Surface exposed epitopes and structural heterogeneity of in vivo formed transthyretin amyloid fibrils

We have investigated the structure of in vivo formed transthyretin (TTR) amyloid deposits by using antisera raised against short linear sequences of the TTR molecule. In immunohistochemistry, antisera anti-TTR41-50 and anti-TTR115-124-a reacted specifically with both wildtype ATTR and ATTR V30M material, whereas only anti-TTR41-50 recognized ATTR Y114C material. Similar results were obtained by ELISA analysis of ATTR V30M and ATTR Y114C vitreous amyloid, where the anti-TTR115-124-a antiserum failed to react with ATTR Y114C material. Moreover, neither of the antisera recognized natively structured TTR present in pancreatic alpha cells. Our results strongly indicate that the TTR molecule undergoes structural changes during fibrillogenesis in vivo. The finding of a structural difference between wildtype ATTR and ATTR V30M material on one hand and ATTR Y114C material on the other suggests that the fibril formation pathway of these ATTR variants may differ in vivo.     

Effects of FXR in foam-cell formation and atherosclerosis development

Farnesoid X receptor (FXR), a bile-acid-activated member of the nuclear receptor superfamily, is essential in regulating bile-acid, cholesterol, and triglyceride homeostasis. Disruption of the FXR gene in mice results in a proatherosclerotic lipid profile with increased serum cholesterols and triglycerides. However, the role of FXR in foam-cell formation and atherosclerosis development remains unclear. The current study showed that the peritoneal macrophages isolated from FXR-null mice took up less oxidized LDL-cholesterol (oxLDL-C), which was accompanied by a marked reduction in CD36 expression in these cells. This result appears to be FXR-independent, as FXR was not detected in the peritoneal macrophages. To assess to what extent FXR modulates atherosclerosis development, FXR/ApoE double-null mice were generated. Female mice were used for atherosclerosis analysis. Compared to ApoE-null mice, the FXR/ApoE double-null mice were found to have less atherosclerotic lesion area in the aorta, despite a further increase in the serum cholesterols and triglycerides. Our results indicate that disruption of the FXR gene could attenuate atherosclerosis development, most likely resulting from reduced oxLDL-C uptake by macrophages. Our study cautions the use of serum lipid levels as a surrogate marker to determine the efficiency of FXR modulators in treating hyperlipidemia.     

Tissue inhibitors of metalloproteinases-1 (TIMP-1) and -2(TIMP-2) are major serum factors that stimulate the TIMP-1 gene in human gingival fibroblasts

We demonstrate in this study that both TIMP-1 and TIMP-2 are major serum factors that stimulate the induction of TIMP-1 mRNA in quiescent human gingival fibroblasts (Gin-1 cells) at mid-G1 (6-9 h after serum stimulation) of the cell cycle, but not that of TIMP-2. When we chased the secretion of both TIMP proteins into culture medium containing 10% FCS freed of both TIMPs, TIMP-2 secretion rose to the level in 10% FCS after 24 h, but TIMP-1 secretion remained at a fairly low level even after 3 days, thus reflecting a contrastive difference in the induction of both TIMP mRNAs. The stimulating activity of TIMP-1 on the expression of the TIMP-1 gene switched over to inhibitory activity, when the TIMP-1 concentration in the culture medium exceeded about 30 ng/ml. The depletion of TIMP-1 and TIMP-2 from FCS affected remarkably the induction of c-jun and c-fos mRNAs, but not that of c-ets-1 mRNA. TIMP-1 and TIMP-2-dependent expression of AP-1 protein was further demonstrated by using nuclear extracts of Gin-1 cells in an electrophoretic mobility shift assay.     

Docosahexaenoic acid enrichment can reduce L929 cell necrosis induced by tumor necrosis factor

We previously reported that docosahexaenoic acid (DHA) attenuated tumor necrosis factor (TNF)-induced apoptosis in human monocytic U937 cells (J. Nutr. 130: 1095-1101, 2000). In the present study, we examined the effects of DHA and other polyunsaturated fatty acids (PUFA) on TNF-induced necrosis, another mode of cell death, using L929 murine fibrosarcoma cells. After preincubation with PUFA conjugated with BSA for 24 h, cells were treated with TNF or TNF+actinomycin D (Act D). Preincubation of cells with DHA enriched this polyunsaturated acid in the phospholipids and attenuated cell death induced by either TNF or TNF+Act D. When cells were treated with TNF alone, DNA laddering was not detected, and cells were coincidently stained with both annexin V-FITC and propidium iodide, indicating that the death mode was necrotic. TNF+Act D predominantly induced necrosis, although concurrent apoptotic cell death was also observed in this case. Preincubation with oleic acid, linoleic acid or 20:3(n-3) did not affect TNF-induced necrosis. Conversely, supplementation with n-3 docosapentaenoic acid (DPAn-3) or eicosapentaenoic acid (EPA) reduced necrotic cell death, but to a lesser extent in comparison with DHA. Unlike the case of U937 cell apoptosis, arachidonic acid (AA) significantly attenuated L929 cell necrosis, and 20:3(n-6) or 22:4(n-6) showed similar or less activity, respectively. Statistical evaluation indicated that the order of effective PUFA activity was DHA>DPAn-3> or =EPA>AA approximately 20:3(n-6)> or =22:4(n-6). One step desaturation, C2 elongation or C2 cleavage within the n-6 or n-3 fatty acid group was probably very active in L929 cells, because AA, synthesized from 20:3(n-6) or 22:4(n-6), and C22 fatty acids, synthesized from AA or EPA, were preferentially retained in cellular phospholipids. These observations suggested that attenuation of TNF-induced necrosis by the supplementation of various C20 or C22 polyunsaturated fatty acids is mainly attributable to the enrichment of three kinds of polyunsaturated fatty acids, i.e., DHA, DPAn-3 or AA, in phospholipids. Among these fatty acids, DHA was the most effective in the reduction of L929 necrosis as observed in the case of U937 apoptosis. This suggests that DHA-enriched membranes can protect cell against TNF irrespective of death modes and that membranous DHA may abrogate the death signaling common to necrosis and apoptosis.     

Extrachromosomal Histone H2B Mediates Innate Antiviral Immune Responses Induced by Intracellular Double-Stranded DNA

Fragments of double-stranded DNA (dsDNA) forming a right-handed helical structure (B-DNA) stimulate cells to produce type I interferons (IFNs). While an adaptor molecule, IFN-β promoter stimulator 1 (IPS-1), mediates dsDNA-induced cellular signaling in human cells, the underlying molecular mechanism is not fully understood. Here, we demonstrate that the extrachromosomal histone H2B mediates innate antiviral immune responses in human cells. H2B physically interacts with IPS-1 through the association with a newly identified adaptor, CIAO (COOH-terminal importin 9-related adaptor organizing histone H2B and IPS-1), to transmit the cellular signaling for dsDNA but not immunostimulatory RNA. Extrachromosomal histone H2B was biologically crucial for cell-autonomous responses to protect against multiplication of DNA viruses but not an RNA virus. Thus, the present findings provide evidence indicating that the extrachromosomal histone H2B is engaged in the signaling pathway initiated by dsDNA to trigger antiviral innate immune responses.Fragments of nucleic acids derived from either infectious agents or host cells activate cell-autonomous responses to inhibit multiplication of certain viruses by inducing type I interferon (IFN) production (5). Such effects are more evident when double-stranded DNA (dsDNA) is transduced into the intracellular compartment by use of a transfection agent or electroporation method, suggesting that the DNA sensing system recognizes aberrant DNA fragments inside the cell (6, 21, 23). dsDNA forming a right-handed helical structure, i.e., B-DNA, has a greater ability to induce type I IFNs than Z-DNA, which has a left-handed zig-zag structure (6). dsDNA activates type I IFN production in a wide variety of cell types, including immune cells, such as dendritic cells and macrophages, and nonimmune cells, such as fibroblasts, epithelial cells, and thyroid cells (6, 23). Such effects of dsDNA were corroborated by the observation in mice deficient for DNase II, in which intracellular accumulation of undegraded DNA fragments resulted in hyperproduction of IFN-β, dysregulation of erythropoiesis, and symptoms resembling rheumatoid arthritis (12, 28). The loss-of-function mutation of the DNase I gene has been found in patients with systemic lupus erythematosus (SLE) and, in fact, DNase I^−/− mice manifest SLE-like symptoms with anti-DNA antibody (Ab) production (18, 27).The immunostimulatory property of dsDNA is quite similar to that of immunostimulatory RNA (isRNA), such as dsRNA and 5′-triphosphate RNA (2, 6). Indeed, the signaling pathways engaged by dsDNA in part are shared with those for isRNA. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) directly associate with isRNA and trigger signaling, while it has been demonstrated that RIG-I does not directly interact with dsDNA but mediates its signaling in a human hepatoma cell line, Huh7 (2). IFN-β promoter stimulator 1 (IPS-1, also known as mitochondrial antiviral signaling), mediates the downstream signaling induced by dsDNA or isRNA in humans, while IPS-1 solely mediates isRNA but not dsDNA signaling in mice (2, 6, 15, 22). In contrast, TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKi) are essential for dsDNA- or isRNA-induced type I IFN production in both humans and mice (2, 6). While examining distinct molecules involved in dsDNA-mediated but not isRNA-mediated upstream signaling, Z-DNA binding protein 1 (ZBP1, also known as DNA-dependent activator of IFN regulatory factors [DAI]) was identified as a candidate cytosolic DNA sensor, at least in a mouse connective tissue cell line, L929, although its in vivo role was dispensable (7, 24, 26). Recently, a PYHIN family member, Absent in melanoma 2 (AIM2) protein, was shown to associate with an inflammasome signaling adaptor, apoptosis-associated speck-like protein containing a CARD (ASC), and to play a critical role for caspase 1 activation and interleukin-1β (IL-1β) secretion in response to dsDNA (1, 3, 4, 20).In the present study, we show that extrachromosomal histone H2B is responsible for the dsDNA-induced type I IFN production in human cells and for the innate immune response to DNA virus infection.     

Selective detection and transport of fully matched DNA by DNA-loaded microtubule and kinesin motor protein

DNA-loaded microtubules (MTs) moving on a kinesin motor protein-coated substrate can selectively hybridize with a target fully matched DNA over single-base mismatched DNA and transport it. This technique is capable of collecting target biomolecules toward one point site to design new methodology of DNA analysis.     

In situ SUMOylation analysis reveals a modulatory role of RanBP2 in the nuclear rim and PML bodies

SUMO modification plays a critical role in a number of cellular functions including nucleocytoplasmic transport, gene expression, cell cycle and formation of subnuclear structures such as promyelocytic leukemia (PML) bodies. In order to identify the sites where SUMOylation takes place in the cell, we developed an in situ SUMOylation assay using a semi-intact cell system and subsequently combined it with siRNA-based knockdown of nucleoporin RanBP2, also known as Nup358, which is one of the known SUMO E3 proteins. With the in situ SUMOylation assay, we found that both nuclear rim and PML bodies, besides mitotic apparatuses, are major targets for active SUMOylation. The ability to analyze possible SUMO conjugation sites would be a valuable tool to investigate where SUMO E3-like activities and/or SUMO substrates exist in the cell. Specific knockdown of RanBP2 completely abolished SUMOylation along the nuclear rim and dislocated RanGAP1 from the nuclear pore complexes. Interestingly, the loss of RanBP2 markedly reduced the number of PML bodies, in contrast to other, normal-appearing nuclear compartments including the nuclear lamina, nucleolus and chromatin, suggesting a novel link between RanBP2 and PML bodies. SUMOylation facilitated by RanBP2 at the nuclear rim may be a key step for the formation of a particular subnuclear organization. Our data imply that SUMO E3 proteins like RanBP2 facilitate spatio-temporal SUMOylation for certain nuclear structure and function.     

Factor determining the antigenic type of interferons produced in human lymphoblastoid cell lines

The factor that determines the antigenic type of IFN produced in human lymphoblastoid cell lines was examined using live Sendai virus, ultraviolet (UV)-irradiated virus, HANA spikes exposed on L cells persistently infected with Sendai virus (L-HVJ) and poly-inosinic acid poly-cytidylic acid (poly I: C). When Sendai virus was irradiated with UV-light for 300 sec, its abilities to infect chicken eggs and induce IFN were diminished, but its HA activity was unaffected. HANA spikes exposed on L-HVJ could not induce IFN in human lymphoblastoid cell lines, although they induced IFN in mouse spleen cells. These results suggest that the induction of IFN in human lymphoblastoid cells is closely related to viral nucleic acid. Poly I: C also induced IFN in some human lymphoblastoid cell lines in which IFN production is induced by Sendai virus. The antigenic types of IFN induced by poly I: C were the same as those induced by Sendai virus. These results suggest that the antigenic type of IFN produced depends on the nature of the IFN producer cells rather than on the kind of IFN inducer.