Protein Methyltransferase Research Articles

Biochemical and docking analysis of substrate interactions with polyisoprenylated methylated protein methyl esterase.

Polyisoprenylated proteins (PPs) methylation by polyisoprenylated protein methyl transferase (PPMTase) is counteracted by polyisoprenylated methylated protein methyl esterase (PMPMEase). This is the only reversible step of the polyisoprenylation pathway as the relative amounts of the acid and ester forms are determined by the two competing reactions. Since PMPMEase and PPMTase may influence both the structural/functional conformations of PPs, a thorough study of these enzymes is essential to our understanding of the structural/functional features of PPs. PMPMEase has been reported under such pseudonyms as human carboxylesterase 1 (hCE1) because of its apparent broad substrate spectrum. The current study aimed to show the complementarity between its active site and the polyisoprenylated substrates that it metabolizes. Kinetics analysis was conducted with N-, S- and O-substituted substrates using porcine liver PMPMEase and docking analysis using Arguslab. Consistent with the biochemical analysis, the S-ethyl analog yielded an AScore binding energy of -11.32 compared to -13.48, -14.88, -16.15, and -16.81 kcal/mol for S-prenyl (C-5), S-trans-geranyl (C-10), S-trans,trans-farnesyl (C-15) and S-all trans-geranylgeranyl (C-20), respectively. The all trans-geranylgeranyl moiety provides the optimal size for active site interactions. The data reveal that the trans,trans-farnesyl and all trans-geranylgeranyl groups, which are reminiscent of endogenous PPs modifications, have the highest affinity for PMPMEase. Since PPs such as monomeric G-proteins are oncogenic, PMPMEase may be viewed as a viable target for anticancer drug development. The analyses reveal the important structural elements for the design of specific PMPMEase inhibitors to serve in the modulation of oncogenic PPs activities. The results also show that hCE1's repertoire of substrates extends beyond xenobiotics to include PPs as its endogenous substrates.

Calmodulin methyltransferase is an evolutionarily conserved enzyme that trimethylates Lys-115 in calmodulin

Calmodulin (CaM) is a key mediator of calcium-dependent signalling and is subject to regulatory post-translational modifications, including trimethylation of Lys-115. In this paper, we identify a class I, non-SET domain protein methyltransferase, calmodulin-lysine N-methyltransferase (EC 2.1.1.60). A polypeptide chosen from a fraction enriched in calmodulin methyltransferase activity was trypsinized and analysed by tandem mass spectrometry. The amino-acid sequence obtained identified conserved, homologous proteins of unknown function across a wide range of species, thus implicating a broad role for lysine methylation in calcium-dependent signalling. Encoded by c2orf34, the human homologue is a component of two related multigene deletion syndromes in humans. Human, rat, frog, insect and plant homologues were cloned and Escherichia coli-recombinant proteins catalysed the formation of a trimethyllysyl residue at position 115 in CaM, as verified by product analyses and mass spectrometry.

Enzymatic Site‐Specific Functionalization of Protein Methyltransferase Substrates with Alkynes for Click Labeling

Pass and click: Protein methylation is an important posttranslational modification. Because the methyl group is a poor reporter group, new methods are needed to analyze methyltransferase substrates. A S-adenosyl-L-methionine-based cofactor was synthesized and used for the site-specific functionalization of proteins with alkynes by methyltransferases (first step) and subsequent labeling through CuAAC click chemistry (second step; see scheme).

Identification of protein N-terminal methyltransferases in yeast and humans.

Protein modification by methylation is important in cellular function. We show here that the Saccharomyces cerevisiae YBR261C/TAE1 gene encodes an N-terminal protein methyltransferase catalyzing the modification of two ribosomal protein substrates, Rpl12ab and Rps25a/Rps25b. The YBR261C/Tae1 protein is conserved across eukaryotes; all of these proteins share sequence similarity with known seven beta-strand class I methyltransferases. Wild-type yeast cytosol and mouse heart cytosol catalyze the methylation of a synthetic peptide (PPKQQLSKY) that contains the first eight amino acids of the processed N-terminus of Rps25a/Rps25b. However, no methylation of this peptide is seen in yeast cytosol from a DeltaYBR261C/tae1 deletion strain. Yeast YBR261C/TAE1 and the human orthologue METTL11A genes were expressed as fusion proteins in Escherichia coli and were shown to be capable of stoichiometrically dimethylating the N-terminus of the synthetic peptide. Furthermore, the YBR261C/Tae1 and METTL11A recombinant proteins methylate variants of the synthetic peptide containing N-terminal alanine and serine residues. However, methyltransferase activity is largely abolished when the proline residue in position 2 or the lysine residue in position 3 is substituted. Thus, the methyltransferases described here specifically recognize the N-terminal X-Pro-Lys sequence motif, and we suggest designating the yeast enzyme Ntm1 and the human enzyme NTMT1. These enzymes may account for nearly all previously described eukaryotic protein N-terminal methylation reactions. A number of other yeast and human proteins also share the recognition motif and may be similarly modified. We conclude that protein X-Pro-Lys N-terminal methylation reactions catalyzed by the enzymes described here may be widespread in nature.

Differentially expressed genes after hyper- and hypo-salt stress in the halophilic archaeonMethanohalophilus portucalensis

Methanohalophilus portucalensis FDF1 can grow over a range of external NaCl concentrations, from 1.2 to 2.9 mol/L. Differential gene expression in response to long-term hyper-salt stress (3.1 mol/L of NaCl) and hypo-salt stress (0.9 mol/L of NaCl) were compared by differential display RT-PCR. Fourteen differentially expressed genes responding to long-term hyper- or hypo-salt stress were detected, cloned, and sequenced. Several of the differentially expressed genes were related to the unique energy-acquiring methanogenesis pathway in this organism, including the transmembrane protein MttP, cobalamin biosynthesis protein, methenyl-H4MPT cyclohydrolase and monomethylamine methyltransferase. One signal transduction histidine kinase was identified from the hyper-salt stress cultures. Moreover, 3 known stress-response gene homologues - the DNA mismatch repair protein, MutS, the universal stress protein, UspA, and a member of the protein-disaggregating multichaperone system, ClpB - were also detected. The transcriptional analysis of these long-term salt stress response and adaptation-related genes for cells immediately after salt stress indicated that the expression of the energy metabolism genes was arrested during hyper-salt shock, while the chaperone clpB gene was stimulated by both hypo- and hyper-salt shock.

Fatty Acid Suppression of Cell Proliferation is Associated with the Inhibition of Polyisoprenylated Methylated Protein Methyl Esterase

Protein polyisoprenylation is essential for the activities of oncogenic proteins such as Ras. The final, reversible step of polyisoprenylation pathway is catalyzed by an ester forming polyisoprenylated protein methyl transferase and polyisoprenylated methylated protein methyl esterase (PMPMEase). Various free fatty acids (FFAs) were analyzed to test whether their beneficial health effects are associated with PMPMEase inhibition. The potent inhibitory FFAs were further analyzed for their effects on cell viability. Arachidonic acid (AA) and lauric acid (LA) inhibited PMPMEase with 0.19 and 6.5 ìM, respectively. AA was over 63‐fold more inhibitory than Prostaglandin A2 (Ki of 12 ìM) while prostaglandin E2 and long chain saturated fatty acids were not inhibitory even at 1 mM. AA and LA inhibited the viability of human neuroblastoma cells with EC50 values of 11 and 25 ìM respectively, after 72 h exposure. Therefore AA and other PUFAs may be involved in the regulation of cell proliferation and that the elevated levels of COX‐2 in tumors convert PUFAs which inhibit PMPMEase at physiological concentrations, to prostanoids that are ineffective at inhibiting both PMPMEase and thus cell proliferation. NSAIDs may thus owe their anticancer effects through their ability to protect the PMPMEase‐ and cell proliferation‐inhibiting PUFAs from COX‐2 metabolism.Grant Funding Source: NIH/NIGMS/SCORE (GM 08111‐35) & NIH/NCRR (G12 RR0 3020)

Polyisoprenylation potentiates the inhibitory and cell degenerative potency of sulfonyl fluorides towards polyisoprenylated methylated protein methyl esterase

Polyisoprenylated protein methyl transferase catalyses the esterification of polyisoprenylated proteins (PP) whereas polyisoprenylated methylated protein methyl esterase (PMPMEase) hydrolyses the ester bond. Significant changes in their enzymatic activity may alter PP function causing disease. Prior studies show that PMPMEase is Sus scrofa carboxylesterase, which hints at its susceptibility to serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF). PMSF was used as a prototypical compound to develop high affinity specific inhibitors of PMPMEase. Pseudo first‐order kinetics revealed an over 680‐fold increase in kobs/[I] (M−1s−1) values from PMSF (6), S‐trans,trans‐farnesyl (L‐28, 2000), to S‐trans‐geranylated (L‐23, 4100) 2‐thioethanesulfonyl fluorides. The compounds induced the degeneration of human neuroblastoma SH‐SY5Y cells with EC50 values of 49, 130 and >1000 μM for L‐28, L‐23 and PMSF, respectively. The increase in affinity with the polyisoprenyl derivatization corroborates the observed substrate specificity and the reported hydrophobicity of the PMPMEase active site. These results suggest that (1) PMPMEase is a key enzyme for PP metabolism, (2) regulation of its activity is essential for maintaining cell viability, (3) abnormal activities may be involved in degenerative diseases and cancers and (4) its specific inhibitors may be useful in combating cancers.

Inhibition of polyisoprenylated protein metabolism may explain the apoptotic properties of farnesol and related flavors

Polyisoprenylation is the modification of about 2% of eukaryotic proteins involving the reaction of trans, trans‐farnesyl or all trans‐geranylgeranyl groups to a C‐terminal cysteine. The –COOH of the cysteine is methylated by polyisoprenylated protein methyl transferase (PPMTase) into esters which are then hydrolyzed by polyisoprenylated methylated protein methyl esterase (PMPMEase). Because aberrant polyisoprenylated protein activity is associated with ~30% of cancers, compounds that modulate their metabolism may be useful as anticancer agents. Farnesol and related compounds are natural food components or flavors that are derived from citrus, ginger, teas and chamomile. They are structurally related to the polyisoprenyl moieties of polyisoprenylated proteins and may thus inhibit PMPMEase. When PMPMEase was incubated with the PMPMEase substrate, RD‐PNB, farnesol, trans, trans‐farnesol, citronellal, α‐Ionone, eugenol and geraniol inhibited the enzyme activity with Ki values of 1.9, 5.5, 6.6, 3, 15 and 45 μM, respectively. Farnesol and trans, trans‐farnesol induced the degeneration of human lung A549, colorectal Caco‐2 and neuroblastoma SH‐SY5Y cancer cells with EC50 values of 18 and 19, 14 and 19 and 265 and 465 μM, respectively. These suggest that the apoptotic effects of these compounds may be associated with PMPMEase inhibition, suggesting the latter may be a viable anticancer target.

Biochemical and Docking Analysis of Polyisoprenylated Methylated Protein Methyl Esterase with Substrates

Polyisprenylated proteins (PP) methylation by polyisoprenylated protein methyl transferase (PPMTase) is counteracted by polyisoprenylated methylated protein methyl esterase (PMPMEase), making it the only reversible step of the polyisoprenylation pathway. Since PMPMEase and PPMTase may influence both the structural and functional conformations of PP, a thorough study of these enzymes is essential to our understanding of the physiology of PP. PMPMEase has been reported under various pseudonyms because of its apparent broad substrate metabolism. The current study aimed to reveal its role as the endogenous polyisoprenylation‐specific esterase. A series of N‐, S‐ and O‐substituted substrates were analyzed by Michaelis‐Menten kinetics using porcine liver PMPMEase and in silico determination of binding affinities using Arguslab. Consistent with the biochemical analysis, the S‐ethyl analog yielded an AScore binding energy of −11.32 compared to −13.48, −14.88, −16.15 and −16.81 kcal/mol for S‐prenyl (C‐5), S‐trans‐geranyl (C‐10), S‐trans, trans‐farnesyl (C‐15) and S‐all trans‐geranylgeranyl (C‐20), respectively. As judged by the KM and AScore values, substrates with the natural modifications have the highest affinity for PMPMEase. The results demonstrate that PP are the likely endogenous substrates for PMPMEase.

Arginine Methylation of Vasa Protein Is Conserved across Phyla

Recent studies have uncovered an unexpected relationship between factors that are essential for germline development in Drosophila melanogaster: the arginine protein methyltransferase 5 (dPRMT5/Csul/Dart5) and its cofactor Valois, methylate the Piwi family protein Aub, enabling it to bind Tudor. The RNA helicase Vasa is another essential protein in germline development. Here, we report that mouse (mouse Vasa homolog), Xenopus laevis, and D. melanogaster Vasa proteins contain both symmetrical and asymmetrical dimethylarginines. We find that dPRMT5 is required for the production of sDMAs of Vasa in vivo. Furthermore, we find that the mouse Vasa homolog associates with Tudor domain-containing proteins, Tdrd1 and Tdrd6, as well as the Piwi proteins, Mili and Miwi. Arginine methylation is thus emerging as a conserved and pivotal post-translational modification of proteins that is essential for germline development.

Sites Inferred by Metabolic Background Assertion Labeling (SIMBAL): adapting the Partial Phylogenetic Profiling algorithm to scan sequences for signatures that predict protein function

BackgroundComparative genomics methods such as phylogenetic profiling can mine powerful inferences from inherently noisy biological data sets. We introduce Sites Inferred by Metabolic Background Assertion Labeling (SIMBAL), a method that applies the Partial Phylogenetic Profiling (PPP) approach locally within a protein sequence to discover short sequence signatures associated with functional sites. The approach is based on the basic scoring mechanism employed by PPP, namely the use of binomial distribution statistics to optimize sequence similarity cutoffs during searches of partitioned training sets.ResultsHere we illustrate and validate the ability of the SIMBAL method to find functionally relevant short sequence signatures by application to two well-characterized protein families. In the first example, we partitioned a family of ABC permeases using a metabolic background property (urea utilization). Thus, the TRUE set for this family comprised members whose genome of origin encoded a urea utilization system. By moving a sliding window across the sequence of a permease, and searching each subsequence in turn against the full set of partitioned proteins, the method found which local sequence signatures best correlated with the urea utilization trait. Mapping of SIMBAL "hot spots" onto crystal structures of homologous permeases reveals that the significant sites are gating determinants on the cytosolic face rather than, say, docking sites for the substrate-binding protein on the extracellular face. In the second example, we partitioned a protein methyltransferase family using gene proximity as a criterion. In this case, the TRUE set comprised those methyltransferases encoded near the gene for the substrate RF-1. SIMBAL identifies sequence regions that map onto the substrate-binding interface while ignoring regions involved in the methyltransferase reaction mechanism in general. Neither method for training set construction requires any prior experimental characterization.ConclusionsSIMBAL shows that, in functionally divergent protein families, selected short sequences often significantly outperform their full-length parent sequence for making functional predictions by sequence similarity, suggesting avenues for improved functional classifiers. When combined with structural data, SIMBAL affords the ability to localize and model functional sites.

Abstract C71: Inhibition of polyisoprenylated methylated protein methylesterase by anticancer drugs

Abstract Polyisoprenylated methylated protein methylesterase (PMPMEase) is a little explored enzyme of the polyisoprenylation (isoprenylation) pathway (PP). Polyisoprenylation is a post-translational modification that is important for the function of such proteins as the heterotrimeric G proteins, Ras and Rab families of proteins. PMPMEase hydrolyzes a methyl ester formed by the S-adenosyl-L-methionine (SAM)-dependent enzyme, polyisoprenylated protein methyltransferase (PPMTase). PMPMEase's regulatory role is suggested by the fact it hydrolyzes the ester products of PPMTase. PMPMEase is a serine hydrolase, a class of hydrolytic enzymes that attack electron-deficient carbons of amide and ester bonds with the alkoxide ion of their catalytic serines. Because alkylating agents function through an aziridinium ion with an electron-deficient carbon atom, it is hypothesized that they may inhibit PMPMEase through active site adduct formation. Alkylating agents have been suspected of having other mechanisms of action against cells besides impeding replication via the cross-linking of DNA strands. The crystal structure of the human form of PMPMEase, human carboxylesterase 1, with tamoxifen bound to its active site has been solved, also suggesting its possible inhibition. Polyisoprenylated proteins are involved in processes that regulate cell viability. Mutations and overexpression of such proteins are implicated in about 30% of cancers. This study considers the possibility that the agents also reduce malignant cell growth by disrupting growth signaling pathways. Cyclophosphamide is an organophosphorous alkylating agent. Organophosphorous compounds irreversibly inhibit PMPMEase and other serine esterases. Busulfan, cyclophosphamide, mechlorethamine and tamoxifen were tested for porcine liver PMPMEase inhibition. At 1 mM concentrations, cyclophosphamide, mechlorethamine and tamoxifen inhibited 62, 35 and 55% of the PMPMEase activity. The results show that the anticancer drugs may owe part of their pharmacological activity to their modulation of oncogenic polyisoprenylated protein function through their inhibition of PMPMEase. Citation Information: Cancer Res 2009;69(23 Suppl):C71.

Methyltetrahydrofolate:corrinoid/iron−sulfur Protein Methyltransferase (MeTr): Protonation State of the Ligand and Active-Site Residues

Methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) catalyzes the transfer of the N5-methyl group from N5-methyltetrahydrofolate (CH(3)THF) to the cobalt center of a corrinoid/iron-sulfur protein, a reaction similar to that of cobalamin-dependent methionine synthase (MetH). For such a reaction to occur, CH(3)THF is expected to be activated by a stereospecific protonation at the N5 position. It has been shown experimentally that binding to MeTr is associated with a pK(a) increase and proton uptake. The enzyme could achieve this by binding the unprotonated form of CH(3)THF, followed by specific protonation at the correct orientation. Here we have used computational approaches to investigate the protonation state of the ligand and active-site residues in MeTr. First, quantum mechanical (QM) methods with the PCM solvation model were used to predict protonation positions and pK(a) values of pterin, folate, and their analogues in an aqueous environment. After a reliable calibration of computational and experimental results was obtained, the effect of the protein environment was then considered. Different protonation states of CH(3)THF and active-site aspartic residues (D75 and D160) were investigated using QM calculations of active-site fragment complexes and the perturbed quantum atom (PQA) approach within QM/MM simulations. The final free energy results indicate that the N5 position of the tetrahydropterin ring is the preferred protonation position of CH(3)THF when bound to the active site of MeTr, followed by Asp160. We also found that the active-site environment is likely to increase the pK(a) of N5 by about 3 units, leading to proton uptake upon CH(3)THF binding, as observed experimentally for MeTr. Some implications of the results are discussed for the MetH mechanism.

A Continuous Protein Methyltransferase (G9a) Assay for Enzyme Activity Measurement and Inhibitor Screening

The authors describe a continuous protein methylation assay using the G9a protein lysine methyltransferase and its substrate protein WIZ (widely interspaced zinc finger motifs). The assay is based on the coupling of the biotinylated substrate protein to streptavidin-coated FlashPlates and the transfer of radioactive methyl groups from the S-adenosyl-L-methionine to the substrate. The reaction progress is monitored continuously by proximity scintillation counting. The assay is very accurate, convenient, well suited for automation, and highly reproducible with standard errors in the range of 5%. Because of few pipetting steps and continuous data readout, it is ideal for high-throughput applications such as screening of inhibitors, testing many enzyme variants, or analyzing differences in methylation rates of different substrates under various conditions. By using this new assay, the IC(50) of AdoHcy and the G9a inhibitor BIX-01294 were determined for methylation of the G9a nonhistone substrate WIZ.

Expression of RIZ1 protein (Retinoblastoma‐interacting zinc‐finger protein 1) in prostate cancer epithelial cells changes with cancer grade progression and is modulated in vitro by DHT and E2

The nuclear protein methyl-transferase Retinoblastoma-interacting zinc-finger protein 1 (RIZ1) is considered to be a downstream effector of estrogen action in target tissues. Silencing of RIZ1 expression is common in many tumors. We analyzed RIZ1 expression in normal and malignant prostate tissue and evaluated whether estradiol (E2) or dihydrotestosterone (DHT) treatment modulated RIZ1 in cultured prostate epithelial cells (PEC). Moreover, we studied the possible involvement of RIZ1 in estrogen action on the EPN prostate cell line, constitutively expressing both estrogen receptor (ER)-alpha and beta. RIZ1 protein, found in the nucleus of normal PECs by immunohistochemistry, was progressively lost in cancer tissues as the Gleason score increased and was only detected in the cytoplasmic compartment. RIZ1 transcript levels, as assayed by semi-quantitative RT-PCR in primary PEC cultures, were significantly reduced in cancer cells (P < 0.05). In EPN DHT treatment significantly increased RIZ1 transcript and protein levels (P < 0.05); E2 induced a reduction of S phase without significant changes of RIZ1 expression. In E2-treated EPN cell extracts RIZ co-immunoprecipitated with ERbeta and ERalpha. Our data demonstrate that RIZ1 is expressed in normal PECs and down-regulated in cancer cells, with a switch of its sub-cellular localization from the nucleus to the cytoplasm upon cancer grade progression. RIZ1 expression levels in the PECs were modulated by DHT or E2 treatment in vitro. Furthermore, the E2 effects on ER-expressing prostate cells involve RIZ1, which confirms a possible role for ER-mediated pathways in a non-classic E(2)-target tissue.

A Methyltransferase-independent Function for Rmt3 in Ribosomal Subunit Homeostasis

Schizosaccharomyces pombe Rmt3 is a member of the protein-arginine methyltransferase (PRMT) family and is the homolog of human PRMT3. We previously characterized Rmt3 as a ribosomal protein methyltransferase based on the identification of the 40 S Rps2 (ribosomal protein S2) as a substrate of Rmt3. RMT3-null cells produce nonmethylated Rps2 and show mis-regulation of the 40 S/60 S ribosomal subunit ratio due to a small subunit deficit. For this study, we have generated a series of RMT3 alleles that express various amino acid substitutions to characterize the functional domains of Rmt3 in Rps2 binding, Rps2 arginine methylation, and small ribosomal subunit production. Notably, catalytically inactive versions of Rmt3 restored the ribosomal subunit imbalance detected in RMT3-null cells. Consistent with a methyltransferase-independent function for Rmt3 in small ribosomal subunit production, the expression of an Rps2 variant in which the identified methylarginine residues were substituted with lysines showed normal levels of 40 S subunit. Importantly, substitutions within the zinc finger domain of Rmt3 that abolished Rps2 binding did not rescue the 40 S ribosomal subunit deficit of RMT3-null cells. Our findings suggest that the Rmt3-Rps2 interaction, rather than Rps2 methylation, is important for the function of Rmt3 in the regulation of small ribosomal subunit production.

Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability

Piwi family proteins are essential for germline development and bind piwi-interacting RNAs (piRNAs 1 2 3). The grandchildless gene aub of Drosophila melanogaster encodes the piRNA-binding protein Aub that is essential for formation of primordial germ cells (PGCs) 4. Here we report that mouse, Xenopus laevis and Drosophila melanogaster Piwi family proteins contain symmetrical dimethylarginines (sDMAs). We find that Piwi proteins are expressed in X. laevis oocytes and we identify numerous X. laevis piRNAs. We report that the Drosophila homolog of protein methyltransferase 5 (dPRMT5, csul/dart5), which is also the product of a grandchildless gene 5, 6, is required for arginine methylation of Drosophila Piwi, Ago3 and Aub proteins, in vivo. Loss of dPRMT5 activity leads to reduction of piRNAs and in particular of Ago3 and Aub protein levels and accumulation of retrotransposons in the Drosophila ovary. Our studies explain the relationship between aub and dPRMT5 (csul/dart5) genes by demonstrating that dPRMT5 is the enzyme that methylates Aub. Our findings underscore the significance of sDMA modification of Piwi proteins in the germline and suggest an interacting pathway of genes that are required for piRNA function and PGC specification.

Methed-Up FOXOs Can't In-Akt-ivate

In this issue of Molecular Cell, Yamagata et al. (2008) identify a role for the protein methyltransferase, PRMT1, in Akt-dependent regulation of the FOXO1 transcription factor and demonstrate another level of control in this critical metabolic and cell survival signaling pathway.

The human interferon-regulated ISG95 protein interacts with RNA polymerase II and shows methyltransferase activity

A major mechanism of cellular resistance to viral invasion involves genes from the interferon signaling pathway, called ISGs (interferon stimulated genes). Global transcriptional profiling studies have linked increased expression of ISG95 (KIAA0082) to response to interferon treatment and viral infection, suggesting that it may be part of the cellular defense against viral replication. In this work, we show that the ISG95 promoter can drive interferon-induced transcription of a reporter gene in Vero cells. Recombinant ISG95 shows RNA- and S-adenosyl-methionine binding and protein methyltransferase activity in vitro. ISG95 interacts with the C-terminal domain of RNA polymerase II, which is consistent with its nuclear localization and with the predicted function of the WW domain found in the C-terminal region of ISG95. The results presented in this work indicate that ISG95 is part of the interferon response pathway and functions in the pre-mRNA processing events mediated by the C-terminal domain of the RNA polymerase II.

Protein Methyltransferase Activities in Commercial In vitro Translation Systems

Protein arginine methylation is a well-known post-translational modification that has been shown to occur in rabbit reticulocyte in vitro translation lysates (RRL); however, it is not known whether this is a general feature of in vitro-produced proteins from other eukaryotic cell-free translation systems, particularly insect-derived lysates (ICL). Because methylation can affect protein localization, RNA binding and protein-protein interactions this may be of great importance as in vitro-produced proteins are often used in assays of protein function. Here, I report the presence of base-stable and base-labile methyltransferase activities in RRL, ICL and wheat germ in vitro extracts (WGE). Indeed, the presence of CARM1 in RRL and ICL and a class II protein arginine methyltransferase activity (PRMT5/7) is documented in all three systems. Additionally, the lysine methyltransferase that modifies eukaryotic elongation factor 1A (eEF-1A) was detected in ICL and WGE. Importantly, using a defined set of substrates under identical conditions I show that all three in vitro systems contain different complements of the various methyltransferases. These data suggest that three systems can be used in a complementary fashion to investigate the effect(s) of post-translational modification on protein function.