Sorting of lipoproteins to the outer membrane in E. coli

Escherichia coli lipoproteins are anchored to the periplasmic surface of the inner or outer membrane depending on the sorting signal. An ATP-binding cassette (ABC) transporter, LolCDE, releases outer membrane-specific lipoproteins from the inner membrane, causing the formation of a complex between the released lipoproteins and the periplasmic molecular chaperone LolA. When this complex interacts with outer membrane receptor LolB, the lipoproteins are transferred from LolA to LolB and then localized to the outer membrane. The structures of LolA and LolB are remarkably similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta-barrel and an alpha-helical lid. Structural differences between the two proteins reveal the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB. Strong inner membrane retention of lipoproteins occurs with Asp at position 2 and a few limited residues at position 3. The inner membrane retention signal functions as a Lol avoidance signal and inhibits the recognition of lipoproteins by LolCDE, thereby causing their retention in the inner membrane. The positive charge of phosphatidylethanolamine and the negative charge of Asp at position 2 are essential for Lol avoidance. The Lol avoidance signal is speculated to cause the formation of a tight lipoprotein-phosphatidylethanolamine complex that has five acyl chains and therefore cannot be recognized by LolCDE.     

Bifidobacterium suppresses IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells

Sixteen heat-killed bifidobacteria isolated from human intestine and a probiotic strain Lactobacillus GG were tested for their ability to influence IgE-mediated degranulation of rat basophilic leukemia (RBL-2H3) cells in vitro . The bifidobacteria suppressed IgE-mediated degranulation of RBL-2H3 cells by 1.6–56.4% in a strain-dependent manner. Bifidobacteria from healthy infants expressed high inhibitory effects on IgE-mediated degranulation (41–55%), while those from allergic infants varied greatly in their effects against degranulation. Bifidobacteria taxonomically identified as Bifidobacterium bifidum exhibited much stronger inhibitory effects against IgE-mediated degranulation than those taxonomically identified as B. adolescentis ( P < 0.05).These results indicate that the intestinal bifidobacteria might be one of factors influencing IgE-mediated allergic responses.     

Presence of multiple copies of capsulation loci in invasive Haemophilus influenzae type b (Hib) strains in Japan before introduction of the Hib conjugate vaccine

Despite the effectiveness of the Hib vaccine, multiple amplification of the capb locus contributes to vaccine failure. However, there has been no report on the effect of Hib locus amplification in Japan. We examined 24 Hib strains from Japanese children with invasive diseases due to Hib. Although all strains showed the same capb sequence, Southern blot analysis showed that four strains (16.7%) harbored multiple copies (more than two) of the capb locus. Careful analysis of the locus in circulating Hib strains is necessary now that the Hib vaccine has been introduced into Japan.     

Genetic analysis of the mode of interplay between an ATPase subunit and membrane subunits of the lipoprotein-releasing ATP-binding cassette transporter LolCDE

The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the inner membrane. Thus, functional interaction between LolD and LolC/E is critically important for coupling of ATP hydrolysis to the lipoprotein release reaction. LolD contains a characteristic sequence called the LolD motif, which is highly conserved among LolD homologs but not other ABC transporters of E. coli. The LolD motif is suggested to be a region in contact with LolC/E, judging from the crystal structures of other ABC transporters. To determine the functions of the LolD motif, we mutagenized each of the 32 residues of the LolD motif and isolated 26 dominant-negative mutants, whose overexpression arrested growth despite the chromosomal lolD(+) background. We then selected suppressor mutations of the lolC and lolE genes that correct the growth defect caused by the LolD mutations. Mutations of the lolC suppressors were mainly located in the periplasmic loop, whereas ones of lolE suppressors were mainly located in the cytoplasmic loop, suggesting that the mode of interaction with LolD differs between LolC and LolE. Moreover, the LolD motif was found to be critical for functional interplay with LolC/E, since some LolD mutations lowered the ATPase activity of LolCDE without affecting that of LolD.     

Roles of the hydrophobic cavity and lid of LolA in the lipoprotein transfer reaction in Escherichia coli

LolA, a periplasmic chaperone, binds to outer membrane-specific lipoproteins released from the inner membrane through the action of an ATP-binding cassette transporter, LolCDE and then transfers them to the outer membrane receptor LolB, thereby mediating the inner to outer membrane transport of lipoproteins. The crystal structure of free LolA revealed that it has an internal hydrophobic cavity, which is surrounded by hydrophobic residues and closed by a lid comprising alpha-helices. The hydrophobic cavity most likely represents the binding site for the lipid moiety of a lipoprotein. It is speculated that the lid undergoes opening and closing upon the binding and transfer of lipoproteins, respectively. To determine the functions of the hydrophobic cavity and lid in detail, 14 residues involved in the formation of these structures were subjected to random mutagenesis. Among the obtained 21 LolA derivatives that did not support growth, 14 were active as to the binding of lipoproteins but defective in the transfer of lipoproteins to LolB, causing the periplasmic accumulation of a lipoprotein as a complex with a LolA derivative. A LolA derivative, I93G, bound lipoproteins faster than wild-type LolA did, whereas it did not transfer associated lipoproteins to LolB. When I93G and wild type LolA co-existed, lipoproteins were bound only to I93G; which therefore exhibited a dominant negative property. Another derivative, L59R, was also defective in the transfer of lipoproteins to LolB but did not exhibit a dominant negative property. Taken together, these results indicate that both the hydrophobic cavity and the lid are critically important for not only the binding of lipoproteins but also their transfer.     

MARCH-V is a novel mitofusin 2- and Drp1-binding protein able to change mitochondrial morphology

Mitofusins and Drp1 are key components in mitochondrial membrane fusion and division, but the molecular mechanism underlying the regulation of their activities remains to be clarified. Here, we identified human membrane-associated RING-CH (MARCH)-V as a novel transmembrane protein of the mitochondrial outer membrane. Immunoprecipitation studies demonstrated that MARCH-V interacts with mitofusin 2 (MFN2) and ubiquitinated forms of Drp1. Overexpression of MARCH-V promoted the formation of long tubular mitochondria in a manner that depends on MFN2 activity. By contrast, mutations in the RING finger caused fragmentation of mitochondria. We also show that MARCH-V promotes ubiquitination of Drp1. These results indicate that MARCH-V has a crucial role in the control of mitochondrial morphology by regulating MFN2 and Drp1 activities.     

Identification of viral induced genes in Ig+ leucocytes of Japanese flounder Paralichthys olivaceus, by differential hybridisation with subtracted and un-subtracted cDNA probes

Up-regulated genes of leucocytes expressing immunoglobulin (Ig+ leucocytes) of hirame rhabdovirus (HRV)-infected Japanese flounder were identified by differential hybridisation, using subtracted and un-subtracted cDNA probes. Ig+ leucocytes were separated from apparently healthy and HRV-infected Japanese flounder by the magnetic beads antibody method using mouse anti-Japanese flounder Ig monoclonal antibody (mab). A cDNA library was constructed from HRV-infected Japanese flounder leucocytes, and was screened with subtracted cDNA probes enriched in genes up-regulated by HRV infection. Fifty cDNAs were isolated for further analysis. These included cDNAs coding for homologues of interferon-inducible 56K protein (IFI56), Stat3, CEF-10, RGS5, inducible poly(A) binding protein, prolylcarboxylpeptidase, basigin III (Ig superfamily), MUC-18 (Ig superfamily), proteasome-nexin 1 (SERPIN), herpes virus entry mediator (TNFR family), collagenase III, gelatinase-b, megakaryocyte stimulating factor, Rab8-interacting protein, IgM, IgD and 20 unknown cDNA clones. The majority of these identified genes are reported for the first time in fish. From leucocytes mRNA for homologues of IFI56, CEF-10, Stat3, SERPIN and inducible poly (A) binding protein expression was shown to increase following HRV infection.     

Exercise-induced muscle damage impairs insulin signaling pathway associated with IRS-1 oxidative modification

Strenuous exercise induces delayed-onset muscle damage including oxidative damage of cellular components. Oxidative stress to muscle cells impairs glucose uptake via disturbance of insulin signaling pathway. We investigated glucose uptake and insulin signaling in relation to oxidative protein modification in muscle after acute strenuous exercise. ICR mice were divided into sedentary and exercise groups. Mice in the exercise group performed downhill running exercise at 30 m/min for 30 min. At 24 hr after exercise, metabolic performance and insulin-signaling proteins in muscle tissues were examined. In whole body indirect calorimetry, carbohydrate utilization was decreased in the exercised mice along with reduction of the respiratory exchange ratio compared to the rested control mice. Insulin-stimulated uptake of 2-deoxy-[(3)H]glucose in damaged muscle was decreased after acute exercise. Tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and phosphatidyl-3-kinase/Akt signaling were impaired by exercise, leading to inhibition of the membrane translocation of glucose transporter 4. We also found that acute exercise caused 4-hydroxy-nonenal modification of IRS-1 along with elevation of oxidative stress in muscle tissue. Impairment of insulin-induced glucose uptake into damaged muscle after strenuous exercise would be related to disturbance of insulin signal transduction by oxidative modification of IRS-1.