DNA and protein methyltransferases inhibition by adenosine dialdehyde reduces the proliferation and migration of breast and lung cancer cells by downregulating autophagy.

Protein and DNA methylation is involved in various biological functions such as signal transmission, DNA repair, and gene expression. Abnormal regulation of methyltransferases has been linked to multiple types of cancer, but its link to autophagy and carcinogenesis in breast and lung cancer is not fully understood. We utilized UALCAN, a web tool, to investigate breast and lung cancer database from The Cancer Genome Atlas. We found that 17 methyltransferases are upregulated in breast and/or lung cancer. We investigated the effect of methylation inhibition on two breast cancer cell lines (MDA-MB-231 and MCF-7) and two lung cancer cell lines (H292 and A549) by treating them with the indirect methyltransferase inhibitor adenosine dialdehyde (AdOx). We found that the migration ability of all cell lines was decreased, and the growth rate of MDA-MB-231, MCF-7 and H292 was also decreased after AdOx treatment. These results were correlated with an inhibition of the autophagy in MDA-MB-231, MCF-7 and H292 cell lines, since AdOx treatment induced a decreased expression of ATG7, a reduced ratio LC3-II/LC3-I and an increased p62 level. These findings suggest that inhibiting cells' methylation ability could be a potential target for breast and lung cancer treatment.

The substitution at residue 218 of the NS5 protein methyltransferase domain of Tembusu virus impairs viral replication and translation and may triggers RIG-I-like receptor signaling.

Flavivirus RNA cap-methylation plays an important role in viral infection, proliferation, and escape from innate immunity. The methyltransferase (MTase) of the flavivirus NS5 protein catalyzes viral RNA methylation. The E218 amino acid of the NS5 protein MTase domain is one of the active sites of flavivirus methyltransferase. In flaviviruses, the E218A mutation abolished 2'-O methylation activity and significantly reduced N-7 methylation activity. Tembusu virus (TMUV, genus Flavivirus) was a pathogen that caused neurological symptoms in ducklings and decreased egg production in laying ducks. In this study, we focused on a comprehensive understanding of the effects of the E218A mutation on TMUV characteristics and the host immune response. E218A mutation reduced TMUV replication and proliferation, but did not affect viral adsorption and entry. Based on a TMUV replicon system, we found that the E218A mutation impaired viral translation. In addition, E218A mutant virus might be more readily recognized by RIG-I-like receptors to activate the corresponding antiviral immune signaling than WT virus. Together, our data suggest that the E218A mutation of TMUV MTase domain impairs viral replication and translation and may activates RIG-I-like receptor signaling, ultimately leading to a reduction in viral proliferation.

Interleukin-4 and interleukin-13 enhance CCL26 production in a human keratinocyte cell line, HaCaT cells.

Eotaxin-2/CCL24 and eotaxin-3/CCL26 are CC chemokines and their receptor, CC chemokine receptor 3 is preferentially expressed on eosinophils. It was reported that vascular endothelial cells and dermal fibroblasts produced CCL26. However, the regulation of CCL24 and CCL26 production in keratinocytes has not been well documented. We investigated the expression and production of CCL24 and CCL26 in the human keratinocyte cell line, HaCaT cells. Reverse transcription and polymerase chain reaction was performed using these cells and Enzyme-linked immunosorbent assay was carried out using supernatant of these cells. The production of CCL24 in HaCaT cells was slightly enhanced by IL-4 and that of CCL26 was strongly enhanced by IL-4 and IL-13. Furthermore, TNF-alpha generated a synergistic effect on IL-4 enhanced CCL26 production. Dexamethasone, IFN-gamma and the p38 mitogen-activated protein kinase inhibitor SB202190 inhibited IL-4 enhanced CCL26 production. IL-4 enhanced production of CCL26 was inhibited by leflunomide and JAK inhibitor 1, but not by JAK3 inhibitor, which indicates that it is mediated by JAK1-STAT6-dependent pathway. This result also strongly suggests the involvement of the type 2 IL-4 receptor in IL-4 enhanced production of CCL26. These results suggest that keratinocytes are involved in the migration of CC chemokine receptor 3 positive cells such as eosinophils in a Th2-dominant situation like atopic dermatitis.

A protein methyl transferase, PRMT5, selectively blocks oncogenic ras-p21 mitogenic signal transduction.

A Janus-2 (JAK-2) binding protein, JBP1, has been found to function as an arginine methyl transferase and is now designated PRMT5. Co-injection of plasmids encoding this protein together with oncogenic (Val 12-containing) ras-p21 protein into Xenopus leavis oocytes results in strong inhibition of oncogenic p21-induced oocyte maturation. This inhibition appears to be dependent on the methyl transferase function since a partially active R368A mutant shows diminished ability to inhibit Val 12-p21-induced oocyte maturation, and an almost totally inactive GAGRG (365-369) deletion mutant fails to inhibit Val 12-p21-induced maturation. In contrast, PRMT5 (JBP1) does not inhibit insulin-induced oocyte maturation. Since insulin-induced maturation depends on activation of cellular ras-p21, PRMT5 does not appear to inhibit the wild-type p21 protein. We also find that arginine methyl transferase inhibitors strongly block oncogenic ras-p21-activated, but not insulin-activated, wild-type ras-p21-induced oocyte maturation. Thus signaling by oncogenic p21 appears to involve methyltransferases uniquely. Surprisingly, the active site peptide, Gly-Arg-Gly, strongly suppresses insulin-induced maturation but has no effect on Val 12-p21-induced maturation. This peptide may therefore be useful in defining steps in the wild-type ras pathway.

The amino terminus of PKA catalytic subunit--a site for introduction of posttranslational heterogeneities by deamidation: D-Asp2 and D-isoAsp2 containing isozymes.

Conserved deamidation of PKA catalytic subunit isozymes Calpha and Cbeta--more than 25% at Asn2 in vivo in both cases--has been shown to yield Asp2- and isoAsp2-containing isozymes (Jedrzejewski PT, Girod A, Tholey A, König N, Thullner S, Kinzel V, Bossemeyer D, 1998, Protein Sci 7:457-469). Isoaspartate formation in proteins in vivo is indicative of succinimide intermediates involved in both the initial deamidation reaction as well as the "repair" of isoAsp to Asp by the action of protein L-isoaspartyl (D-aspartyl) O-methyl transferase (PIMT). L-Succinimide is prone to racemization to D-succinimide, which may hydrolyze to D-isoAsp- and D-Asp-containing diastereomers with, respectively, no and poor substrate character for PIMT. To analyze native PKA catalytic subunit from cardiac muscle for these isomers the N-terminal tryptic peptides (T1) of the enzyme were analyzed following procedures refined specifically with a set of corresponding synthetic peptides. The methods combined high resolution high-performance liquid chromatography and a new mass spectrometric procedure for the discrimination between Asp- and isoAsp-residues in peptides (Lehmann et al., 2000). The results demonstrate the occurrence of D-isoAsp- and D-Asp-containing T1 fragments in addition to the L-isomers. The small amount of the L-isoAsp isomer, representing only part of the D-isoAsp isomer, and the relatively large amounts of the L-Asp and D-Asp isomers argues for an effective action of PIMT present in cardiac tissue.

Enzymatic trimethylation of residue-72 lysine in cytochrome c. Effect on the total structure.

A highly purified protein methylase III from Neurospora crassa or Saccharomyces cerevisiae specifically methylates a single lysine residue of position 72 of horse heart cytochrome c. The enzymatically methylated cytochrome c has been separated from the unmethylated counterpart species by isoelectric focusing. Simultaneously, the pI values of these two species were found to be 9.49 and 10.03, respectively. Since methyl substitution increases the basicity associated with the epsilon-amino group of lysine residues, the observed decrease in pI value is in opposition to the predicted increase. Space-filling models revealed the possibility of a hydrogen bond between the oxygen of amide of residue-70 asparagine and the epsilon-amino nitrogen of residue-72 lysine in unmethylated horse heart cytochrome C. the enzymatic methylation of residue-72 lysine tends to dissociate this hydrogen bond, thereby possibly inducing the shift of 'effective charge' of the protein molecule. This paper also deals with the pI values of cytochromes c from 13 different sources, determined by the isoelectric focusing technique.