Increased methylation of the cytosolic 20-kD protein is accompanied by liver regeneration in a hepatectomized rat

Arginine methylation has been implicated in the signal transduction pathway leading to cell growth. Here we show that a regenerating rat liver following partial hepatectomy exhibited elevated methyltransferase activity as shown by increased methylation of a subset of endogenous proteins in vitro. The 20-kDa protein was shown to be a major cytosolic protein undergoing methylation in regenerating hepatocytes. Methylation of the 20-kDa protein peaked at 1 d following partial hepatectomy, which gradually declined to a basal level within the next 14 d. Likewise, methylation of exogenously added bulk histones followed the similar time kinetics as the 20-kDa protein, reflecting time-dependent changes in methyltransferase activity in regenerating hepatocytes. Presence of exogenously added bulk histone in the in vitro methylation assay resulted in dose-dependent inhibition of methylation of the 20-kDa protein. All the histone subtypes tested, histone 1, 2A, 2B, 3 or 4, were able to inhibit methylation of the 20-kDa protein while addition of cytochrome C, a-lactalbumin, carbonic anhydrase, bovine serum albumin, and g globulin minimally affected methylation of the 20-kDa protein. Since methylation of the 20-kDa protein preceded proliferation of hepatocytes upon partial hepatectomy, it is tempting to speculate that the methylated 20-kDa protein by activated histone-specific methyltransferase may be involved in an early signal critical for liver regeneration.     

Increased lysine N-methylation of a 23-kDa protein during hepatic regeneration

The methylation of a 23-kDa nuclear protein increased after partial hepatectomy and methylation returned to basal levels after the initial stage of regeneration. The methylating enzyme was partially purified from rat liver by ammonium sulfate precipitation, DEAE-anion exchange chromatography and Butyl-Sepharose chromatography. The 23-kDa protein was purified from a nuclear fraction of liver tissue with SP-Sepharose. When the 23-kDa protein was methylated with the partially purified methyltransferase and analyzed on C(18) high performance liquid chromatography (HPLC), the methylated acceptor amino acid was monomethyl lysine (MML). Previously, only arginine N-methylation of specific substrate proteins has been reported during liver regeneration. However, in this report, we found that lysine N-methylation increased during early hepatic regeneration, suggesting that lysine N-methylation of the 23-kDa nuclear protein may play a functional role in hepatic regeneration. The methyltransferase did not methylate other proteins such as histones, hnRNPA1, or cytochrome C, suggesting the enzyme is a 23-kDa nuclear protein- specific lysine N-methyltransferase.     

Proteomics analysis of in vitro protein methylation during Src-induced transformation

Src, a nonreceptor tyrosine kinase, was the first oncogene identified from an oncogenic virus. Mechanistic studies of Src-induced transformations aid in understanding the pathologic processes underlying tumorigenesis and may provide new strategies for cancer therapy. Although several pathways and protein modifications are reportedly involved in Src-induced transformation, the detailed mechanisms of their regulation remain unclear. Protein methylation is an important PTM that is widely involved in cellular physiology. In this study, we determined if protein methylation was involved in Src activation and which methylated proteins were associated with this activity. Using in vitro methylation and 2-DE analysis of viral Src (v-Src)-transformed rat kidney epithelial cells (RK3E), several known and novel methylated proteins were identified based on their changes in methylation signal intensity upon transformation. Among these, elongation factor 2 (EF-2), heterogeneous nuclear ribonucleoprotein K (hnRNP K), and β-tubulin protein expressions remained unchanged, indicating that their altered methylation levels were due to Src activation. In addition, the altered expression of β-actin, vimentin, and protein phosphatase 2, catalytic subunit (PPP2C) as well as protein phosphatase 2, catalytic subunit methylation were also confirmed in RK3E cells transformed with a human oncogenic Src mutant (Src531), supporting their association with Src-induced transformation in human cancer. Together, we showed putative involvement of protein methylation in Src activation and our identification of methylated proteins provides important targets for extensively studying Src-induced transformations.     

Detection of the changes in cellular proteins in regenerating rat liver by high-resolution two-dimensional electrophoresis

The changes in cellular proteins in regenerating rat liver after partial hepatectomy were examined by high-resolution two-dimensional electrophoresis. The cellular proteins in regenerating rat livers were separated into two fractions (soluble and insoluble protein fractions) and the proteins in each fraction were analysed by means of two-dimensional electrophoresis. A rapid increase in three proteins and a rapid decrease in two proteins were detected after partial hepatectomy. The changes in these proteins were in parallel with the regeneration rate of liver, suggesting a close relationship with the proliferation of liver after partial hepatectomy.     

Specificity analysis-based identification of new methylation targets of the SET7/9 protein lysine methyltransferase

We applied peptide array methylation to determine an optimized target sequence for the SET7/9 (KMT7) protein lysine methyltransferase. Based on this, we identified 91 new peptide substrates from human proteins, many of them better than known substrates. We confirmed methylation of corresponding protein domains in?vitro and in?vivo with a high success rate for strongly methylated peptides and showed methylation of nine nonhistone proteins (AKA6, CENPC1, MeCP2, MINT, PPARBP, ZDH8, Cullin1, IRF1, and [weakly] TTK) and of H2A and H2B, which more than doubles the number of known SET7/9 targets. SET7/9 is inhibited by phosphorylation of histone and nonhistone substrate proteins. One lysine in the MINT protein is dimethylated in?vitro and in?vivo demonstrating that the product pattern created by SET7/9 depends on the amino acid sequence context of the target site.     

Cysteine carboxyl O-methylation of human placental 23 kDa protein

C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling.     

Arginylation and methylation double up to regulate nuclear proteins and nuclear architecture in vivo

Protein arginylation and arginine methylation are two posttranslational modifications of emerging importance that involve Arg residues and their modifications. To test a hypothesis that posttranslationally added arginines can be methylated, we used high-precision mass spectrometry and metabolic labeling to find whether posttranslationally added arginines can serve as methylation sites. We identified?a number of proteins in?vivo, on which posttranslationally added Arg have undergone mono- and dimethylation. This double modification predominantly affects the chromatin-containing nuclear fraction and likely plays an important regulatory role in chromatin-associated proteins. Moreover, inhibition of arginylation and Arg methylation results in?a significant reduction of the nucleus size in cultured cells, suggesting changes in chromatin compaction and nuclear architecture. Our findings suggest?a functional link between protein regulation by arginylation and methylation that affects nuclear structure in?vivo.     

Studies on DNA sequence specific binding proteins for 5' flanking upstream region of CPSI gene.

Two specific carbamyl phosphate synthetase I gene binding nuclear proteins (M. W. 109 kD and 74 kD) have been determined in the rat liver by the protein blotting technique (Southwestern blot assay). The result shows that they are not present in the normal rat spleen and F-26 rat hepatoma cell. The Bal31 nuclease deletion in the CPSI gene 5' upstream region proves that the binding sites for 109 kD and 74 kD are respectively located in the regions of -38 bp to -4 bp and -113 bp to -38 bp. The binding proteins may be the liver-specific ones of the CPSI gene, which are related to hepatocyte differentiation and hepatocarcinogenesis.     

Rapid Electrochemical Assessment of Tumor Suppressor Gene Methylations in Raw Human Serum and Tumor Cells and Tissues Using Immunomagnetic Beads and Selective DNA Hybridization

We report a rapid and sensitive electrochemical strategy for the detection of gene‐specific 5‐methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5‐methylcytosines (5‐mC) are used for the capture of any 5‐mC methylated single‐stranded (ss)DNA sequence. A flanking region next to the 5‐mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen‐printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O‐6‐methylguanine‐DNA methyltransferase (MGMT) promoter region. The method is applied in the 45‐min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U‐87 glioblastoma cells and paraffin‐embedded brain tumor tissues without any amplification and pretreatment step.     

Cell cycle protein profile of the hepatic stellate cells(HSCs)in dimethylnitrosamine-induced rat hepatic fibrosis

Cell cycle regulating proteins are known to have close relation with the proliferation of the mammalian cells. In injured liver, the number of HSCs is increased from proliferation. However, the expression of cell cycle proteins of HSCs during proliferation remains unevaluated. Therefore, cell cycle protein profiles of HSCs were studied in dimethyl-nitrosamine (DMN)-induced rat liver fibrosis model. Sprague-Dawley rats were intraperitoneally injected of DMN and the animals were sacrificed every week up to 4 weeks. HSCs were separated and the number of the cells in S phase was counted to evaluate the cell proliferation by flow cytometry. The expression of cyclin A, cyclin B, cyclin D1, cdk2, cdk4, cdc2, proliferating cell nuclear antigen (PCNA), p21(Cip/WAF1), and p27 was examined with immunoblotting analysis. Portion of S-phase cells peaked 7days after DMN injection. At that time, cyclin A, and PCNA showed significant increase in HSCs compared to untreated HSCs (114% and 116%, respectively, P<0.001). p21(Cip/WAF1) was decreased significantly in DMN-treated HSCs compared to control cells (88%, P<0.001). The increase of cyclin A, and PCNA and the decrease of p21(Cip/WAF1) seem to play important roles in the proliferation of HSCs during the early period of DMN treatment.     

Two-dimensional electrophoresis of liver proteins: characterization of a drug-induced hepatomegaly in rats

Two-dimensional electrophoresis (2-DE) of liver proteins was applied to further characterize an unusual drug-induced increase in hepatocellular rough endoplasmic reticulum (RER) in Sprague-Dawley rats given a substituted pyrimidine derivative. Absolute liver weights of drug-treated rats (9.9 +/- 0.4 g) increased above vehicle-treated controls (7.2 +/- 0.2 g) by 37%. Light microscopy revealed diffuse granular basophilia of the hepatocellular cytoplasm, uncharacteristic of hepatocytes and suggested cells rich in ribosomes, which was confirmed by electron microscopy. Immunostaining for cell proliferation, viz., 5-bromo-2'-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA), indicated marked hepatocellular proliferative activity. 2-DE of solubilized liver using an ISO-DALT gel system indicated significant (p<0.001) quantitative changes in at least 17 liver proteins (12 increased, 5 decreased) compared to controls. The protein with the largest increase was homologous to acute-phase reactant, contrapsin-like protein inhibitor-6. Other markedly upregulated proteins were methionine adenosyltransferase, a catalyst in methionine/ATP metabolism and mitochondrial HMG-CoA synthase, involved in cholesterol synthesis. The complementary strategies of 2-DE coupled either with database spot mapping or protein isolation and amino acid sequencing successfully identified a subset of proteins from xenobiotic-damaged rodent livers, the expression of which differed from controls. However, the current bioinformatics platform for rodent hepatic proteins and limited knowledge of specific protein functionality restricted application of this proteomics profile to further define a mechanistic basis for this unusual hepatotoxicity.     

Functional Component Isolated from Phaseolus vulgaris Lectin Exerts In Vitro and In Vivo Anti-Tumor Activity Through Potentiation of Apoptosis and Immunomodulation

Phytohemagglutinin (PHA), the lectin purified from red kidney bean (Phaseolus vulgaris), is a well-known mitogen for human lymphocyte. Because it has obvious anti-proliferative and anti-tumor activity, PHA may serve as a potential antineoplastic drug in future cancer therapeutics. However, the literature is also replete with data on detrimental effects of PHA including oral toxicity, hemagglutinating activity, and immunogenicity. There is a critical need to evaluate the functional as well as the toxic components of PHAs to assist the rational designs of treatment with it. In this report, we performed SDS-PAGE to identify components of PHA-L, the tetrameric isomer of PHA with four identical L-type subunits, and then characterized biological function or toxicity of the major protein bands through in vitro experiments. It was found that the protein appearing as a 130 kD band in SDS-PAGE gel run under the condition of removal of β-mercaptoethanol from the sample buffer together with omission of a heating step could inhibit tumor cell growth and stimulate lymphocyte proliferation, while most of the 35 kD proteins are likely non-functional impurity proteins and 15 kD protein may be related to hemolytic effect. Importantly, the 130 kD functional protein exhibits promising in vivo anti-tumor activity in B16-F10 melanoma C57 BL/6 mouse models, which may be achieved through potentiation of apoptosis and immunomodulation. Altogether, our results suggest that PHA-L prepared from crude extracts of red kidney bean by standard strategies is a mixture of many ingredients, and a 130 kD protein of PHA-L was purified and identified as the major functional component. Our study may pave the way for PHA-L as a potential anticancer drug.     

Quantitative DNA methylation analysis by fluorescent polymerase chain reaction single-strand conformation polymorphism using an automated DNA sequencer

A novel DNA methylation assay technique, termed bisulfite single-strand conformation polymorphism (bisulfite-SSCP), is a combination of sodium-bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. After bisulfite treatment followed by PCR amplification, methylated and unmethylated alleles can be simultaneously separated in a nondenaturing gel using an automated DNA sequencer. Using bisulfite-SSCP, methylation of hMLH1 was detected in a quantitative manner. This method is not only simple, quick, accurate, and quantitative, but detailed information about methylation is also available with less work. Methylation analysis of large numbers of samples for multiple loci will be facilitated by bisulfite-SSCP.     

Solvation effects in the methylation of barbituric acid and its derivatives by diazomethane

Methylation of barbituric acid and its N-methylderivatives by diazomethane in ethers and methanol occurs only at the oxygen atom of the -dicarbonyl fragment. The resulting 6-methoxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidine and its derivatives are methylated at both the oxygen and nitrogen atoms; relative to ethers, methanol facilitates a greater degree of methylation at the nitrogen atom.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 11, pp. 1523–1526, November, 1987.     

Analysis of arginine and lysine methylation utilizing peptide separations at neutral pH and electron transfer dissociation mass spectrometry

Arginine and lysine methylation are widespread protein post-translational modifications. Peptides containing these modifications are difficult to retain using traditional reversed-phase liquid chromatography because they are intrinsically basic/hydrophilic and often fragment poorly during collision induced fragmentation (CID). Therefore, they are difficult to analyze using standard proteomic workflows. To overcome these caveats, we performed peptide separations at neutral pH, resulting in increased retention of the hydrophilic/basic methylated peptides before identification using MS/MS. Alternatively trifluoroacetic acid (TFA) was used for increased trapping of methylated peptides. Electron-transfer dissociation (ETD) mass spectrometry was then used to identify and characterize methylated residues. In contrast to previous reports utilizing ETD for arginine methylation, we observed significant amount of side-chain fragmentation. Using heavy methyl stable isotope labeling with amino acids in cell culture it was shown that, similar to CID, a loss of monomethylamine or dimethylamine from the arginine methylated side-chain during ETD can be used as a diagnostic to determine the type of arginine methylation. CID of lysine methylated peptides does not lead to significant neutral losses, but ETD is still beneficial because of the high charge states of such peptides. The developed LC MS/MS methods were successfully applied to tryptic digests of a number of methylated proteins, including splicing factor proline-glutamine-rich protein (SFPQ), RNA and export factor-binding protein 2 (REF2-I) and Sul7D, demonstrating significant advantages over traditional LC MS/MS approaches.     

Chemical agents for binding post-translationally methylated lysines and arginines

Post-translational methylation, discovered more than half a century ago, encodes information in the form of a structural modification on a peptide or protein. The addition of a CH₃ group is one of the most subtle covalent modifications that exist in biology. In spite of this, recent years have revealed the many profound functional effects that arise from protein methylation in the cell. In an effort to open the doors to new assays and detection methods that would enable new basic and applied research into methylation pathways, chemical agents that can recognise and bind to methylated sites are now being pursued. In this review, we describe the supramolecular approaches to the recognition of methylated amino acids, peptides and proteins that have arisen in the last few years.     

Site-specific mapping and time-resolved monitoring of lysine methylation by high-resolution NMR spectroscopy

Methylation and acetylation of protein lysine residues constitute abundant post-translational modifications (PTMs) that regulate a plethora of biological processes. In eukaryotic proteins, lysines are often mono-, di-, or trimethylated, which may signal different biological outcomes. Deconvoluting these different PTM types and PTM states is not easily accomplished with existing analytical tools. Here, we demonstrate the unique ability of NMR spectroscopy to discriminate between lysine acetylation and mono-, di-, or trimethylation in a site-specific and quantitative manner. This enables mapping and monitoring of lysine acetylation and methylation reactions in a nondisruptive and continuous fashion. Time-resolved NMR measurements of different methylation events in complex environments including cell extracts contribute to our understanding of how these PTMs are established in vitro and in vivo.     

Formation of methylated oxyarsenicals and thioarsenicals in wild-type and arsenic (+3 oxidation state) methyltransferase knockout mice exposed to arsenate

Arsenic (+3 oxidation state) methyltransferase (As3mt) plays a central role in the enzymatically catalyzed conversion of inorganic arsenic into methylated metabolites. Most studies of the metabolism and disposition of arsenicals following exposure to inorganic arsenic focus on the formation and fate of methylated oxyarsenicals. However, recent research has shown methylated thioarsenicals to be another important class of metabolites of inorganic arsenic. Here, we report on the presence of methylated oxy- and thioarsenicals in urine and liver from wild-type mice that efficiently methylate inorganic arsenic and from As3mt knockout mice that lack arsenic methyltransferase activity. Following a single oral dose of 0.5 mg of arsenic as arsenate/kg body weight, urine from wild-type mice contained methylated oxyarsenicals and unknown arsenicals. Further analysis identified one unknown arsenical in urine of wild-type mice as dimethylmonothioarsinic acid. In addition, another unknown arsenical in urine of wild-type mice that occurred in the urine of about 20 % of arsenate-treated mice. The presence of low levels of methylated arsenicals in liver digests of As3mt knockout mice may reflect the activity of other methyltransferases or the absorption of methylated arsenicals formed by the microbiota of the gastrointestinal tract. The lack of methylated thioarsenicals in urine of As3mt knockout mice suggests a close link between the processes that form methylated oxy- and thioarsenicals.