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Isoprenyl cysteine carboxyl methyltransferase (ICMT) catalyzes the last step of the prenylation pathway. Previously, we found that high ICMT levels enhance tumorigenesis in vivo and that its expression is repressed by the p53 tumor suppressor. Based on evidence suggesting that some ICMT substrates affect invasive traits, we wondered if this enzyme may promote metastasis. In this work, we found that ICMT overexpression enhanced lung metastasis in vivo. Accordingly, ICMT overexpression also promoted cellular functions associated with aggressive phenotypes such as migration and invasion in vitro. Considering that some ICMT substrates are involved in the regulation of actin cytoskeleton, we hypothesized that actin-rich structures, associated with invasion and metastasis, may be affected. Our findings revealed that ICMT enhanced the formation of invadopodia. Additionally, by analyzing cancer patient databases, we found that ICMT is overexpressed in several tumor types. Furthermore, the concurrent expression of ICMT and CTTN, which encodes a crucial component of invadopodia, showed a significant correlation with clinical outcome. In summary, our work identifies ICMT overexpression as a relevant alteration in human cancer that promotes the development of metastatic tumors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The HEMK2 protein methyltransferase has been described as glutamine methyltransferase catalyzing ERF1-Q185me1 and lysine methyltransferase catalyzing H4K12me1. Methylation of two distinct target residues is unique for this class of enzymes. To understand the specific catalytic adaptations of HEMK2 allowing it to master this chemically challenging task, we conducted a detailed investigation of the substrate sequence specificities of HEMK2 for Q- and K-methylation. Our data show that HEMK2 prefers methylation of Q over K at peptide and protein level. Moreover, the ERF1 sequence is strongly preferred as substrate over the H4K12 sequence. With peptide SPOT array methylation experiments, we show that Q-methylation preferentially occurs in a G-Q-X 3 -R context, while K-methylation prefers S/T at the first position of the motif. Based on this, we identified novel HEMK2 K-methylation peptide substrates with sequences taken from human proteins which are methylated with high activity. Since H4K12 methylation by HEMK2 was very low, other protein lysine methyltransferases were examined for their ability to methylate the H4K12 site. We show that SETD6 has a high H4K12me1 methylation activity (about 1000-times stronger than HEMK2) and this enzyme is mainly responsible for H4K12me1 in DU145 prostate cancer cells., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Adult skeletal muscle is composed of thousands of fibers (also called myofibers) that contract thus allowing voluntary movements. Following an injury, muscle stem cells, surrounding the myofibers, activate, proliferate, and differentiate to form de novo myofibers. These steps constitute a process called adult (or regenerative) myogenesis. This process is possible thanks to various transcription factors sequentially expressed and regulated by epigenetic factors that modulate the chromatin and therefore lead to the regulation of gene expression. Gene expression changes consequently affect the fate of muscle stem cells. Histone Lysine Methyltransferases methylate some histones involved in the repression of gene expression. We describe here the role of SETDB1 during adult myogenesis, notably its role during muscle stem cell differentiation., (© 2023 médecine/sciences – Inserm.)

Histidine (His) residues are methylated in various proteins, but their roles and regulation mechanisms remain unknown. Here, we show that carnosine N-methyltransferase 1 (CARNMT1), a known His methyltransferase of dipeptide carnosine (βAla-His), is a major His N1-position-specific methyltransferase. We found that 52 His sites in 20 proteins underwent CARNMT1-mediated methylation. The consensus methylation site for CARNMT1 was identified as Cx(F/Y)xH, a C3H zinc finger (C3H ZF) motif. CARNMT1-deficient and catalytically inactive mutant mice showed embryonic lethality. Among the CARNMT1 target C3H ZF proteins, RNA degradation mediated by Roquin and tristetraprolin (TTP) was affected by CARNMT1 and its enzymatic activity. Furthermore, the recognition of the 3' splice site of the CARNMT1 target C3H ZF protein U2AF1 was perturbed, and pre-mRNA alternative splicing (AS) was affected by CARNMT1 deficiency. These findings indicate that CARNMT1-mediated protein His methylation, which is essential for embryogenesis, plays roles in diverse aspects of RNA metabolism by targeting C3H ZF-type RNA-binding proteins and modulating their functions, including pre-mRNA AS and mRNA degradation regulation., (© 2023 Shimazu et al.; Published by Cold Spring Harbor Laboratory Press.)

Protein posttranslation modifications (PTMs) are a critical regulatory mechanism of protein function. Protein α-N-terminal (Nα) methylation is a conserved PTM across prokaryotes and eukaryotes. Studies of the Nα methyltransferases responsible for Να methylation and their substrate proteins have shown that the PTM involves diverse biological processes, including protein synthesis and degradation, cell division, DNA damage response, and transcription regulation. This review provides an overview of the progress toward the regulatory function of Να methyltransferases and their substrate landscape. More than 200 proteins in humans and 45 in yeast are potential substrates for protein Nα methylation based on the canonical recognition motif, XP[KR]. Based on recent evidence for a less stringent motif requirement, the number of substrates might be increased, but further validation is needed to solidify this concept. A comparison of the motif in substrate orthologs in selected eukaryotic species indicates intriguing gain and loss of the motif across the evolutionary landscape. We discuss the state of knowledge in the field that has provided insights into the regulation of protein Να methyltransferases and their role in cellular physiology and disease. We also outline the current research tools that are key to understanding Να methylation. Finally, challenges are identified and discussed that would aid in unlocking a system-level view of the roles of Να methylation in diverse cellular pathways., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lysine methylation is an abundant posttranslational modification, which has been most intensively studied in the context of histone proteins, where it represents an important epigenetic mark. Lysine methylation of histone proteins is primarily catalyzed by SET-domain methyltransferases (MTases). However, it has recently become evident that also another MTase family, the so-called seven-β-strand (7BS) MTases, often denoted METTLs (methyltransferase-like), contains several lysine (K)-specific MTases (KMTs). These enzymes catalyze the attachment of up to three methyl groups to lysine residues in specific substrate proteins, using S-adenosylmethionine (AdoMet) as methyl donor. About a decade ago, only a single human 7BS KMT was known, namely the histone-specific DOT1L, but 15 additional 7BS KMTs have now been discovered and characterized. These KMTs typically target a single nonhistone substrate that, in most cases, belongs to one of the following three protein groups: components of the cellular protein synthesis machinery, mitochondrial proteins, and molecular chaperones. This article provides an extensive overview and discussion of the human 7BS KMTs and their biochemical and biological roles., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal domain (BET) family of proteins. Brd4 regulates human papillomavirus (HPV) transcription, genome replication, and segregation by binding to the E2 protein. The SETD6 methyltransferase binds to and methylates Brd4 at lysine 99. We investigated the interactions of SETD6 and Brd4 with E2 and their role in HPV transcription. SETD6 coimmunoprecipitated with the E2 transactivation domain, and its depletion in CIN612 episomal cells reduced human papillomavirus type 31 (HPV-31) transcription, whereas depletion of SETD6 in integrated HPV cell lines had no effect on viral gene expression. The mutant Brd4 K99R (bearing a change of K to R at position 99), which cannot be methylated by SETD6, displayed decreased binding to HPV-31 E2, suggesting that SETD6 methylation of Brd4 also influences E2 association with the Brd4 protein. Using chromatin immunoprecipitation, SETD6 was detected at the enhancer region of the HPV long control region. We propose that methylation of Brd4 at K99 by SETD6 is an important mechanism for E2-Brd4 association and HPV transcriptional activation. IMPORTANCE Human papillomaviruses (HPV) cause cervical, anogenital, and oral cancers. Brd4 plays an important role in the HPV life cycle. SETD6 was recently shown to methylate Brd4. The current study demonstrates that methylation of Brd4 by SETD6 in HPV-episomal cells is required for the activation of viral transcription. This study illustrates a novel regulatory mechanism involving E2, Brd4, and SETD6 in the HPV life cycle and provides insight into the multiple roles of Brd4 in viral pathogenesis.

Among the protein lysine methyltransferases family members, it appears that SETD6 is highly similar and closely related to SETD3. The two methyltransferases show high similarity in their structure, which raised the hypothesis that they share cellular functions. Using a proteomic screen, we identified 52 shared interacting-proteins. Gene Ontology (GO) analysis of the shared proteins revealed significant enrichment of proteins involved in transcription. Our RNA-seq data of SETD6 KO and SETD3 KO HeLa cells identified ∼100 up-regulated and down-regulated shared genes. We have also identified a substantial number of genes that changed dramatically in the double KO cells but did not significantly change in the single KO cells. GO analysis of these genes revealed enrichment of apoptotic genes. Accordingly, we show that the double KO cells displayed high apoptotic levels, suggesting that SETD6 and SETD3 inhibit apoptosis. Collectively, our data strongly suggest a functional link between SETD6 and SETD3 in the regulation of apoptosis., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Flavivirus RNA cap-methylation plays an important role in viral infection, proliferation, and escape from innate immunity. The methyltransferase (MTase) of the flavivirus NS5 protein catalyzes viral RNA methylation. The E218 amino acid of the NS5 protein MTase domain is one of the active sites of flavivirus methyltransferase. In flaviviruses, the E218A mutation abolished 2'-O methylation activity and significantly reduced N-7 methylation activity. Tembusu virus (TMUV, genus Flavivirus) was a pathogen that caused neurological symptoms in ducklings and decreased egg production in laying ducks. In this study, we focused on a comprehensive understanding of the effects of the E218A mutation on TMUV characteristics and the host immune response. E218A mutation reduced TMUV replication and proliferation, but did not affect viral adsorption and entry. Based on a TMUV replicon system, we found that the E218A mutation impaired viral translation. In addition, E218A mutant virus might be more readily recognized by RIG-I-like receptors to activate the corresponding antiviral immune signaling than WT virus. Together, our data suggest that the E218A mutation of TMUV MTase domain impairs viral replication and translation and may activates RIG-I-like receptor signaling, ultimately leading to a reduction in viral proliferation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Gliomas are one of the most common and lethal brain tumors among adults. One process that contributes to glioma progression and recurrence is the epithelial to mesenchymal transition (EMT). EMT is regulated by a set of defined transcription factors which tightly regulate this process, among them is the basic helix-loop-helix family member, TWIST1. Here we show that TWIST1 is methylated on lysine-33 at chromatin by SETD6, a methyltransferase with expression levels correlating with poor survival in glioma patients. RNA-seq analysis in U251 glioma cells suggested that both SETD6 and TWIST1 regulate cell adhesion and migration processes. We further show that TWIST1 methylation attenuates the expression of the long-non-coding RNA, LINC-PINT, thereby promoting EMT in glioma. Mechanistically, TWIST1 methylation represses the transcription of LINC-PINT by increasing the occupancy of EZH2 and the catalysis of the repressive H3K27me3 mark at the LINC-PINT locus. Under un-methylated conditions, TWIST1 dissociates from the LINC-PINT locus, allowing the expression of LINC-PINT which leads to increased cell adhesion and decreased cell migration. Together, our findings unravel a new mechanistic dimension for selective expression of LINC-PINT mediated by TWIST1 methylation., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

PRDF1 and RIZ1 homology domain containing (PRDMs) are a subfamily of Krüppel-like zinc finger proteins controlling key processes in metazoan development and in cancer. PRDMs exhibit unique dualities: (a) PR domain/ZNF arrays-their structure combines a SET-like domain known as a PR domain, typically found in methyltransferases, with a variable array of C2H2 zinc fingers (ZNF) characteristic of DNA-binding transcription factors; (b) transcriptional activators/repressors-their physiological function is context- and cell-dependent; mechanistically, some PRDMs have a PKMT activity and directly catalyze histone lysine methylation, while others are rather pseudomethyltransferases and act by recruiting transcriptional cofactors; (c) oncogenes/tumor suppressors-their pathological function depends on the specific PRDM isoform expressed during tumorigenesis. This duality is well known as the 'Yin and Yang' of PRDMs and involves a complex regulation of alternative splicing or alternative promoter usage, to generate full-length or PR-deficient isoforms with opposing functions in cancer. In conclusion, once their dualities are fully appreciated, PRDMs represent a promising class of targets in oncology by virtue of their widespread upregulation across multiple tumor types and their somatic dispensability, conferring a broad therapeutic window and limited toxic side effects. The recent discovery of a first-in-class compound able to inhibit PRDM9 activity has paved the way for the identification of further small molecular inhibitors able to counteract PRDM oncogenic activity., (© 2021 Federation of European Biochemical Societies.)

Purpose: The poor outcomes in glioblastoma necessitate new therapeutic target. Isoprenylcysteine carboxyl methyltransferase (ICMT), a unique enzyme of the final step of prenylation that modifies activities of oncogenic proteins, represents a promising target for many cancers., Methods: Expression pattern, function and downstream pathway of ICMT in glioblastoma were analyzed using immunohistochemistry, ELISA, cellular assays and immunoblotting method. Combinatory effect was analyzed using Chou-Talalay approach., Results: Upregulation of ICMT expression is a common phenomenon in glioblastoma patients regardless of clinicopathological characteristics. Gain-of-function and loss-of-function analysis support the role of ICMT in glioblastoma growth and survival but not migration. Importantly, pharmacological inhibitors of ICMT are effectively against glioblastoma cells while sparing normal neuron cells, and furthermore that they act synergistically with chemotherapeutic drugs. Consistently, ICMT inhibitor UCM-1336 significantly inhibits glioblastoma growth without causing toxicity in mice. Mechanistic studies demonstrate that Ras/Raf/Mek/Erk rather than Ras/PI3K/Akt/mTOR is the downstream pathway of ICMT-mediated glioblastoma growth., Conclusions: Our findings provide the proof-of-concept of pharmacologically targeting ICMT in the treatment of glioblastoma via deactivation of Ras/Raf/Mek/Erk., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Steroid receptors (SRs) are members of the nuclear hormonal receptor family, many of which are transcription factors regulated by ligand binding. SRs regulate various human physiological functions essential for maintenance of vital biological pathways, including development, reproduction, and metabolic homeostasis. In addition, aberrant expression of SRs or dysregulation of their signaling has been observed in a wide variety of pathologies. SR activity is tightly and finely controlled by post-translational modifications (PTMs) targeting the receptors and/or their coregulators. Whereas major attention has been focused on phosphorylation, growing evidence shows that methylation is also an important regulator of SRs. Interestingly, the protein methyltransferases depositing methyl marks are involved in many functions, from development to adult life. They have also been associated with pathologies such as inflammation, as well as cardiovascular and neuronal disorders, and cancer. This article provides an overview of SR methylation/demethylation events, along with their functional effects and biological consequences. An in-depth understanding of the landscape of these methylation events could provide new information on SR regulation in physiology, as well as promising perspectives for the development of new therapeutic strategies, illustrated by the specific inhibitors of protein methyltransferases that are currently available., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

Posttranslational methylation of amino acid side chains in proteins mainly occurs on lysine, arginine, glutamine, and histidine residues. It is introduced by different protein methyltransferases (PMTs) and regulates many aspects of protein function including stability, activity, localization, and protein/protein interactions. Although the biological effects of PMTs are mediated by their methylation substrates, the full substrate spectrum of most PMTs is not known. For many PMTs, their activity on a particular potential substrate depends, among other factors, on the peptide sequence containing the target residue for methylation. In this protocol, we describe the application of SPOT peptide arrays to investigate the substrate specificity of PMTs and identify novel substrates. Methylation of SPOT peptide arrays makes it possible to study the methylation of many different peptides in one experiment at reasonable costs and thereby provides detailed information about the specificity of the PMT under investigation. In these experiments, a known substrate sequence is used as template to design a SPOT peptide array containing peptides with single amino acid exchanges at all positions of the sequence. Methylation of the array with the PMT provides detailed preferences for each amino acid at each position in the substrate sequence, yielding a substrate sequence specificity profile. This information can then be used to identify novel potential PMT substrates by in silico data base searches. Methylation of novel substrate candidates can be validated in SPOT arrays at peptide level, followed by validation at protein level in vitro and in cells., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Dynamic posttranslational modifications to canonical histones that constitute the nucleosome (H2A, H2B, H3, and H4) control all aspects of enzymatic transactions with DNA. Histone methylation has been studied heavily for the past 20 years, and our mechanistic understanding of the control and function of individual methylation events on specific histone arginine and lysine residues has been greatly improved over the past decade, driven by excellent new tools and methods. Here, we will summarize what is known about the distribution and some of the functions of protein methyltransferases from all major eukaryotic supergroups. The main conclusion is that protein, and specifically histone, methylation is an ancient process. Many taxa in all supergroups have lost some subfamilies of both protein arginine methyltransferases (PRMT) and the heavily studied SET domain lysine methyltransferases (KMT). Over time, novel subfamilies, especially of SET domain proteins, arose. We use the interactions between H3K27 and H3K36 methylation as one example for the complex circuitry of histone modifications that make up the "histone code," and we discuss one recent example (Paramecium Ezl1) for how extant enzymes that may resemble more ancient SET domain KMTs are able to modify two lysine residues that have divergent functions in plants, fungi, and animals. Complexity of SET domain KMT function in the well-studied plant and animal lineages arose not only by gene duplication but also acquisition of novel DNA- and histone-binding domains in certain subfamilies., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

DNA damage is a double-edged sword for cancer cells. On the one hand, DNA damage-induced genomic instability contributes to cancer development; on the other hand, accumulating damage compromises proliferation and survival of cancer cells. Understanding the key regulators of DNA damage repair machinery would benefit the development of cancer therapies that induce DNA damage and apoptosis. In this study, we found that isoprenylcysteine carboxylmethyltransferase (ICMT), a posttranslational modification enzyme, plays an important role in DNA damage repair. We found that ICMT suppression consistently reduces the activity of MAPK signaling, which compromises the expression of key proteins in the DNA damage repair machinery. The ensuing accumulation of DNA damage leads to cell cycle arrest and apoptosis in multiple breast cancer cells. Interestingly, these observations are more pronounced in cells grown under anchorage-independent conditions or grown in vivo. Consistent with the negative impact on DNA repair, ICMT inhibition transforms the cancer cells into a "BRCA-like" state, hence sensitizing cancer cells to the treatment of PARP inhibitor and other DNA damage-inducing agents., (© 2021 Tang et al.)

Cells synthesize proteins using 20 standard amino acids and expand their biochemical repertoire through intricate enzyme-mediated post-translational modifications (PTMs). PTMs can either be static and represent protein editing events or be dynamically regulated as a part of a cellular response to specific stimuli. Protein histidine methylation (Hme) was an elusive PTM for over 5 decades and has only recently attracted considerable attention through discoveries concerning its enzymology, extent, and function. Here, we review the status of the Hme field and discuss the implications of Hme in physiological and cellular processes. We also review the experimental toolbox for analysis of Hme and discuss the strengths and weaknesses of different experimental approaches. The findings discussed in this review demonstrate that Hme is widespread across cells and tissues and functionally regulates key cellular processes such as cytoskeletal dynamics and protein translation. Collectively, the findings discussed here showcase Hme as a regulator of key cellular functions and highlight the regulation of this modification as an emerging field of biological research., Competing Interests: Conflict of interest The author declares that he has no conflicts of interest with the contents of this article., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Protein methyltransferases (PMTs) regulate many aspects of normal and disease processes through substrate methylation, with S-adenosyl-L-methionine (SAM) as a cofactor. It has been challenging to elucidate cellular protein lysine and arginine methylation because these modifications barely alter physical properties of target proteins and often are context dependent, transient, and substoichiometric. To reveal bona fide methylation events associated with specific PMT activities in native contexts, we developed the live-cell Bioorthogonal Profiling of Protein Methylation (lcBPPM) technology, in which the substrates of specific PMTs are labeled by engineered PMTs inside living cells, with in situ-synthesized SAM analogues as cofactors. The biorthogonality of this technology is achieved because these SAM analogue cofactors can only be processed by the engineered PMTs-and not native PMTs-to modify the substrates with distinct chemical groups. Here, we describe the latest lcBPPM protocol and its application to reveal proteome-wide methylation and validate specific methylation events. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Live-cell labeling of substrates of protein methyltransferases GLP1 and PRMT1 with lcBPPM-feasible enzymes and SAM analogue precursors Support Protocol: Gram-scale synthesis of Hey-Met Basic Protocol 2: Click labeling of lcBPPM cell lysates with a biotin-azide probe Alternate Protocol: Click labeling of small-scale lcBPPM cell lysates with a TAMRA-azide dye for in-gel fluorescence visualization Basic Protocol 3: Enrichment of biotinylated lcBPPM proteome with streptavidin beads Basic Protocol 4: Proteome-wide identification of lcBPPM targets with mass spectrometry Basic Protocol 5: Validation of individual lcBPPM targets by western blot., (© 2021 Wiley Periodicals LLC.)

Mitochondrial oxidative phosphorylation (OXPHOS) is a vital regulator of tumor metastasis. However, the mechanisms governing OXPHOS to facilitate tumor metastasis remain unclear. In this study, we discovered that arginine 21(R21) and lysine 108 (K108) of mitochondrial ribosomal protein S23 (MRPS23) was methylated by the protein arginine methyltransferase 7 (PRMT7) and SET-domain-containing protein 6 (SETD6), respectively. R21 methylation accelerated the poly-ubiquitin-dependent degradation of MRPS23 to a low level. The MRPS23 degradation inhibited OXPHOS with elevated mtROS level, which consequently increased breast cancer cell invasion and metastasis. In contrast, K108 methylation increased MRPS23 stability, and K108 methylation coordinated with R21 methylation to maintain a low level of MRPS23, which was in favor of supporting breast cancer cell survival through regulating OXPHOS. Consistently, R21 and K108 methylation was correlated with malignant breast carcinoma. Significantly, our findings unveil a unique mechanism of controlling OXPHOS by arginine and lysine methylation and point to the impact of the PRMT7-SETD6-MRPS23 axis during breast cancer metastasis.

Protein methylation occurs primarily on lysine and arginine, but also on some other residues, such as histidine. METTL18 is the last uncharacterized member of a group of human methyltransferases (MTases) that mainly exert lysine methylation, and here we set out to elucidate its function. We found METTL18 to be a nuclear protein that contains a functional nuclear localization signal and accumulates in nucleoli. Recombinant METTL18 methylated a single protein in nuclear extracts and in isolated ribosomes from METTL18 knockout (KO) cells, identified as 60S ribosomal protein L3 (RPL3). We also performed an RPL3 interactomics screen and identified METTL18 as the most significantly enriched MTase. We found that His-245 in RPL3 carries a 3-methylhistidine (3MH; τ-methylhistidine) modification, which was absent in METTL18 KO cells. In addition, both recombinant and endogenous METTL18 were found to be automethylated at His-154, thus further corroborating METTL18 as a histidine-specific MTase. Finally, METTL18 KO cells displayed altered pre-rRNA processing, decreased polysome formation and codon-specific changes in mRNA translation, indicating that METTL18-mediated methylation of RPL3 is important for optimal ribosome biogenesis and function. In conclusion, we have here established METTL18 as the second human histidine-specific protein MTase, and demonstrated its functional relevance., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Leishmania major is the main causative agent of cutaneous leishmaniasis in the Old World. In Leishmania parasites, the lack of transcriptional control is mostly compensated by post-transcriptional mechanisms. Methylation of arginine is a conserved post-translational modification executed by Protein Arginine Methyltransferase (PRMTs). The genome from L. major encodes five PRMT homologs, including the cytosolic protein associated with several RNA-binding proteins, LmjPRMT7. It has been previously reported that LmjPRMT7 could impact parasite infectivity. In addition, a more recent work has clearly shown the importance of LmjPRMT7 in RNA-binding capacity and protein stability of methylation targets, demonstrating the role of this enzyme as an important epigenetic regulator of mRNA metabolism. In this study, we unveil the impact of PRMT7-mediated methylation on parasite development and virulence. Our data reveals that higher levels of LmjPRMT7 can impair parasite pathogenicity, and that deletion of this enzyme rescues the pathogenic phenotype of an attenuated strain of L. major. Interestingly, lesion formation caused by LmjPRMT7 knockout parasites is associated with an exacerbated inflammatory reaction in the tissue correlated with an excessive neutrophil recruitment. Moreover, the absence of LmjPRMT7 also impairs parasite development within the sand fly vector Phlebotomus duboscqi. Finally, a transcriptome analysis shed light onto possible genes affected by depletion of this enzyme. Taken together, this study highlights how post-transcriptional regulation can affect different aspects of the parasite biology., Competing Interests: The authors have declared that no competing interests exist.

Isoprenylcysteine carboxyl methyltransferase (ICMT) is the third of three enzymes that sequentially modify the C-terminus of CaaX proteins, including RAS. Although all four RAS proteins are substrates for ICMT, each traffics to membranes differently by virtue of their hypervariable regions that are differentially palmitoylated. We found that among RAS proteins, NRAS was unique in requiring ICMT for delivery to the PM, a consequence of having only a single palmitoylation site as its secondary affinity module. Although not absolutely required for palmitoylation, acylation was diminished in the absence of ICMT. Photoactivation and FRAP of GFP-NRAS revealed increase flux at the Golgi, independent of palmitoylation, in the absence of ICMT. Association of NRAS with the prenyl-protein chaperone PDE6δ also required ICMT and promoted anterograde trafficking from the Golgi. We conclude that carboxyl methylation of NRAS is required for efficient palmitoylation, PDE6δ binding, and homeostatic flux through the Golgi, processes that direct delivery to the plasma membrane., (© 2021 Ahearn et al.)

A farnesylated and methylated form of prelamin A called progerin causes Hutchinson-Gilford progeria syndrome (HGPS). Inhibiting progerin methylation by inactivating the isoprenylcysteine carboxylmethyltransferase (ICMT) gene stimulates proliferation of HGPS cells and improves survival of Zmpste24 -deficient mice. However, we don't know whether Icmt inactivation improves phenotypes in an authentic HGPS mouse model. Moreover, it is unknown whether pharmacologic targeting of ICMT would be tolerated by cells and produce similar cellular effects as genetic inactivation. Here, we show that knockout of Icmt improves survival of HGPS mice and restores vascular smooth muscle cell numbers in the aorta. We also synthesized a potent ICMT inhibitor called C75 and found that it delays senescence and stimulates proliferation of late-passage HGPS cells and Zmpste24 -deficient mouse fibroblasts. Importantly, C75 did not influence proliferation of wild-type human cells or Zmpste24 -deficient mouse cells lacking Icmt , indicating drug specificity. These results raise hopes that ICMT inhibitors could be useful for treating children with HGPS., Competing Interests: XC, HY, MK, GR, ME, JH, TW, YL, ET, SS, IA, MP, CW, MI, MB No competing interests declared, (© 2021, Chen et al.)

Background: Oral squamous cell carcinoma (OSCC) is one of the most comment types of oral malignancies. SET-domain-containing protein 6 (SETD6) was recently identified as an important regulator of multiple signaling pathways through methylating protein substrates. Meanwhile, SETD6 is known to participate in multiple cancers. However, the role of SETD6 in OSCC remains unclear., Methods: Gene and protein expressions in OSCC cells or tissues were detected by RT-qPCR and western blot, respectively. In addition, CCK-8 assay was used to test the cell viability. A transwell assay was performed to measure cell migration and invasion. Flow cytometry was used to test cell apoptosis and cycle. Meanwhile, methylation-specific PCR (MSP) was used to detect the status of promoter methylation., Results: SETD6 was significantly upregulated in OSCC tissues. In addition, knockdown of SETD6 notably inhibited the proliferation and induced the apoptosis of OSCC cells. Furthermore, silencing of SETD6 notably suppressed the migration and invasion of OSCC cells. Meanwhile, SETD6 siRNA significantly inhibited the promoter methylation of RelA (NF-κB p65) and PAK4. Furthermore, SETD6 siRNA induced G1 arrest in OSCC cells., Conclusion: Knockdown of SETD6 inhibits the tumorigenesis of OSCC by suppressing promoter methylation of PAK4 and RelA. Therefore, our study might shed new light on exploring strategies for the treatment of OSCC.

Methylation is a common posttranslational modification of arginine and lysine in eukaryotic proteins. Methylproteomes are best characterized for higher eukaryotes, where they are functionally expanded and evolved complex regulation. However, this is not the case for protist species evolved from the earliest eukaryotic lineages. Here, we integrated bioinformatic, proteomic, and drug-screening data sets to comprehensively explore the methylproteome of Giardia duodenalis-a deeply branching parasitic protist. We demonstrate that Giardia and related diplomonads lack arginine-methyltransferases and have remodeled conserved RGG/RG motifs targeted by these enzymes. We also provide experimental evidence for methylarginine absence in proteomes of Giardia but readily detect methyllysine. We bioinformatically infer 11 lysine-methyltransferases in Giardia, including highly diverged Su(var)3-9, Enhancer-of-zeste and Trithorax proteins with reduced domain architectures, and novel annotations demonstrating conserved methyllysine regulation of eukaryotic elongation factor 1 alpha. Using mass spectrometry, we identify more than 200 methyllysine sites in Giardia, including in species-specific gene families involved in cytoskeletal regulation, enriched in coiled-coil features. Finally, we use known methylation inhibitors to show that methylation plays key roles in replication and cyst formation in this parasite. This study highlights reduced methylation enzymes, sites, and functions early in eukaryote evolution, including absent methylarginine networks in the Diplomonadida. These results challenge the view that arginine methylation is eukaryote conserved and demonstrate that functional compensation of methylarginine was possible preceding expansion and diversification of these key networks in higher eukaryotes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Isoprenylcysteine carboxylmethyltransferase (ICMT) has been reported to regulate the inflammatory response through the Ras/MAPK/AP-1 pathway. Nevertheless, the potential of ICMT inhibitors as therapeutic agents against inflammatory diseases has not been examined. Therefore, in this study, we investigated the anti-inflammatory properties of two ICMT inhibitors, cysmethynil (CyM) and 3-methoxy-N-[2-2,2,6,6-tetramethyl-4-phenyltetrahydropyran-4-yl)ethyl]aniline (MTPA), using in vitro analyses and in vivo analyses (lipopolysaccharide (LPS)/D-GalN-triggered hepatitis and DSS-induced colitis mouse models). CyM and MTPA inhibited the production of nitric oxide (NO) and prostaglandin E (PGE) 2 and the expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α and interleukin (IL)-1β in LPS-induced RAW264.7 cells and peritoneal macrophages without cytotoxicity. CyM also reduced AP-1-mediated luciferase activity in LPS-stimulated RAW264.7 cells and MyD88- and TRIF-expressing HEK293 cells. In addition, CyM and MTPA suppressed the translocation of Ras to the cell membrane and ER as well as phosphorylation of Ras-dependent AP-1 signaling molecules including Raf, MEK1/2, ERK p38, and JNK. Consistent with these results, CyM diminished the expression of inflammatory genes (COX-2, TNF-α, IL-1β, and IL-6), AP-1-Luc activity, and phosphorylation of Ras-mediated signaling enzymes in Ras-overexpressing HEK 293 cells. Moreover, CyM and MTPA ameliorated symptoms of hepatitis and colitis in mice and restrained the ICMT/Ras-dependent AP-1 pathway in inflammatory lesions of the mouse model systems. Taken together, our results indicate that CyM and MTPA alleviate the LPS-induced ICMT/Ras/AP-1 signaling pathway, thereby inhibiting the inflammatory response as promising anti-inflammatory drugs., (Copyright © 2020 Elsevier Inc. All rights reserved.)

P21-activated kinase 4 (PAK4), a member of serine/threonine kinases family is over-expressed in numerous cancer tumors and is associated with oncogenic cell proliferation, migration and invasion. Our recent work demonstrated that the SET-domain containing protein 6 (SETD6) interacts with and methylates PAK4 at chromatin in mammalian cells, leading to activation of the Wnt/β-catenin signaling pathway. In our current work, we identified lysine 473 (K473) on PAK4 as the primary methylation site by SETD6. Methylation of PAK4 at K473 activates β-catenin transcriptional activity and inhibits cell adhesion. Specific methylation of PAK4 at K473 also attenuates paxillin localization to focal adhesions leading to overall reduction in adhesion-related features, such as filopodia and actin structures. The altered adhesion of the PAK4 wild-type cells is accompanied with a decrease in the migrative and invasive characteristics of the cells. Taken together, our results suggest that methylation of PAK4 at K473 plays a vital role in the regulation of cell adhesion and migration.

Methyltransferases are a superfamily of enzymes that transfer methyl groups to proteins, nucleic acids, and small molecules. Traditionally, these enzymes have been shown to carry out a specific modification (mono-, di-, or trimethylation) on a single, or limited number of, amino acid(s). The largest subgroup of this family, protein methyltransferases, target arginine and lysine side chains of histone molecules to regulate gene expression. Although there is a large number of functional studies that have been performed on individual methyltransferases describing their methylation targets and effects on biological processes, no analyses exist describing the spatial distribution across tissues or their differential expression in the diseased heart. For this review, we performed tissue profiling in protein databases of 199 confirmed or putative methyltransferases to demonstrate the unique tissue-specific expression of these individual proteins. In addition, we examined transcript data sets from human heart failure patients and murine models of heart disease to identify 40 methyltransferases in humans and 15 in mice, which are differentially regulated in the heart, although many have never been functionally interrogated. Lastly, we focused our analysis on the largest subgroup, that of protein methyltransferases, and present a newly emerging phenomenon in which 16 of these enzymes have been shown to play dual roles in regulating transcription by maintaining the ability to both activate and repress transcription through methyltransferase-dependent or -independent mechanisms. Overall, this review highlights a novel paradigm shift in our understanding of the function of histone methyltransferases and correlates their expression in heart disease.

Diabetic nephropathy (DN), a serious complication of hyperglycemia, is one of the most common causes of end-stage renal disease (ESRD). Glomerular podocyte injury is a major mechanism that leads to DN. However, the mechanisms underlying podocyte injury are ambiguous. In this study, we sought to investigate the contribution of SET domain-containing protein 6 (SETD6) to the pathogenesis of podocyte injury induced by glucose (GLU) and palmitic acid (PA), as well as the underlying mechanisms. Our results showed that GLU and PA treatment significantly decreased SETD6 expression in mouse podocytes. Besides, Cell Counting Kit-8 (CCK-8) and flow cytometry assay demonstrated that silencing of SETD6 silence obviously enhanced cell viability, and suppressed apoptosis in GLU and PA-induced podocytes. We also discovered that downregulation of SETD6 suppressed GLU and PA-induced ROS generation and podocyte mitochondrial dysfunction. Nrf2-Keap1 signaling pathway was involved in the effect of SETD6 on mitochondrial dysfunction. Taken together, silencing of SETD6 protected mouse podocyte against apoptosis and mitochondrial dysfunction through activating Nrf2-Keap1 signaling pathway. Therefore these data provide new insights into new potential therapeutic targets for DN treatment.

Bacterial soft rot caused by the bacteria Dickeya and Pectobacterium is a destructive disease of vegetables, as well as ornamental plants. Several management options exist to help control these pathogens. Because of the limited success of these approaches, there is a need for the development of alternative methods to reduce losses. In this study, we evaluated the effect of potassium tetraborate tetrahydrate (PTB) on the growth of six Dickeya and Pectobacterium spp. Disc diffusion assays showed that Dickeya spp. and Pectobacterium spp. differ in their sensitivity to PTB. Spontaneous PTB-resistant mutants of Pectobacterium were identified and further investigation of the mechanism of PTB resistance was conducted by full genome sequencing. Point mutations in genes cpdB and supK were found in a single Pectobacterium atrosepticum PTB-resistant mutant. Additionally, point mutations in genes prfB (synonym supK ) and prmC were found in two independent Pectobacterium brasiliense PTB-resistant mutants. prfB and prmC encode peptide chain release factor 2 and its methyltransferase, respectively. We propose the disruption of translation activity due to PTB leads to Pectobacterium growth inhibition. The P. atrosepticum PTB-resistant mutant showed altered swimming motility. Disease severity was reduced for P. atrosepticum -inoculated potato stems sprayed with PTB. We discuss the potential risk of selecting for bacterial resistance to this chemical.

Lysine (Lys) residues in proteins undergo a wide range of reversible post-translational modifications (PTMs), which can regulate enzyme activities, chromatin structure, protein-protein interactions, protein stability, and cellular localization. Here we discuss the "writers," "erasers," and "readers" of some of the common protein Lys PTMs and summarize examples of their major biological impacts. We also review chemical biology approaches, from small-molecule probes to protein chemistry technologies, that have helped to delineate Lys PTM functions and show promise for a diverse set of biomedical applications., Competing Interests: Declaration of Interests P.A.C. is a cofounder of Acylin and has been a scientific advisor for Abbvie, which has had therapeutics programs targeting p300 and CBP., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Silica cell walls of diatoms have attracted attention as a source of nanostructured functional materials and have immense potential for a variety of applications. Previous studies of silica cell wall formation have identified numerous involved proteins, but most of these proteins are species-specific and are not conserved among diatoms. However, because the basic process of diatom cell wall formation is common to all diatom species, ubiquitous proteins and molecules will reveal the mechanisms of cell wall formation. In this study, we assembled de novo transcriptomes of three diatom species, Nitzschia palea, Achnanthes kuwaitensis, and Pseudoleyanella lunata, and compared protein-coding genes of five genome-sequenced diatom species. These analyses revealed a number of diatom-specific genes that encode putative endoplasmic reticulum-targeting proteins. Significant numbers of these proteins showed homology to silicanin-1, which is a conserved diatom protein that reportedly contributes to cell wall formation. These proteins also included a previously unrecognized SET domain protein methyltransferase family that may regulate functions of cell wall formation-related proteins and long-chain polyamines. Proteomic analysis of cell wall-associated proteins in N. palea identified a protein that is also encoded by one of the diatom-specific genes. Expression analysis showed that candidate genes were upregulated in response to silicon, suggesting that these genes play roles in silica cell wall formation. These candidate genes can facilitate further investigations of silica cell wall formation in diatoms.

Cancer stem cells possess the capacity for self-renewal and resistance to chemotherapy. It is therefore crucial to understand the molecular regulators of stemness in the quest to develop effective cancer therapies. TAZ is a transcription activator that promotes stem cell functions in post-development mammalian cells; suppression of TAZ activity reduces or eliminates cancer stemness in select cancers. Isoprenylcysteine carboxylmethyltransferase (ICMT) is the unique enzyme of the last step of posttranslational prenylation processing pathway that modifies several oncogenic proteins, including RAS. We found that suppression of ICMT results in reduced self-renewal/stemness in KRAS-driven pancreatic and breast cancer cells. Silencing of ICMT led to significant reduction of TAZ protein levels and loss of self-renewal ability, which could be reversed by overexpressing mutant KRAS, demonstrating the functional impact of ICMT modification on the ability of KRAS to control TAZ stability and function. Contrary to expectation, YAP protein levels appear to be much less susceptible than TAZ to the regulation by ICMT and KRAS, and YAP is less consequential in regulating stemness characteristics in these cells. Further, we found that the ICMT-dependent KRAS regulation of TAZ was mediated through RAF, but not PI3K, signaling. Functionally, we demonstrate that a signaling cascade from ICMT modification of KRAS to TAZ protein stability supports cancer cell self-renewal abilities in both in vitro and in vivo settings. In addition, studies using the proof-of-concept small molecule inhibitors of ICMT confirmed its role in regulating TAZ and self-renewal, demonstrating the potential utility of targeting ICMT to control aggressive KRAS-driven cancers.

Therapeutic intervention of proteins participating in chromatin-mediated signaling with small-molecules is a novel option to reprogram expression networks for restraining disease states. Protein methyltransferases form the prominent family of such proteins regulating gene expression via epigenetic mechanisms thereby representing novel targets for pharmacological intervention. Disruptor of telomeric silencing, hDot1L is the only non-SET domain containing histone methyltransferase that methylates histone H3 at lysine 79. H3K79 methylation mediated by hDot1L plays a crucial role in mixed lineage leukemia (MLL) pathosis. MLL fusion protein mediated mistargeting of DOT1L to aberrant gene locations results in ectopic H3K79 methylation culminating in aberrant expression of leukemogenic genes like HOXA9 and MEIS1. hDOT1L has thus been proposed as a potential target for therapeutic intervention in MLL. This review presents the general overview of hDOT1L and its functional role in distinct biological processes. Furthermore, we discuss various therapeutic strategies against hDOT1L as a promising drug target to vanquish therapeutically challenging MLL.

In this study, we investigated the functional role of isoprenylcysteine carboxyl methyltransferase (ICMT) and its methylatable substrate Ras in Toll-like receptor (TLR)-activated macrophages and in mouse inflammatory disease conditions. ICMT and RAS expressions were strongly increased in macrophages under the activation conditions of TLRs by lipopolysaccharide (LPS, a TLR4 ligand), pam3CSK (TLR2), or poly(I:C) (TLR3) and in the colons, stomachs, and livers of mice with colitis, gastritis, and hepatitis. The inhibition and activation of ICMT and Ras through genetic and pharmacological approaches significantly affected the activation of interleukin-1 receptor-associated kinase (IRAK)s, tumor necrosis factor receptor associated factor 6 (TRAF6), transforming growth factor-β-activated kinase 1 (TAK1), mitogen-activated protein kinase (MAPK), and MAPK kinases (MAPKKs); translocation of the AP-1 family; and the expressions of inflammation-related genes that depend on both MyD88 and TRIF. Interestingly, the Ras/ICMT-mediated inflammatory reaction critically depends on the TIR domains of myeloid differentiation primary response 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF). Taken together, these results suggest that ICMT and its methylated Ras play important roles in the regulation of inflammatory responses through cooperation with the TIR domain of adaptor molecules.

The maternal nucleolus plays an indispensable role in zygotic genome activation (ZGA) and early embryonic development in mice. During oocyte-to-embryo transition, the nucleolus is subject to substantial transformation. Despite the primary role of the nucleolus is ribosome biogenesis, accumulating evidence has uncovered its functions in various other cell processes. However, the regulation of nucleolar maturation and ribosome biogenesis and the molecules involved remain unclear during early embryonic development. In this study, we observed that nucleolar protein 2 (NOP2) is restrictedly localized within the nucleolus, first detected in the late two-cell embryos, and increases to a peak level at the eight-cell stage in mice. RNAi-mediated NOP2 depletion leads to a developmental arrest during the morula-to-blastocyst transition. RNA-seq analyses reveal that 208 genes are differentially expressed, including multiple lineage-specific genes and several genes encoding ribosome proteins. Indeed, we observe a failure of the first lineage specification with reduced TEA domain transcription factor 4(TEAD4) (trophectoderm-specific), tir na nog (NANOG), and kruppel-like factor 4 (KLF4) (inner cell mass-specific). Importantly, by Transmission Electron Microscopy (TEM), we noted a decrease in the ratio of the nucleolus size and an increase in the ratio of the size of the nucleolus precursor body, suggesting the nucleolar maturation is disrupted. Moreover, both qPCR and Fluorescence In Situ Hybridization (FISH) data showcase a significant decrease in the abundance of ribosome RNAs. Similarly, NOP2 depletion causes reduced developmental potential and decreased rRNA level in bovine early embryos, suggesting a functional conservation of NOP2 in mammals. Taken together, these results suggest that NOP2 is required for mammalian preimplantation development, presumably by regulating nucleolar maturation and ribosome biogenesis., (© 2019 Federation of American Societies for Experimental Biology.)

Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins' sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins' sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily., Competing Interests: The authors declare no competing interest.

Although central to regulating the access to genetic information, most lysine methyltransferases remain poorly characterised relative to other family of enzymes. Herein, I report new substrates for the lysine methyltransferase SETD6. Based on the SETD6-catalysed site on the histone variant H2AZ, I identified similar sequences in the canonical histones H2A, H3, and H4 that are modified by SETD6 in vitro , and putative non-histone substrates. I herein expend the repertoire of substrates for methylation by SETD6.

Protein histidine methylation is a rarely studied posttranslational modification in eukaryotes. Although the presence of N-methylhistidine was demonstrated in actin in the early 1960s, so far, only a limited number of proteins containing N-methylhistidine have been reported, including S100A9, myosin, skeletal muscle myosin light chain kinase (MLCK 2), and ribosomal protein Rpl3. Furthermore, the role of histidine methylation in the functioning of the protein and in cell physiology remains unclear due to a shortage of studies focusing on this topic. However, the molecular identification of the first two distinct histidine-specific protein methyltransferases has been established in yeast (Hpm1) and in metazoan species (actin-histidine N-methyltransferase), giving new insights into the phenomenon of protein methylation at histidine sites. As a result, we are now beginning to recognize protein histidine methylation as an important regulatory mechanism of protein functioning whose loss may have deleterious consequences in both cells and in organisms. In this review, we aim to summarize the recent advances in the understanding of the chemical, enzymological, and physiological aspects of protein histidine methylation., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Covering: 2012 to 2019HemN-like radical S-adenosyl-l-methionine (SAM) enzymes have been recently disclosed to catalyze diverse chemically challenging reactions from primary to secondary metabolic pathways. In this highlight, we summarize the reaction examples catalyzed by HemN-like enzymes to date and the enzymatic mechanisms reported. From the recent mechanistic investigations, we reason that there is a shared initiating mechanism wherein a characteristic SAM methylene radical is proposed to abstract a hydrogen atom from an sp 3 carbon or add onto an sp 2 carbon center although variations occur thereafter from reaction to reaction, as well as providing a brief insight into some future prospects.

ChuW, ChuX, and ChuY are contiguous genes downstream from a single promoter that are expressed in the enteric pathogen Escherichia coli O157:H7 when iron is limiting. These genes, and the corresponding proteins, are part of a larger heme uptake and utilization operon that is common to several other enteric pathogens, such as Vibrio cholerae . The aerobic degradation of heme has been well characterized in humans and several pathogenic bacteria, including E. coli O157:H7, but only recently was it shown that ChuW catalyzes the anaerobic degradation of heme to release iron and produce a reactive tetrapyrrole termed "anaerobilin". ChuY has been shown to function as an anaerobilin reductase, in a role that parallels biliverdin reductase. In this work we have employed biochemical and biophysical approaches to further interrogate the mechanism of the anaerobic degradation of heme. We demonstrate that the iron atom of the heme does not participate in the catalytic mechanism of ChuW and that S- adenosyl-l-methionine binding induces conformational changes that favor catalysis. In addition, we show that ChuX and ChuY have synergistic and additive effects on the turnover rate of ChuW. Finally, we have found that ChuS is an effective source of heme or protoporphyrin IX for ChuW under anaerobic conditions. These data indicate that ChuS may have dual functionality in vivo. Specifically, ChuS serves as a heme oxygenase during aerobic metabolism of heme but functions as a cytoplasmic heme storage protein under anaerobic conditions, akin to what has been shown for PhuS (45% sequence identity) from Pseudomonas aeruginosa.

Isoprenylcysteine carboxylmethyltransferase (Icmt) which catalyzes the final step of prenylation of many oncoproteins, such as Ras. Despite studies on Icmt and its regulation in biological activities of various cancers, little is known on the expression, function and mechanisms of the impact of Icmt on hepatocellular carcinoma (HCC). We report here the findings that Icmt is critical for HCC growth, migration, survival and chemoresistance by multiple oncogenic pathways. Expression analysis on primary patient and cell line samples demonstrated that Icmt protein level was significantly higher in the majority (∼70%) of HCC tissues and cells than corresponding normal counterparts. Icmt depletion inhibited growth, survival and migration in HCC cells, and augmented the inhibitory effects of doxorubicin. Consistently, Icmt also inhibited growth, and migration, and induced apoptosis in HCC cells that are resistant to doxorubicin. In contrast, Icmt overexpression promoted growth and migration in normal liver cells. Mechanistically, Icmt inhibition suppressed Ras/Raf/Mek/Erk signaling and epithelial-mesenchymal transition (EMT) in HCC cells. Several different approaches demonstrated that Icmt was critical for HCC biological activities with the predominant role in cell response to chemotherapy. This previously unappreciated function of Icmt can be targeted to enhance chemotherapy in particular those HCC patients with high Icmt expression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cockayne syndrome is an accelerated aging disorder, caused by mutations in the CSA or CSB genes. In CSB-deficient cells, poly (ADP ribose) polymerase (PARP) is persistently activated by unrepaired DNA damage and consumes and depletes cellular nicotinamide adenine dinucleotide, which leads to mitochondrial dysfunction. Here, the distribution of poly (ADP ribose) (PAR) was determined in CSB-deficient cells using ADPr-ChAP (ADP ribose-chromatin affinity purification), and the results show striking enrichment of PAR at transcription start sites, depletion of heterochromatin and downregulation of H3K9me3-specific methyltransferases SUV39H1 and SETDB1. Induced-expression of SETDB1 in CSB-deficient cells downregulated PAR and normalized mitochondrial function. The results suggest that defects in CSB are strongly associated with loss of heterochromatin, downregulation of SETDB1, increased PAR in highly-transcribed regions, and mitochondrial dysfunction., (Published by Oxford University Press on behalf of Nucleic Acids Research 2019.)

Many neuropsychiatric risk genes contribute to epigenetic regulation but little is known about specific chromatin-associated mechanisms governing the formation of neuronal connectivity. Here we show that transcallosal connectivity is critically dependent on C11orf46, a nuclear protein encoded in the chromosome 11p13 WAGR risk locus. C11orf46 haploinsufficiency was associated with hypoplasia of the corpus callosum. C11orf46 knockdown disrupted transcallosal projections and was rescued by wild type C11orf46 but not the C11orf46 R236H mutant associated with intellectual disability. Multiple genes encoding key regulators of axonal development, including Sema6a, were hyperexpressed in C11orf46-knockdown neurons. RNA-guided epigenetic editing of Sema6a gene promoters via a dCas9-SunTag system with C11orf46 binding normalized SEMA6A expression and rescued transcallosal dysconnectivity via repressive chromatin remodeling by the SETDB1 repressor complex. Our study demonstrates that interhemispheric communication is sensitive to locus-specific remodeling of neuronal chromatin, revealing the therapeutic potential for shaping the brain's connectome via gene-targeted designer activators and repressor proteins.

Development of chemo-resistance in nasopharyngeal carcinoma (NPC) poses the therapeutic challenge and its mechanisms are still poorly understood. In this work, we demonstrate that targeting isoprenylcysteine carboxylmethyltransferase (Icmt) is a therapeutic strategy to overcome NPC chemo-resistance. We found that Icmt mRNA and protein levels were increased in NPC cells after prolonged exposure to chemotherapy. Using pharmacological inhibitor cysmethynil or genetic siRNA approaches, we showed that Icmt inhibition was more effective against chemoresistant compared to chemosensitive NPC cells, suggesting that chemoresistant NPC cells is more dependent on Icmt function. The combination of Icmt inhibition with 5-FU or cisplatin resulted in greater efficacy than single chemotherapeutic agent alone in NPC. Notably, we demonstrated that the in vitro observations were translatable to in vivo NPC cancer xenograft mouse model. Mechanism analysis indicated that Icmt inhibition decreased Ras and RhoA activities, leading to the suppression of Ras and RhoA-mediated downstream signaling in NPC cells. The reverse of the inhibitory effects of cysmethynil by constitutively active Ras suggests that Ras is the critical effector of Icmt in NPC cells. Our work is the first to show that Icmt plays an important role in the development of NPC chemoresistance. Our findings also suggest that targeting Icmt represents a promising strategy to inhibit Ras function., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Lysine methylation is a common posttranslational modification of nuclear and cytoplasmic proteins but is also present in mitochondria. The human protein denoted "family with sequence similarity 173 member B" (FAM173B) was recently uncovered as a mitochondrial lysine (K)-specific methyltransferase (KMT) targeting the c-subunit of mitochondrial ATP synthase (ATPSc), and was therefore renamed ATPSc-KMT. We here set out to investigate the biochemical function of its yet uncharacterized paralogue FAM173A. We demonstrate that FAM173A localizes to mitochondria, mediated by a noncanonical targeting sequence that is partially retained in the mature protein. Immunoblotting analysis using methyllysine-specific antibodies revealed that FAM173A knock-out (KO) abrogates lysine methylation of a single mitochondrial protein in human cells. Mass spectrometry analysis identified this protein as adenine nucleotide translocase (ANT), represented by two highly similar isoforms ANT2 and ANT3. We found that methylation occurs at Lys-52 of ANT, which was previously reported to be trimethylated. Complementation of KO cells with WT or enzyme-dead FAM173A indicated that the enzymatic activity of FAM173A is required for ANT methylation at Lys-52 to occur. Both in human cells and in rat organs, Lys-52 was exclusively trimethylated, indicating that this modification is constitutive, rather than regulatory and dynamic. Moreover, FAM173A-deficient cells displayed increased mitochondrial respiration compared with FAM173A-proficient cells. In summary, we demonstrate that FAM173A is the long-sought KMT responsible for ANT methylation at Lys-52, and point out the functional significance of Lys-52 methylation in ANT. Based on the established naming nomenclature for KMTs, we propose to rename FAM173A to ANT-KMT (gene name ANTKMT )., (© 2019 Małecki et al.)

Prader-Willi syndrome (PWS) is a complex and multisystem neurobehavioral disorder. The molecular mechanism of PWS is deficiency of paternally expressed gene gene or genes from the chromosome 15q11-q13. Due to imprinted gene regulation, the same genes in the maternal chromosome 15q11-q13 are structurally intact but transcriptionally repressed by an epigenetic mechanism. The unique molecular defect underlying PWS renders an exciting opportunity to explore epigenetic-based therapy to reactivate the expression of repressed PWS genes from the maternal chromosome. Inactivation of H3K9m3 methyltransferase SETDB1 and zinc finger protein ZNF274 results in reactivation of SNRPN and SNORD116 cluster from the maternal chromosomes in PWS patient iPSCs and iPSC-derived neurons, respectively. High content screening of small molecule libraries using cells derived from transgenic mice carrying the SNRPN-EGFP fusion protein has discovered that inhibitors of EHMT2/G9a, a histone 3 lysine 9 methyltransferase, are capable of reactivating expression of paternally expressed SNRPN and SNORD116 from the maternal chromosome, both in cultured PWS patient-derived fibroblasts and in a PWS mouse model. Treatment with an EMHT2/G9a inhibitor also rescues perinatal lethality and failure to thrive phenotypes in a PWS mouse model. These findings present the first evidence to support a proof-of-principle for epigenetic-based therapy for the PWS in humans., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Protein α-N-terminal methylation is catalyzed by protein N-terminal methyltransferases. The prevalent occurrence of this methylation in ribosomes, myosin, and histones implies its function in protein-protein interactions. Although its full spectrum of function has not yet been outlined, recent discoveries have revealed the emerging roles of α-N-terminal methylation in protein-chromatin interactions, DNA damage repair, and chromosome segregation. Herein, an overview of the discovery of protein N-terminal methyltransferases and functions of α-N-terminal methylation is presented. In addition, substrate recognition, mechanisms, and inhibition of N-terminal methyltransferases are reviewed. Opportunities and gaps in protein α-N-terminal methylation are also discussed., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Objective: MicroRNAs (miRNAs) affect almost all cell behaviors of human cancers including gastric cancer (GC). However, the biological role of miR-411 in GC remains to be elucidated., Materials and Methods: We determined the expression level of miR-411 in GC cell lines. After synthetic miRNAs or SET domain containing 6 (SETD6) expression vectors transfection, cell proliferation, colony formation, and migration were examined. Moreover, the target of miR-411 was identified by luciferase activity reporter assay and Western blot assay., Results: Results revealed that the miR-411 expression was significantly down-regulated in GC cell lines compared with normal colonic epithelial cells. Overexpression of miR-411 inhibits GC cell proliferation, colony formation, and migration, whereas the overexpression of SETD6 promotes cell proliferation, colony formation, and migration. Moreover, SETD6 was identified as a putative target of miR-411. In addition, we showed miR-411 regulates GC cell behaviors by targeting SETD6. The overexpression of SETD6 promoted the activation of the nuclear factor (NF)-κB signaling pathway., Conclusions: These results suggested that miR-411 functions as a tumor suppressor in GC through targeting SETD6/NF-κB pathway. Targeting miR-411 may be a novel clue for GC treatment.