Protein Methylation Research Articles

Antimicrobial resistance in Klebsiella pneumoniae: identification of bacterial DNA adenine methyltransferase as a novel drug target from hypothetical proteins using subtractive genomics.

Klebsiella pneumoniae is a gram-negative bacterium that is known for causing infection innosocomial settings. As reported by the World Health Organization, carbapenem-resistantEnterobacteriaceae, a category that includes K. pneumoniae, are classified as an urgentthreat, and the greatest concern is that these bacterial pathogens may acquire genetictraits that make them resistant towards antibiotics. The last class of antibiotics, carbapenems, are not able to combat these bacterial pathogens, allowing them to clonally expandantibiotic-resistant strains. Most antibiotics target essential pathways of bacterial cells;however, these targets are no longer susceptible to antibiotics. Hence, in our study, we focused on a hypothetical protein in K. pneumoniae that contains a DNA methylation proteindomain, suggesting a new potential site as a drug target. DNA methylation regulates theattenuation of bacterial virulence. We integrated computational-aided drug design by using a bioinformatics approach to perform subtractive genomics, virtual screening, and fingerprint similarity search. We identified a new potential drug, koenimbine, which could bea novel antibiotic.

Abnormal methylation caused by folic acid deficiency in neural tube defects.

Neural tube closure disorders, including anencephaly, spina bifida, and encephalocele, cause neural tube defects (NTDs). This congenital disability remained not only a major contributor to the prevalence of stillbirths and neonatal deaths but also a significant cause of lifelong physical disability in surviving infants. NTDs are complex diseases caused by multiple etiologies, levels, and mechanisms. Currently, the pathogenesis of NTDs is considered to be associated with both genetic and environmental factors. Here, we aimed to review the research progress on the etiology and mechanism of NTDs induced by methylation modification caused by folic acid deficiency. Folic acid supplementation in the diet is reported to be beneficial in preventing NTDs. Methylation modification is one of the most important epigenetic modifications crucial for brain neurodevelopment. Disturbances in folic acid metabolism and decreased S-adenosylmethionine levels lead to reduced methyl donors and methylation modification disorders. In this review, we summarized the relationship between NTDs, folic acid metabolism, and related methylation of DNA, imprinted genes, cytoskeletal protein, histone, RNA, and non-coding RNA, so as to clarify the role of folic acid and methylation in NTDs and to better understand the various pathogenesis mechanisms of NTDs and the effective prevention.

Multi-omics pan-cancer study of cuproptosis core gene FDX1 and its role in kidney renal clear cell carcinoma.

The mechanism of copper-induced cellular death was newly discovered and termed cuproptosis. Inducing cuproptosis in cancer cells is well anticipated for its curative potential in treating tumor diseases. However, ferredoxin 1 (FDX1), the core regulatory gene in cuproptosis, is rarely studied, and the regulation of FDX1 in tumor biology remains obscure. A comprehensive pan-cancer analysis of FDX1 is needed. Thirty-three types of tumors were included with paired normal tissues in The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) datasets. The interaction between transcription, protein, phosphorylation, and promoter methylation levels was analyzed. Survival, immune infiltration, single-cell FDX1 expression, FDX1-related tumor mutational burden (TMB), microsatellite instability (MSI), stemness, tumor immune dysfunction and exclusion (TIDE), and immunotherapy-related analyses were performed. FDX1 protein expression was assessed by kidney renal clear cell carcinoma (KIRC) tissue microarray immunohistochemistry. The function of FDX1 in KIRC was further explored by experiments in 786-O cell lines in vitro. FDX1 is highly expressed in 15 tumor types and lowly expressed in 11 tumor types. The corresponding changes in protein expression, phosphorylation, and promoter methylation level of FDX1 have been described in several tumors. Survival analysis showed that FDX1 was related to favorable or poor overall survival in eight tumors and progression-free survival in nine tumors. Immune infiltration and single-cell analysis indicated the indispensable role of FDX1 expression in macrophages and monocytes. Multiple established immunotherapy cohorts suggested that FDX1 may be a potential predictor of treatment effects for tumor patients. Tissue microarray analysis showed decreased FDX1 expression in KIRC patients' tumor tissues. Knockdown of FDX1 resulted in the downregulation of cuproptosis in kidney renal clear tumor cells. Mechanistically, the FDX1-associated gene expression signature in KIRC is related to the enrichment of genes involved in the tricarboxylic acid (TCA) cycle, NOTCH pathway, etc. Several NOTCH pathway genes were differentially expressed in the high- and low-FDX1 groups in KIRC. Our analysis showed that the central regulatory gene of cuproptosis, FDX1, has differential expression and modification levels in various tumors, which is associated with cellular function, immune modulation, and disease prognosis. Thus, FDX1-dependent cuproptosis may serve as a brand-new target in future therapeutic approaches against tumors.

One-Carbon and Polyamine Metabolism as Cancer Therapy Targets

Cancer metabolic reprogramming is essential for maintaining cancer cell survival and rapid replication. A common target of this metabolic reprogramming is one-carbon metabolism which is notable for its function in DNA synthesis, protein and DNA methylation, and antioxidant production. Polyamines are a key output of one-carbon metabolism with widespread effects on gene expression and signaling. As a result of these functions, one-carbon and polyamine metabolism have recently drawn a lot of interest for their part in cancer malignancy. Therapeutic inhibitors that target one-carbon and polyamine metabolism have thus been trialed as anticancer medications. The significance and future possibilities of one-carbon and polyamine metabolism as a target in cancer therapy are discussed in this review.

Potential association of eEF1A dimethylation at lysine 55 in the basal area of Helicobacter pylori-eradicated gastric mucosa with the risk of gastric cancer: a retrospective observational study

BackgroundAlthough eradication therapy for chronic Helicobacter pylori (H. pylori) reduces the risk of gastric cancer (GC), its effectiveness is not complete. Therefore, it is also critically important to identifying those patients who remain at high risk after H. pylori eradication therapy. Accumulation of protein methylation is strongly implicated in cancer, and recent study showed that dimethylation of eEF1A lysine 55 (eEF1AK55me2) promotes carcinogenesis in vivo. We aimed to investigate the relationship between eEF1A dimethylation and H. pylori status, efficacy of eradication therapy, and GC risk in H. pylori-eradicated mucosa, and to reveal the potential downstream molecules of eEF1A dimethylation.MethodsRecords of 115 patients (11 H. pylori-negative, 29 H. pylori-positive, 75 post-eradication patients) who underwent upper gastrointestinal endoscopy were retrospectively reviewed. The eEF1A dimethyl level was evaluated in each functional cell type of gastric mucosa by immunofluorescent staining. We also investigated the relationship between eEF1AK55me2 downregulation by CRISPR/Cas9 mediated deletion of Mettl13, which is known as a dimethyltransferase of eEF1AK55me2.ResultsThe level of eEF1A dimethylation significantly increased in the surface and basal areas of H. pylori-positive mucosa compared with the negative mucosa (surface, p = 0.0031; basal, p = 0.0036, respectively). The eEF1A dimethyl-levels in the surface area were significantly reduced by eradication therapy (p = 0.005), but those in the basal area were maintained even after eradication therapy. Multivariate analysis revealed that high dimethylation of eEF1A in the basal area of the mucosa was the independent factor related to GC incidence (odds ratio = 3.6611, 95% confidence interval = 1.0350–12.949, p = 0.0441). We also showed the relationship between eEF1A dimethylation and expressions of reprogramming factors, Oct4 and Nanog, by immunohistochemistry and in vitro genome editing experiments.ConclusionsThe results indicated that H. pylori infection induced eEF1A dimethylation in gastric mucosa. The accumulation of dimethyl-eEF1A in the basal area of the mucosa might contribute to GC risk via regulation of reprograming factors in H. pylori eradicated-gastric mucosa.

SETD2 regulates the methylation of translation elongation factor eEF1A1 in clear cell renal cell carcinoma.

SET domain-containing protein 2 (SETD2) is commonly mutated in renal cell carcinoma. SETD2 methylates histone H3 as well as a growing list of non-histone proteins. Initially, we sought to explore SETD2-dependent changes in lysine methylation of proteins in proximal renal tubule cells. Subsequently, we focused on changes in lysine methylation of the translation elongation factor eEF1A1. To accomplish these objectives, we initially performed a systems-wide analysis of protein lysine-methylation and expression in wild type (WT) and SETD2-knock out (KO) kidney cells and later focused our studies on eEF1A1 as well as the expression of lysine methyltransferases that regulate its lysine methylation. We observed decreased lysine methylation of the translation elongation factor eEF1A1. EEF1AKMT2 and EEF1AKMT3 are known to methylate eEF1A1, and we show here that their expression is dependent on SET-domain function of SETD2. Globally, we observe differential expression of hundreds of proteins in WT versus SETD2-KO cells, including increased expression of many involved in protein translation. Finally, we observe decreased progression free survival and loss of EEF1AKMT2 gene expression in SETD2-mutated tumors predicted to have loss of function of the SET domain. Overall, these data suggest that SETD2-mutated ccRCC, via loss of enzymatic function of the SET domain, displays dysregulation of protein translation as a potentially important component of the transformed phenotype.

Abstract A080: METTL21A inhibits pancreatic ductal adenocarcinoma tumorigenesis through methylation of HSPA1/8

Abstract Background: Our work focuses on the role of METTL21A, a member of the little studied seven β-strand family of candidate lysine methyltransferases (KMTs), and its role in the regulation of pancreatic ductal adenocarcinoma (PDAC) development. Using a meta-analysis of publicly available gene expression datasets, we found that METTL21A is significantly downregulated in PDAC versus normal tissue and the reduced expression predicts poor patient survival. Thus, there are intriguing correlations connecting METTL21A to PDAC pathogenesis; however, to date, there are no data on the role of METTL21A in cancer and an enzymatic function for METTL21A is poorly recognized. Methods: To test the hypothesis that METTL21A plays a role in PDAC initiation and progression, we generated conditional Mettl21a knockout mice and crossed to PDAC models: p48Cre/+ KrasG12D/+ (Kras vs. Kras;Mettl21a) and p48Cre/+ KrasG12D/+ p53fl/fl (Kras;p53 vs. Kras;p53;Mettl21a). Furthermore, we studied the impact of METTL21A depletion on human PDAC cell lines and PDX growth in vivo and in vitro. To identify the substrate of METTL21A enzymatic activity we performed methylation assays followed by mass spectrometry analysis. Results: Histopathological analysis of pancreatic tissues of Kras and Kras;Mettl21a mice at 6 months of age revealed that loss of METTL21A significantly accelerates pancreatic cancer initiation. Additional studies using Kras;p53 and Kras;p53;Mettl21a mice demonstrated that METTL21A depletion accelerates PDAC progression and results in a significantly shorter overall survival. In congruence with those observations, knockdown of METTL21A in human PDAC cell lines leads to the robust increase in cell proliferation, enhances colony formation ability in vitro and xenograft growth in vivo. In contrast, cells rescued with ectopic expression of wildtype METTL21A but not with enzyme-dead mutant METTL21A showed significant growth impairment in vitro and in vivo. Next, our in vivo methylation assays identified HSPA1 and HSPA8 (members of the HSP70 protein family) as substrates of METTL21A in PDAC cells. Our subsequent analysis showed that METTL21a specifically tri-methylates HSPA1 and HSPA8 at lysine 561 (K561me3). Next, to directly evaluate the role of METTL21A mediated methylation of HSPA1/A8 at K561 in regulating cancer phenotype, we knocked out HSPA1/A8 and complemented with expression of wildtype or K561A mutant HSPA1/A8 in PDAC cell lines. We found that only methylation-resistant HSPA1/A8 increased cell proliferation and tumor growth in vivo. Together, these results argue that in PDAC, the principal physiologic activity of METTL21A is generation of HSPA1/A8 K561me3, which suppresses cancer growth. Conclusion: Our research revealed that METTL21A is a novel tumor suppressor that inhibits the initiation and progression of PDAC through methylation of the heat-shock proteins HSPA1/8 substrate-binding domain. Citation Format: Xiaojie Yang, Mohamad Zoabi, Simone Hausmann, Natasha M. Flores, Xiaoyin Lu, Jibo Wu, Ana Morales-Benitez, Or Gozani, Pawel K. Mazur. METTL21A inhibits pancreatic ductal adenocarcinoma tumorigenesis through methylation of HSPA1/8 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A080.

The Role of Hottip Lncrna m6a Modification in AML Genome Organization and Leukemogenesis

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with the dysregulation of HOX genes. MLLr+ AML is characterized by aberrant expression of HOX genes and the HOX cofactor MEIS1. HOTTIP, a HOXA-associated long non-coding RNA (lncRNA), is significantly up-regulated in MLLr+ AML. Aberrant overexpression of Hottip dramatically perturbs hematopoietic stem cell (HSC) self-renewal leading to mouse AML-like disease through maintaining a leukemic profiling. Recent studies show that N6-methyladenosine (m6A), as the most prevalent internal modification in eukaryotic coding and non-coding RNAs, plays important roles in normal and malignant hematopoiesis. There are several potential m6A modified sites in HOTTIP exon regions and 3'UTR region by re-analyzing m6A-methylated RNA immunoprecipitation sequencing (m6A-meRIP-seq) data in leukemia. However, it is still unclear whether HOTTIP m6A modification plays a critical role in AML genome organization and AML leukemogenesis. Our HOTTIP ChIRP-MASS Spectrometry (ChIRP-MS) data shows that HOTTIP directly interacts with m6A methyltransferase METTL3/METTL14 and CTCF/cohesin chromatin organization proteins in MOLM13 AML cells. To investigate whether HOTTIP lncRNA is methylated by METTL3, we perform m6A-eCLIP-seq and METTL3-eCLIP-seq in MOLM13 cells. Our data indicates that there are several m6A modified sites co-bound with METTL3 in HOTTIP exon and 3'UTR regions. Our m6A-eCLIP-qPCR data further confirms that depletion of METTL3 significantly decreases HOTTIP m6A modification levels at HOTTIP RNA m6A core motif GGACU regions (g1, g2 and g3) by comparing shScramble and shMETTL3 cells. It suggests that HOTTIP lncRNA contains specific m6A modified sites methylated by METTL3. Next, to further examine whether and which HOTTIP m6A sites is required for the interaction of HOTTIP RNA with CTCF protein, we have performed in vitro RNA methylation and RNA binding protein assay by synthesizing biotinylated HOTTIP RNA fragments in g1, g2 and g3 regions. Subsequently, these biotin-HOTTIP-g1/g2/g3 RNAs are methylated with METTL3/METTL14 recombinant proteins and then incubated with GST control or GST-CTCF fusion in vitro. Our data shows that m6A modification of HOTTIP-g2 or HOTTIP-g3 RNA fragment enhances its interaction with GST-CTCF protein, suggesting that HOTTIP m6A modification plays a critical role in coordinating with CTCF-mediated genome organization. To investigate the function of HOTTIP m6A modification in AML leukemogenesis, we have generated CRISPR-dCas13-ALKBH5 (dALK) system to specifically demethylate HOTTIP m6A sites (g1, g2 and g3) by co-transfecting gRNA vector and the catalytically inactive Cas13 (dCas13) fused with m6A demethylase AlkB homolog 5 (ALKBH5) vector. Targeting dALK to specific m6A site in HOTTIP lncRNA results in site-specific loss of m6A modification. Although loss of HOTTIP m6A modification never affects the stability and transcription of HOTTIP in dALK clones, loss of HOTTIP m6A modification significantly affects the transcription of leukemic signature genes HOXA9/13 and WNT target (β-catenin and MYC etc) in dALK-g2 or dALK-g3 cells. Additionally, our HOTTIP ChIRP-qPCR shows that loss of HOTTIP m6A modification affects HOTTIP binding at WNT target loci (β-catenin and MYC loci) in dALK-g2 or dALK-g3 cells. Critically, our data find that loss of HOTTIP m6A modification significantly impairs cell proliferation and cell cycle in dALK-g2 or dALK-g3 cells compared with ctrl cells. To further elucidate whether HOTTIP m6A modification is required for AML leukemogenesis in vivo, we xenograft ctrl and dALK clones into NOD-scid IL2Rgammanull (NSG) mice. Our data indicates that mice transplanted with ctrl or dALK-g1 cells dies around 40 days after transplantation, while those receiving dALK-g2 or dALK-g3 cells survives significantly longer up to 55 days. Critically, we also find that ctrl recipient mice have significantly higher human CD45+ cell chimerism in the BM than mice receiving dALK-g2 or dALK-g3 AML cells. Thus, loss of the specific HOTTIP m6A modification site reduces the AML leukemic burden in vivo. In summary, this research will provide a prospective insight for the role of HOTTIP-m6A-CTCT axis in AML genome organization and leukemogenesis.

Tumor cell-derived asymmetric dimethylarginine regulates macrophage functions and polarization

BackgroundAsymmetric dimethylarginine (ADMA), which is significantly elevated in the plasma of cancer patients, is formed via intracellular recycling of methylated proteins and serves as a precursor for resynthesis of arginine. However, the cause of ADMA elevation in cancers and its impact on the regulation of tumor immunity is not known.MethodsThree mouse breast cell lines (normal breast epithelial HC11, breast cancer EMT6 and triple negative breast cancer 4T1) and their equivalent 3D stem cell culture were used to analyze the secretion of ADMA using ELISA and their responses to ADMA. Bone marrow-derived macrophages and/or RAW264.7 cells were used to determine the impact of increased extracellular ADMA on macrophage-tumor interactions. Gene/protein expression was analyzed through RNAseq, qPCR and flow cytometry. Protein functional analyses were conducted via fluorescent imaging (arginine uptake, tumor phagocytosis) and enzymatic assay (arginase activity). Cell viability was measured via MTS assay and/or direct cell counting using Countess III FL system.ResultsFor macrophages, ADMA impaired proliferation and phagocytosis of tumor cells, and even caused death in cultures incubated without arginine. ADMA also led to an unusual macrophage phenotype, with increased expression of arginase, cd163 and cd206 but decreased expression of il10 and dectin-1. In contrast to the severely negative impacts on macrophages, ADMA had relatively minor effects on proliferation and survival of mouse normal epithelial HC11 cells, mouse breast cancer EMT6 and 4T1 cells, but there was increased expression of the mesenchymal markers, vimentin and snail2, and decreased expression of the epithelial marker, mucin-1 in EMT6 cells. When tumor cells were co-cultured ex vivo with tumor antigen in vivo-primed splenocytes, the tumor cells secreted more ADMA and there were alterations in the tumor cell arginine metabolic landscape, including increased expression of genes involved in arginine uptake, metabolism and methylation, and decreased expression of a gene that is responsible for arginine demethylation. Additionally, interferon-gamma, a cytokine involved in immune challenge, increased secretion of ADMA in tumor cells, a process attenuated by an autophagy inhibitor.ConclusionOur results suggest initial immune attack promotes autophagy in tumor cells, which then secrete ADMA to manipulate macrophage polarization favoring tumor tolerance.

TMIC-20. SERINE AND ONE-CARBON METABOLISM ADAPTING GLIOMA CELLS TO LOW GLUTAMINE MICROENVIRONMENT

Abstract Cancer cells optimize nutrient utilization to supply energetic and biosynthetic pathways. This metabolic process also includes redox maintenance and epigenetic regulation through nucleic acid and protein methylation, which enhance tumorigenicity and clinical resistance. However, less is known about how cancer cells exhibit metabolic flexibility to sustain cell growth and survival from low nutrient microenvironment. Here, we find that serine and glycine levels were higher in low-nutrient regions of tumors in glioblastoma multiforme (GBM) patients than they were in other regions. Metabolic and functional studies in malignant glioma cells demonstrated that serine availability and one-carbon metabolism support glioma cell survival following glutamine deprivation. Serine synthesis was mediated through autophagy rather than glycolysis. Gene expression analysis identified upregulation of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) to regulate one-carbon metabolism in malignant glioma cells and GBM patient-derived sphere cells. In clinical samples, MTHFD2 expression was highest in the nutrient-poor areas around “pseudopalisading necrosis.” Genetic and pharmacological suppression of MTHFD2 caused tumor cell death and growth inhibition of glioma cells upon glutamine deprivation as well as autophagy inhibition did. These results may have important implications for serine-dependent one-carbon metabolism for glioma cells to survive glutamine starvation and suggest a new therapeutic strategy for patients with malignant glioma.

CSIG-20. PROTEOGENOMICS PROFILING REVEALS ENRICHED PROTEIN TRANSLATION REGULATORS AS NEW THERAPEUTIC TARGETS IN DIPG

Abstract Diffuse intrinsic pontine glioma (DIPG) is a devastating brain tumor arising in the brainstems of children. Current therapies are ineffective resulting in a median survival rate of less than one year and it is the leading cause of brain tumor-related death in children. A novel mutation in histone H3 protein (H3K27M) was recently identified as a genetic initiation event and affects global K27 trimethylation on histone H3 proteins and DNA methylation. The epigenetic changes caused by H3K27M mutation suggest the existence of an H3K27M-specific transcriptome and proteome. We investigated DIPG tissues at the multi-omics level including total proteome, phosphoproteome, methylproteome and metabolome by mass spectrometry. A total of 30 patient tumours were profiled to identify differentially expressed proteins, differentially phosphorylated proteins, and differentially methylated proteins in tumour tissues compared to normal brains. We identified 2995 proteins that are differentially regulated, suggesting changes in key oncogenic pathways including negative regulation of apoptosis, translation, and metabolic pathways such as methionine salvage and TCA cycle in DIPG. The deregulation of these metabolic pathways due to the differentially expressed proteins in DIPG cells was confirmed by metabolomics studies. Protein phosphorylation and protein methylation profiling of DIPG implicated that translation-related proteins were the most highly modified (post-translationally) proteins in DIPG tissues. Furthermore, protein translation measured by CyTOF showed higher translation rates in DIPG and immortalized astrocytes carrying H3K27M than their WT counterparts. We investigated the functional consequence of knockdown of the highest methylated translation regulator EEF1A1 and its methyltransferase METTL13. ShRNA knockdown of both, EEF1A and METTL13 in DIPG cells significantly reduced the cell growth. Multi-omics analysis of DIPG highlighted regulation of protein translation as a potential therapeutic target.

TMET-09. LOSS OF MAT2A COMPROMISES METHIONINE METABOLISM AND REPRESENTS A VULNERABILITY IN H3K27M MUTANT GLIOMAS

Abstract H3K27-mutant diffuse midline gliomas (DMGs) are defined as grade IV tumors by the World Health Organization. DMGs are inoperable and resistant to chemo/radio therapies. Median survival ranges from 8-11 months, with 2% of patients surviving beyond 5 years. H3K27M mutations lead to global epigenetic and transcriptional reprogramming driven by global loss of negative transcriptional regulator H3K27 trimethylation (H3K27me3). Loss of H3K27me3 is an initiating event in gliomagenesis. This disease lacks appropriate models to predict disease biology and response to treatment. Therefore, we developed a novel syngeneic H3K27M mouse model. An unbiased integrated systems biology approach identified that H3K27M but not isogenic controls relied on the amino acid methionine and the enzyme Methionine Adenosyltransferase 2A (MAT2A). MAT2A is a central regulator of one-carbon metabolism by converting methionine to S-adenosylmethionine (SAM), the universal methyl-donor for protein and nucleotide methylation reactions. In complementary genetic approaches, we applied these findings to patient-derived cell lines with the H3K27M mutation. We hypothesize that MAT2A abrogation, genetic/pharmacological, would alter DMG viability by disrupting the methylome. The current MAT2A sensitivity paradigm is based on Methylthioadenosine Phosphorylase (MTAP) deletion through a synthetic lethal mechanism. We provide a novel mechanism whereby H3K27M cells are sensitive to MAT2A loss, independent of MTAP and through Adenosylmethionine Decarboxylase 1 (AMD1) overexpression disrupting MAT2A regulation. This results in H3K27M cells having lower MAT2A protein levels, conferring a sensitivity by inhibiting residual MAT2A. Genetic/pharmacological aberrations to MAT2A resulted in reduced proliferation. Parallel H3K36me3 ChIP and RNA-sequencing identified loss of oncogenic and developmental transcriptional programs associated with MAT2A loss. In vivo syngeneic and patient-derived xenograft models with both inducible MAT2A knockdown or methionine restricted diets showed extended survival. These results suggest novel interactions between methionine metabolism and the epigenome of H3K27M gliomas and provide evidence that MAT2A, presents exploitable therapeutic vulnerabilities in histone mutant gliomas.

Heavy Methyl SILAC Metabolic Labeling of Human Cell Lines for High-Confidence Identification of R/K-Methylated Peptides by High-Resolution Mass Spectrometry.

Protein methylation is a widespread post-translational modification (PTM) involved in several important biological processes including, but not limited to, RNA splicing, signal transduction, translation, and DNA repair. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered today the most versatile and accurate technique to profile PTMs with high precision and proteome-wide depth; however, the identification of protein methylations by MS is still prone to high false discovery rates. In this chapter, we describe the heavy methyl SILAC metabolic labeling strategy that allows high-confidence identification of in vivo methyl-peptides by MS-based proteomics. We provide a general protocol that covers the steps of heavy methyl labeling of cultured cells, protein sample preparation, LC-MS/MS analysis, and downstream computational analysis of the acquired MS data.

Methylated ZNF582: a therapeutic target in oral cancer

Tweetable abstract Zinc finger proteins control the transcription of downstream genes that are implicated in migration, invasion, cell death and proliferation. More mechanistic research on ZNF582 is needed to ascertain how this protein's methylation regulates the inflammatory pathway in oral cancer.

Bioinformatics analyses of rice calmodulin-lysine N-methyltransferases gene (OsCaM KMT) and characterization of its promoter from Oryza sativa L. sub sp. indica through transgenic approach

• Cloning of OsCaM KMT gene and promoter region from “Swarna”, an indica rice variety. • Presence of OsCAM KMT transcript in meristematic zone of rice plants, with higher expression in root tips suggesting they might be involved in auxin responsiveness. • Promoter analysis of OsCaM KMT::GUS construct depicted strong expression of GUS reporter gene in root tip, lateral roots and young leaf tip region. • Sequencing result indicated single nucleotide difference between the OsCAM KMT CDS from indica and japonica rice variety, resulting in silent mutation. Calmodulin-lysine N-methyltransferases (CaM KMT) belong to a family of protein lysine methyltransferases that catalyzes methylation of non-histone proteins. Lysine present at 116 position in the protein sequence of calmodulin is apparently methylated by CaM KMT in most species, however not characterized in rice. In the present study rice calmodulin-lysine N-methyltransferase ( OsCaM KMT ) gene has been isolated from an indica rice variety ‘Swarna’. Derived amino acid sequences of CaM KMT gene from rice and other plants were used in constructing a phylogenetic tree to show the evolutionary relationship among those species. Motif analysis along with comparative protein modeling was done to find out structural relationships among rice, Arabidopsis, and human CaM KMT. Gene expression analysis revealed differential tissue specific expression of OsCaM KMT . The highest transcript expression was revealed in the root tip followed by flower and new leaf, while, the least expression was found in the old leaf. In silico analysis was performed considering the 2 kilobases (kb) upstream promoter region of OsCaM KMT gene to find out various transcription factor binding sites. Analyses of the putative cis-regulatory elements revealed their involvement in tissue specific and stress dependent responses. For further characterization, the promoter region (2 kb) of OsCaM KMT gene was isolated, cloned, and sequenced. Histochemical GUS detection of transgenic rice depicted strong OsCaM KMT promoter expression in midribs, stomata, and tip of leaf tissues, central cylinder as well as tip parts of the primary root and lateral roots, along with significant expression in pollen grains, ovary, and vascular bundles. OsCaM KMT gene expression was corroborated by the histological GUS detection in OsCaM KMT promoter:: GUS fusion construct. Although several putative microRNAs (miRNAs) has been identified targeting OsCaM KMT coding DNA sequence (CDS) and UTR region, further studies are required to elucidate their tissue-specific regulation.

Multifactorial feature extraction and site prognosis model for protein methylation data.

Integrated studies (multi-omics studies) comprising genetic, proteomic and epigenetic data analyses have become an emerging topic in biomedical research. Protein methylation is a posttranslational modification that plays an essential role in various cellular activities. The prediction of methylation sites (arginine and lysine) is vital to understand the molecular processes of protein methylation. However, current experimental techniques used for methylation site predictions are tedious and expensive. Hence, computational techniques for predicting methylation sites in proteins are necessary. For predicting methylation sites, various computational methods have been proposed in recent years. Most existing methods require structural and evolutionary information for retrieving features, acquiring this information is not always convenient. Thus, we proposed a novel method, called multi-factorial feature extraction and site prognosis model (MufeSPM), for the prediction of protein methylation sites based on information theory features (Renyi, Shannon, Havrda-Charvat and Arimoto entropy), amino acid composition and physicochemical properties acquired from protein methylation data. A random forest algorithm was used to predict methylation sites in protein sequences. This paper also studied the impact of different features and classifiers on arginine and lysine methylation data sets. For the R methylation data set, MufeSPM yielded 82.45%($\pm $ 3.47) accuracy, and for the K methylation data set, it provided an average accuracy of 71.94%($\pm $ 2.12). Additionally, the area under the receiver operating characteristic curve for different classifiers in predicting methylation site was provided. The experimental results signify that MufeSPM performs better than the state-of-the-art predictors.

Artificial optimization of bamboo Ppmar2 transposase and host factors effects on Ppmar2 transposition in yeast.

Mariner-like elements (MLEs) are promising tools for gene cloning, gene expression, and gene tagging. We have characterized two MLE transposons from moso bamboo, Ppmar1 and Ppmar2. Ppmar2, is smaller in size and has higher natural activities, thus making it a more potential genomic tool compared to Ppmar1. Using a two-component system consisting of a transposase expression cassette and a non-autonomous transposon cotransformed in yeast, we investigated the transposition activity of Ppmar2 and created hyperactive transposases. Five out of 19 amino acid mutations in Ppmar2 outperformed the wild-type in terms of catalytic activities, especially with the S347R mutant having 6.7-fold higher transposition activity. Moreover, 36 yeast mutants with single-gene deletion were chosen to screen the effects of the host factors on Ppmar2NA transposition. Compared to the control strain (his3Δ), the mobility of Ppmar2 was greatly increased in 9 mutants and dramatically decreased in 7 mutants. The transposition ability in the efm1Δ mutant was 15-fold higher than in the control, while it was lowered to 1/66 in the rtt10Δ mutant. Transcriptomic analysis exhibited that EFM1 defection led to the significantly impaired DDR2, HSP70 expression and dramatically boosted JEN1 expression, whereas RTT10 defection resulted in significantly suppressed expression of UTP20, RPA190 and RRP5. Protein methylation, chromatin and RNA transcription may affect the Ppmar2NA transposition efficiency in yeast. Overall, the findings provided evidence for transposition regulation and offered an alternative genomic tool for moso bamboo and other plants.

Hypermethylation of RASSF1A gene in pediatric rhabdoid tumor of the kidney and clear cell sarcoma of the kidney.

Among pediatric renal tumors, rhabdoid tumor of the kidney (RTK) and clear cell sarcoma of the kidney (CCSK) are rare and associated with an unfavorable prognosis, while congenital mesoblastic nephroma (CMN) is associated with a good prognosis. Methylation of the Ras association domain-containing protein 1 isoform A (RASSF1A) promoter has been reported to correlate with a poor prognosis in patients with Wilms tumors, while its methylation status is unclear in other types of pediatric renal tumors. DNA methylation of the RASSF1A promoter in several pediatric renal tumors was analyzed with pyrosequencing. In order to clarify the correlation between expression of RASSF1A and DNA methylation of its promoter, the RTK cell line was treated with 5-Aza-2'-deoxycytidine (5-Aza-dC). RASSF1A was overexpressed in the RTK cell line to evaluate its functional effects. Quantitative methylation analysis demonstrated hypermethylation in the RASSF1A promoter region in RTK and CCSK, but not CMN. The 5-Aza-dC treatment induced demethylation of the RASSF1A promoter as well as increased RASSF1A mRNA expression. The transduction of RASSF1A has an effect on the suppression of viability and proliferation of RTK cells. DNA methylation-mediated deficiency of RASSF1A might be involved in the development and aggressiveness of some pediatric renal tumors and correlated with a poor prognosis.

Droplet digital polymerase chain reaction assay for methylated ring finger protein 180 in gastric cancer.

BACKGROUNDGastric cancer (GC) is one of the most prevalent malignant tumors that endangers human health. Early diagnosis is essential for improving the prognosis and survival rate of GC patients. Ring finger protein 180 (RNF180) is involved in the regulation of cell differentiation, proliferation, apoptosis, and tumorigenesis, and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC. Thus, methylated RNF180 can be used as a potential biomarker for GC diagnosis.AIMTo use droplet digital polymerase chain reaction (ddPCR) to quantify the methylation level of the RN180 gene. A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.METHODSThe primer and probe were designed and selected, the conversion time of bisulfite was optimized, the ddPCR system was adjusted by primer concentration, amplification temperature and amplification cycles, and the detection limit of ddPCR was determined.RESULTSThe best conversion time for blood DNA was 2 h 10 min, and that for plasma DNA was 2 h 10 min and 2 h 30 min. The results of ddPCR were better when the amplification temperature was 56 °C and the number of amplification cycles was 50. Primer concentrations showed little effect on the assay outcome. Therefore, the primer concentration could be adjusted according to the reaction system and DNA input. The assay required at least 0.1 ng of input DNA.CONCLUSIONIn summary, a ddPCR assay was established to detect methylated RNF180, which is expected to be a new diagnostic biomarker for GC.

A methylation-dependent checkpoint by SETD7 promotes myocardial ischemic injury in mice and men

Abstract Background Despite appropriate revascularization strategies, a significant number of patients with myocardial infarction (MI) develop ischemic heart failure suggesting that breakthrough therapies are yet to be approved in this setting. Methylation of non-histone proteins is emerging as a central regulatory mechanism in health and disease. The methyltransferase SETD7 has been shown to methylate and alter the function of a variety of proteins in vitro, however, its function in the heart is poorly understood. Purpose To determine the role of SETD7 in myocardial ischemic injury. Methods Neonatal rat ventricular myocytes (NRVM) were exposed to normal glucose levels or glucose deprivation (GD) for 15 h, in the presence of the selective SETD7 inhibitor (R)-PFI-2 or its inactive enantiomer (S)-PFI-2. Western blot and real-time PCR were employed to investigate the effects of energy stress on SETD7 and the Hippo pathway, while apoptosis and oxidative stress were assessed by Caspase-3 activity assay and mitoSOX staining. YAP transcriptional activity was assessed by chromatin immunoprecipitation assay (ChIP) while its localization and methylation were examined by confocal microscopy and immunoblotting, respectively. SETD7 knockout (SETD7−/−) mice and wild-type (WT) littermates underwent myocardial ischemia-reperfusion (I/R) injury (1h coronary ligation /24 h of reperfusion) followed by assessment of cardiac function by echocardiography. Left ventricular (LV) myocardial samples were collected from I/R mice and patients with ischemic cardiomyopathy (ICM), and isolated cardiomyocytes were treated with (R)-PFI-2. Finally, SETD7 expression was also assessed in peripheral blood mononuclear cells (PBMCs) from patients with ST-elevation MI (STEMI). Results SETD7 was activated upon energy deprivation in cultured NRVMs and methylated YAP, leading to its cytosolic retention and impaired transcription of antioxidant genes MnSOD and CAT. Pharmacological inhibition of SETD7 by (R)-PFI-2 restored YAP nuclear localization thus preventing mitochondrial reactive oxygen species (mtROS) and apoptosis. SETD7 deletion in mice attenuated I/R injury, mtROS and LV dysfunction by restoring YAP-dependent transcriptional programs. SETD7/YAP dysregulation was also observed in LV specimens from ICM patients. Moreover, in cardiomyocytes isolated from I/R mice and ICM patients, (R)-PFI-2 restored YAP nuclear localization, prevented mtROS accumulation while improving myofibrillar protein contractility and Ca2+ sensitivity. Finally, SETD7 was upregulated in PBMCs from STEMI patients and negatively correlated with the expression of MnSOD and CAT. Conclusions SETD7-dependent methylation of YAP is an important mechanism underpinning myocardial oxidative stress and apoptosis during ischemia. Pharmacological modulation of SETD7 by (R)-PFI-2 may represent a potential therapeutic approach to prevent myocardial ischemic damage through modulation of the Hippo pathway. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Zurich