to expand monkey and human ES cells-derived VPC with the proper differentiation stage in a large quantity. Here we investigated the therapeutic potential of human VPC-derived EC and MC for vascular regeneration.

Pathway for differentiation of human embryonic stem cells to vascular cell components and their potential for vascular regeneration.

Objective: We demonstrated previously that mouse embryonic stem (ES) cell-derived vascular endothelial growth factor receptor-2 (VEGF-R2)-positive cells can differentiate into both vascular endothelial cells and mural cells. This time, we investigated kinetics of differentiation of human ES cells to vascular cells and examined their potential as a source for vascular regeneration.

Methods And Results: Unlike mouse ES cells, undifferentiated human ES cells already expressed VEGF-R2, but after differentiation, a VEGF-R2-positive but tumor rejection antigen 1-60 (TRA1-60)-negative population emerged.

Adrenomedullin/cyclic AMP pathway induces Notch activation and differentiation of arterial endothelial cells from vascular progenitors.

Objective: The acquisition of arterial or venous identity is highlighted in vascular development. Previously, we have reported an embryonic stem (ES) cell differentiation system that exhibits early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors. In this study, we constructively induced differentiation of arterial and venous endothelial cells (ECs) in vitro to elucidate molecular mechanisms of arterial-venous specification.

The neuroprotective and vasculo-neuro-regenerative roles of adrenomedullin in ischemic brain and its therapeutic potential.

Adrenomedullin (AM) is a vasodilating hormone secreted mainly from vascular wall, and its expression is markedly enhanced after stroke. We have revealed that AM promotes not only vasodilation but also vascular regeneration. In this study, we focused on the roles of AM in the ischemic brain and examined its therapeutic potential.

Roles of thromboxane A(2) and prostacyclin in the development of atherosclerosis in apoE-deficient mice.

Production of thromboxane (TX) A2 and PG I2/prostacyclin (PGI2) is increased in patients with atherosclerosis. However, their roles in atherogenesis have not been critically defined. To examine this issue, we cross-bred atherosclerosis-prone apoE-deficient mice with mice deficient in either the TXA receptor (TP) or the PGI receptor (IP).

Adrenomedullin provokes endothelial Akt activation and promotes vascular regeneration both in vitro and in vivo.

We previously reported that adrenomedullin (AM), a vasodilating hormone secreted from blood vessels, promotes proliferation and migration of human umbilical vein endothelial cells (HUVECs). In this study, we examined the ability of AM to promote vascular regeneration. AM increased the phosphorylation of Akt in HUVECs and the effect was inhibited by the AM antagonists and the inhibitors for protein kinase A (PKA) or phosphatidylinositol 3-kinase (PI3K).

Different differentiation kinetics of vascular progenitor cells in primate and mouse embryonic stem cells.

Background: We demonstrated that vascular endothelial growth factor receptor 2 (VEGF-R2)-positive cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells and mural cells to suffice as vascular progenitor cells (VPCs). Here we examined whether VPCs occur in primate ES cells and investigated the differences in VPC differentiation kinetics between primate and mouse ES cells.

Methods And Results: In contrast to mouse ES cells, undifferentiated monkey ES cells expressed VEGF-R2.

Significance and therapeutic potential of the natriuretic peptides/cGMP/cGMP-dependent protein kinase pathway in vascular regeneration.

Natriuretic peptides (NPs), which consist of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), are characterized as cardiac or vascular hormones that elicit their biological effects by activation of the cGMPcGMP-dependent protein kinase (cGK) pathway. We recently reported that adenoviral gene transfer of CNP into rabbit blood vessels not only suppressed neointimal formation but also accelerated reendothelialization, a required step for endothelium-dependent vasorelaxation and antithrombogenicity. Accordingly, we investigated the therapeutic potential of the NPscGMPcGK pathway for vascular regeneration.

Effective contribution of transplanted vascular progenitor cells derived from embryonic stem cells to adult neovascularization in proper differentiation stage.

We demonstrated that Flk-1(+) cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells (ECs) and mural cells (MCs) to suffice as vascular progenitor cells (VPCs). In the present study, we investigated the importance of the stage of ES cell differentiation on effective participation in adult neovascularization. We obtained Flk-1(+) LacZ-expressing undifferentiated VPCs.