Identification of nucleobase chemical modifications that reduce the hepatotoxicity of gapmer antisense oligonucleotides

Currently, gapmer antisense oligonucleotide (ASO) therapeutics are under clinical development for the treatment of various diseases, including previously intractable human disorders; however, they have the potential to induce hepatotoxicity. Although several groups have reported the reduced hepatotoxicity of gapmer ASOs following chemical modifications of sugar residues or internucleotide linkages, only few studies have described nucleobase modifications to reduce hepatotoxicity. In this study, we introduced single or multiple combinations of 17 nucleobase derivatives, including four novel derivatives, into hepatotoxic locked nucleic acid gapmer ASOs and examined their effects on hepatotoxicity. The results demonstrated successful identification of chemical modifications that strongly reduced the hepatotoxicity of gapmer ASOs. This approach expands the ability to design gapmer ASOs with optimal therapeutic profiles.     

mTORC1-independent translation control in mammalian cells by methionine adenosyltransferase 2A and S-adenosylmethionine

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (SAM). As the sole methyl-donor for methylation of DNA, RNA, and proteins, SAM levels affect gene expression by changing methylation patterns. Expression of MAT2A, the catalytic subunit of isozyme MAT2, is positively correlated with proliferation of cancer cells; however, how MAT2A promotes cell proliferation is largely unknown. Given that the protein synthesis is induced in proliferating cells and that RNA and protein components of translation machinery are methylated, we tested here whether MAT2 and SAM are coupled with protein synthesis. By measuring ongoing protein translation via puromycin labeling, we revealed that MAT2A depletion or chemical inhibition reduced protein synthesis in HeLa and Hepa1 cells. Furthermore, overexpression of MAT2A enhanced protein synthesis, indicating that SAM is limiting under normal culture conditions. In addition, MAT2 inhibition did not accompany reduction in mechanistic target of rapamycin complex 1 activity but nevertheless reduced polysome formation. Polysome-bound RNA sequencing revealed that MAT2 inhibition decreased translation efficiency of some fraction of mRNAs. MAT2A was also found to interact with the proteins involved in rRNA processing and ribosome biogenesis; depletion or inhibition of MAT2 reduced 18S rRNA processing. Finally, quantitative mass spectrometry revealed that some translation factors were dynamically methylated in response to the activity of MAT2A. These observations suggest that cells possess an mTOR-independent regulatory mechanism that tunes translation in response to the levels of SAM. Such a system may acclimate cells for survival when SAM synthesis is reduced, whereas it may support proliferation when SAM is sufficient.     

nanos1: a mouse nanos gene expressed in the central nervous system is dispensable for normal development

A mouse nanos (nanos1) gene was cloned and its function was examined by generating a gene-knockout mouse. The nanos1 gene encodes an RNA-binding protein, which contains a putative zinc-finger motif that exhibits similarity with other nanos-class genes in vertebrates and invertebrates. Although nanos1 is not detected in primordial germ cells, it is observed in seminiferous tubules of mature testis. Interestingly, maternally expressed nanos1 is observed in substantial amounts in oocytes, but the amount of maternal RNA is rapidly reduced after fertilization, and the transient zygotic nanos1 expression is observed in eight-cell embryos. At 12.5 days postcoitum, nanos1 is re-expressed in the central nervous system and the expression continues in the adult brain, in which the hippocampal formation is the predominant region. The nanos1 -deficient mice develop to term without any detectable abnormality and they are fertile. No significant neural defect is observed in terms of their behavior to date.     

A possible role of Atg8 homologs as a scaffold for signal transduction

Atg8 and its homologs are essential for autophagosome formation in various species. In animal cells, Atg8 homologs have an additional function in clearance of damaged organelles and bacteria, acting as a landmark for selective autophagy. We have recently shown that OATL1, a Rab-GTPase-activating protein (Rab-GAP), is a novel binding partner of Atg8 homologs in mammalian cells, but to our surprise, it is not a substrate of autophagy. Further analysis indicates that OATL1 is involved in the fusion between autophagosomes and lysosomes through its GAP activity and its Atg8 homolog binding activity. Our findings suggest a novel function of Atg8 homologs as a scaffold for signal transduction that regulates autophagosomal maturation.     

Structure of human lysosomal membrane glycoprotein 1. Assignment of disulfide bonds and visualization of its domain arrangement

The amino acid sequence of one of the major lysosomal membrane glycoproteins, lysosome-associated membrane protein 1 (lamp-1), was deduced from its cDNA sequence (Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S. R. (1988) J. Biol. Chem. 263, 18920-18928). This amino acid sequence suggests that lamp-1 contains a hinge-like structure and could form disulfide bridges that are observed in the immunoglobulin superfamily. To test this possibility, we have determined the positions of the disulfide bridges by isolating and sequencing cystine-containing peptides which contain disulfide bridges. The results indicate that disulfide arrangement of lamp-1 is different from that of immunoglobulins. Each molecule contains, in total, four loops formed by disulfide bonds, and each loop contains 36-39 amino acid residues. However, none of the disulfide bonds connects two domains that are separated by a hinge-like structure. The results indicate that the hinge region has no ordered structure, and the relative positions of the two domains can be altered in space. Examination of the ultrastructure of lamp-1 by electron microscopy showed that the hinge-like structure actually functions as a hinge. These results indicate that the lamp-1 molecule represents a novel family of glycoproteins with unique structural properties.     

Risk factors for bone loss in patients with rheumatoid arthritis treated with biologic disease-modifying anti-rheumatic drugs

Objective

Osteoporosis is a complication of rheumatoid arthritis. We examined the risk factors for bone loss in rheumatoid arthritis patients receiving biological disease-modifying anti-rheumatic drugs. Lumbar spine and femoral neck bone mineral density was measured at two time points in 153 patients with rheumatoid arthritis managed with biological disease-modifying anti-rheumatic drugs. We examined patients’ variables to identify risk factors for least significant reduction of bone mineral density.

Results

Least significant reduction of lumbar spine bone mineral density (≤ ? 2.4%) was seen in 13.1% of patients. Least significant reduction of femoral neck bone mineral density (≤ ? 1.9%) was seen in 34.0% of patients. Multiple logistic regression analysis showed that a risk factor for least significant reduction of the lumbar spine was high-dose methylprednisolone use. Multiple regression analysis showed that a risk factor for least significant reduction of the femoral neck was short disease duration. Our findings showed that a risk factor for femoral neck bone mineral density reduction was a short disease duration. These findings suggest that rheumatoid arthritis patients receiving treatment with biological disease-modifying anti-rheumatic drugs may benefit from earlier osteoporosis treatments to prevent femoral neck bone loss.

     

Functional conservation between fission yeast moc1/sds23 and its two orthologs, budding yeast SDS23 and SDS24, and phenotypic differences in their disruptants

The moc1/sds23 gene was isolated to induce sexual development of a sterile strain due to overexpression of adenylate cyclase in Schizosaccharomyces pombe. Here, we studied the functional conservation between moc1/sds23 and its two orthologs SDS23 and SDS24 in Saccharomyces cerevisiae. We observed that the temperature sensitivity, salt tolerance, cell morphology, and sterility of the Deltamoc1 mutant in S. pombe were recovered by expressing either S. cerevisiae SDS23 or SDS24. We found that deletion of both SDS23 and SDS24 resulted in the production of a large vacuole that was reversed by the expression of S. pombe moc1/sds23. In these ways we found that S. pombe Moc1/Sds23 and S. cerevisiae SDS23p or SDS24p are functional homologs. In addition we found that the Deltasds23 Deltasds24 diploid strain reduces cell separation in forming pseudohyphal-like growth in S. cerevisiae. Thus S. pombe moc1/sds23 and S. cerevisiae SDS23 or SDS24 are interchangeable with each other, but their disruptants are phenotypically dissimilar.     

Enantioselectivity of bunitrolol 4‐hydroxylation is reversed by the change of an amino acid residue from valine to methionine at position 374 of cytochrome P450‐2D6

The enantioselectivity of 4‐hydroxylation of bunitrolol (BTL), a β‐adrenoceptor blocking drug, was studied in microsomes from human liver, human hepatoma (Hep G2) cells expressing CYP2D6, and lymphoblastoid cells expressing CYP2D6. Kinetics in human liver microsomes showed that the Vmax value for (+)‐BTL was 2.1‐fold that of (−)‐BTL, and that the Km value for (+)‐BTL was lower than that for the (−)‐antipode, resulting in the intrinsic clearance (Vmax/Km) of (+)‐BTL being 2.1‐fold over its (−)‐antipode. CYP2D6 (CYP2D6‐met) expressed in Hep G2 cells had a methionine residue at position 373 of the amino acid sequence and a rat‐type N‐terminal peptide (MELLNGTGLWSM) instead of the human‐type (MGLEALVPLAVIV), and showed enantioselectivity of [(+)‐BTL < (−)‐BTL] for the rate of BTL 4‐hydroxylation. In contrast, enantioselectivity [(+)‐BTL > (−)‐BTL] for Hep G2‐CYP2D6 (CYP2D6‐val) with a human‐type N‐terminal peptide that had a valine residue at 374, which corresponds to the methionine of the CYP2D6‐met variant, was the same as that for human liver microsomes. We further confirmed that CYP2D6‐met and CYP2D6‐val expressed in human lymphoblastoid cells, both of which have methionine and valine, respectively, at position 374 and a human‐type N‐terminal peptide, exhibited the same enantioselectivities as those obtained from CYP2D6‐met and CYP2D6‐val expressed in the Hep G2 cell system. These results indicate that the amino acid at 374 of CYP2D6 is one of the key factors influencing the enantioselectivity of BTL 4‐hydroxylation. Chirality 11:1–9, 1999. © 1999 Wiley‐Liss, Inc.     

Alterations in cardiac function and subcellular membrane activities after hypervitaminosis D3

The present study was designed to induce massive accumulation of calcium in the myocardium and to evaluate the effect of calcium overload on myocardial contractile function and biochemical activity of cardiac subcellular membranes. Rats were treated with an oral administration of 500,000 units/kg of vitamin D₃ for 3 consecutive days, and their hearts were sampled on the 5th day for biochemical analysis. On the 4th and 5th days, heart rate, mean aortic pressure, left ventricular systolic pressure and left ventricular dP/dt were significantly lowered in vitamin D₃-treated rats, demonstrating the existence of appreciable myocardial contractile dysfunction. Marked increases in the myocardial calcium (67-fold increase) and mitochondrial calcium contents (24-fold increase) were observed by hypervitaminosis D3. Mitochondrial oxidative phosphorylation and ATPase activity were significantly reduced by this treatment. A decline in sarcolemmal Na⁺, K⁺-ATPase activity was also observed, while relatively minor or insignificant changes in calcium uptake and ATPase activities of sarcoplasmic reticulum were detectable. Electron microscopic examination revealed calcium deposits in the mitochondria after vitamin D₃ treatment. The results suggest that hypervitaminosis D₃ produces massive accumulation of calcium in the myocardium, particularly in the cardiac mitochondrial membrane, which may induce an impairment in the mitochondrial function and eventually may lead to a failure in the cardiac contractile function.