The microcin J25 beta-hairpin region is important for antibiotic uptake but not for RNA polymerase and respiration inhibition

The antibiotic microcin J25 (MccJ25) was cleaved by hydrolysis with thermolysin giving a two-chain peptide (MccJ25-Th19) of 10 and 9 amino acid residues. MccJ25-Th19 with deep modifications in beta-hairpin region had no effect on Escherichia coli growth, but still inhibited RNA polymerase in vitro and oxygen consumption in Salmonella strains. MccJ25-Th19 showed antibiotic activity on E. coli transformed with plasmids containing either fhuA or sbmA genes, which code for proteins involved in MccJ25 transport. These results suggest that an intact beta-hairpin region is crucial for MccJ25 import but not for inhibition of E. coli RNA polymerase or oxygen consumption in Salmonella strains.     

Microcin J25-Ga induces apoptosis in mammalian cells by inhibiting mitochondrial RNA-polymerase

MccJ25, an antimicrobial peptide, was unable to cause apoptosis of COS-7 cells in spite of inducing reactive-oxygen species overproduction as well as cytochrome c release from isolated mitochondria. Surprisingly, MccJ25-Ga, an amidated variant of MccJ25 that displays similar anti-mitochondrial effects, did induce apoptosis in COS-7. The only difference found between the activities of these peptides was the unpredicted inhibition of mitochondrial RNA synthesis by MccJ25-Ga. These results led us to hypothesize that both mitochondrial RNA polymerase and mitochondrial membrane might be the molecular targets of MccJ25-Ga in mitochondria and this combined effect may lead to apoptosis.     

The antibacterial threaded-lasso peptide capistruin inhibits bacterial RNA polymerase

Capistruin, a ribosomally synthesized, post-translationally modified peptide produced by Burkholderia thailandensis E264, efficiently inhibits growth of Burkholderia and closely related Pseudomonas strains. The functional target of capistruin is not known. Capistruin is a threaded-lasso peptide (lariat peptide) consisting of an N-terminal ring of nine amino acids and a C-terminal tail of 10 amino acids threaded through the ring. The structure of capistruin is similar to that of microcin J25 (MccJ25), a threaded-lasso antibacterial peptide that is produced by some strains of Escherichia coli and targets DNA-dependent RNA polymerase (RNAP). Here, we show that capistruin, like MccJ25, inhibits wild type E. coli RNAP but not mutant, MccJ25-resistant, E. coli RNAP. We show further that an E. coli strain resistant to MccJ25, as a result of a mutation in an RNAP subunit gene, exhibits resistance to capistruin. The results indicate that the structural similarity of capistruin and MccJ25 reflects functional similarity and suggest that the functional target of capistruin, and possibly other threaded-lasso peptides, is bacterial RNAP.     

The Ile13 residue of microcin J25 is essential for recognition by the receptor FhuA, but not by the inner membrane transporter SbmA

Entry of the peptide antibiotic microcin J25 (MccJ25) into target cells is mediated by the outer membrane receptor FhuA and the inner membrane protein SbmA. The latter also transports MccB17 into the cell cytoplasm. Comparison of MccJ25 and MccB17 revealed a tetrapeptide sequence (VGIG) common to both antibiotics. We speculated that this structural feature in MccJ25 could be a motif recognized by SbmA. To test this hypothesis, we used a MccJ25 variant in which the isoleucine in VGIG (position 13 in the MccJ25 sequence) was replaced by lysine (I13K). In experiments in which the FhuA receptor was bypassed, the substituted microcin showed an inhibitory activity similar to that of the wild-type peptide. Moreover, MccJ25 interfered with colicin M uptake by FhuA in a competition assay, while the I13K mutant did not. From these results, we propose that the Ile¹³ residue is only required for interaction with FhuA, and that VGIG is not a major recognition element by SbmA.     

Cytochromes bd-I and bo3 are essential for the bactericidal effect of microcin J25 on Escherichia coli cells

Microcin J25 has two targets in sensitive bacteria, the RNA polymerase, and the respiratory chain through inhibition of cellular respiration. In this work, the effect of microcin J25 in E. coli mutants that lack the terminal oxidases cytochrome bd-I and cytochrome bo₃ was analyzed. The mutant strains lacking cytochrome bo₃ or cytochrome bd-I were less sensitive to the peptide. In membranes obtained from the strain that only expresses cytochrome bd-I a great ROS overproduction was observed in the presence of microcin J25. Nevertheless, the oxygen consumption was less inhibited in this strain, probably because the oxygen is partially reduced to superoxide. There was no overproduction of ROS in membranes isolated from the mutant strain that only express cytochrome bo₃ and the inhibition of the cellular respiration was similar to the wild type. It is concluded that both cytochromes bd-I and bo₃ are affected by the peptide. The results establish for the first time a relationship between the terminal oxygen reductases and the mechanism of action of microcin J25.     

Arg 901 in the AE1 C-terminal tail is involved in conformational change but not in substrate binding

In our previous paper, we demonstrated that Arg 901 in the C-terminal tail of human AE1 (band 3, anion exchanger 1) had a functional role in conformational change during anion exchange. To further examine how Arg 901 is involved in conformational change, we expressed various Arg 901 mutants and alanine mutants of the C-terminal tail (from Leu 886 to Val 911) on the plasma membrane of Saccharomyces cerevisiae and evaluated the kinetic parameters of sulfate ion transport. As a result, Vmax decreased as the hydrophobicities of the 901st and peripheral hydrophilic residues increased, indicating that the hydrophobicity of the C-terminal residue is involved in the conformational change. We also found the alkali and protease resistance of the C-terminal region after Arg 901 modification with hydroxyphenylglyoxal (HPG) or phenylglyoxal (PG), a hydrophobic reagent. These results suggested that the increased hydrophobicity of the C-terminal region around Arg 901 leads to inefficient conformational change by the newly produced hydrophobic interaction.     

Redox-active tyrosine residue in the microcin J25 molecule

Microcin J25 (MccJ25) is a 21 amino acid lasso-peptide antibiotic produced by Escherichia coli and composed of an 8-residues ring and a terminal ‘tail’ passing through the ring. We have previously reported two cellular targets for this antibiotic, bacterial RNA polymerase and the membrane respiratory chain, and shown that Tyr9 is essential for the effect on the membrane respiratory chain which leads to superoxide overproduction. In the present paper we investigated the redox behavior of MccJ25 and the mutant MccJ25 (Y9F). Cyclic voltammetry measurements showed irreversible oxidation of both Tyr9 and Tyr20 in MccJ25, but infrared spectroscopy studies demonstrated that only Tyr9 could be deprotonated upon chemical oxidation in solution. Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H₂O₂ was demonstrated by Electron Paramagnetic Resonance Spectroscopy; this radical was not detected when the reaction was carried out with the MccJ25 (Y9F) mutant. These results show that the essential Tyr9, but not Tyr20, can be easily oxidized and form a tyrosyl radical.     

CD150 (SLAM) is a receptor for measles virus but is not involved in viral contact-mediated proliferation inhibition

Measles virus (MV) interacts with cellular receptors on the surface of peripheral blood lymphocytes (PBL) which mediate virus binding and uptake. Simultaneously, the direct contact of the viral glycoproteins with the cell surface induces a negative signal blocking progression to the S phase of the cell cycle, resulting in a pronounced proliferation inhibition. We selected a monoclonal antibody (MAb 5C6) directed to the surface of highly MV-susceptible B cells (B95a), which inhibits binding to and infection of cells with MV wild-type and vaccine strains. By screening a retroviral cDNA library from human splenocytes (ViraPort; Stratagene) with this antibody, we cloned and identified the recognized molecule as signaling lymphocytic activation molecule (SLAM; CD150), which is identical to the MV receptor recently found by H. Tatsuo et al. (Nature 406:893-897, 2000). After infection of cells, and after surface contact with MV envelope proteins, SLAM is downregulated from the cell surface of activated PBL and cell lines. Although anti-SLAM and/or anti-CD46 antibodies block virus binding, they do not interfere with the contact-mediated proliferation inhibition. In addition, the cell-type-specific expression of SLAM does not correlate with the sensitivity of cells for proliferation inhibition. The data indicate that proliferation inhibition induced by MV contact is independent of the presence or absence of the virus-binding receptors SLAM and CD46.     

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.     

NbALY916 is involved in potato virus X P25-triggered cell death in Nicotiana benthamiana

Systemic necrosis often occurs during viral infection of plants and is thought mainly to be the result of long-term stress induced by viral infection. Potato virus X (PVX) encodes the P25 pathogenicity factor that triggers a necrotic reaction during PVX-potato virus Ysynergistic coinfection. In this study, we discovered that NbALY916, a multifunctional nuclear protein, could interact with P25. When NbALY916 expression was reduced by tobacco rattle virus (TRV)-based virus-induced gene silencing, the accumulation of P25 was increased, which would be expected to cause more severe necrosis. However, silencing of NbALY916 reduced the extent of cell death caused by P25. Furthermore, we found that overexpression of NbALY916 increased the accumulation of H₂O₂ and triggered more extensive cell death when coexpressed with P25, even though accumulation of P25 was itself reduced by the increased expression of NbALY916. Furthermore, transient expression of P25 specifically induced the expression of NbALY916 mRNA, but not the mRNAs of three other ALYs in Nicotiana benthamiana. In addition, we showed that silencing of NbALY916 or transient overexpression of NbALY916 affected the infection of PVX in N. benthamiana. Our results reveal that NbALY916 has an antiviral role that, in the case of PVX, operates by inducing the accumulation of H₂O₂ and mediating the degradation of P25.     

Aggregation of detergent-insoluble tau is involved in neuronal loss but not in synaptic loss

Neurofibrillary tangles (NFTs), which consist of highly phosphorylated tau, are hallmarks of neurodegenerative diseases including Alzheimer disease (AD). In neurodegenerative diseases, neuronal dysfunction due to neuronal loss and synaptic loss accompanies NFT formation, suggesting that a process associated with NFT formation may be involved in neuronal dysfunction. To clarify the relationship between the tau aggregation process and synapse and neuronal loss, we compared two lines of mice expressing human tau with or without an aggregation-prone P301L mutation. P301L tau transgenic (Tg) mice exhibited neuronal loss and produced sarcosyl-insoluble tau in old age but did not exhibit synaptic loss and memory impairment. By contrast, wild-type tau Tg mice neither exhibited neuronal loss nor produced sarcosyl-insoluble tau but did exhibit synaptic loss and memory impairment. Moreover, P301L tau was less phosphorylated than wild-type tau, suggesting that the tau phosphorylation state is involved in synaptic loss, whereas the tau aggregation state is involved in neuronal loss. Finally, increasing concentrations of insoluble tau aggregates leads to the formation of fibrillar tau, which causes NFTs to form.     

CDC25B is involved in the centrosomal microtubule nucleation in two‐cell stage mouse embryos

CDC25B has been demonstrated to activate the complex of CDK1/Cyclin B and trigger mitosis. We have recently demonstrated that p‐CDC25B‐Ser351 is located at the centrosomes of mouse oocytes and contributes to the release of mouse oocytes from prophase I arrest. But much less is known about CDC25B function at the centrosome in two‐cell stage mouse embryos. Here we investigate the effect of CDC25B regulating the microtubules nucleation. Microinjection of anti‐CDC25B antibody caused aberrant microtubule nucleation. In addition, embryos injected with anti‐CDC25B antibody showed the marked absence of microtubule repolymerization and Nek2 foci after nocodazole washout. CDC25B overexpression caused microtubule‐organizing center (MTOC) overduplication. Moreover, overexpression of CDC25B–?65 mutant resulted in the loss of CDC25B localization in the perinuclear region and made CDC25B less efficient in inducing mitosis. We additionally identified that CDC25B is responsible for the pericentrin localization to the MTOC. Our data suggest an important role of CDC25B for microtubule nucleation and organization. N‐terminal of CDC25B is required for regulating the microtubule dynamics and mitotic function.     

The membrane-associated progesterone-binding protein 25-Dx is expressed in brain regions involved in water homeostasis and is up-regulated after traumatic brain injury

After traumatic brain injury, progesterone has important neuroprotective effects in the nervous system. There is better functional outcome and less oedema formation in pseudopregnant rat females (high levels of endogenous progesterone) than in males. In addition to intracellular progesterone receptors, membrane binding sites of the hormone such as 25-Dx may also be involved in neuroprotection. In the present study we investigated the distribution of the membrane-associated progesterone-binding protein 25-Dx in rat brain. Immunohistochemical analysis showed that 25-Dx is particularly abundant in the hypothalamic area, circumventricular organs, and ependymal cells of the lateral walls of the third and lateral ventricles. A strong signal was also detected in the meninges. Double immunofluorescence immunolabelling and confocal microscopy showed that 25-Dx is co-expressed with vasopressin in neurones of the paraventricular, supraoptic and retrochiasmatic nuclei. Levels of 25-Dx expression were higher in pseudopregnant females than in males. After traumatic brain injury, 25-Dx expression was up-regulated in neurones and induced in astrocytes, which play an important role in regulating water and ion homeostasis. The expression of 25-Dx in structures involved in CSF production (choroid plexus) and in osmoregulation (circumventricular organs, hypothalamus and meninges), and its up-regulation after brain damage, point to a novel and potentially important role of this progesterone-binding protein in the maintenance of water homeostasis after traumatic brain injury.     

1 alpha, 25-Dihydroxyvitamin D3 directly induces fusion of alveolar macrophages by a mechanism involving RNA and protein synthesis, but not DNA synthesis

The results of our present study indicate that 1 alpha, 25-dihydroxyvitamin D3[1 alpha, 25(OH)2D3] directly induces fusion of mouse alveolar macrophages without any participation of T-lymphocytes by a mechanism involving RNA and protein synthesis but not DNA synthesis. We have reported that 1 alpha, 25(OH)2D3 induces fusion of alveolar macrophages by a direct mechanism and by a spleen cell-mediated indirect mechanism [(1983) Proc. Natl. Acad. Sci. USA 80, 5583-5587]. Alveolar macrophages pretreated with or without anti-Thy 1.2 antibody and complement fused similarly when they were incubated with 1 alpha, 25(OH)2D3. The vitamin suppressed DNA synthesis, but it significantly enhanced RNA and protein synthesis. The 1 alpha, 25(OH)2D3-induced fusion was blocked by adding actinomycin D or cycloheximide, but not by hydroxyurea.     

Poly (ADP-ribose) polymerase (PARP) is not involved in base excision repair but PARP inhibition traps a single-strand intermediate

Base excision repair (BER) represents the most important repair pathway of endogenous DNA lesions. Initially, a base damage is recognized, excised and a DNA single-strand break (SSB) intermediate forms. The SSB is then ligated, a process that employs proteins also involved in SSB repair, e.g. XRCC1, Ligase III and possibly PARP1. Here, we confirm the role of XRCC1 and PARP in direct SSB repair. Interestingly, we uncover a synthetic lethality between XRCC1 deficiency and PARP inhibition. We also treated cells with alkylating agent dimethyl sulfate (DMS) and monitored the SSB intermediates formed during BER. DMS-induced SSBs were quickly repaired in wild-type cells; while a rapid accumulation of SSBs was observed in cells where post-incision repair was blocked by a PARP inhibitor or by XRCC1 deficiency (EM9 cells). Interestingly, DMS-induced SSBs did not accumulate in PARP1 siRNA depleted cells, demonstrating that PARP1 is not required for efficient completion of BER. Based on these results we suggest no immediate role for PARP1 in BER, but that PARP inhibitors trap PARP on the SSB intermediate formed during BER. Unexpectedly, addition of PARP inhibitor 2 h after DMS treatment still increased SSB levels indicating ongoing repair even at this late time point.     

Mechanism of adenylate kinase. Histidine-36 is not directly involved in catalysis, but protects cysteine-25 and stabilizes the tertiary structure

Several previous reports on muscle adenylate kinase (AK) have suggested that histidine-36 (His-36) is located in the binding site of adenosine 5'-triphosphate (ATP) and is involved in catalysis. We have tested the role of His-36 using site-specific mutagenesis on chicken muscle AK expressed in Escherichia coli. Three mutant proteins (H36Q, H36N, and H36G) were obtained by substituting His-36 with glutamine, asparagine, and glycine, respectively. Steady-state kinetic studies showed that the mutants have similar kinetic properties to those of the wild-type (WT) AK, which suggested that His-36 is not directly involved in catalysis. However, His-36 is likely to interact with or protect cysteine-25 (Cys-25) on the basis of the following evidence: The crystal structure of porcine muscle AK revealed a close proximity between His-36 and Cys-25; the mutants were unstable during purification (the order of stability was WT greater than H36Q greater than H36N greater than H36G); the H36G mutant readily dimerized; the sulfhydryl groups of mutants became more reactive (WT less than H36Q less than H36N) toward 5,5'-dithiobis(2-nitrobenzoic acid). Furthermore, His-36 was found to stabilize the tertiary structure of AK on the basis of guanidine hydrochloride induced denaturation studies, which showed that the conformational stability decreases in the order WT greater than H36Q greater than H36N.(ABSTRACT TRUNCATED AT 250 WORDS)