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. We screened 25 non-glycosylated GPCRs for functional receptor production in the methylotrophic yeast Pichia pastoris using specific ligand-receptor binding assays. We found that five clones were expressed at greater than 10 pmol/mg, 9 clones at 1-10 pmol/mg and 11 clones at less than 1 pmol/mg of membrane protein. Further optimization of culture parameters including culture scale, induction time, pH and temperature enabled us to achieve expression of a functional human muscarinic acetylcholine receptor subtype 2 (CHRM2) with a B(max) value of 51.2 pmol/mg of membrane protein. Approximately 1.9 mg of the human CHRM2 was produced from a 1-L culture.

Interferon-ß Mediates Signaling Pathways Uniquely Regulated in Hepatic Stellate Cells and Attenuates the Progression of Hepatic Fibrosis in a Dietary Mouse Model.

The results of clinical and experimental studies suggest that type I interferons (IFNs) may have direct antifibrotic activity in addition to their antiviral properties. However, the mechanisms are still unclear; in particular, little is known about the antifibrotic activity of IFN-ß and how its activity is distinct from that of IFN-α. Using DNA microarrays, we demonstrated that gene expression in TWNT-4 cells, an activated human hepatic stellate cell line, was remarkably altered by IFN-ß more than by IFN-α. Integrated pathway enrichment analyses revealed that a variety of IFN-ß-mediated signaling pathways are uniquely regulated in TWNT-4 cells, including those related to cell cycle and Toll-like receptor 4 (TLR4) signaling. To investigate the antifibrotic activity of IFN-ß and the involvement of TLR4 signaling in vivo, we used mice fed a choline-deficient l-amino acid-defined diet as a model of nonalcoholic steatohepatitis-related hepatic fibrosis. In this model, the administration of IFN-ß significantly attenuated augmentation of the area of liver fibrosis, with accompanying transcriptional downregulation of the TLR4 adaptor molecule MyD88. Our results provide important clues for understanding the mechanisms of the preferential antifibrotic activity of IFN-ß and suggest that IFN-ß itself, as well as being a modulator of its unique signaling pathway, may be a potential treatment for patients with hepatic fibrosis in a pathogenesis-independent manner.

Reconstitution in vitro of V1 complex of Thermus thermophilus V-ATPase revealed that ATP binding to the A subunit is crucial for V1 formation.

Vacuolar-type H(+)-ATPase (V-ATPase or V-type ATPase) is a multisubunit complex comprised of a water-soluble V(1) complex, responsible for ATP hydrolysis, and a membrane-embedded V(o) complex, responsible for proton translocation. The V(1) complex of Thermus thermophilus V-ATPase has the subunit composition of A(3)B(3)DF, in which the A and B subunits form a hexameric ring structure. A central stalk composed of the D and F subunits penetrates the ring. In this study, we investigated the pathway for assembly of the V(1) complex by reconstituting the V(1) complex from the monomeric A and B subunits and DF subcomplex in vitro. Assembly of these components into the V(1) complex required binding of ATP to the A subunit, although hydrolysis of ATP is not necessary. In the absence of the DF subcomplex, the A and B monomers assembled into A(1)B(1) and A(3)B(3) subcomplexes in an ATP binding-dependent manner, suggesting that ATP binding-dependent interaction between the A and B subunits is a crucial step of assembly into V(1) complex. Kinetic analysis of assembly of the A and B monomers into the A(1)B(1) heterodimer using fluorescence resonance energy transfer indicated that the A subunit binds ATP prior to binding the B subunit. Kinetics of binding of a fluorescent ADP analog, N-methylanthraniloyl ADP (mant-ADP), to the monomeric A subunit also supported the rapid nucleotide binding to the A subunit.

Rotation scheme of V1-motor is different from that of F1-motor.

V(1), a water-soluble portion of vacuole-type ATPase (V-ATPase), is an ATP-driven rotary motor, similar to F(1)-ATPase. Hydrolysis of ATP is coupled to unidirectional rotation of the central rotor D and F subunits relative to the A(3)B(3) cylinder. In this study, we analyzed the rotation kinetics of V(1) in detail. At low ATP concentrations, the D subunit rotated stepwise, pausing every 120 degrees . The dwell time between steps revealed that V(1) consumes one ATP per 120 degrees step. V(1) generated torque of approximately 35 pN nm, slightly lower than the approximately 46 pN nm measured for F(1). Noticeably, the angles for both ATP cleavage and binding were apparently the same in V(1), in sharp contrast to F(1), which cleaves ATP at 80 degrees posterior to the binding of ATP. Thus, the mechanochemical cycle of V(1) has marked differences to that of F(1).