Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex.

To facilitate selfish replication, viruses halt host gene expression in various ways. The nuclear export of mRNA is one such process targeted by many viruses. SARS-CoV-2, the etiological agent of severe acute respiratory syndrome, also prevents mRNA nuclear export. In this study, Nsp14, a bifunctional viral replicase subunit, was identified as a novel inhibitor of mRNA nuclear export. Nsp14 induces poly(A)+ RNA nuclear accumulation and the dissolution/coalescence of nuclear speckles. Genome-wide gene expression analysis revealed the global dysregulation of splicing and 3'-end processing defects of replication-dependent histone mRNAs by Nsp14. These abnormalities were also observed in SARS-CoV-2-infected cells. A mutation introduced at the guanine-N7-methyltransferase active site of Nsp14 diminished these inhibitory activities. Targeted capillary electrophoresis-mass spectrometry analysis (CE-MS) unveiled the production of N7-methyl-GTP in Nsp14-expressing cells. Association of the nuclear cap-binding complex (NCBC) with the mRNA cap and subsequent recruitment of U1 snRNP and the stem-loop binding protein (SLBP) were impaired by Nsp14. These data suggest that the defects in mRNA processing and export arise from the compromise of NCBC function by N7-methyl-GTP, thus exemplifying a novel viral strategy to block host gene expression.

Importins: Diverse roles in male fertility.

Regulated nucleocytoplasmic transport is central to the changes in gene expression that underpin cellular development and homeostasis, including in the testis, and proteins in the importin family are the predominant facilitators of cargo transport through the nuclear envelope. Reports documenting cell-specific profiles of importin transcripts and proteins during spermatogenesis led us to hypothesize that importins facilitate developmental switches in the testis. More recently, importins have been shown to serve additional functions, both inside and outside the nucleus; these include acting as subcellular scaffolding, mediating cellular stress responses, and controlling transcription. This paper seeks to provide an overview and update on the functions of importin proteins, with a focus on testis development and spermatogenesis. We present an extended survey of importins by combining published single cell RNAseq data with immunohistochemistry on developing and adult mouse testes. This approach reinforces and broadens knowledge of importins in biological processes, including in spermatogenesis and during testis development, revealing additional avenues for impactful investigations.

ACAGT-007a, an anti-cancer compound that modulates ERK MAPK signaling, induces nuclear enrichment of phosphorylated ERK in T3M4 pancreatic cancer cells.

The extracellular-signal-regulated-kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT-007a (GT-7), an anti-cancer compound, stimulates ERK phosphorylation, thereby inducing growth inhibition and apoptosis in T3M4 pancreatic cancer cells. However, how GT-7 stimulates ERK phosphorylation and induces apoptosis in ERK-active T3M4 cells remains unclear. To look into the mechanism, we performed a spatiotemporal analysis of ERK phosphorylation mediated by GT-7 in T3M4 cells. The immunoblotting showed that GT-7 stimulates ERK phosphorylation within 1 h, which was more remarkable after 2 h. Importantly, apoptosis induction as evaluated by the cleaved Caspase-3 was observed only after 2-h incubation with GT-7. The immunofluorescence staining revealed the enrichment of phosphorylated ERK (phospho-ERK) in the nucleus upon 1-h incubation with GT-7. Fractionation experiments showed that GT-7 increases phospho-ERK levels in the cytoplasm within 1 h, whereas nuclear phospho-ERK accumulation is observed after 2-h incubation with GT-7. MEK inhibition by U0126 significantly diminishes nuclear phospho-ERK distribution and apoptosis induction stimulated by GT-7. Thus, GT-7 may initiate the induction of ERK phosphorylation in the cytoplasm, which leads to phospho-ERK enrichment in the nucleus. This nuclear phospho-ERK accumulation by GT-7 precedes and may underlie apoptosis induction in T3M4.

Extracellular acidification augments sclerostin and osteoprotegerin production by Ocy454 mouse osteocytes.

Osteocytes sense the microenvironmental stimuli, including mechanical stress, and regulate bone resorption by osteoclasts and bone formation by osteoblasts. Diabetes and cancer metastasis to bone raise l-lactic acid in the bone tissue, causing acidification. Here, we investigated the effects of l-lactic acid and extracellular acidification on the function of mouse Ocy454 osteocytes. L- and d-lactic acid with low chiral selectivity and acidification of the medium raised the production of sclerostin and osteoprotegerin by Ocy454 cells. The mRNA expression of their genes increased after either treatment of L- and d-lactic acid or acidification of the medium. Furthermore, the conditioned medium of Ocy454 cells cultured in an acidic environment suppressed the induction of alkaline phosphatase activity in MC3T3-E1 cells, which was recovered by the anti-sclerostin antibody. While it is reported that HDAC5 inhibits the transcription of the sclerostin gene, extracellular acidification reduced the nuclear localization of HDAC5 in Ocy454 cells. While calmodulin kinase II (CaMKII) is known to phosphorylate and induce extranuclear translocation of HDAC5, KN-62, an inhibitor of CaMKII lowered the expression of the sclerostin gene in Ocy454 cells. Collectively, extracellular acidification is a microenvironmental factor that modulates osteocyte functions.

8-Nitro-cGMP suppresses mineralization by mouse osteoblasts.

Nitric oxide and reactive oxygen species regulate bone remodeling, which occurs via bone formation and resorption by osteoblasts and osteoclasts, respectively. Recently, we found that 8-nitro-cGMP, a second messenger of nitric oxide and reactive oxygen species, promotes osteoclastogenesis. Here, we investigated the formation and function of 8-nitro-cGMP in osteoblasts. Mouse calvarial osteoblasts were found to produce 8-nitro-cGMP, which was augmented by tumor necrosis factor-α (10 ng/ml) and interleukin-1ß (1 ng/ml). These cytokines suppressed osteoblastic differentiation in a NO synthase activity-dependent manner. Exogenous 8-nitro-cGMP (30 µmol/L) suppressed expression of osteoblastic phenotypes, including mineralization, in clear contrast to the enhancement of mineralization by osteoblasts induced by 8-bromo-cGMP, a cell membrane-permeable analog of cGMP. It is known that reactive sulfur species denitrates and degrades 8-nitro-cGMP. Mitochondrial cysteinyl-tRNA synthetase plays a crucial role in the endogenous production of RSS. The expression of osteoblastic phenotypes was suppressed by not only exogenous 8-nitro-cGMP but also by silencing of the Cars2 gene, indicating a role of endogenous 8-nitro-cGMP in suppressing the expression of osteoblastic phenotypes. These results suggest that 8-nitro-cGMP is a negative regulator of osteoblastic differentiation.

Zoledronate promotes inflammatory cytokine expression in human CD14-positive monocytes among peripheral mononuclear cells in the presence of γδ T cells.

Bisphosphonates distributed to bone exert toxic effects specifically towards osteoclasts. On the other hand, intravenous administration of a nitrogen-containing bisphosphonate (N-BP) such as zoledronate induces acute-phase reactions (APRs), including influenza-like fever 1 day later, indicating an interaction with immunocompetent cells circulating blood. Although it has been reported that activation of γδ T cells is pivotal to induce an APR following treatment with zoledronate, downstream events, including the production of inflammatory cytokines after activation of γδ T cells, remain obscure. We investigated the effects of zoledronate on inflammatory cytokine expression in human peripheral blood mononuclear cells (PBMCs) in vitro. While zoledronate induced mRNA expressions of tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 and interferon-γ (IFN-γ) in PBMC, depletion of γδ T cells abolished that zoledronate-induced expression of those cytokines, indicating the necessity of γδ T cells for expression induction by zoledronate. However, which types of cells were responsible for the production of those cytokines in blood remained unclear. As it is generally accepted that monocytes and macrophages are primary sources of inflammatory cytokines, CD14+ cells from PBMC were exposed to zoledronate in the presence of PBMC, which resulted in induced expression of mRNAs for IL-1ß, IL-6 and IFN-γ, but not for TNF-α. These results indicate that CD14+ cells are responsible, at least in part, for the production of IL-1ß, IL-6 and IFN-γ in blood exposed to zoledronate. This suggests that CD14+ cells play an essential role in the occurrence of APRs following N-BP administration.

Genetic loss of importin α4 causes abnormal sperm morphology and impacts on male fertility in mouse.

Importin α proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin α4, which is encoded by the Kpna4 gene (Kpna4-/- ), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin α3 (Kpna3-/- ) were fertile. In vitro fertilization and sperm motility assays demonstrated that sperm from Kpna4-/- mice had significantly reduced quality and motility. In addition, acrosome reaction was also impaired in Kpna4-/- mice. Transmission electron microscopy revealed striking defects, including abnormal head morphology and multiple axoneme structures in the flagella of Kpna4-/- mice. A five-fold increase in the frequency of abnormalities in Kpna4-/- mice compared to WT mice indicates the functional importance of importin α4 in normal sperm development. Moreover, Nesprin-2, which is a component of the linker of nucleus and cytoskeleton complex, was expressed at lower levels in sperm from Kpna4-/- mice and was localized with abnormal axonemes, suggesting incorrect formation of the nuclear membrane-cytoskeleton structure during spermiogenesis. Proteomics analysis of Kpna4-/- testis showed significantly altered expression of proteins related to sperm formation, which provided evidence that genetic loss of importin α4 perturbed chromatin status. Collectively, these findings indicate that importin α4 is critical for establishing normal sperm morphology in mice, providing new insights into male germ cell development by highlighting the requirement of importin α4 for normal fertility.

Total Synthesis of Dictyodendrins A-F by the Gold-Catalyzed Cascade Cyclization of Conjugated Diyne with Pyrrole.

Invited for the cover of this issue is Hiroaki Ohno and co-workers at Kyoto University and National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN). The image depicts gold catalysis which promotes the domino reaction to push the switch for the diversity-directed total synthesis. Read the full text of the article at 10.1002/chem.202001950.

Total Synthesis of Dictyodendrins A-F by the Gold-Catalyzed Cascade Cyclization of Conjugated Diyne with Pyrrole.

The total synthesis of dictyodendrins A-F was achieved by using the gold(I)-catalyzed annulation of a conjugated diyne with N-Boc-pyrrole for direct construction of the pyrrolo[2,3-c]carbazole scaffold. Late-stage functionalization of the resulting pyrrolo[2,3-c]carbazole to introduce various substituents provided divergent access to dictyodendrins. Some dictyodendrin analogues exhibited inhibitory activities toward CDK2/CycA2 and GSK3.

Correction: Development of oligonucleotide-based antagonists of Ebola virus protein 24 inhibiting its interaction with karyopherin alpha 1.

Correction for 'Development of oligonucleotide-based antagonists of Ebola virus protein 24 inhibiting its interaction with karyopherin alpha 1' by Keisuke Tanaka et al., Org. Biomol. Chem., 2018, 16, 4456-4463.

Development of oligonucleotide-based antagonists of Ebola virus protein 24 inhibiting its interaction with karyopherin alpha 1.

The investigation of protein-protein interactions (PPIs) and the preparation of antagonists are important for determining whether certain proteins are suitable medical targets. In the present study, we used the capillary electrophoresis-systematic evolution of ligands by exponential enrichment to generate natural and artificial nucleic acid aptamers targeting Ebola virus protein 24 (eVP24), demonstrating that artificial aptamers, synthesised utilising a uridine analogue with an adenine residue at its C5 position, exhibited activities exceeding those of natural ones. To confirm the functionality of the as-prepared aptamers, their abilities to inhibit the PPIs of eVP24 were determined by capillary electrophoresis and bio-layer interferometry, and the obtained results unambiguously demonstrated that these aptamers interacted with the functional site of eVP24 and were thus good antagonists.

Extracellular matrix loss in chondrocytes after exposure to interleukin-1ß in NADPH oxidase-dependent manner.

Osteoarthritis is a degenerative joint disease caused by excessive death of chondrocytes and loss of the extracellular matrix (ECM) in articular cartilage. We previously reported that reactive oxygen species (ROS) generated by the NADPH oxidase (NOX) isoform NOX-2 are involved in chondrocyte death induced by interleukin-1ß (IL-1ß). In this study, we investigate the role of NOX-2 in the production and degradation of ECM by chondrocytes. Although IL-1ß lowered the mRNA expression of type II collagen (Col2a1) and aggrecan (Acan) in mouse chondrocyte-like ATDC5 cells, RNA silencing of Nox2 did not change the mRNA expression of these major components of the ECM of cartilage. Hence, NOX-2 is not involved in the IL-1ß-induced suppression of ECM production. On the other hand, the NOX inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), the ROS scavenger N-acetylcysteine and an antisense oligodeoxynucleotide for Nox2 prevented the loss of proteoglycan induced by IL-1ß in highly differentiated ATDC5 cells. Furthermore, AEBSF did not affect the expression of hyaluronidase-1 and -2, whereas it suppressed hyaluronidase activity in culture medium. IL-1ß-induced intra- and extracellular acidification was also suppressed by AEBSF, as was the antisense oligodeoxynucleotide for Nox2. Since hyaluronidase activity is known to be higher under acidic conditions, NOX-2 probably contributes to ECM loss by the activation of hyaluronidase through acidification.

Nanoindentation time-dependent deformation/recovery suggestive of methylglyoxal induced glycation in calcified nodules.

Although empirical findings have indicated increase in bone fracture risk in type 2 diabetes patients, that has yet to be proven by results obtained at the material level. Here, we report evidence showing nanoscale time-dependent deformation/recovery of in vitro calcified nodules mimicking bone turnover in type 2 diabetes in respect to methylglyoxal (MG)-induced glycation. Nanoindentation test results revealed that calcified nodules cultured with MG did not show adequate dimensional recovery, despite a large creep rate during constant load indentation testing. This lesser recovery is likely based on the linear matrix polymerization network formed by advanced glycation end products (AGEs) as a secondary product of MG. Since elevated serum MG and abnormal bone turnover related to the amount of AGEs are observed in cases of type 2 diabetes, this time-dependent behavior may be one of the factors of the bone fracture mechanism at the material level in affected patients.

Retinoblastoma-binding Protein 4-regulated Classical Nuclear Transport Is Involved in Cellular Senescence.

Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma-binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin α/ß-mediated nuclear import. RBBP4 accelerates the release of importin ß1 from importin α via competitive binding to the importin ß-binding domain of importin α in the presence of RanGTP. Therefore, it facilitates importin α/ß-mediated nuclear import. We showed that the importin α/ß pathway is down-regulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin ß1 to bind to importin α, and it demonstrates that the loss of this factor can trigger cellular senescence.

Functional characterization of importin α8 as a classical nuclear localization signal receptor.

Importin α8 has recently been identified as an importin α family member based on its primary structure and binding ability to importin ß1 and to several karyophilic proteins. However, there has been no experimental evidence that importin α8 actually functions in the nuclear transport of classical nuclear localization signal (cNLS)-containing cargo. Here, using an in vitro transport assay, we demonstrate that purified recombinant importin α8 can transport SV40T antigen cNLS-containing cargo into the nucleus of HeLa cells, in conjunction with importin ß1. Pull-down assays, followed by mass spectrometry analysis, identified 179 putative importin α8-binding proteins, only 62 of which overlap with those of importin α1, the closest importin α family member. Among the importin α8-binding candidates, we showed that DNA damage-binding protein 2 (DDB2) was actually transported into the nucleus via the importin α8/ß1 pathway. Furthermore, we found that the other subtypes of importin α, which were also identified as importin α8-binding candidates, indeed form heterodimers with importin α8. Notably, we found that these importin α8-containing heterodimers were more stable in the presence of cNLS-substrates than heterodimers containing importin α1. From these findings, we propose that importin α8 functions as a cNLS receptor with distinct cargo specificity, and that heterodimerization by importin α8 is a novel regulatory mode of cNLS binding, in addition to the autoinhibitory regulation by the importin ß binding domain.

Nuclear retention of importin α coordinates cell fate through changes in gene expression.

Various cellular stresses including oxidative stress induce a collapse of the Ran gradient, which causes accumulation of importin α in the nucleus and a subsequent block of nuclear protein import. However, it is unknown whether accumulated importin α performs roles in the nucleus after its migration in response to stress. In this study, we found that nuclear-retained importin α2 binds with DNase I-sensitive nuclear component(s) and exhibits selective upregulation of mRNA encoding Serine/threonine kinase 35 (STK35) by microarray analysis. Chromatin immunoprecipitation and promoter analysis demonstrated that importin α2 can access to the promoter region of STK35 and accelerate its transcription in response to hydrogen peroxide exposure. Furthermore, constitutive overexpression of STK35 proteins enhances caspase-independent cell death under oxidative stress conditions. These results collectively reveal that nuclear-localized importin α2 influences gene expression and contributes directly to cell fate outcomes including non-apoptotic cell death.

Enhanced and suppressed mineralization by acetoacetate and ß-hydroxybutyrate in osteoblast cultures.

It is known that diabetes aggravates alveolar bone loss associated with periodontitis. While insulin depletion increases the blood concentration of ketone bodies, i.e., acetoacetate and ß-hydroxybutyrate, their roles in bone metabolism have not been much studied until today. We investigated the effects of acetoacetate and ß-hydroxybutyrate on mineralization of extracellular matrix in cultures of mouse osteoblastic MC3T3-E1 cells and primary mouse osteoblasts in the presence and absence of bone morphogenetic protein-2. Acetoacetate potentiated alkaline phosphatase activity in MC3T3-E1 cells in a concentration-dependent manner, ranging from physiological to pathological concentrations (0.05-5 mmol/L). In contrast, ß-hydroxybutyrate lowered it in the same experimental settings. Mineralization in cultures of these cells was also up-regulated by acetoacetate and down-regulated by ß-hydroxybutyrate. Similar results were obtained in cultures of mouse primary osteoblasts. Neither alkaline phosphatase mRNA nor its protein expression in MC3T3-E1 cells was affected by acetoacetate or ß-hydroxybutyrate, indicating that these ketone bodies control the enzyme activity of alkaline phosphatase in osteoblasts and hence their mineralization bi-directionally. Finally, either gene silencing of monocarboxylate transporter-1, a major transmembrate transporter for ketone bodies, nullified the effects of ketone bodies on alkaline phosphatase activity in MC3T3-E1 cells. Collectively, we found that ketone bodies bidirectionally modulates osteoblast functions, which suggests that ketone bodies are important endogenous factors that regulate bone metabolism in both physiological and pathological situations.

hCAS/CSE1L regulates RAD51 distribution and focus formation for homologous recombinational repair.

Homologous recombinational repair (HR) is one of the major repair systems for DNA double-strand breaks. RAD51 is a key molecule in HR, and the RAD51 concentration in the cell nucleus increases after DNA damage induction. However, the mechanism that regulates the intracellular distribution of RAD51 is still unclear. Here, we show that hCAS/CSE1L associates with RAD51 in human cells. We found that hCAS/CSE1L negatively regulates the nuclear protein level of RAD51 under normal conditions. hCAS/CSE1L is also required to repress the DNA damage-induced focus formation of RAD51. Moreover, we show that hCAS/CSE1L plays roles in the regulation of the HR activity and in chromosome stability. These findings suggest that hCAS/CSE1L is responsible for controlling the HR activity by directly interacting with RAD51.

Oxygenated Static Preservation of Donation after Cardiac Death Liver Grafts Improves Hepatocyte Viability and Function.

BACKGROUND: Cell therapy, such as hepatocyte transplantation (HTx), is promising for the treatment of metabolic liver diseases or as a bridge to orthotopic liver transplantation in patients with fulminant liver failure. However, one of the limitations of this therapy is the shortage of donors. The present study aims to investigate whether the two-layer method (TLM) of cold preservation with oxygenation improves the viability and activity of hepatocytes from rat donation after cardiac death (DCD) donors compared with results obtained with the University of Wisconsin (UW) solution. Moreover, we evaluated the hepatocyte function after culture or transplantation into the spleen. MATERIALS AND METHODS: We used male Sprague-Dawley rats for this study. The DCD model was induced by phrenotomy after injecting heparin. We assigned rats based on warm ischemia times of 15 and 30 min to groups S and L, respectively. Each group (n = 5) was then subdivided as follows: (1) group S: not preserved (S/N), preserved by TLM for 3 h (S/TLM3) and 12 h (S/TLM12), and in the UW solution for 3 h (S/UW3) and 12 h (S/UW12), and (2) group L: not preserved (L/N), preserved by TLM for 3 h (L/TLM3) and 12 h (L/TLM12), and in the UW solution for 3 h (L/UW3) and 12 h (L/UW12). The cell viability and function of isolated DCD hepatocytes were analyzed for culture or HTx into the spleen. RESULTS: The viability and ATP levels of DCD hepatocytes significantly improved after TLM compared with the values after preservation in cold UW solution in group S/N (p < 0.059). The levels of albumin production and urea synthesis by hepatocytes after culture were significantly higher in groups S/TLM3 and S/TLM12 than in groups S/UW3 and S/UW12 (p < 0.05), respectively. Further, serum albumin levels after HTx were also markedly higher in groups S/TLM3 and S/TLM12 than in groups S/UW3 and S/UW12. The morphological features revealed that cultured and transplanted hepatocytes remained clearly viable and maintained an expression for specific hepatic function, such as the production of albumin and glycogen. CONCLUSION: This novel method of oxygenated cold preservation of DCD livers can expand the hepatocyte donor pool for HTx and establish a wider application of this developing technique.

Porphyromonas gingivalis-derived lysine gingipain enhances osteoclast differentiation induced by tumor necrosis factor-α and interleukin-1ß but suppresses that by interleukin-17A: importance of proteolytic degradation of osteoprotegerin by lysine gingipain.

Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity (i.e. arginine (Rgps) and lysine (Kgps) gingipains). We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara, R., Miyamoto, Y., Takami, M., Imamura, T., Potempa, J., Yoshimura, K., and Kamijo, R. (2009) Lysine-specific gingipain promotes lipopolysaccharide- and active-vitamin D3-induced osteoclast differentiation by degrading osteoprotegerin. Biochem. J. 419, 159-166). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-17A, in inflammatory conditions, such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1ß in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1ß were less susceptible, whereas IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of receptor activator of nuclear factor κB ligand. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in the development of osteoclastogenesis and bone loss in periodontitis.