Expression of receptors for neuregulins, ErbB2, ErbB3 and ErbB4, in developing mouse cerebellum.

We have examined the expression of ErbB2, ErbB3 and ErbB4 in developing mouse cerebellum. ErbB2, ErbB3 and ErbB4 were all expressed in granule cells during cerebellar development. However, the expression pattern for each ErbB receptor changed with the developmental stage. Variations of signal transduction pathway through combinations of these ErbB receptors might have important roles in controlling cerebellar postnatal development.

Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bond formation system in aerobically growing Escherichia coli cells.

DsbA, the disulfide bond catalyst of Escherichia coli, is a periplasmic protein having a thioredoxin-like Cys-30-Xaa-Xaa-Cys-33 motif. The Cys-30-Cys-33 disulfide is donated to a pair of cysteines on the target proteins. Although DsbA, having high oxidizing potential, is prone to reduction, it is maintained essentially all oxidized in vivo. DsbB, an integral membrane protein having two pairs of essential cysteines, reoxidizes DsbA that has been reduced upon functioning. It is not known, however, what might provide the overall oxidizing power to the DsbA-DsbB disulfide bond formation system. We now report that E. coli mutants defective in the hemA gene or in the ubiA-menA genes markedly accumulate the reduced form of DsbA during growth under the conditions of protoheme deprivation as well as ubiquinone/menaquinone deprivation. Disulfide bond formation of beta-lactamase was impaired under these conditions. Intracellular state of DsbB was found to be affected by deprivation of quinones, such that it accumulates first as a reduced form and then as a form of a disulfide-linked complex with DsbA. This is followed by reduction of the bulk of DsbA molecules. These results suggest that the respiratory electron transfer chain participates in the oxidation of DsbA, by acting primarily on DsbB. It is remarkable that a cellular catalyst of protein folding is connected to the respiratory chain.

Roles of disulfide bonds in bacterial alkaline phosphatase.

Alkaline phosphatase of Escherichia coli (a homodimeric protein found in the periplasmic space) contains two intramolecular disulfide bonds (Cys-168-Cys-178 and Cys-286-Cys-336) that are formed after export to the periplasmic space. The location-specific folding character of this enzyme allowed its wide usage as a reporter of protein localization in prokaryotic cells. To study the roles of disulfide bonds in alkaline phosphatase, we eliminated each of them by Cys to Ser mutations. Intracellular stability of alkaline phosphatase decreased in the absence of either one or both of the disulfide bonds. The mutant proteins were stabilized in a DegP protease-deficient strain, allowing accumulation at significant levels and subsequent characterization. A mutant protein that lacked the N-terminally located disulfide bond (Cys-168-Cys-178) was found to have Cys-286 and Cys-336 residues disulfide-bonded, to have a dimeric structure, and to have almost full enzymatic activity. Nevertheless, the mutant protein lost the trypsin-resistant conformation that is characteristically observed for the wild-type enzyme. In contrast, mutants lacking Cys-286 and Cys-336 were monomeric and inactive. These results indicate that the Cys-286-Cys-336 disulfide bond is required and is sufficient for correctly positioning the active site region of this enzyme, but such an active conformation is still insufficient for the conformational stability of the enzyme. Thus, a fully active state of this enzyme can be formed without full protein stability, and the two disulfide bonds differentially contribute to these properties.

Roles of cysteine residues of DsbB in its activity to reoxidize DsbA, the protein disulphide bond catalyst of Escherichia coli.

BACKGROUND: DsbA, a periplasmic protein, catalyses the disulphide bond formation of other cell surface proteins in E. coli. Reoxidation of DsbA for catalytic turn over is assured by DsbB, a membrane protein with four essential cysteine residues facing the periplasm. We and others previously reported that the reactive Cys30 residue of DsbA forms a mixed disulphide with DsbB in the absence of its partner Cys33 residue. RESULTS: Under the medium condition in which the DsbA mutant lacking Cys33 forms a mixed disulphide only with DsbB, we examined cysteine mutants of epitope-tagged DsbB for their ability to form the complex. It was shown that Cys104 of DsbB is absolutely required while other three cysteines are also required for maximum interaction. Examination of the redox states of cysteines in wild-type and mutant DsbB suggested that Cys104 and Cys130 form a disulphide bond which will be transferred to DsbA. In agreement with this notion, DsbB mutants lacking one of the N-terminally located cysteines retain weak DsbB activity in vivo. The primary role of the N-terminally located thioredoxin-like motif of DsbB is probably to reoxidize Cys104 and Cys130. CONCLUSIONS: We propose the following reaction cycle. DsbB is initially oxidized (State A in Summary Figure). Disulphide interaction between Cys30 of DsbA and Cys104 of DsbB should then trigger the recycling reaction of DsbA (State B), allowing over all electron transfer from newly secreted protein via DsbA (Cys30/Cys33) to DsbB in which intrachain electron flow from Cys104/Cys130 (State C) to Cys41/Cys44 (State D) may occur.

DsbA-DsbB interaction through their active site cysteines. Evidence from an odd cysteine mutant of DsbA.

Formation of disulfide bonds in Escherichia coli envelope proteins is facilitated by the Dsb system, which is thought to consist of at least two components, a periplasmic soluble enzyme (DsbA) and a membrane-bound factor (DsbB). Although it is believed that DsbA directly oxidizes substrate cysteines and DsbB reoxidizes DsbA to allow multiple rounds of reactions, direct evidence for the DsbA-DsbB interaction has been lacking. We examined intracellular activities of mutant forms of DsbA, DsbA30S and DsbA33S, in which one of its active site cysteines (Cys30 or Cys33, respectively) has been replaced by serine. The DsbA33S protein was found to dominantly interfere with the disulfide bonds formation and to form intermolecular disulfide bonds with numerous other proteins when cells were grown in media containing low molecular weight disulfides such as GSSG. In the absence of added GSSG, DsbA33S protein remained specifically disulfide-bonded with DsbB. These in vivo results not only confirm the previous findings that Cys30 of DsbA is hyper-reactive in vitro but provide evidence that DsbA indeed interacts selectively with DsbB. We propose that the Cys30-mediated DsbA-DsbB complex represents an intermediate state of DsbA-DsbB recycling reaction that has been fixed because of the absence of Cys33 on DsbA.

Redox states of DsbA in the periplasm of Escherichia coli.

DsbA is a periplasmic, disulfide bond formation factor of E. coli. We studied in vivo redox states of its active site cysteines. When periplasmic contents were prepared from iodoacetic acid-treated cells, according to the previously published procedures, variable but major proportions of DsbA were in the reduced form. We found that this was due to an artificial reduction that occurred after cell disruption; even purified and oxidized DsbA underwent reduction when incubated with cell extracts in the absence of any added reducing agent. Such DsbA-reducing activities were detected in both the periplasmic and the cytoplasmic fractions. To circumvent the artifact, we analyzed redox states of DsbA under denaturing conditions. Now virtually all the DsbA molecules were detected as oxidized or reduced in the dsbB+ background or in the dsbB- background, respectively. Using the improved method, we also examined redox states of DsbA when it was overproduced, and followed the oxidation/reduction pathway that DsbA follows after biosynthesis. It is suggested that newly synthesized DsbA is rapidly oxidized by pre-existing DsbA, while oxidation of mature (functional) DsbA requires DsbB, whose roles might include that of antagonizing the actions of DsbA-reducing enzyme(s).

Study on the detection of implantation site in rabbits.

In this study, an alkaline treatment was applied to the observation of implantation site in rabbits. Embryos in pregnant rabbits were stabbed to death on days 7, 8, 9, 10 and 18 of gestation. These animals were sacrificed on day 29. The uteri were removed, immersed in 2% sodium hydroxide and fixed with 10% buffered formalin. After the alkaline treatment, all of the implantation sites were clearly recognizable as white tissue since the other parts became fairly transparent. Even after the formalin fixation, all the implantation sites were similarly detectable. The mean area of implantation sites visibly increased after day 10. From all the results, the simple and accurate procedure including alkaline treatment was concluded to be useful for estimating implantation and pregnancy conditions in rabbits.