Methylation Of Non-histone Proteins Research Articles

Chromosome Gene, Hsl7 Gene in Vitro Studies

Chromosome gene can expression many functions in human body. Eukaryotic cells are presence in yeast and human, in order to more understanding human gene, we use yeast as a model to research. The ORF (open reading frame) position of the Hsl7 gene is in yeast Saccharomyces YBR133C between 501804bp~504287bp of Chromosome II, with a total length of 2483bp, and the protein size is 95 kDa. Hsl7 gene is a methyltransferase. Protein-arginine N-methyltransferase involved in protein catabolism and regulation of the G2/M transition of mitosis; localizes to the septin collar of the bud neck, and the outer plaque of the spindle pole body in the G1 phase of the cell cycle (Saccharomyces Genome Database), Hsl7 (histone synthetic lethal 7) represent protein methylation, protein methylation is a type of post-translational modification that involves the addition of methyl groups to proteins, Protein methylation, including histone methylation and non-histone protein methylation, is one of the most important forms of posttranscriptional and epigenetic modifications.

Role of methyltransferase Set7 in obesity-related endothelial activation: functions beyond histone methylation

Abstract Introduction Obesity is associated with environmental changes such as hyperglycemia, hyperinsulinemia, inflammation, and elevated free fatty acids, all of which compromise endothelial function1,2. We have previously reported that in individuals with type 2 diabetes, lysine methyltransferase Set7 is involved in hyperglycemia-induced endothelial damage through monomethylation of histone 3 at lysine 4 (H3K4me1)3. Apart from such histone-driven epigenetic modulation, Set7-induced methylation of non-histone proteins is emerging as a novel mechanism underlying the (dys)regulation of many biological processes4. Purpose To investigate the role of Set7-related endothelial damage in cells exposed to different obesity-associated stimuli and to determine its impact on the protein methylome in these settings. Methods Human aortic endothelial cells (HAECs) were exposed to high concentrations of glucose, insulin, TNFa, or fatty acids (palmitate or oleate). Set7 overexpression, silencing, and inhibition studies were also performed in these cells. Gene and protein expression were assessed by RT-PCR and immunoblotting respectively, whereas production of the reactive oxygen species (ROS) was measured by electron spin resonance spectroscopy. Specific antibodies were used for the enrichment of lysine mono-, di-, and tri-methylated proteins and LC-MS/MS proteomic analysis was carried out for their identification. Results Each obesity model in HAECs increased the expression of nearly all the inflammatory markers tested and, except for TNFa, they also increased the expression of Set7. Significant changes in the global levels of H3K4me1, as well as of methylated proteins were also observed in these cells. Studies overexpressing a mutant form of Set7, which is lacking the SET domain, revealed that Set7 methyltransferase activity is required to define the protein methylome in HAECs. Similar results were obtained after Set7 silencing and inhibition. Despite no differences were observed in the production of ROS when modulating Set7 expression in HAECs, overexpression of an active form of Set7 and its silencing/inhibition resulted in a consistent up- and down-regulation, respectively, of several inflammatory mediators and adhesion molecules. Finally, proteomics analysis identified, for the first time, the protein methylome of endothelial cells, comprising almost 2,700 methylated proteins involved in essential biological processes such as RNA metabolism and cytoskeleton. Conclusions Our results indicate that Set7 may contribute to inflammatory changes in response to obesogenic insults. Moreover, our findings uncover a dual role of methyltransferase Set7 in governing the methylation of both histone and non-histone proteins in endothelial cells. These preliminary findings open a new field of research aiming to further elucidate the molecular mechanisms underlying the modulation of the protein methylome in the endothelium and its role in endothelial damage.

PRMT6 methylation of STAT3 regulates tumor metastasis in breast cancer

Overcoming distant metastasis stands as a paramount challenge in enhancing the outcomes of breast cancer treatments. Thus, delving deeper into comprehending the intricate mechanisms underlying breast cancer metastasis becomes imperative, offering potential avenues for pioneering therapeutic approaches. PRMT6, an arginine N-methyltransferase, possesses the ability to methylate both histone and non-histone proteins. It has been reported that methylation of non-histone proteins impacts their cellular localization, stability, and activation, consequently influencing tumor progression. However, the extent to which PRMT6-mediated non-histone protein methylation influences cancer cell metastasis, particularly in the context of breast cancer, remains elusive. In this study, we established that PRMT6 exerted a positive regulatory influence on breast cancer metastasis through both in vivo and in vitro experiments. Mechanistically, we innovatively revealed that PRMT6 asymmetrically di-methylated STAT3 at arginine 729 (STAT3 R729me2a). This modification proved indispensable for STAT3’s membrane localization, its interaction with JAK2, STAT3 Y705 phosphorylation, and PRMT6-driven cancer cell metastasis. From a clinical perspective, we unearthed the promising potential of STAT3 R729me2a as a robust prognostic marker for predicting the overall survival time of breast cancer patients. In terms of therapeutic intervention, we demonstrated the significant capability of the PRMT6 inhibitor, EPZ020411, to curtail breast cancer metastasis both in vivo and in vitro. In sum, our study unveils the pivotal biological role of PRMT6-mediated STAT3 R729me2a in breast cancer metastasis and underscores the prospective utility of PRMT6 inhibitors as effective therapeutic strategies against STAT3-driven metastatic breast cancer.

Non-histone protein methylation in Trypanosoma cruzi epimastigotes.

Post-translational methylation of proteins, which occurs in arginines and lysines, modulates several biological processes at different levels of cell signaling. Recently, methylation has been demonstrated in the regulation beyond histones, for example, in the dynamics of protein-protein and protein-nucleic acid interactions. However, the presence and role of non-histone methylation in Trypanosoma cruzi, the etiologic agent of Chagas disease, has not yet been elucidated. Here, we applied mass spectrometry-based-proteomics (LC-MS/MS) to profile the methylproteome of T. cruzi epimastigotes, describing a total of 1252 methyl sites in 824 proteins. Functional enrichment and protein-protein interaction analysis show that protein methylation impacts important biological processes of the parasite, such as translation, RNA and DNA binding, amino acid, and carbohydrate metabolism. In addition, 171 of the methylated proteins were previously reported to bear phosphorylation sites in T. cruzi, including flagellar proteins and RNA binding proteins, indicating that there may be an interplay between these different modifications in non-histone proteins. Our results show that a broad spectrum of functions is affected by methylation in T. cruzi, indicating its potential to impact important processes in the biology of the parasite and other trypanosomes.

Emerging Role of SMYD Family of Proteins in Human Tumorigenesis

Protein lysine methylation is a post-translational modification (PTM) that promotes protein complex formation to regulate DNA replication, gene expression, and repair mechanisms. The Su(Var)3–9, Enhancer-of-zeste and Trithorax (SET) and Myeloid, Nervy, and DEAF-1 (MYND) domain-containing proteins SMYD are lysine methyltransferases that catalyze the methylation of various histone and non-histone proteins. There are five members of this SMYD family, and all of these have conserved SET and MYND domains. The SET domain is divided into two segments by the MYND domain (the S-sequence and a core SET domain). SMYD Family performs a key role in numerous biological functions, including growth, development, apoptosis, and proliferation. SMYD family members are associated with skeletal and cardiac muscle physiology and pathology. Several studies have shown that aberrant lysine methylation plays a significant role in oncogenesis. Recently, the SMYD family has gained importance for its role in various mechanisms involved in cancer development and progressions, such as methylation and modification of tumor suppressor proteins (p53 and pRb), transcriptional factors (STAT3, NF-κB), nuclear proteins (PARP1), chaperons (Hsp90), protein kinases (MAPK, ERK), and cell cycle regulatory proteins (CDKN2). SMYD family proteins drive oncogenesis, lead the way to metastasis, and develop chemoresistance, allowing cancer cells to grow, invade the neighboring tissues, and resist therapeutics. In this review, we summarize SMYD family members' role in different cancers by focusing on their histone and non-histone methylation targets and illustrating the mechanism of SMYD family-mediated oncogenesis.

Abstract 3089: XNW5004: a novel EZH2 inhibitor efficacious in multiple cancer xenograft models as a single agent and in combination studies

Abstract Enhancer of zeste homolog 2 (EZH2) is the histone methyltransferase component of polycomb repressive complex 2 (PRC2) that can regulate gene expression by H3K27 trimethylation (H3K27Me3). EZH2 also mediates PRC2-dependent non-histone protein methylation or directly methylates non-histone proteins to regulate gene expression independent of PRC2. EZH2 plays an important role in regulation of cell cycle, autophagy, apoptosis and cell senescence. Mutated or overexpressed EZH2 associates with cancer initiation, progression, drug resistance and immunity regulation in cancer. XNW5004 is a rationally designed selective EZH2 inhibitor which potently inhibits both wild type and mutant EZH2. XNW5004 significantly suppresses level of H3K27Me3, arrests cell cycle in G1/S phase and promotes apoptosis in tumor. XNW5004 exhibits strong antitumor activity in EZH2-mutant lymphoma xenograft models, such as Karpas422 (Y641N), Pfeiffer (A677G) and SU-DHL-10 (Y641F). Since inhibition of EZH2 synthetically lethal in SWI/SNF subunits mutated cancers, XNW5004 exhibits dose-dependent efficacy in solid tumor models such as G401 (SMARCB1 deficient) and COV434 (SMARCA2/SMARCA4 deficient) cancer cell lines and xenografts. In prostate cancer cells, EZH2 can bind to the promoter of androgen receptor (AR) gene and activate AR expression. In PARPi-treated cancer cells, inhibition of PARP (poly-ADP-ribose polymerase) diminishes PARylation of EZH2 to result in reactivation of EZH2 and drug resistance to PARPi. In tumor microenvironment, inhibition of EZH2 promotes differentiation of T cells and NK cells, enhances antigen presentation ability of dendritic cells, and increases cytokine/chemokine secretion to recruit T cells into tumor tissue. Based on the above mechanisms, XNW5004 could potentially improve efficacy of ARi, PARPi and anti-PD-1. We have verified the synergistic effects of those combinations in multiple solid tumor xenograft models. In pharmacokinetic studies, XNW5004 has excellent oral bioavailability and exposure. XNW5004 shows good toxicological profile as well, has a wide therapeutic window in rats and dogs, and the risk of clinical safety was controllable. In summary, XNW5004 is a potent EZH2 inhibitor with promising efficacy, pharmacokinetic and safety properties. XNW5004 is expected to be a potent therapeutic candidate in cancer treatment and is now in Phase II clinical trial for both liquid and solid tumors. Citation Format: Yonghan Hu, Wei Peng, Zhe Zhang, Zhenwei Wu, Xi Chen, Yan Huang, Xin Li, Cungang Liu, Xiaojun Liu, Qifeng Shi, Xufang Wang, Wengui Wang, Haiyang Wei, Yuchuan Wu, Jun Xian, Linfeng Xu, Lina Zhang, Jinfeng Zhao, Meijie Le, Jing Qiang. XNW5004: a novel EZH2 inhibitor efficacious in multiple cancer xenograft models as a single agent and in combination studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3089.

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.

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

Histone modification and histone modification-targeted anti-cancer drugs in breast cancer: Fundamentals and beyond.

Dysregulated epigenetic enzymes and resultant abnormal epigenetic modifications (EMs) have been suggested to be closely related to tumor occurrence and progression. Histone modifications (HMs) can assist in maintaining genome stability, DNA repair, transcription, and chromatin modulation within breast cancer (BC) cells. In addition, HMs are reversible, dynamic processes involving the associations of different enzymes with molecular compounds. Abnormal HMs (e.g. histone methylation and histone acetylation) have been identified to be tightly related to BC occurrence and development, even though their underlying mechanisms remain largely unclear. EMs are reversible, and as a result, epigenetic enzymes have aroused wide attention as anti-tumor therapeutic targets. At present, treatments to restore aberrant EMs within BC cells have entered preclinical or clinical trials. In addition, no existing studies have comprehensively analyzed aberrant HMs within BC cells; in addition, HM-targeting BC treatments remain to be further investigated. Histone and non-histone protein methylation is becoming an attractive anti-tumor epigenetic therapeutic target; such methylation-related enzyme inhibitors are under development at present. Consequently, the present work focuses on summarizing relevant studies on HMs related to BC and the possible mechanisms associated with abnormal HMs. Additionally, we also aim to analyze existing therapeutic agents together with those drugs approved and tested through pre-clinical and clinical trials, to assess their roles in HMs. Moreover, epi-drugs that target HMT inhibitors and HDAC inhibitors should be tested in preclinical and clinical studies for the treatment of BC. Epi-drugs that target histone methylation (HMT inhibitors) and histone acetylation (HDAC inhibitors) have now entered clinical trials or are approved by the US Food and Drug Administration (FDA). Therefore, the review covers the difficulties in applying HM-targeting treatments in clinics and proposes feasible approaches for overcoming such difficulties and promoting their use in treating BC cases.

Role of EZH2 in adipogenesis and obesity: Current state of the art and implications - A review.

Obesity is characterized by excessive accumulation of adiposity and has been implicated in a strong predisposition to metabolic disorders and cancer, constituting one of the major public health issues worldwide. The formation of new mature adipocytes through differentiation of progenitor or precursor cells during adipogenesis can lead to the expansion of adipose tissue. Recent studies have revealed that the intrinsic risk of obesity arises not only through genetic variants but also through epigenetic predisposition. Enhancer of zeste homolog 2 (EZH2) is an enzymatic catalytic component of polycomb repressive complex 2 that acts as an epigenetic modulator in the regulation of gene expression. EZH2 can modulate the expression of its target genes by the trimethylation of Lys-27 in histone 3 or methylation of non-histone proteins. Emerging evidence has shown the important role played by EZH2 in adipogenesis and obesity. This review provides the latest knowledge about the involvement of EZH2 in the process of adipogenesis and obesity involving adipocyte differentiation, extract key concepts, and highlight open questions toward a better understanding of EZH2 function and the molecular mechanisms underlying obesity.

The Epigenetic Regulation of Nonhistone Proteins by SETD7: New Targets in Cancer.

Epigenetic modifications are essential mechanism by which to ensure cell homeostasis. One such modification is lysine methylation of nonhistone proteins by SETD7, a mono-methyltransferase containing SET domains. SETD7 methylates over 30 proteins and is thus involved in various classical pathways. As such, SETD7 has been implicated in both the basic functions of normal tissues but also in several pathologies, such as cancers. In this review, we summarize the current knowledge of SETD7 substrates, especially transcriptional-related proteins and enzymes, and their putative roles upon SETD7-mediated methylation. We focus on the role of SETD7 in cancers, and speculate on the possible points of intervention and areas for future research.

Methylation of the Hippo effector YAP by the methyltransferase SETD7 drives myocardial ischaemic injury: a translational study.

Methylation of non-histone proteins is emerging as a central regulatory mechanism in health and disease. The methyltransferase SETD7 has shown to methylate and alter the function of a variety of proteins in vitro; however, its function in the heart is poorly understood. The present study investigates the role of SETD7 in myocardial ischaemic injury. Experiments were performed in neonatal rat ventricular myocytes (NRVMs), SETD7 knockout mice (SETD7-/-) undergoing myocardial ischaemia/reperfusion (I/R) injury, left ventricular (LV) myocardial samples from patients with ischaemic cardiomyopathy (ICM), and peripheral blood mononuclear cells (PBMCs) from patients with ST-elevation MI (STEMI). We show that SETD7 is activated upon energy deprivation in cultured NRVMs and methylates the Hippo pathway effector YAP, leading to its cytosolic retention and impaired transcription of antioxidant genes manganese superoxide dismutase (MnSOD) and catalase (CAT). Such impairment of antioxidant defence was associated with mitochondrial reactive oxygen species (mtROS), organelle swelling, and apoptosis. Selective pharmacological inhibition of SETD7 by (R)-PFI-2 restored YAP nuclear localization, thus preventing mtROS, mitochondrial damage, and apoptosis in NRVMs. In mice, genetic deletion of SETD7 attenuated myocardial I/R injury, mtROS, and LV dysfunction by restoring YAP-dependent transcription of MnSOD and CAT. Moreover, in cardiomyocytes isolated from I/R mice and ICM patients, (R)-PFI-2 prevented mtROS accumulation, while improving Ca2+-activated tension. Finally, SETD7 was up-regulated in PBMCs from STEMI patients and negatively correlated with MnSOD and CAT. We show a methylation-dependent checkpoint regulating oxidative stress during myocardial ischaemia. SETD7 inhibition may represent a valid therapeutic strategy in this setting.

Methyltransferase SET domain family and its relationship with cardiovascular development and diseases.

Abnormal epigenetic modification is closely related to the occurrence and development of cardiovascular diseases. The SET domain (SETD) family is an important epigenetic modifying enzyme containing SETD. They mainly affect gene expression by methylating H3K4, H3K9, H3K36 and H4K20. Additionally, the SETD family catalyzes the methylation of non-histone proteins, thereby affects the signal transduction of signal transduction and activator of transcription (STAT) 1, Wnt/β-catenin, hypoxia-inducible factor (HIF)-1α and Hippo/YAP pathways. The SETD family has the following regulatory effects on cardiovascular development and diseases: regulating coronary artery formation and cardiac development; protecting cardiac tissue from ischemia reperfusion injury; regulating inflammation, oxidative stress and apoptosis in cardiovascular complications of diabetes; participating in the formation of pulmonary hypertension; regulating thrombosis, cardiac hypertrophy and arrhythmia. This article summarizes the basic structures, expression regulation mechanisms and the role of existing SETD family members in cardiovascular development and diseases, in order to provide a basis for understanding the molecular mechanism of cardiovascular disease and exploring the therapeutic targets.

EHMT1/EHMT2 in EMT, cancer stemness and drug resistance: emerging evidence and mechanisms.

Metastasis, therapy failure and tumour recurrence are major clinical challenges in cancer. The interplay between tumour-initiating cells (TICs) and epithelial-mesenchymal transition (EMT) drives tumour progression and spread. Recent advances have highlighted the involvement of epigenetic deregulation in these processes. The euchromatin histone lysine methyltransferase 1 (EHMT1) and euchromatin histone lysine methyltransferase 2 (EHMT2) that primarily mediate histone 3 lysine 9 di-methylation (H3K9me2), as well as methylation of non-histone proteins, are now recognised to be aberrantly expressed in many cancers. Their deregulated expression is associated with EMT, cellular plasticity and therapy resistance. In this review, we summarise evidence of their myriad roles in cancer metastasis, stemness and drug resistance. We discuss cancer-type specific molecular targets, context-dependent mechanisms and future directions of research in targeting EHMT1/EHMT2 for the treatment of cancer.

Abstract 10477: Targeting the Methyltransferase Setd7 Prevents Myocardial Ischemic Injury: A Translational Study

Introduction: 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 shown to methylate and alter the function of a variety of proteins in vitro, however its function in the heart is poorly understood. Hypothesis: In the present study we sought to determine the role of SETD7 in myocardial ischemic injury. Methods: Experiments were performed in neonatal rat ventricular myocytes (NRVM), SETD7 knockout mice (SETD7-/-) undergoing myocardial ischemia/reperfusion (I/R) injury, rats undergoing permanent left anterior descending (LAD) artery ligation, myocardial samples from patients with and without ischemic cardiomyopathy (ICM) as well as peripheral blood mononuclear cells from patients with ST elevation MI (STEMI) and age-matched healthy controls. Results: We show that SETD7 is activated upon energy deprivation in cultured NRVMs and methylates the Hippo pathway effector YAP, leading to its cytosolic retention and impaired transcription of antioxidant genes. 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 rats with permanent LAD ligation and LV specimens from ICM patients. Of note, (R)-PFI-2 treatment prevented titin oxidation and myofilament stiffness in cardiomyocytes isolated from I/R mice and patients with ICM. Finally, SETD7 was upregulated in STEMI patients and its expression negatively correlated with antioxidant genes. Conclusions: In the present study we show that SETD7-dependent methylation of YAP is required for its inactivation, thus leading to myocyte oxidative stress and apoptosis. Therefore, we identified SETD7 as a valid and druggable therapeutic target to protect the heart during ischemia.

Targeting the methyltransferase setd7 prevents myocardial ischemic injury: a translational study

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 shown to methylate and alter the function of a variety of proteins in vitro, however its function in the heart is poorly understood. Purpose In the present study we sought to determine the role of SETD7 in myocardial ischemic injury. Methods Neonatal rat ventricular myocytes (NRVM) were exposed to 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 mitochondrial swelling. YAP activity was assessed through chromatin immunoprecipitation assay (ChIP), its localization was examined by confocal microscopy while mono-methylation was assessed by immunoblotting. Expression of YAP-dependent antioxidant genes was assessed by western blot. SETD7 knockout (SETD7−/−) mice and wild-type (WT) littermates underwent ischemia/reperfusion (I/R) injury. Rats underwent permanent ligation of left anterior descending coronary artery (MI). Left ventricular (LV) myocardial samples were collected from mice undergoing I/R injury and patients with ischemic cardiomyopathy (ICM) and treated ex-vivo with (R)-PFI-2. SETD7 and antioxidant genes expression was assessed in peripheral blood mononuclear cells (PBMCs) from patients with ST-elevation MI (STEMI). Results We show that SETD7 is activated upon energy deprivation in cultured NRVMs and methylates the Hippo pathway effector YAP, leading to its cytosolic retention and impaired transcription of antioxidant genes. 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 rats with MI and LV specimens from ICM patients. Of note, (R)-PFI-2 treatment prevented titin oxidation and myofilament stiffness in cardiomyocytes isolated from I/R mice and patients with ICM. Finally, SETD7 was upregulated in STEMI patients and its expression negatively correlated with antioxidant genes. Conclusions Targeting SETD7 may represent a valid therapeutic strategy to protect the heart during ischemia. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Zurich

Searching for methyllysine-binding aromatic cages

Methylation of lysine residues plays crucial roles in a wide variety of cell signaling processes. While the biological importance of recognition of methylated histones by reader domains in the cell nucleus is well established, the processes associated with methylation of non-histone proteins, particularly in the cytoplasm of the cell, are not well understood. Here, we describe a search for potential methyllysine readers using a rapid structural motif-mining algorithm Erebus, the PDB database, and knowledge of the methyllysine binding mechanisms.

The lysine methyltransferase SETD2 is a dynamically expressed regulator of early neural crest development

SETD2 is a histone H3 lysine 36 (H3K36) tri-methylase that is upregulated in response to neural crest induction. Because the H3K36 di-methylase NSD3 and cytoplasmic non-histone protein methylation are necessary for neural crest development, we investigated the expression and requirement for SETD2 in the neural crest. SetD2 is expressed throughout the chick blastoderm beginning at gastrulation. Subsequently, SetD2 mRNA becomes restricted to the neural plate, where it is strongly and dynamically expressed as neural tissue is regionalized and cell fate decisions are made. This includes expression in premigratory neural crest cells, which is downregulated prior to migration. Likely due to the early onset of its expression, SETD2 morpholino knockdown does not significantly alter premigratory Sox10 expression or neural crest migration; however, both are disrupted by a methyltransferase mutant SETD2 construct. These results suggest that SETD2 activity is essential for early neural crest development, further demonstrating that lysine methylation is an important mechanism regulating the neural crest.

Long noncoding RNA-dependent methylation of nonhistone proteins.

In the last decade, an intriguing new paradigm of regulation has emerged in which some transcripts longer than 200 nucleotides and no coding potential, long noncoding RNA (lncRNAs), exhibit the capability to control posttranslational modifications of nonhistone proteins in both invertebrates and vertebrates. The extent of such a regulation is still largely unknown. We performed a systematic review to identify and evaluate the potential impact of lncRNA-dependent methylation of nonhistone proteins. Collectively, these lncRNAs primarily act as scaffolds upon which methyltransferases (MTases) and targets are brought in proximity. In this manner, the N-MTase activity of EZH2, protein arginine-MTase 1/4/5, and SMYD2 is exploited to modulate the stability or the compartmentalization of several nonhistone proteins with roles in cell signaling, gene expression, and RNA processing. Moreover, these lncRNAs can indirectly affect the methylation of nonhistone proteins by transcriptional or posttranscriptional regulation of MTases. Strikingly, the lncRNAs/MTases/nonhistone proteins networking seem to be relevant to carcinogenesis and neurological disorders. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

PRMT5 promotes colorectal cancer growth by interaction with MCM7.

Protein arginine methyltransferase 5 (PRMT5) is a type of methyltransferase enzyme that can catalyse arginine methylation of histones and non‐histone proteins. Accumulating evidence indicates that PRMT5 promotes cancer development and progression. However, its function in colorectal cancer (CRC) is poorly understood. In this study, we revealed the oncogenic roles of PRMT5 in CRC. We found that PRMT5 promoted CRC cell proliferation, migration and invasion in vitro and in vivo. We identified minichromosome maintenance‐7 (MCM7) as the direct PRMT5‐binding partner. A co‐immunoprecipitation (co‐IP) assay indicated that PRMT5 physically interacted with MCM7 and that the direct binding domain was located between residues 1‐248 in MCM7. In addition, our results from analysis of 99 CRC tissues and 77 adjacent non‐cancerous tissues indicated that the PRMT5 and MCM7 expression levels were significantly higher in CRC tissues than in control tissues, which was further confirmed by bioinformatic analysis using TCGA and GEO datasets. We also found that MCM7 promoted CRC cell proliferation, migration and invasion in vitro. Furthermore, we observed that increased PRMT5 expression predicted unfavourable patient survival in CRC patients and in the subgroup of patients with a tumour size of ≤5 cm. These data suggested that PRMT5 and MCM7 might be novel potential targets for the treatment of CRC.