Two-Step Mechanism of Cyclin B Degradation Initiated by Proteolytic Cleavage with the 26 S Proteasome in Fish.

To complete meiosis II, cyclin B is degraded in a short period by the inactivation of M-phase promoting factor (MPF). Previously, we showed that the destruction of cyclin B was initiated by the ubiquitin-independent proteolytic activity of the 26 S proteasome through an initial cut in the N-terminus of cyclin (at K57 in the case of goldfish cyclin B). We hypothesized that this cut allows cyclin to be ubiquitinated for further destruction by the ubiquitin-dependent proteolytic pathway, which leads to MPF inactivation. In this study, we aimed to identify the ubiquitination site for further degradation. The destruction of cyclin B point mutants in which lysine residues in a lysine-rich stretch following the cut site of cyclin B had been mutated was analyzed. All the lysine point mutants except K57R (a point mutant in which K57 was substituted with arginine) were susceptible to proteolytic cleavage by the 26 S proteasome. However, the degradation of the K77R and K7677R mutants in Xenopus egg extracts was significantly slower than the degradation of other mutants, and a 42 kDa truncated form of cyclin B was detected during the onset of the degradation of these mutants. The truncated form of recombinant cyclin B, an N-terminal truncated cyclin BΔ57 produced as cut by the 26 S proteasome, was not further cleaved by the 26 S proteasome but rather degraded in Xenopus egg extracts. The injection of the K57R, K77R and K7677R cyclin B proteins stopped cleavage in Xenopus embryos. From the results of a series of experiments, we concluded that cyclin B degradation involves a two-step mechanism initiated by initial ubiquitin-independent cleavage by the 26 S proteasome at lysine 57 followed by its ubiquitin-dependent destruction by the 26 S proteasome following ubiquitination at lysine 77.

Establishment of procedures for studying mPR-interacting agents and physiological roles of mPR.

More than 10years have passed since the discovery of membrane progestin receptors (mPRs). Although the identification of mPR genes in various organisms and mPR expression patterns have been described since then, the precise physiological roles of mPRs are still unclear, except their function as a receptor for maturation-inducing steroid in fish. The wide distribution of mPRs suggests variable actions for progestins through mPRs in the tissues. Information about the physiological roles of mPRs, such as roles in the progression of breast cancer and T-cell proliferation, has gradually accumulated recently. These results suggest that mPRs are possible targets for new pharmaceuticals. We established a cell line that was transformed with cDNAs for mPRα and a recombinant luciferase gene named GloSensor. The cells can be used for monitoring the effects of ligands on mPRα based on intracellular cyclic adenosine monophosphate (cAMP) levels. Studies using these cell lines indicated that the cAMP concentration is decreased by ligands for mPRα. The results provide support for previous results suggesting that mPRα is coupled to inhibitory G protein (Gi). We also established screening methods that make it possible to screen ligands for mPR. Recently, we succeeded in expressing and purifying recombinant mPR protein in the yeast Pichia pastoris. Relatively large amounts of mPR protein with hormonal binding activity can be purified by our method. The recombinant protein will be applicable to establishing a molecular probe to detect mPR-interacting agents. To obtain decisive evidence for the roles of mPRs, we are establishing strains of medaka fish that are deficient in mPRs. In medaka, four subtypes of mPR genes (α, ß, γ, and α2) have been identified. By reverse genetic screening, we have selected three to four strains in which a point mutation has been induced in the coding sequence of the mPR subtypes. However, homozygous mutants of each mPR gene showed no phenotype. The results suggested that mPR genes share redundancy. We are currently producing double and triple mutants of the mPR subtypes. The physiological roles of mPRs will be demonstrated using the mutant medaka strains.

Expression and Purification of Human Membrane Progestin Receptor α (mPRα).

Membrane progestin receptors (mPRs) are responsible for mediating the rapid, nongenomic activity of progestins and belong to the G protein-coupled receptor (GPCR) family. mPRs are also considered as attractive proteins to draw a new medicinal approach. In this study, we optimized a procedure for the expression and purification of recombinant human mPRα protein (hmPRα) by a methylotropic yeast, Pichia pastoris, expression system. The protein expressed in crude membrane fractions exhibited a binding affinity of Kd = 3.8 nM and Bmax = 288.8 fmol/mg for progesterone. These results indicated that the hmPRα expressed in yeast was active. Solubilized hmPRα was purified through three column chromatography steps. A nickel-nitrilotriacetic acid (Ni-NTA) column was first used, and the mPRα proteins were then bound to cellulose resin with free amino groups (Cellufine Amino) and finally passed through an SP-Sepharose column. The optimization of expression and purification conditions resulted in a high yield of purified hmPRα (1.3-1.5 mg from 1 L culture). The purified hmPRα protein demonstrated progesterone binding (Kd = 5.2 nM and Bmax = 111.6 fmol/mg). The results indicated that we succeeded in solubilizing and purifying hmPRα in an active form. Sufficient amount of active hmPRα protein will support the establishment of applications for the screening of ligands for mPRα.

Cell-based assay of nongenomic actions of progestins revealed inhibitory G protein coupling to membrane progestin receptor α (mPRα).

Previously, we established cell lines stably producing goldfish membrane progestin receptor α (goldfish mPRα) proteins, which mediate steroidal nongenomic actions. In this study, we transfected these cell lines (MDA-MD-231) with cDNAs encoding a recombinant luciferase gene (GloSensor). These cells can be used for monitoring the effects of ligands that bind to mPR by means of luminescence, the intensity of which reflects intracellular cyclic adenosine monophosphate (cAMP) levels. Luminescence intensity of the cells increased significantly when cells were treated with forskolin, strong activator of adenylyl cyclase. Then, we established a strategy to measure changes in luminescence that correlated with the actions of the ligands. The actions of ligands were measurable by the prevention of stimulation caused by forskolin after ligand stimulation. The studies using these cell lines indicated that cAMP concentrations were decreased specifically by the mPR ligands 17α,20ß-dihydroxy-4-pregnen-3-one, diethylstilbestrol and progesterone. Furthermore, pertussis toxin inhibited the decrease in cAMP levels caused by mPR ligands. These results support evidence from previous results that mPRα is coupled to an inhibitory G protein.