Proteomic identification of arginine-methylated proteins in colon cancer cells and comparison of messenger RNA expression between colorectal cancer and adjacent normal tissues.

PURPOSE: Identification of type I protein arginine methyltransferase (PRMT) substrates and their functional significance during tumorigenesis is becoming more important. The present study aimed to identify target substrates for type I PRMT using 2-dimensional (2D) gel electrophoresis (GE) and 2D Western blotting (WB). METHODS: Using immunoblot analysis, we compared the expression of type I PRMTs and endogenous levels of arginine methylation between the primary colorectal cancer (CRC) and adjacent noncancerous tissues paired from the same patient. To identify arginine-methylated proteins in HCT116 cells, we carried out 2D-GE and 2D-WB with a type I PRMT product-specific antibody (anti-dimethyl-arginine antibody, asymmetric [ASYM24]). Arginine-methylated protein spots were identified by mass spectrometry, and messenger RNA (mRNA) levels corresponding to the identified proteins were analyzed using National Center for Biotechnology Information (NCBI) microarray datasets between the primary CRC and noncancerous tissues. RESULTS: Type I PRMTs and methylarginine-containing proteins were highly maintained in CRC tissues compared to noncancerous tissues. We matched 142 spots using spot analysis software between a Coomassie blue (CBB)-stained 2D gel and 2D-WB, and we successfully identified 7 proteins that reacted with the ASYM24 antibody: CACYBP, GLOD4, MAPRE1, CCT7, TKT, CK8, and HSPA8. Among these proteins, the levels of 4 mRNAs including MAPRE1, CCT7, TKT, and HSPA8 in CRC tissues showed a statistically significant increase compared to noncancerous tissues from patients using the NCBI microarray datasets. CONCLUSION: Our results indicate that the method shown here is useful in identifying arginine-methylated proteins, and significance of arginine modification in the proteins identified here should be further identified during CRC development.

A Comprehensive Analysis of Symmetric Arginine Dimethylation in Colorectal Cancer Tissues Using Immunoaffinity Enrichment and Mass Spectrometry.

Protein arginine methyltransferase 5 (PRMT5) is a major enzyme responsible for generating monomethyl and symmetric dimethyl arginine in proteins. PRMT5 is essential for cell viability and development, and its overexpression is observed in a variety of cancers. In the present study, it is found that levels of PRMT5 protein and symmetric arginine dimethylation in colorectal cancer (CRC) tissues are increased compared to those in adjacent noncancerous tissues. Using immunoaffinity enrichment of methylated peptides combined with high-resolution mass spectrometry, a total of 147 symmetric dimethyl-arginine (SDMA) sites in 94 proteins are identified, many of which are RNA binding proteins and enzymes. Quantitative analysis comparing CRC and normal tissues reveals significant increase in the symmetric dimethylation of 70 arginine sites in 46 proteins and a decrease in that of four arginine sites in four proteins. Among the 94 proteins identified in this study, it is confirmed that KH-type splicing regulatory protein is a target of PRMT5 and highly expressed in CRC tissues compared to noncancerous tissues. This study is the first comprehensive analysis of symmetric arginine dimethylation using clinical samples and extends the number of known in vivo SDMA sites. The data obtained are available via ProteomeXchange with the identifier PXD015653.

Proteome-wide identification of arginine methylation in colorectal cancer tissues from patients.

BACKGROUND: Protein arginine methylation reaction is catalyzed by protein arginine methyltransferase (PRMT) and the modification is implicated in various diseases including cancer. Currently, thousands of arginine methylation sites have been identified using high-resolution mass spectrometry-based proteomics technology. However, identification of arginine methylation using clinical samples at proteome level has not been reported yet. The objective of the present study was to identify, monomethyl-arginine (MMA) and asymmetric dimethyl-arginine (ADMA) sites in colorectal cancer (CRC) tissues at proteome level. METHODS: Pooled CRC tissue samples from 10 patients with stage II and III were digested by trypsin and these digests were further processed and lyophilized. Using monomethyl- or asymmetric dimethyl arginine (MMA or ADMA, respectively) motif kits, methylarginine-containing peptides were enriched and subsequently analyzed by high-resolution LC-MS/MS. DLD1 and HCT116 colon cancer cells were treated with type I PRMTs inhibitor (MS023) alone or combined with SN-38, and the effect of the drugs on CRC cell proliferation and apoptosis was measured by water-soluble tetrazolium salt (WST-1) assay and FACS analysis, respectively. RESULTS: In the present study, 455 MMA sites of 272 proteins and 314 ADMA sites of 155 proteins were identified from CRC tissues acquired from patients. In addition, 216 methylation sites and 75 substrates for PRMTs were newly identified. These results reveal the significant presence of MMA and ADMA sites on nucleic acid binding proteins and protein complexes involved in transcription. To investigate the effect of protein arginine methylation in CRC proliferation and apoptosis, MS023 was treated to two CRC cell lines. After 48 h treatment with various concentrations of MS023, CRC cell proliferation was significantly suppressed, with concomitant apoptosis induction. Furthermore, MS023 treatment significantly enhanced the inhibitory effect of SN-38 on CRC cell proliferation. CONCLUSION: This work reports the first comprehensive analysis of arginine methylation with clinical sample and suggests that type I PRMTs are potential therapeutic targets for drug discovery in CRC.

The prognostic significance of protein arginine methyltransferase 6 expression in colon cancer.

Protein arginine methylation is involved in cellular differentiation and proliferation. Recently, aberrant expression of protein arginine methyltransferases, which are responsible for the methylation reaction, has been reported in various types of cancer. However, there is no clear evidence regarding the prognostic value of abnormal PRMT6 expression in colorectal cancer or the effect of PRMT6 regulation on CRC cells. We investigated the expression patterns of PRMT6 in patients with stage II and III CRC. We detected nuclear expression of PRMT6 in 23.7% of carcinoma samples by immunohistochemistry. Among the clinicopathological parameters, the ratio of poorly differentiated cancer cells was approximately two-fold higher in patients with PRMT6-positive disease than in those with PRMT6-negative disease (p = 0.002). Patients with PRMT6-positive CRC had a shorter disease-free survival than those with PRMT6-negative CRC in both univariate and multivariate analyses (p = 0.018 and p = 0.035, respectively). siRNA-mediated inhibition of PRMT6 expression in CRC cells induced p21WAF1/CIP1 overexpression and suppressed cell growth and colony-forming ability. Concomitantly, apoptosis was induced in PRMT6-suppressed CRC cells. These data suggest that PRMT6 can serve as a biomarker for unfavorable prognosis and as a therapeutic target in CRC.

Elevated Pressure Enhanced Trail-Induced Apoptosis in Hepatocellular Carcinoma Cells Via Erk1/2-Inactivation.

The high frequency of intrinsic resistance to TNF-related apoptosisinducing ligand (TRAIL) in tumor cell lines has necessitated the development of strategies to sensitize tumors to TRAIL-induced apoptosis. We previously showed that elevated pressure applied as a mechanical stressor enhanced TRAIL-mediated apoptosis in human lung carcinoma cells in vitro and in vivo. This study focused on the effect of elevated pressure on the sensitization of TRAIL-resistant cells and the underlying mechanism. We observed elevated pressure-induced sensitization to TRAIL-mediated apoptosis in Hep3B cells, accompanied by the activation of several caspases and the mitochondrial signaling pathway. Interestingly, the enhanced apoptosis induced by elevated pressure was correlated with suppression of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) phosphorylation and CREB without any change to other MAPKs. Phosphorylation of Bcl-2-associated death promoter (BAD) also decreased, leading to inhibition of the mitochondrial pathway. To confirm whether the activation of pERK1/2 plays a key role in the TRAIL-sensitizing effect of elevated pressure, Hep3B cells were pre-treated with the ERK1/2-specific inhibitor PD98059 instead of elevated pressure. Co-treatment with PD98059 and TRAIL augmented TRAIL-induced apoptosis and decreased BAD phosphorylation. The inhibition of ERK1/2 activation by elevated pressure and PD98059 also reduced BH3 interacting-domain death agonist (BID), thereby amplifying apoptotic stress at the mitochondrial level. Our results suggest that elevated pressure enhances TRAIL-induced apoptosis of Hep3B cells via specific suppression of ERK1/2 activation among MAPKs.

PRMT4 is involved in insulin secretion via the methylation of histone H3 in pancreatic ß cells.

The relationship between protein arginine methyltransferases (PRMTs) and insulin synthesis in ß cells is not yet well understood. In the present study, we showed that PRMT4 expression was increased in INS-1 and HIT-T15 pancreatic ß cells under high-glucose conditions. In addition, asymmetric dimethylation of Arg17 in histone H3 was significantly increased in both cell lines in the presence of glucose. The inhibition or knockdown of PRMT4 suppressed glucose-induced insulin gene expression in INS-1 cells by 81.6 and 79% respectively. Additionally, the overexpression of mutant PRMT4 also significantly repressed insulin gene expression. Consistently, insulin secretion induced in response to high levels of glucose was decreased by both PRMT4 inhibition and knockdown. Moreover, the inhibition of PRMT4 blocked high-glucose-induced insulin gene expression and insulin secretion in primary pancreatic islets. These results indicate that PRMT4 might be a key regulator of high-glucose-induced insulin secretion from pancreatic ß cells via H3R17 methylation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induced cell-specific drug transporters with acquired cisplatin resistance in cisplatin sensitive cancer cells.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can induce drug transporter genes such as the ATP-binding cassette G member 2 (ABCG2), which contributes to multidrug resistance. We investigated the effect of TCDD pretreatment on drug transporters induction from cancer cells of various origins. Cell viabilities after treatment of cisplatin were measured to evaluate acquiring cisplatin resistance by TCDD. Acquring cisplatin resistance was found only in cisplatin senstivie cancer cells including gastric SNU601, colon LS180, brain CRT-MG and lymphoma Jurkat cells which showed a significant increase in cell viability after combined treatment with TCDD and cisplatin. High increase of ABCG2 gene expression was found in SNU601 and LS180 cells with a mild increase in the expression of the ABCC3, ABCC5,and SLC29A2 genes in SNU601 cells, and of major vault protein (MVP) in LS180 cells. The AhR inhibitor kaempferol suppressed the upregulation of ABCG2 expression and reversed the TCDD-induced increase in cell viability in LS180 cells. However, in CRT-MG cells, other transporter genes including ABCC1, ABCC5, ABCA3, ABCA2, ABCB4, ABCG1, and SLC29A1 were up-regulated. These findings suggested the acquiring cisplatin resistance by TCDD associated with cancer cell-type-specific induction of drug transporters.

E3 ubiquitin ligase, WWP1, interacts with AMPKα2 and down-regulates its expression in skeletal muscle C2C12 cells.

It is known that the activity of AMP-activated protein kinase (AMPKα2) was depressed under high glucose conditions. However, whether protein expression of AMPKα2 is also down-regulated or not remains unclear. In this study, we showed that the expression of AMPKα2 was down-regulated in cells cultured under high glucose conditions. Treatment of proteasome inhibitor, MG132, blocked high glucose-induced AMPKα2 down-regulation. Endogenous AMPKα2 ubiquitination was detected by immunoprecipitation of AMPKα2 followed by immunoblotting detection of ubiquitin. The yeast-two hybrid (YTH) approach identified WWP1, an E3 ubiquitin ligase, as the AMPKα2-interacting protein in skeletal muscle cells. Interaction between AMPKα2 and WWP1 was validated by co-immunoprecipitation. Knockdown of WWP1 blocked high glucose-induced AMPKα2 down-regulation. The overexpression of WWP1 down-regulated AMPKα2. In addition, the expression of WWP1 is increased under high glucose culture conditions in both mRNA and protein levels. The level of AMPKα2 was down-regulated in the quadriceps muscle of diabetic animal model db/db mice. Expression of WWP1 blocked metformin-induced glucose uptake. Taken together, our results demonstrated that WWP1 down-regulated AMPKα2 under high glucose culture conditions via the ubiquitin-proteasome pathway.

Tissue-specific and age-dependent expression of protein arginine methyltransferases (PRMTs) in male rat tissues.

Protein arginine methyltransferases (PRMTs) generate asymmetric and symmetric dimethyl-arginines by catalyzing the transfer of methyl groups from S: -adenosyl-L-methionine to arginines in target proteins. Previously, we observed that the expression and activity of PRMTs were significantly down-regulated in replicatively senescent fibroblasts compared to young fibroblasts. In this study, we determined the level of three PRMT family members (PRMT1, PRMT4, and PRMT5) and the arginine methylation status in eight tissues from 6- and 24-month-old rats. We observed tissue-specific down-regulation of individual PRMT members in testis, thymus, kidney, lung, and heart from 24-month-old as compared to 6-month-old rats. Specifically, we observed reduced levels of PRMT1 in thymus and lung, reduced levels of PRMT4 in testis, thymus, and hearts, and reduced levels of PRMT5 in all five tissues. PRMT enzyme activity on histones generally correlated with PRMT expression. Furthermore, we observed a reduction in asymmetric and symmetric dimethylation on proteins in aged thymus and lung, and a reduction in symmetric dimethylation in aged testes relative to the testes harvested from young rats. These results suggest that individual PRMT proteins have tissue-specific functions and are regulated in a tissue-specific and age-dependent manner.

Proteomic identification and comparative analysis of asymmetrically arginine-methylated proteins in immortalized, young and senescent cells.

Protein-arginine methylation is one of the modifications that yields mono and dimethyl (asymmetric or symmetric) arginine residues in proteins. Previously, we found that asymmetric arginine methylation is decreased proportionately with a decrease of cell proliferation potential of cells, and such arginine methylation is greatest in immortalized cells, followed by normal young cells, and lowest in replicatively senescent cells. Using an asymmetric dimethyl-arginine-specific antibody, we identified arginine-methylated proteins in these cell types by immunoprecipitation and 2-D immunoblotting followed by MS. As a result, arginine methylation of chaperone molecules and RNA-binding proteins was differentially regulated between immortalized or young cells and senescent cells. Immortalized cells had significantly higher levels of methyl-accepting proteins, such as cleavage stimulation factor 2 (CstF2) and heterogeneous nuclear ribonucleoprotein (hnRNP) R, than young cells. However, senescent cells contained hypomethylated CstF2, hnRNP K, and chaperone containing TCP1 subunit 7, as well as decreased hnRNP R level. Further, significant reduction of arginine modification in CstF2 and chaperone containing TCP1 subunit 7 was observed in prematurely senescent fibroblasts, induced by treatment with adenosine dialdehyde, a transmethylation inhibitor, or subcytotoxic concentration of H(2)O(2). These results suggest that asymmetric modification of RNA-binding proteins and molecular chaperones plays an essential role in maintaining cell proliferation capability.

Regulation of post-translational protein arginine methylation during HeLa cell cycle.

BACKGROUND: Post-translational arginine methylation which modifies protein-arginyl residues by protein arginine methyltransferase (PRMT) was investigated during synchronized HeLa cell cycle. METHODS: The lysates of cells synchronized at each stage were subjected to one and/or two dimensional electrophoresis followed by Western immunoblot using against anti-asymmetric-dimethyl-arginine (ASYM24), anti-symmetric-dimethyl-arginine (SYM10), and subclasses of PRMTs, including PRMT1, PRMT3, PRMT4 (CARM1), PRMT5, PRMT6, and PRMT7 antibodies. RESULTS: Proteins with approximate molecular masses of 80 kDa, 68 kDa, and 64 kDa, containing asymmetric-dimethyl-arginine (aDMA) were increased at G0/G1 to G1, which lasted until S phase. In addition, 25 kDa protein of symmetric-dimethyl-arginine (sDMA) was also markedly up-regulated from G0/G1 to G1. The levels of PRMT3, PRMT6 and PRMT7 were concurrently increased during the cell cycle. Two-dimensional gel electrophoresis followed by MALDI-TOF-MS was identified as aDMA-80 kDa and aDMA-68 kDa proteins as heterogeneous nuclear ribonucleoprotein R (hnRNPR), aDMA-64 kDa proteins as cleavage stimulation factor 64 kDa subunit (CstF-64), and sDMA-25 kDa protein as triosephosphate isomerase (TPI). The levels of increased aDMA of hnRNPR were reduced, when HeLa cells were transfected with siRNA for PRMT1, and the aDMA of CstF-64 with siRNA for PRMT3, while depletion of PRMT5 down-regulated sDMA of TPI. CONCLUSION: Protein arginine dimethylations of hnRNPR, CstF-64, and TPI were regulated during HeLa cell cycle by respective PRMTs. GENERAL SIGNIFICANCE: These results suggest that regulation of arginine dimethylation of hnRNPR, CstF-64, and TPI at G0/G1 to G1 are most likely to modulate the cellular growth and proliferation in HeLa cell cycle.

Down-regulation of asymmetric arginine methylation during replicative and H2O2-induced premature senescence in WI-38 human diploid fibroblasts.

Protein arginine methylation is one of the post-translational modifications which yield monomethyl and dimethyl (asymmetric or symmetric) arginines in proteins. In the present study, we investigated the status of protein arginine methylation during human diploid fibroblast senescence. When the expression of protein arginine methyltransferases (PRMTs), namely PRMT1, PRMT4, PRMT5 and PRMT6 was examined, a significant reduction was found in replicatively senescent cells as well as their catalytic activities against histone mixtures compared with the young cells. Furthermore, when the endogenous level of arginine-dimethylated proteins was determined, asymmetric modification (the product of type I PRMTs including PRMT1, PRMT4 and PRMT6) was markedly down-regulated. In contrast, both up- and down-regulations of symmetrically arginine-methylated proteins (the product of type II PRMTs including PRMT5) during replicative senescence were found. Furthermore, when young fibroblasts were induced to premature senescence by sub-cytotoxic H2O2 treatment, results similar to replicative senescence were obtained. Finally, we found that SV40-mediated immortalized WI-38 and HeLa cell lines maintained a higher level of asymmetrically modified proteins as well as type I PRMTs than young fibroblasts. These results suggest that the maintenance of asymmetric modification in the expressed target proteins of type I PRMTs might be critical for cellular proliferation.

Comparison of the effects of 40% oxygen and two atmospheric absolute air pressure conditions on stress-induced premature senescence of normal human diploid fibroblasts.

The pressure during hyperbaric oxygen treatment may increase oxygen toxicity via an augmented oxygen pressure in the gas. Nevertheless, only a few reports have been published on the effect of cells grown under 2 atmospheric absolute (ATA) pressure. To evaluate the effect of pressure on oxygen toxicity and to study effects in addition to oxygen toxicity, we designed an experiment to compare the effects of normobaric mild hyperoxia (NMH, 40% oxygen) and hyperbaric air condition (HA, air with 2 ATA) on human diploid fibroblasts (HDF) in a hyperbaric incubator. HDFs in both the NMH and the HA condition had a similar oxidative stress response and exhibited premature senescence. To investigate differences in gene profiling in cells grown in the NMH and HA conditions, samples from cells exposed to each condition were applied to microarrays. We found no expression difference in genes related to aging and deoxyribonucleic acid damage, but the expression of genes including cell adhesion, stress response, and transcription were significantly increased in fibroblasts that were responsive to pressure. Among 26 statistically reliable genes, the expression of apoptosis related genes such as ADAM22, Bax, BCL2L14, and UBD, as well as tumor suppressor-related genes like Axin2 and ATF, and also mitogen-activated protein kinase-related genes like mitogen-activated protein kinase kinase kinase 1, histamine receptor, and RAB24, were significantly changed in cells responsive to pressure-induced oxidative stress.

Multimerization of expressed protein-arginine methyltransferases during the growth and differentiation of rat liver.

Protein-arginine methylation is a posttranslational modification which yields monomethyl and dimethyl (asymmetric or symmetric) arginines in proteins. We investigated the expressions of PRMT1 and PRMT5 in relation to their catalytic activities in rat liver during growth and differentiation as well as in the pancreas. Western immunoblot analysis revealed that both PRMT1 and PRMT5 proteins were expressed in the cytosol of liver and pancreas with molecular mass of about 42 kDa and 72 kDa, respectively. However, on molecular sieve chromatography, the enzyme activities were eluted at about 500 kDa for PRMT5 and 440 kDa for PRMT1, indicating that the multimer complex of these expressed monomers were catalytically active. While the 500 kDa complex methylated predominantly myelin basic protein (MBP), the 440 kDa complex methylated hnRNP A1 protein. In fetal rat liver, the amount of expressed 42 kDa PRMT1 protein and the enzyme activity to methylate hnRNPA1 protein were 2- to 3-fold and 4- to 5-fold higher, respectively, than those of post-natal livers. While the 72 kDa PRMT5 protein was consistently expressed, its activity varied only about 2-fold. However, PRMT5 to methylate MBP showed one distinct peak at around the 20th day post-natal. Furthermore, while the PRMT1 enzyme activity increased more than 10-fold after 3 days of 70% partial hepatectomy, the amount of expressed PRMT1 protein was only about 3.2-fold higher than the control livers. In summary, we observed that PRMTs are catalytically active only in the form of multimers, but not as a dimer or tetramer of the expressed subunit. Furthermore, the amount of expressed PRMT protein, determined by Western immunoblot, did not correlate with the amount of their catalytic activity, and thus, some uncharacterized additional factor(s) may multimerize PRMTs to express catalytic activities in vivo.

Increased methylation of endogenous 20-kDa protein in HIT beta-cell during insulin secretion.

Enzymatic methylation of endogenous proteins in clonal pancreatic beta-cell, HIT-T15, was investigated. When cell extract incubated with S-adenosyl-L-[methyl-3H]methionine was subjected to SDS-PAGE followed by fluorography, endogenous 20-kDa protein was highly [methyl-3H]-labeled. The increase of methylation was correlated with insulin secretion, when the cells were treated with secretagogue; at 5.5mM glucose, insulin secretion increased by 2.5-fold, while the 20-kDa methylation to about 3.2-fold. In the case of forskolin, another secretagogue, at 0.1mM, the methylation increased by approximately 4.5-fold. This increase of 20-kDa methylation was inhibited when the cells were treated with 3mM EGTA to inhibit insulin secretion by depleting extracellular calcium ion, indicating intercausal relation between methylation and insulin secretion. The [methyl-3H]-labeled amino acids were identified by thin layer chromatography as N(G)-methylated arginines. While arginyl residues in Gly-Arg-Gly sequence are known to be posttranslationally methylated, a synthetic nonapeptide, GGRGRGRGG, competed with the 20-kDa methylation; at 1 and 10 micro M nonapeptides, 62% and 78% of 20-kDa methylation were inhibited, respectively. Furthermore, Western immunoblot analysis of HIT cell extract against GGRGRGRGG antibodies strongly immunoreacted with the 20-kDa protein. These results suggested that methylation of the endogenous 20-kDa protein might play some role in insulin secretion.