Apoptosis observed in BALB/3T3 cells having ingested Staphylococcus aureus.

Staphylococcus aureus was previously shown to be internalized by murine fibroblast. We examined the intracellular events of S. aureus ingested by BALB/3T3 cells. After uptake of strains A191 and A151, isolates from atopic lesion, and a laboratory strain, Cowan I, for 1 hr, BALB/3T3 cells were incubated with 1.25 microg/ml lysostaphin. Laddering of the DNA in multiples of approximately 180 bp occurred within 4 hr following bacterial addition in BALB/3T3 cells infected with A191 and within 18 hr in BALB/3T3 cells infected with A151: histochemical staining by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method revealed that the rate of the fragmentation of nucleic DNA in Cowan I-infected BALB/3T3 cells at 21 hr following bacterial addition was 0.52 +/- 0.25%, significantly higher than that in the control cells. Transmission electron micrographs of BALB/3T3 cells at 4 hr following A191 addition showed that the apoptotic features, including electron-dense nucleus and plasma membrane blebbing, occurred in some cells in which many staphylococci escaped the endosome and went on to cell division. At the same time, A151 organisms enclosed with endosome membrane were static in the intact BALB/3T3 cells. The significant increase of A191 was confirmed by counting intracellular live bacteria during 2- to 6-hr incubation. These results suggest that internalized S. aureus escapes the endosome, multiplies and induces apoptosis in the fibroblast cell.     

Replication-dependent cytotoxicity and Spartan-mediated repair of trapped PARP1–DNA complexes

The antitumor activity of poly(ADP-ribose) polymerase inhibitors (PARPis) has been ascribed to PARP trapping, which consists in tight DNA–protein complexes. Here we demonstrate that the cytotoxicity of talazoparib and olaparib results from DNA replication. To elucidate the repair of PARP1–DNA complexes associated with replication in human TK6 and chicken DT40 lymphoblastoid cells, we explored the role of Spartan (SPRTN), a metalloprotease associated with DNA replication, which removes proteins forming DPCs. We find that SPRTN-deficient cells are hypersensitive to talazoparib and olaparib, but not to veliparib, a weak PARP trapper. SPRTN-deficient cells exhibit delayed clearance of trapped PARP1 and increased replication fork stalling upon talazoparib and olaparib treatment. We also show that SPRTN interacts with PARP1 and forms nuclear foci that colocalize with the replicative cell division cycle 45 protein (CDC45) in response to talazoparib. Additionally, SPRTN is deubiquitinated and epistatic with translesion synthesis (TLS) in response to talazoparib. Our results demonstrate that SPRTN is recruited to trapped PARP1 in S-phase to assist in the excision and replication bypass of PARP1–DNA complexes.     

Involvement of the cytoplasmic juxtamembrane region of matriptase in its exclusive localization to the basolateral membrane domain of Madin–Darby canine kidney epithelial cells

Matriptase is a type II transmembrane serine protease. This protease is strongly expressed in simple epithelial cells such as enterocytes and kidney tubular cells in which the plasma membranes are separated into apical and basolateral domains. Although matriptase was found previously to occur exclusively on the basolateral membrane of enterocytes, the underlying mechanism of localization is unclear. In the present study, a full-length rat matriptase and a chimera consisting of the cytoplasmic and transmembrane regions of the protease and green fluorescent protein (designated as 1–86GFP) were found to localize exclusively to the basolateral membrane domain when expressed in Madin–Darby canine kidney epithelial cells. Mutagenesis analysis of 1–86GFP revealed that the matriptase cytoplasmic juxtamembrane amino acid residues (Lys45, Val47, and Arg50) play a role in mediating the localization in the cells. This study provides the first evidence that matriptase carries information for its localization in simple epithelia.     

Bis-THF motif of acetogenin binds to the third matrix-side loop of ND1 subunit in mitochondrial NADH-ubiquinone oxidoreductase

Natural acetogenins are among the most potent inhibitors of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I). Our photoaffinity labeling study suggested that the hydroxylated bis-THF ring moiety of acetogenins binds at "site A" in the third matrix-side loop connecting the fifth and sixth transmembrane helices in the ND1 subunit [Kakutani et al. (2010) Biochemistry 49, 4794-4803]. Nevertheless, since this proposition was led using a photoreactive Δlac-acetogenin derivative, it needs to be directly verified using a natural acetogenin-type probe. We therefore conducted photoaffinity labeling using a photoreactive natural acetogenin mimic ([(125)I]diazinylated natural acetogenin, [(125)I]DANA), which has a small photolabile diazirine group, in place of a hydroxy group, attached to the bis-THF ring moiety. Analysis of the photocross-linked protein in bovine heart submitochondrial particles unambiguously revealed that [(125)I]DANA binds to the membrane subunit ND1 with high specificity. The photocross-linking was completely blocked in the presence of just a 5-fold excess of bullatacin, indicating that [(125)I]DANA is an excellent mimic of natural acetogenins and hence binds to the site that accommodates natural products. Careful examination of the fragmentation patterns of the cross-linked ND1 generated by different proteases and their combinations indicated that the cross-linked residue is predominantly located at the supposed site A in the third matrix-side loop.     

Chemical modifications of respiratory complex I for structural and functional studies

Studies on chemical modifications of bacterial and mitochondrial complex I by synthetic chemical probes as well as endogenous chemicals have provided useful information on the structural and functional aspects of this enzyme. We herein reviewed recent studies that investigated chemical modifications of complex I by endogenous chemicals (e.g. Cys-S-nitrosation, Cys-S-glutathionylation, and Ser-O-phosphorylation) and synthetic reagents (e.g. Cys-SH modification by SH-reagents and the cross-linking of nearby subunits by bifunctional cross-linkers). We also reviewed recent photoaffinity labeling studies using complex I inhibitors, which can be recognized as “site-specific modification” by synthetic chemicals. In addition, we discussed the possibility of site-specific modification by various functional probes via ligand-directed tosylate (LDT) chemistry as a promising approach for unique biophysical studies on complex I.     

Searching for Genomic Region of High-Fat Diet-Induced Type 2 Diabetes in Mouse Chromosome 2 by Analysis of Congenic Strains

SMXA-5 mice are a high-fat diet-induced type 2 diabetes animal model established from non-diabetic SM/J and A/J mice. By using F2 intercross mice between SMXA-5 and SM/J mice under feeding with a high-fat diet, we previously mapped a major diabetogenic QTL (T2dm2sa) on chromosome 2. We then produced the congenic strain (SM.A-T2dm2sa (R0), 20.8–163.0 Mb) and demonstrated that the A/J allele of T2dm2sa impaired glucose tolerance and increased body weight and body mass index in the congenic strain compared to SM/J mice. We also showed that the combination of T2dm2sa and other diabetogenic loci was needed to develop the high-fat diet-induced type 2 diabetes. In this study, to narrow the potential genomic region containing the gene(s) responsible for T2dm2sa, we constructed R1 and R2 congenic strains. Both R1 (69.6–163.0 Mb) and R2 (20.8–128.2 Mb) congenic mice exhibited increases in body weight and abdominal fat weight and impaired glucose tolerance compared to SM/J mice. The R1 and R2 congenic analyses strongly suggested that the responsible genes existed in the overlapping genomic interval (69.6–128.2 Mb) between R1 and R2. In addition, studies using the newly established R1A congenic strain showed that the narrowed genomic region (69.6–75.4 Mb) affected not only obesity but also glucose tolerance. To search for candidate genes within the R1A genomic region, we performed exome sequencing analysis between SM/J and A/J mice and extracted 4 genes (Itga6, Zak, Gpr155, and Mtx2) with non-synonymous coding SNPs. These four genes might be candidate genes for type 2 diabetes caused by gene-gene interactions. This study indicated that one of the genes responsible for high-fat diet-induced diabetes exists in the 5.8 Mb genomic interval on mouse chromosome 2.