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A large-scale protein interaction network is carried out by pull-down assay in Escherichia coli using His-tagged bait proteins. It provides a framework for elucidation of cellular activities, targeted-drug design, and whole cell engineering.

Gingipains are cysteine proteinases that are responsible for the virulence of Porphyromonas gingivalis. Recent studies have shown that P. gingivalis is trapped within autophagic compartments of infected cells, where it promotes survival. In this study we investigated the role of gingipains in the intracellular trafficking and survival of this bacterium in human aortic endothelial cells and any possible involvement of these enzymes in the autophagic pathway. Although autophagic events were enhanced by infection with either wild-type (WT) P. gingivalis strains (ATCC 33277, 381, and W83) or an ATCC 33277 mutant lacking gingipains (KDP136), we have found that more than 90% of intracellular WT and KDP136 colocalized with cathepsin B, a lysosome marker, and only a few of the internalized cells colocalized with LC3, an autophagosome marker, during the 0.5- to 4-h postinfection period. This was further substantiated by immunogold electron microscopic analyses, thus implying that P. gingivalis evades the autophagic pathway and instead directly traffics to the endocytic pathway to lysosomes. At the late stages after infection, WT strains in phagolysosomes retained their double-membrane structures. KDP136 in these compartments, however, lost its double-membrane structures, representing a characteristic feature of its vulnerability to rupture. Together with the ultrastructural observations, we found that the number of intracellular viable WT cells decreased more slowly than that of KDP136 cells, thus suggesting that gingipains contribute to bacterial survival, but not to trafficking, within the infected cells.

ABSTRACTGingipains are cysteine proteinases that are responsible for the virulence of Porphyromonas gingivalis. Recent studies have shown that P. gingivalisis trapped within autophagic compartments of infected cells, where it promotes survival. In this study we investigated the role of gingipains in the intracellular trafficking and survival of this bacterium in human aortic endothelial cells and any possible involvement of these enzymes in the autophagic pathway. Although autophagic events were enhanced by infection with either wild-type (WT) P. gingivalisstrains (ATCC 33277, 381, and W83) or an ATCC 33277 mutant lacking gingipains (KDP136), we have found that more than 90% of intracellular WT and KDP136 colocalized with cathepsin B, a lysosome marker, and only a few of the internalized cells colocalized with LC3, an autophagosome marker, during the 0.5- to 4-h postinfection period. This was further substantiated by immunogold electron microscopic analyses, thus implying that P. gingivalisevades the autophagic pathway and instead directly traffics to the endocytic pathway to lysosomes. At the late stages after infection, WT strains in phagolysosomes retained their double-membrane structures. KDP136 in these compartments, however, lost its double-membrane structures, representing a characteristic feature of its vulnerability to rupture. Together with the ultrastructural observations, we found that the number of intracellular viable WT cells decreased more slowly than that of KDP136 cells, thus suggesting that gingipains contribute to bacterial survival, but not to trafficking, within the infected cells.

We cloned a ∼70 kDa rat mitochondrial outer membrane protein (OM70)with a sequence identity of 28.1% and 20.1% with N. crassa and S. cerevisiae Tom70, respectively. Even with this low sequence identity,however, the proteins share a remarkable structural similarity: they have 7-10 tetratricopeptide repeat motifs and are anchored to the outer membrane through the N-terminal transmembrane domain with the bulk portion located in the cytosol. Antibodies against OM70 inhibited import of preproteins, such as the ADP/ATP carrier and rTOM40, that use internal targeting signals but not the import of cleavable presequence-containing preproteins. Blue native gel electrophoresis and immunoprecipitation of digitoninsolubilized mitochondrial outer membranes revealed that OM70 was loosely associated with the ∼400 kDa translocase complex of the mitochondrial outer membrane, which contains rTOM22 and rTOM40. A yeast two-hybrid system demonstrated that OM70 interacted with rTOM20 and rTOM22 through the cytoplasmic domains. Thus, OM70 is a functional homologue of fungal Tom70 and functions as a receptor of the preprotein import machinery of the rat mitochondrial outer membrane. Furthermore, the N-terminal 66 residue region of OM70, which comprises a hydrophilic 41 residue N-terminal domain, a 22 residue transmembrane domain and three arginine residues, is sufficient to act as a mitochondria-targeting signal, and the arginine cluster is crucial for this function.

The fifth component of the COP9 signalosome complex, Jab1/CSN5, directly binds to and induces specific down-regulation of the cyclin-dependent kinase inhibitor p27 (p27Kip1). Nuclear-cytoplasmic translocation plays an important role because leptomycin B (LMB), a chemical inhibitor of CRM1-dependent nuclear export, prevents p27 degradation mediated by Jab1/CSN5. Here we show that Jab1/CSN5 functions as an adaptor between p27 and CRM1 to induce nuclear export and subsequent degradation. Jab1/CSN5, but not p27, contains a typical leucine-rich nuclear export signal (NES) sequence conserved among different species, through which CRM1 bound to Jab1/CSN5 in an LMB-sensitive manner. Alteration of conserved leucine residues to alanine within Jab1/CSN5-NES abolished the interaction with CRM1 in vitroand impaired LMB-sensitive nuclear export and the ability to induce p27 breakdown in cultured cells. A Jab1/CSN5 truncation mutant lacking NES reversed p27 down-regulation induced by the full-length Jab1/CSN5, indicating that this mutant functions as a dominant negative (DN-Jab1). Introduction of DN-Jab1 into proliferating fibroblasts increased the level of p27 protein, thereby inducing growth arrest of the cells. Random mutagenesis analysis revealed that specific aspartic acid, leucine, and asparagine residues contained in the Jab1/CSN5-binding domain of p27 were required for interaction with Jab1/CSN5 and for down-regulation of p27. Glycerol gradient and cell fractionation experiments showed that at least two different forms of Jab1/CSN5-containing complexes existed within the cell. One is the conventional 450-kDa COP9 signalosome (CSN) complex located in the nucleus, and the other is much smaller (around 100-kDa), containing only a subset of CSN components (CSN4–8 but not CSN1–3), and mainly located in the cytoplasm. Treatment of cells with LMB greatly reduced the level of the smaller complex, suggesting that it originated from the CSN complex by nuclear export. Besides Jab1/CSN5, CSN3, −6, −7, and −8 were capable of inducing p27 down-regulation, when ectopically expressed. These results indicate that cytoplasmic shuttling regulated by Jab1/CSN5 and other CSN components may be a new pathway to control the intracellular abundance of the key cell cycle regulator.

Amino acid substitutions were introduced into a structurally flexible and highly conserved region of Escherichia coliSpoT protein. SpoT protein changed from Asp to Ala at the 293rdposition did not restore cell growth of E. coliCF8295 (ΔrelA, ΔspoT) and did not accumulate ppGpp in the cell, suggesting that the Asp293 is indispensable for ppGpp synthesis of the protein.

A mitochondrial outer membrane protein of ∼22 kDa (1C9-2) was purified from Vero cells assessing immunoreactivity with a monoclonal antibody, and the cDNA was cloned based on the partial amino acid sequence of the trypsin-digested fragments. 1C9-2 had 19–20% sequence identity to fungal Tom22, a component of the preprotein translocase of the outer membrane (the TOM complex) with receptor and organizer functions. Despite such a low sequence identity, both shared a remarkable structural similarity in the hydrophobicity profile, membrane topology in the Ncyt-Cin orientation through a transmembrane domain in the middle of the molecule, and the abundant acidic amino acid residues in the N-terminal domain. The antibodies against 1C9-2 inhibited the import of a matrix-targeted preprotein into isolated mitochondria. Blue native polyacrylamide gel electrophoresis of digitonin-solubilized outer membranes revealed that 1C9-2 is firmly associated with TOM40 in the ∼400-kDa complex, with a size and composition similar to those of the fungal TOM core complex. Furthermore, 1C9-2 complemented the defects of growth and mitochondrial protein import in Δtom22yeast cells. Taken together, these results demonstrate that 1C9-2 is a functional homologue of fungal Tom22 and functions as a component of the TOM complex.

Metastin was first isolated from the human placenta and found as an endogenous ligand for GPR54, a G protein-coupled receptor and shown as a potent stimulator of GnRH/LH release in mammalian species. In female rats, metastin neurons are present in the anteroventral priventricular nucleus (AVPV), known as a target site for the estrogen positive feedback action. Metastin has been considered to be involved in controlling GnRH/LH surges, because KiSS-1 mRNA expression in the nuclei is positively controlled by estrogen. Involvement of AVPV metastin neurons in the surge is also evidenced by the absence of the neuronal population found in the male, which lacks the surge-mode of GnRH/LH release. The surge system or AVPV metastin neurons are known to be muscularized or defeminized by neonatal adrogen/estrogen treatment, but the role of neonatal endogenous androgen in muscularizing the GnRH/LH surge system and AVPV metastin neurons still remains to be elucidated. The purpose of the present study is to clarify the role of neonatal androgen in abolishing LH surge system and the AVPV metastin expression by comparing males castrated at birth (D0 Cast) with female treated with estradiol benzoate at the day 5 of age (D5 EB). Females (AD OVX) and males (AD Cast) gonadectomized in the adult serve as controls. All animals were treated with subcutaneous Silastic E2 implants to produce diestrus E2 levels and then with high level of E2 for 2 or 3 days before samplings to induce surges. The D0 Cast animals showed an apparent surge-like LH release with AVPV metastin expressions at peptide and mRNA levels, which were similar to AD OVX controls. AD Cast and D5 EB did not show LH surges with significantly lower levels of AVPV metastin expressions at peptide and mRNA levels compared with D0 Cast or AD OVX animals. Kiss-1 mRNA expression in the medial preoptic area or GPR54 mRNA expression in the AVPV, arcuate nucleus and mPOA did not show significant differences between any groups. The present study demonstrated that castrating genetical males at birth rescues metastin neuronal expressions in the AVPV and results in the generation of LH surges in the adult. Thus, the neonatal steroid environment induces the sex difference in the metastin expression in the AVPV, which may be associated with the LH surge generation. This research was supported by PROBRAIN of Japan.

It has been reported that prolonged exposure to estrogen at neonatal stage caused persistant diestrus in female rats during adulthood. The present study was performed to determine the effects of the neonatal prolonged estrogen treatment on pulsatile lutenising hormone (LH) release and expression of hypothalamic metastin/kisspeptin, a KiSS-1gene product, and its receptor, GPR54. We focused on the metastin/GPR54 signaling in the brain, because the signaling has been known to play a central role in the regulation of gonadotropin release in several mammals including human, monkeys and rodents. Newborn female Wistar-Imamichi rats were daily treated with subcutaneous injections of estradiol 3-benzoate (EB) dissolved in peanut oil from the day of birth for 10 days. The viginal smears in the animals were daily examined, and ovaries were removed when they became 11-13 weeks of age. The animals were bled for 3 h at 6-min intervals 2 weeks after the ovariectomy to investigate pulsatile LH release. Some of the neonatally EB-treated animals were injected with rat metastin (0.1 or 1 nmol/2µl UPW) into the third ventricle (3V) to investigate the LH response to exogenous metastin. The KiSS-1and GPR54mRNA levels were determined in the hypothalamic arcuate nucleus (ARC), anteroventral paraventlicular nucleus (AVPV) and medial preoptic nucleus (mPOA) in the neonatally EB- or vehicle-treated ovariectomized (OVX) rats. Immunohistochemistry for metastin/kisspeptin-, tyrosine hydroxylase (TH)- and GnRH-immunoreactive cells were performed in the ARC-median eminence region. Neonatally EB-treated female rats showed persistent viginal diestrus after pubertal period with ovary weight being significantly lower than neonatally oil-treated controls. On the other hand, neonatally oil-injected controls showed normal 4-day estrous cycle after puberty. Pulsatile LH secretion was profoundly inhibited in OVX rats with neonatal EB treatment, resulting in significantly lower mean LH levels and frequency of LH pulses compared with those in neonatally oil-treated OVX controls. Immunohistochemistry has shown that few metastin-positive cells were found in the ARC in the neonatally EB-treated OVX rats, whereas many ARC metastin-positive cells were present in neonatally oil-treated OVX controls. KiSS-1mRNA levels in the ARC of the neonatally EB-treated animals was significantly lower than those in oil-injected controls, but GPR54mRNA levels didn't show significantly change in any brain regions determined. No obvious difference was found in TH-positive cells in the ARC or GnRH-ir fibers in the median eminence between the groups. Intravenous metastin/kisspeptin challenge significantly increased plasma LH levels in neonatally EB-treated animals in a dose-dependent manner. The present results show that the prolonged neonatal EB treatment causes a reduction of ARC metastin/kisspeptin neurons in the ARC without affecting GPR54 expression at adult, resulting in the suppression of LH pulses. The results also suggest that ARC metastin neurons may play a role in generating pulsatile GnRH/LH release in female rats. This research was supported by PROBRAIN of Japan.