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.     

Distinct DNA methylation activity of Dnmt3a and Dnmt3b towards naked and nucleosomal DNA

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. De novo type DNA methyltransferases Dnmt3a and Dnmt3b are responsible for creating DNA methylation patterns during embryogenesis and in germ cells. Although their in vitro DNA methylation properties are similar, Dnmt3a and Dnmt3b methylate different genomic DNA regions in vivo. In the present study, we have examined the DNA methylation activity of Dnmt3a and Dnmt3b towards nucleosomes reconstituted from recombinant histones and DNAs, and compared it to that of the corresponding naked DNAs. Dnmt3a showed higher DNA methylation activity than Dnmt3b towards naked DNA and the naked part of nucleosomal DNA. On the other hand, Dnmt3a scarcely methylated the DNA within the nucleosome core region, while Dnmt3b significantly did, although the activity was low. We propose that the preferential DNA methylation activity of Dnmt3a towards the naked part of nucleosomal DNA and the significant methylation activity of Dnmt3b towards the nucleosome core region contribute to their distinct methylation of genomic DNA in vivo.     

Archaeal pre-mRNA splicing: a connection to hetero-oligomeric splicing endonuclease

Eukaryotic Cbf5 is a protein subunit of the small nucleolar RNA-protein complex. Previously, we identified, in archaeal homologs of cbf5 of the crenarchaea, Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii, the first examples of introns of archaeal protein-coding genes. Here, we report the immunological detection of Cbf5 protein of S. tokodaii, the product of the spliced cbf5 mRNA. The hetero-oligomeric splicing endonuclease activity from recombinant S. tokodaii subunits cleaved at the exon-intron boundaries of cbf5 pre-mRNA fragments,suggesting that synthesis of full-length Cbf5 protein requires this activity. Database searches and PCR screens identified additional cbf5 introns in some, but not all sequenced crenarchaeal genomes. The predicted secondary structures of exon-intron boundaries of many of the newly identified intron-containing cbf5 pre-mRNAs contained relaxed forms of the bulge-helix-bulge motif similar to that of S. tokodaii. These observations are consistent with previous reports indicating that subunit composition of the splicing endonuclease contributes to substrate specificity.     

Small molecule inhibitors of alpha-synuclein filament assembly

Alpha-synuclein is the major component of the filamentous inclusions that constitute defining characteristics of Parkinson's disease and other alpha-synucleinopathies. Here we have tested 79 compounds belonging to 12 different chemical classes for their ability to inhibit the assembly of alpha-synuclein into filaments in vitro. Several polyphenols, phenothiazines, porphyrins, polyene macrolides, and Congo red and its derivatives, BSB and FSB, inhibited alpha-synuclein filament assembly with IC(50) values in the low micromolar range. Many compounds that inhibited alpha-synuclein assembly were also found to inhibit the formation of Abeta and tau filaments. Biochemical analysis revealed the formation of soluble oligomeric alpha-synuclein in the presence of inhibitory compounds, suggesting that this may be the mechanism by which filament formation is inhibited. Unlike alpha-synuclein filaments and protofibrils, these soluble oligomeric species did not reduce the viability of SH-SY5Y cells. These findings suggest that the soluble oligomers formed in the presence of inhibitory compounds may not be toxic to nerve cells and that these compounds may therefore have therapeutic potential for alpha-synucleinopathies and other brain amyloidoses.     

Covalent circularization of exogenous RNA during incubation with a wheat embryo cell extract

Cell extracts from wheat embryos have been widely used for mRNA-directed protein production. Here, we found that a significant fraction of exogenous linear RNAs are circularized in a wheat embryo extract. The circularization was seen only in uncapped RNAs. The amount of the circular species reached around 1% of the initial RNA and increased along with an increase in the initial concentration more than proportionally. The circular RNAs were stable but unable to be translated in the extract. The circularization was competitively inhibited in the presence of a known substrate of a wheat embryo RNA ligase. Thus, we cloned the RNA ligase cDNAs. Three isoform sequences were homologous to the other plant RNA ligases. An addition of a cell-free synthesized wheat RNA ligase abolished the inhibition, which indicates a participation of its activity in the circularization. A possible role in RNA metabolism, RNA silencing in particular, is discussed.     

14-3-3 Mediates phosphorylation-dependent inhibition of the interaction between the ubiquitin E3 ligase Nedd4-2 and epithelial Na+ channels

Although recent studies show that the 14-3-3 protein is a negative regulator of ubiquitin E3 protein ligases, the molecular mechanism remains largely unknown. We previously demonstrated that 14-3-3 specifically binds one of the E3 enzymes, Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2), which can be phosphorylated by serum glucocorticoid-inducible protein kinase 1 (SGK1); this binding protects the phosphorylated/inactive hNedd4-2 from phosphatase-catalyzed dephosphorylation [Ichimura, T., et al. (2005) J. Biol. Chem. 280, 13187-13194]. Here we report an additional mechanism of 14-3-3-mediated regulation of hNedd4-2. Using surface plasmon resonance spectrometry, we show that 14-3-3 inhibits the interaction between the WW domains of hNedd4-2 and the PY motif of the epithelial Na(+) channel, ENaC. The inhibition was dose-dependent and was dependent on SGK1-catalyzed phosphorylation of Ser468 located between the WW domains. Importantly, a mutant of hNedd4-2, which can be phosphorylated by SGK1 but cannot bind 14-3-3, reduced SGK1-mediated stimulation of the ENaC-induced current in Xenopus laevis oocytes. In addition, 14-3-3 had similar effects on hNedd4-2 that had been phosphorylated by cAMP-dependent protein kinase (PKA). Our results, together with the recent finding on 14-3-3/parkin interactions [Sato, S., et al. (2006) EMBO J. 25, 211-221], suggest that 14-3-3 suppresses ubiquitin E3 ligase activities by inhibiting the formation of the enzyme/substrate complex.     

Association of the -112A>C polymorphism of the uncoupling protein 1 gene with insulin resistance in Japanese individuals with type 2 diabetes

The -112A>C polymorphism (rs10011540) of the gene for uncoupling protein 1 (UCP1) has been associated with type 2 diabetes mellitus in Japanese individuals. The aim of the present study was to investigate the effects of this polymorphism, as well as the well-known -3826A>G polymorphism (rs1800592), on clinical characteristics of type 2 diabetes. We determined the genotypes of the two polymorphisms in 93 Japanese patients with type 2 diabetes. Intramyocellular lipid content and hepatic lipid content (HLC) were measured by magnetic resonance spectroscopy. No significant differences in age, sex, BMI, or HbA1c level were detected between type 2 diabetic patients with the -112C allele and those without it. However, homeostasis model assessment for insulin resistance (p=0.0089) and HLC (p=0.012) was significantly greater in patients with the -112C allele. We did not detect an association of the -3826A>G polymorphism (rs1800592) of UCP1 gene with any measured parameters. These results suggest that insulin resistance caused by the -112C allele influences the susceptibility to type 2 diabetes.     

Design, construction, and analysis of a novel class of self-folding RNA

RNA can play multiple biological roles through use of its three-dimensional (3-D) structures. Recent advances in RNA structural biology have revealed that complex RNA 3D structures are assemblages of double-stranded helices with a variety of tertiary structural motifs. By employing RNA tertiary structural motifs together with the helices, we designed a novel class of self-folding RNA. In RNA composed of three helices (P1, P2, and P3), P1 interacts with P3 via a tetraloop-receptor interaction and P2 forms consecutive base-triples. Two designed RNAs of this class were prepared and their folding properties indicate that they form defined tertiary structures as designed. These RNAs may be used as modular units for constructing artificial ribozymes or nanometer-scale materials.