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1.
Methods Mol Biol ; 2529: 109-120, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35733012

RESUMO

The catalytic activity of histone methyltransferases is not restricted to histones but also includes noncanonical substrates. Increasing evidence shows that histone methyltransferases methylate themselves, and automethylation has emerged as a self-regulatory mechanism. Here, we introduce experimental procedures to identify automethylation sites of histone methyltransferases and to investigate the function of automethylation in a reconstituted biochemical system and in cellular contexts.


Assuntos
Histonas , Metiltransferases , Histona Metiltransferases , Histonas/metabolismo , Metilação , Proteínas Metiltransferases
2.
Methods Mol Biol ; 2529: 3-40, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35733008

RESUMO

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.


Assuntos
Histonas , Proteínas Metiltransferases , Animais , Arginina/metabolismo , Eucariotos/genética , Eucariotos/metabolismo , Histona-Lisina N-Metiltransferase/química , Histonas/metabolismo , Lisina/metabolismo , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional
3.
Methods Mol Biol ; 2529: 313-325, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35733022

RESUMO

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.


Assuntos
Peptídeos , Proteínas Metiltransferases , Sequência de Aminoácidos , Lisina/metabolismo , Metilação , Metiltransferases/metabolismo , Peptídeos/metabolismo , Proteínas Metiltransferases/metabolismo , Especificidade por Substrato
4.
Redox Biol ; 51: 102282, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35305370

RESUMO

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.


Assuntos
Leucemia Mieloide Aguda , Proteína-Arginina N-Metiltransferases , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Oxirredução , Estresse Oxidativo , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo
5.
J Clin Lab Anal ; 36(5): e24348, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35312113

RESUMO

BACKGROUND: circRNA hsa_circ_0018289-mediated growth and metastasis of CC cells were investigated, as well as the mechanistic pathway. METHODS: Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was carried out to examine the expression of hsa_circ_0018289, microRNA (miR)-1294, and isoprenylcysteine carboxyl methyltransferase (ICMT). CC cell proliferation, migration, and invasion were evaluated by 5-ethynyl-2'-deoxyuridine (EdU) incorporation, colony formation, transwell assays, Western blot analysis of ICMT, and glycolysis-associated proteins. Dual-luciferase reporter or RNA pull-down analysis of the target interaction between miR-1294 and hsa_hsa_circ_0018289 or ICMT. Xenograft model assay was implemented to assess the role of hsa_circ_0018289 in vivo. Immunofluorescence (IHC) was employed to detect the level of Ki-67. RESULTS: Hsa_circ_0018289 was elevated in CC tissues and cells, its deficiency could repress growth, metastasis, and glycolysis of CC cells in vitro, as well as hamper tumor growth in vivo. Hsa_circ_0018289 sponged miR-1294 while miR-1294 bound with ICMT, and the inhibition of miR-1294 or addition of ICMT could partially relieve the effect caused by hsa_circ_0018289 depletion. CONCLUSION: Hsa_circ_0018289 contributes to malignant development by regulating the miR-1294/ICMT axis, affording novel insight into CC therapy.


Assuntos
MicroRNAs , Proteínas Metiltransferases , RNA Circular , Neoplasias do Colo do Útero , Carcinogênese , Proliferação de Células/genética , Feminino , Humanos , MicroRNAs/genética , Proteínas Metiltransferases/genética , RNA Circular/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
6.
Cancer Chemother Pharmacol ; 89(3): 401-411, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35171349

RESUMO

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.


Assuntos
Glioblastoma , Animais , Linhagem Celular Tumoral , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Fosfatidilinositol 3-Quinases , Proteínas Metiltransferases/análise , Proteínas Metiltransferases/metabolismo
7.
Arch Oral Biol ; 134: 105320, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34875442

RESUMO

OBJECTIVE: This research investigated the biological role of isoprenylcysteine carboxyl methyltransferase (ICMT) in tongue squamous cell carcinoma (TSCC) progression meanwhile to explore the conceivable mechanism. METHODS: The mRNA and protein expression were measured using real-time PCR and Western blot. Cell proliferation, apoptosis, cycle distribution, migration and invasion were evaluated by CCK-8 assay, flow cytometry, wound-healing assay and transwell assay. The anti-tumor activity of ICMT silencing was observed in nude mice. RESULTS: Our results indicated that silencing of ICMT-mediated methylation effectively inhibited TSCC cells proliferation in vitro and reduced tumor growth in vivo. Moreover, ICMT knockdown also induced cell apoptosis and cell cycle arrest of both CAL-27 and SCC-4 cells. In addition, CAL-27 and SCC-4 cells migration and invasion were weakened by ICMT siRNA. Mechanistically, ICMT deficiency significantly decreased the K-Ras and RhoA membrane targeting localization, leading to the suppression of K-Ras- and RhoA-mediated downstream signaling in CAL-27 and SCC-4 cells. CONCLUSIONS: Altogether, our findings identified a crucial role played by ICMT in the progression of TSCC and the potential mechanisms by which exerted its effects, indicating that targeting ICMT may represent a promising therapeutic strategy for TSCC.


Assuntos
Carcinoma de Células Escamosas , Neoplasias da Língua , Animais , Linhagem Celular Tumoral , Proliferação de Células , Camundongos , Camundongos Nus , Proteínas Metiltransferases , Transdução de Sinais , Língua
8.
FEBS J ; 289(5): 1256-1275, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-33774927

RESUMO

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.


Assuntos
Epigênese Genética , Proteínas Metiltransferases/metabolismo , Sequência de Aminoácidos , Carcinogênese , Cristalização , DNA/metabolismo , Meiose , Neoplasias/enzimologia , Neoplasias/patologia , Oncogenes , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Metiltransferases/química , Proteínas Metiltransferases/genética , Alinhamento de Sequência , Transdução de Sinais
9.
Endocr Rev ; 43(1): 160-197, 2022 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33955470

RESUMO

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.


Assuntos
Neoplasias , Receptores de Esteroides , Humanos , Metilação , Neoplasias/metabolismo , Proteínas Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Esteroides/metabolismo
10.
Life Sci Alliance ; 4(12)2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34610973

RESUMO

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.


Assuntos
Neoplasias da Mama/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas Metiltransferases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzamidas/farmacologia , Neoplasias da Mama/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Feminino , Vetores Genéticos , Células HEK293 , Humanos , Indazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos SCID , Piperidinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Metiltransferases/genética , RNA Interferente Pequeno/genética , Ribonucleosídeos/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Molecules ; 26(16)2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34443661

RESUMO

Protein methyltransferases are vital to the epigenetic modification of gene expression. Thus, obtaining a better understanding of and control over the regulation of these crucial proteins has significant implications for the study and treatment of numerous diseases. One ideal mechanism of protein regulation is the specific installation of a photolabile-protecting group through the use of photocaged non-canonical amino acids. Consequently, PRMT1 was caged at a key tyrosine residue with a nitrobenzyl-protected Schultz amino acid to modulate protein function. Subsequent irradiation with UV light removes the caging group and restores normal methyltransferase activity, facilitating the spatial and temporal control of PRMT1 activity. Ultimately, this caged PRMT1 affords the ability to better understand the protein's mechanism of action and potentially regulate the epigenetic impacts of this vital protein.


Assuntos
Epigênese Genética/efeitos da radiação , Proteínas Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras/genética , Sequência de Aminoácidos/genética , Aminoácidos , Epigênese Genética/genética , Expressão Gênica/efeitos da radiação , Humanos , Metilação/efeitos da radiação , Proteínas Metiltransferases/efeitos da radiação , Proteína-Arginina N-Metiltransferases/efeitos da radiação , Proteínas Repressoras/efeitos da radiação , Fatores de Transcrição/genética , Tirosina/química , Raios Ultravioleta
12.
J Biol Chem ; 297(4): 101130, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34461099

RESUMO

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.


Assuntos
Histidina/metabolismo , Proteínas Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Citoesqueleto/metabolismo , Humanos , Metilação
13.
Curr Protoc ; 1(8): e213, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34370893

RESUMO

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.


Assuntos
Metionina , S-Adenosilmetionina , Humanos , Metilação , Proteínas Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteoma/metabolismo , Proteínas Repressoras , S-Adenosilmetionina/metabolismo
14.
Sci Adv ; 7(22)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-34039605

RESUMO

The transcriptional coactivator BRD4 has a fundamental role in transcription regulation and thus became a promising epigenetic therapeutic candidate to target diverse pathologies. However, the regulation of BRD4 by posttranslational modifications has been largely unexplored. Here, we show that BRD4 is methylated on chromatin at lysine-99 by the protein lysine methyltransferase SETD6. BRD4 methylation negatively regulates the expression of genes that are involved in translation and inhibits total mRNA translation in cells. Mechanistically, we provide evidence that supports a model where BRD4 methylation by SETD6 does not have a direct role in the association with acetylated histone H4 at chromatin. However, this methylation specifically determines the recruitment of the transcription factor E2F1 to selected target genes that are involved in mRNA translation. Together, our findings reveal a previously unknown molecular mechanism for BRD4 methylation-dependent gene-specific targeting, which may serve as a new direction for the development of therapeutic applications.


Assuntos
Proteínas de Ciclo Celular , Proteínas Nucleares , Proteínas Metiltransferases , Fatores de Transcrição , Proteínas de Ciclo Celular/genética , Cromatina , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas , Proteínas Metiltransferases/genética , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 39(3): 328-335, 2021 Jun 01.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-34041883

RESUMO

OBJECTIVES: The effect of isoprenylcysteine carboxymethyltransferase (ICMT) silencing on the migration and invasion of tongue squamous cell carcinoma was investigated by constructing the small interfering RNA (siRNA) of ICMT. METHODS: Through liposomal transfection, siRNA was transfected into human tongue squamous cell carcinoma CAL-27 and SCC-4 cells (ICMT-siRNA group) with a negative control group (transfected with NC-siRNA) and a blank control group (transfected with a transfection reagent but not with siRNA). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of ICMT and RhoA in each group of cells after transfection and to measure the silencing efficiency. Western blot was applied to examine the expression levels of ICMT, total RhoA, membrane RhoA, ROCK1, matrix metalloproteinase (MMP)-2, and MMP-9 proteins in each group. The migration and invasion abilities were evaluated via wound healing and Transwell motility assays. RESULTS: After CAL-27 and SCC-4 cells were transfected with ICMT-siRNA, the expression levels of ICMT genes and proteins decreased significantly in the experimental group compared with those in the negative and blank control groups (P<0.05). The mRNA and total protein expression levels of RhoA in the two groups were not significantly different (P>0.05). The expression levels of RhoA membrane protein, ROCK1, MMP-2, and MMP-9 decreased (P<0.05). The migration and invasion abilities were inhibited (P<0.05). CONCLUSIONS: The migration and invasion abilities of CAL-27 and SCC-4 cells were reduced significantly after the transfection of ICMT-siRNA, and the involved mechanism might be related to the RhoA-ROCK signaling pathway.


Assuntos
Carcinoma de Células Escamosas , Neoplasias da Língua , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Invasividade Neoplásica , Proteínas Metiltransferases , RNA Interferente Pequeno , Língua , Transfecção , Quinases Associadas a rho
16.
J Int Med Res ; 49(5): 300060520939031, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34038200

RESUMO

BACKGROUND: MicroRNA (miR)-99a-5p acts as a tumor suppressor in several tumors, including bladder cancer and breast cancer, but its biological function in oral squamous cell carcinoma (OSCC) is poorly understood. METHODS: miR-99a-5p expression was determined in OSCC tissues and cell lines using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Cell proliferation was assessed by the Cell Counting Kit-8 assay and colony formation assay. Wound healing and Transwell assays were used to analyze migration and invasion abilities, respectively, in OSCC cells. The luciferase reporter assay, RT-qPCR, and western blotting were used to determine the relationship between miR-99a-5p and isoprenylcysteine carboxylmethyltransferase (ICMT). RESULTS: miR-99a-5p expression in OSCC tissues and cell lines was significantly decreased compared with corresponding controls, and was significantly associated with clinical stage and lymph node metastasis in OSCC. Functional assays revealed that miR-99a-5p overexpression significantly inhibited the proliferation, migration, and invasion abilities of CAL-27 and TCA-8113 OSCC cells. miR-99a-5p was found to directly target ICMT, while ICMT restoration reversed the role of miR-99a-5p in OSCC cells. CONCLUSIONS: Our results indicate that miR-99a-5p-mediates the down-regulation of ICMT, which could be used as a novel potential therapeutic target for OSCC treatment.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , MicroRNAs , Neoplasias Bucais , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Neoplasias Bucais/genética , Proteínas Metiltransferases , Carcinoma de Células Escamosas de Cabeça e Pescoço
17.
Oncogene ; 40(20): 3548-3563, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33927350

RESUMO

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.


Assuntos
Arginina/química , Neoplasias da Mama/metabolismo , Lisina/química , Mitocôndrias/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Humanos , Metilação , Mitocôndrias/patologia , Metástase Neoplásica , Organoides , Fosforilação Oxidativa , Proteínas Metiltransferases/genética , Proteínas Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo
18.
Hua Xi Kou Qiang Yi Xue Za Zhi ; 39(1): 64-73, 2021 Feb 01.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-33723939

RESUMO

OBJECTIVES: This study aimed to explore the effects of silencing isoprenylcysteine carboxyl methyltransfe-rase (Icmt) through small interfering RNA (siRNA) interference on the proliferation and apoptosis of tongue squamous cell carcinoma (TSCC). METHODS: Three siRNA were designed and constructed for the Icmt gene sequence and were then transfected into TSCC cells CAL-27 and SCC-4 to silence Icmt expression. The tested cells were divided as follows: RNA interference groups Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The transfection efficiency of siRNA was detected by the fluorescent group Cy3-labeled siRNA, and the expression of Icmt mRNA was screened by quantitive real-time polymerase chain reaction (qRT-PCR) selected the experimental group for subsequent experiments. The expression of Icmt, RhoA, Cyclin D1, p21, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) were analyzed by Western blot. The proliferation abilities of TSCC cells were determined by cell counting kit-8 assay. The change in apoptosis was detected by AnnexinV-APC/propidium staining (PI) assay. Cell-cycle analysis was conducted by flow cytometry. RESULTS: The expression of Icmt mRNA and protein in TSCC cells significantly decreased after Icmt-siRNA transfection (P<0.05). No significant difference in RhoA mRNA and protein expression was detected (P>0.05), but the expression of RhoA membrane protein decreased compared with the negative control group and blank control groups (P<0.05). Cyclin D1 expression decreased, whereas p21 expression significantly increased and the relative expression of ERK protein in the experimental group did not significantly different that in the control group (P>0.05). However, the phosphorylation level of ERK was significantly reduced (P<0.05). The cell cycles of TSCC CAL-27 and SCC-4 were altered in G1/S, cell proliferation activity was inhibited, and apoptosis was induced (P<0.05). CONCLUSIONS: Silencing Icmt can effectively downregulate its expression in TSCC cells, reduce the RhoA membrane targeting localization and cell proliferation, and induce apoptosis. Thus, Icmt may be a potential gene therapy target for TSCC.


Assuntos
Carcinoma de Células Escamosas , Neoplasias da Língua , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Proteínas Metiltransferases , RNA Interferente Pequeno , Língua
19.
PLoS Negl Trop Dis ; 15(3): e0009230, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33651805

RESUMO

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.


Assuntos
Leishmania major/enzimologia , Leishmaniose Cutânea/patologia , Neutrófilos/fisiologia , Proteínas Metiltransferases/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Leishmania major/genética , Leishmania major/metabolismo , Leishmaniose Cutânea/parasitologia , Camundongos , Proteínas Metiltransferases/genética
20.
Biochemistry ; 60(11): 854-858, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33667085

RESUMO

SAM is a powerful methylating agent, with a methyl group transfer potential matching the phosphoryl group transfer potential of ATP. SAM-dependent N-methyltransferases have evolved to catalyze the modification of specific lysine residues in histones and transcription factors, in addition to generating epinephrine, N-methylnicotinamide, and a quaternary amine (betaine) that is used to maintain osmotic pressure in plants and halophilic bacteria. To assess the catalytic power of these enzymes and their potential susceptibility to transition state and multisubstrate analogue inhibitors, we determined the rates and positions of the equilibrium of methyl transfer from the trimethylsulfonium ion to model amines in the absence of a catalyst. Unlike the methyl group transfer potential of SAM, which becomes more negative with an increase in pH throughout the normal pH range, equilibrium constants for the hydrolytic demethylation of secondary, tertiary, and quaternary amines are found to be insensitive to a change in pH and resemble each other in magnitude, with an average ΔG value of approximately -0.7 kcal/mol at pH 7. Thus, each of the three steps in the mono-, di-, and trimethylation of lysine by SAM is accompanied by a change in free energy of -7.5 kcal/mol in a neutral solution. Arrhenius analysis of the uncatalyzed reactions shows that the unprotonated form of glycine attacks the trimethylsulfonium ion (TMS+) with second-order rates constant of 1.8 × 10-7 M-1 s-1 at 25 °C (ΔH⧧ = 22 kcal/mol, and TΔS⧧ = -6 kcal/mol). Comparable values are observed for the methylation of secondary and tertiary amines, with k25 values of 1.1 × 10-7 M-1 s-1 for sarcosine and 4.3 × 10-8 M-1 s-1 for dimethylglycine. The non-enzymatic methylations of imidazole and methionine by TMS+, benchmarks for the methylation of histidine and methionine residues by SETD3, exhibit k25 values of 3.3 × 10-9 and 1.2 × 10-9 M-1 s-1, respectively. Lysine methylation by SAM, although slow under physiological conditions (t1/2 = 7 weeks at 25 °C), is accelerated 1.1 × 1012 -fold at the active site of a SET domain methyltransferase.


Assuntos
Proteínas Metiltransferases/química , Proteínas Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Domínio Catalítico , Cinética , Metilação , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Especificidade por Substrato
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