Cooperation of the N-terminal Helicase and C-terminal endonuclease activities of Archaeal Hef protein in processing stalled replication forks

Blockage of replication fork progression often occurs during DNA replication, and repairing and restarting stalled replication forks are essential events in all organisms for the maintenance of genome integrity. The repair system employs processing enzymes to restore the stalled fork. In Archaea Hef is a well conserved protein that specifically cleaves nicked, flapped, and fork-structured DNAs. This enzyme contains two distinct domains that are similar to the DEAH helicase family and XPF nuclease superfamily proteins. Analyses of truncated mutant proteins consisting of each domain revealed that the C-terminal nuclease domain independently recognized and incised fork-structured DNA. The N-terminal helicase domain also specifically unwound fork-structured DNA and Holliday junction DNA in the presence of ATP. Moreover, the endonuclease activity of the whole Hef protein was clearly stimulated by ATP hydrolysis catalyzed by the N-terminal domain. These enzymatic properties suggest that Hef efficiently resolves stalled replication forks by two steps, which are branch point transfer to the 5'-end of the nascent lagging strand by the N-terminal helicase followed by template strand incision for leading strand synthesis by the C-terminal endonuclease.     

Extracellular inorganic phosphate regulates gibbon ape leukemia virus receptor-2/phosphate transporter mRNA expression in rat bone marrow stromal cells

In mammalian cells, several observations indicate not only that phosphate transport probably regulates local inorganic phosphate (Pi) concentration, but also that Pi affects normal cellular metabolism, which in turn regulates apoptosis and the process of mineralization. To elucidate how extracellular Pi regulates cellular functions of pre-osteoblastic cells, we investigated the expression of type III sodium (Na)-dependent Pi transporters in rat bone marrow stromal cells and ROB-C26 pre-osteoblastic cells. The mRNA expression level of gibbon ape leukemia virus receptor (Glvr)-2 was increased by the addition of Pi in rat bone marrow stromal cells, but not in ROB-C26 or normal rat kidney (NRK) cells. In contrast, the level of Glvr-1 mRNA was not altered by the addition of extracellular Pi in these cells. The induction of Glvr-2 mRNA by Pi was inhibited in the presence of cycloheximide (CHX). Moreover, mitogen-activated protein kinase (MEK) /extracellular-signal-regulated kinase (ERK) pathway inhibitors; U0126 (1.4-diamino-2, 3-dicyano-1, 4-bis [2-amino-phenylthio] butadiene) and PD98059 (2'-Amino-3'-methoxyflavone) inhibited inducible Glvr-2 mRNA expression, but p38 MEK inhibitor SB203580 [4-(4'-fluorophenyl)-2-(4'-methyl-sulfinylphenyl)-5-(4'pyridyl) imidazole] did not inhibit the induction of Glvr-2 mRNA expression, suggesting that extracellular Pi regulates de novo protein synthesis and MEK/ERK activity in rat bone marrow stromal cells, and through these, induction of Glvr-2 mRNA. Although Pi also induced osteopontin mRNA expression in rat bone marrow stromal cells but not in ROB-C26 and NRK cells, changes in cell viability with the addition of Pi were similar in both cell types. These data indicate that extracellular Pi regulates Glvr-2 mRNA expression, provide insights into possible mechanisms whereby Pi may regulate protein phosphorylation, and suggest a potential role for the Pi transporter in rat bone marrow stromal cells.     

A signal adaptor SLAM-associated protein regulates spontaneous autoimmunity and Fas-dependent lymphoproliferation in MRL-Faslpr lupus mice

Autoantibody production and lymphadenopathy are common features of systemic autoimmune disease. Targeted or spontaneous mutations in the mouse germline have generated many autoimmune models with these features. Importantly, the models have provided evidence for the gene function in prevention of autoimmunity, suggesting an indispensable role for the gene in normal immune response and homeostasis. We describe here pathological and genetic characterizations of a new mutant strain of mice, the mutation of which spontaneously occurred in the Fas-deficient strain, MRL/Mp.Faslpr (MRL/lpr). MRL/lpr is known to stably exhibit systemic lupus erythematosus-like diseases. However, the mutant mice barely displayed autoimmune phenotypes, though the original defect in Fas expression was unchanged. Linkage analysis using (mutant MRL/lpr x C3H/lpr)F2 mice demonstrated a nucleotide insertion that caused loss of expression of small adaptor protein, signaling lymphocyte activation molecule (SLAM)-associated protein (SAP). SAP is known to be a downstream molecule of SLAM family receptors and to mediate the activation signal for tyrosine kinase Fyn. Recent studies have shown pleiotropic roles of SAP in T, B, and NK cell activations and NKT cell development. The present study will provide evidence for an essential role for SAP in the development of autoimmune diseases, autoantibodies, and lymphadenopathy in MRL/lpr lupus mice.     

Identification of a novel tetrapeptide structure of the Mycobacterium avium glycopeptidolipid that functions as a specific target for the host antibody response

Mycobacterium avium complex (MAC) is a group of non-tuberculous mycobacteria that cause tuberculosis-like diseases in humans. Unlike Mycobacterium tuberculosis, MAC expresses high levels of glycopeptidolipids (GPLs) containing a well-defined tetrapeptide-amino alcohol core, composed of D-phenylalanine, D-allo-threonine, D-alanine, and L-alaninol, that is modified with a fatty acid and sugar residues. Surprisingly, however, a careful scrutiny of the mass spectrum of MAC GPLs revealed the presence of ions that could not readily accountable for the known GPL structure. The magnitude of the ions was increased prominently when GPLs were isolated from the valine-supplemented culture, and the ions representing the authentic GPL species were diminished, suggesting the possibility that the basic structure of the peptide backbone might be altered in response to the exogenously added valine. Indeed, further mass spectrometry (MS)/MS and gas chromatography-MS analysis indicated a substitution of D-valine for the N-terminal D-phenylalanine of the tetrapeptide core, and the presence of D-valine and the absence of D-phenylalanine was confirmed by high-performance liquid chromatography, using the derivatized amino acid residues that were released from the tetrapeptide. Finally, specific antibodies to the purified valine-containing GPL species were detected in the serum of a MAC-infected guinea pig. Therefore, these results identify a new molecular species of MAC GPLs with immunogenic potential.     

Sar1 translocation onto the ER-membrane for vesicle budding has different pathways for promotion and suppression of ER-to-Golgi transport mediated through H89-sensitive kinase and ER-resident G protein

ER-to-Golgi protein transport involves transport vesicles of which formation is initiated by assembly of Sar1. The assembly of Sar1 is suppressed by protein kinase inhibitor H89, suggesting that ER-to-Golgi transport is regulated progressively by H89 sensitive kinase. ER-resident G(i2) protein suppresses vesicle formation with inhibition of Sar1 assembly. This study examined whether these promotion and suppression of vesicle transport share the same signal pathway, by examining the effects of G(i/o) protein activator mastoparan 7 (Mp-7) and H89 on Sar1 and Sec23 recruitment onto microsomes. In a cell-free system for Sar1 translocation assay, GTPγS addition induced the translocation of Sar1 onto microsomes. Mp-7 and H89 decreased the Sar1 translocation. Double treatment of Mp-7 and H89 strongly decreased Sar1 translocation. In single and double treatments, however, G(i/o) protein inactivator pertussis toxin (IAP) partially restored the suppressive effect of Mp-7, but had not any effect on H89-induced effect. Then, the assembly of Sec23 onto the microsome was also increased by the addition of GTPγS. Sec23 translocation was decreased by Mp-7 and/or H89 treatment and recovered by IAP pretreatment except for H89 single treatment, similarly to Sar1 translocation in each treatment. Inhibitory effects of H89 and Mp-7on ER-to-Golgi vesicle transport by H89 or Mp-7 were also confirmed in a cell culture system by BFA-dispersion and BFA-reconstruction experiments. These findings indicate that promotion and suppression of ER-to-Golgi vesicle transport are modulated through separate signal pathways.     

Osteocyte network; a negative regulatory system for bone mass augmented by the induction of Rankl in osteoblasts and Sost in osteocytes at unloading

Reduced mechanical stress is a major cause of osteoporosis in the elderly, and the osteocyte network, which comprises a communication system through processes and canaliculi throughout bone, is thought to be a mechanosensor and mechanotransduction system; however, the functions of osteocytes are still controversial and remain to be clarified. Unexpectedly, we found that overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteoblast and osteoclast differentiation were unaffected by BCL2 transgene in vitro. However, the cortical bone mass increased due to enhanced osteoblast function and suppressed osteoclastogenesis at 4 months of age, when the frequency of TUNEL-positive lacunae reached 75%. In the unloaded condition, the trabecular bone mass decreased in both wild-type and BCL2 transgenic mice at 6 weeks of age, while it decreased due to impaired osteoblast function and enhanced osteoclastogenesis in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Rankl and Opg were highly expressed in osteocytes, but Rankl expression in osteoblasts but not in osteocytes was increased at unloading in wild-type mice but not in BCL2 transgenic mice at 4 months of age. Sost was locally induced at unloading in wild-type mice but not in BCL2 transgenic mice, and the dissemination of Sost was severely interrupted in BCL2 transgenic mice, showing the severely impaired osteocyte network. These findings indicate that the osteocyte network is required for the upregulation of Rankl in osteoblasts and Sost in osteocytes in the unloaded condition. These findings suggest that the osteocyte network negatively regulate bone mass by inhibiting osteoblast function and activating osteoclastogenesis, and these functions are augmented in the unloaded condition at least partly through the upregulation of Rankl expression in osteoblasts and that of Sost in osteocytes, although it cannot be excluded that low BCL2 transgene expression in osteoblasts contributed to the enhanced osteoblast function.     

MRGD, a MAS-related G-protein coupled receptor, promotes tumorigenisis and is highly expressed in lung cancer

To elucidate the function of MAS-related GPCR, member D (MRGD) in cancers, we investigated the in vitro and in vivo oncogenic function of MRGD using murine fibroblast cell line NIH3T3 in which MRGD is stably expressed. The expression pattern of MRGD in clinical samples was also analyzed. We found that overexpression of MRGD in NIH3T3 induced focus formation and multi-cellular spheroid formation, and promoted tumors in nude mice. In other words, overexpression of MRGD in NIH3T3 induced the loss of contact inhibition, anchorage-independent growth and in vivo tumorigenesis. Furthermore, it was found that the ligand of MRGD, beta-alanine, enhanced spheroid formation in MRGD-expressing NIH3T3 cells. From investigation of clinical cancer tissues, we found high expression of MRGD in several lung cancers by immunohistochemistry as well as real time PCR. Based on these results, MRGD could be involved in tumorigenesis and could also be a novel anticancer drug target.