RESUMO
Although nanotechnology often addresses biomedical needs, nanoscale tools can also facilitate broad biological discovery. Nanoscale delivery, imaging, biosensing, and bioreactor technologies may address unmet questions at the interface between chemistry and biology. Currently, many chemical biologists do not include nanomaterials in their toolbox, and few investigators develop nanomaterials in the context of chemical tools to answer biological questions. We reason that the two fields are ripe with opportunity for greater synergy. Nanotechnologies can expand the utility of chemical tools in the hands of chemical biologists, for example, through controlled delivery of reactive and/or toxic compounds or signal-binding events of small molecules in living systems. Conversely, chemical biologists can work with nanotechnologists to address challenging biological questions that are inaccessible to both communities. This Perspective aims to introduce the chemical biology community to nanotechnologies that may expand their methodologies while inspiring nanotechnologists to address questions relevant to chemical biology.
Assuntos
Biologia Molecular/tendências , Nanotecnologia/tendências , Animais , Materiais Biocompatíveis , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Enzimas/química , Humanos , Biologia Molecular/métodos , Imagem Molecular/métodos , NanopartículasRESUMO
Short-chain acylations of lysine residues in eukaryotic proteins are recognized as essential posttranslational chemical modifications (PTMs) that regulate cellular processes from transcription, cell cycle, metabolism, to signal transduction. Lysine butyrylation was initially discovered as a normal straight chain butyrylation (Knbu). Here we report its structural isomer, branched chain butyrylation, i.e. lysine isobutyrylation (Kibu), existing as a new PTM on nuclear histones. Uniquely, isobutyryl-CoA is derived from valine catabolism and branched chain fatty acid oxidation which is distinct from the metabolism of n-butyryl-CoA. Several histone acetyltransferases were found to possess lysine isobutyryltransferase activity in vitro, especially p300 and HAT1. Transfection and western blot experiments showed that p300 regulated histone isobutyrylation levels in the cell. We resolved the X-ray crystal structures of HAT1 in complex with isobutyryl-CoA that gleaned an atomic level insight into HAT-catalyzed isobutyrylation. RNA-Seq profiling revealed that isobutyrate greatly affected the expression of genes associated with many pivotal biological pathways. Together, our findings identify Kibu as a novel chemical modification mark in histones and suggest its extensive role in regulating epigenetics and cellular physiology.
Assuntos
Código das Histonas , Isobutiratos/metabolismo , Lisina Acetiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Acil Coenzima A/síntese química , Acil Coenzima A/metabolismo , Acilação , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Células HEK293 , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Isobutiratos/farmacologia , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem , Valina/metabolismo , Fatores de Transcrição de p300-CBPRESUMO
Enzymatic suicide inactivation, a route of permanent enzyme inhibition, is the mechanism of action for a wide array of pharmaceuticals. Here, we developed the first nanosensor that selectively reports the suicide inactivation pathway of an enzyme. The sensor is based on modulation of the near-infrared fluorescence of an enzyme-bound carbon nanotube. The nanosensor responded selectively to substrate-mediated suicide inactivation of the tyrosinase enzyme via bathochromic shifting of the nanotube emission wavelength. Mechanistic investigations revealed that singlet oxygen generated by the suicide inactivation pathway induced the response. We used the nanosensor to quantify the degree of enzymatic inactivation by measuring response rates to small molecule tyrosinase modulators. This work resulted in a new capability of interrogating a specific route of enzymatic death. Potential applications include drug screening and hit-validation for compounds that elicit or inhibit enzymatic inactivation and single-molecule measurements to assess population heterogeneity in enzyme activity.
Assuntos
Monofenol Mono-Oxigenase , Nanotubos de Carbono , Fluorescência , Humanos , Cinética , Monofenol Mono-Oxigenase/metabolismo , NanotecnologiaRESUMO
Protein lysine methylation is a distinct posttranslational modification that causes minimal changes in the size and electrostatic status of lysine residues. Lysine methylation plays essential roles in regulating fates and functions of target proteins in an epigenetic manner. As a result, substrates and degrees (free versus mono/di/tri) of protein lysine methylation are orchestrated within cells by balanced activities of protein lysine methyltransferases (PKMTs) and demethylases (KDMs). Their dysregulation is often associated with neurological disorders, developmental abnormalities, or cancer. Methyllysine-containing proteins can be recognized by downstream effector proteins, which contain methyllysine reader domains, to relay their biological functions. While numerous efforts have been made to annotate biological roles of protein lysine methylation, limited work has been done to uncover mechanisms associated with this modification at a molecular or atomic level. Given distinct biophysical and biochemical properties of methyllysine, this review will focus on chemical and biochemical aspects in addition, recognition, and removal of this posttranslational mark. Chemical and biophysical methods to profile PKMT substrates will be discussed along with classification of PKMT inhibitors for accurate perturbation of methyltransferase activities. Semisynthesis of methyllysine-containing proteins will also be covered given the critical need for these reagents to unambiguously define functional roles of protein lysine methylation.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Lisina/química , Coenzimas/química , Coenzimas/metabolismo , Histona Desmetilases/química , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/classificação , Humanos , Lisina/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Especificidade por SubstratoRESUMO
Transition state stabilization is essential for rate acceleration of enzymatic reactions. Despite extensive studies on various transition state structures of enzymes, an intriguing puzzle is whether an enzyme can accommodate multiple transition states (TSs) to catalyze a chemical reaction. It is experimentally challenging to study this proposition in terms of the choices of suitable enzymes and the feasibility to distinguish multiple TSs. As a paradigm with the protein lysine methyltransferase (PKMT) SET7/9 paired with its physiological substrates H3 and p53, their TSs were solved with experimental kinetic isotope effects as computational constraints. Remarkably, SET7/9 adopts two structurally distinct TSs, a nearly symmetric SN2 and an extremely early SN2, for H3K4 and p53K372 methylation, respectively. The two TSs are also different from those previously revealed for other PKMTs. The setting of multiple TSs is expected to be essential for SET7/9 and likely other PKMTs to act on broad substrates with high efficiency.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , S-Adenosilmetionina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Catálise , Histona-Lisina N-Metiltransferase/química , Histonas/química , Humanos , Cinética , Lisina/química , Lisina/metabolismo , Metilação , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , S-Adenosilmetionina/química , Proteína Supressora de Tumor p53/químicaRESUMO
Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13C KIE of 1.04, an inverse intrinsic α-secondary CD3 KIE of 0.90, and a small but statistically significant inverse CD3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analog Se-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.
Assuntos
Histona-Lisina N-Metiltransferase/química , Isótopos/química , Ligação Competitiva , Catálise , Ciclo Celular , Simulação por Computador , Histonas/química , Humanos , Cinética , Lisina/química , Metilação , Modelos Moleculares , Modelos Teóricos , Metástase Neoplásica , Peptídeos/química , Estrutura Secundária de Proteína , S-Adenosilmetionina/química , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-ß-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.
Assuntos
Antituberculosos/metabolismo , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/metabolismo , Antituberculosos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Benzimidazóis/química , Benzimidazóis/metabolismo , Regulação Bacteriana da Expressão Gênica , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutação , Mycobacterium tuberculosis/genética , Domínios Proteicos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , S-Adenosilmetionina/metabolismoRESUMO
RNA labeling is crucial for the study of RNA structure and metabolism. Herein we report N6-allyladenosine (a6A) as a new small molecule for RNA labeling through both metabolic and enzyme-assisted manners. a6A behaves like A and can be metabolically incorporated into newly synthesized RNAs inside mammalian cells. We also show that human RNA N6-methyladenosine (m6A) methyltransferases METTL3/METTL14 can work with a synthetic cofactor, namely allyl-SAM (S-adenosyl methionine with methyl replaced by allyl) in order to site-specifically install an allyl group to the N6-position of A within specific sequence to generate a6A-labeled RNAs. The iodination of N6-allyl group of a6A under mild buffer conditions spontaneously induces the formation of N1,N6-cyclized adenosine and creates mutations at its opposite site during complementary DNA synthesis of reverse transcription. The existing m6A in RNA is inert to methyltransferase-assisted allyl labeling, which offers a chance to differentiate m6A from A at individual RNA sites. Our work demonstrates a new method for RNA labeling, which could find applications in developing sequencing methods for nascent RNAs and RNA modifications.
Assuntos
Adenosina/análogos & derivados , Bioensaio , Mutação , RNA/genética , RNA/metabolismo , Adenosina/metabolismo , Humanos , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Coloração e Rotulagem/métodosRESUMO
PRMT6 is a type I protein arginine methyltransferase, generating the asymmetric dimethylarginine mark on proteins such as histone H3R2. Asymmetric dimethylation of histone H3R2 by PRMT6 acts as a repressive mark that antagonizes trimethylation of H3 lysine 4 by the MLL histone H3K4 methyltransferase. PRMT6 is overexpressed in several cancer types, including prostate, bladder and lung cancers; therefore, it is of great interest to develop potent and selective inhibitors for PRMT6. Here, we report the synthesis of a potent bisubstrate inhibitor GMS [6'-methyleneamine sinefungin, an analog of sinefungin (SNF)], and the crystal structures of human PRMT6 in complex, respectively, with S-adenosyl-L-homocysteine (SAH) and the bisubstrate inhibitor GMS that shed light on the significantly improved inhibition effect of GMS on methylation activity of PRMT6 compared with SAH and an S-adenosyl-L-methionine competitive methyltransferase inhibitor SNF. In addition, we also crystallized PRMT6 in complex with SAH and a short arginine-containing peptide. Based on the structural information here and available in the PDB database, we proposed a mechanism that can rationalize the distinctive arginine methylation product specificity of different types of arginine methyltransferases and pinpoint the structural determinant of such a specificity.
Assuntos
Arginina/metabolismo , Proteínas Nucleares/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Cristalografia por Raios X , Humanos , Metilação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformação Proteica , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Homologia de Sequência de AminoácidosRESUMO
Resveratrol is a natural polyphenol with plethora of biological activities. Resveratrol has previously shown to decrease DNA-methyltransferase (DNMT) enzymes expression and to reactivate silenced tumor suppressor genes. Currently, it seems that no resveratrol analogs have been developed as DNMT inhibitors. Recently, we reported the synthesis of resveratrol-salicylate derivatives and by examining the chemical structure of these analogs, we proposed that these compounds could exhibit DNMT inhibition especially that they resembled NSC 14778, a compound we previously identified as a DNMT inhibitor by virtual screening. Indeed, using in vitro DNMT inhibition assay, some of the resveratrol-salicylate analogs we screened in this work that showed selective inhibition against DNMT3 enzymes which were greater than resveratrol. A molecular docking study revealed key binding interactions with DNMT3A and DNMT3B enzymes. In addition, the most active analog, 10 showed considerable cytotoxicity against three human cancer cells; HT-29, HepG2 and SK-BR-3, which was greater than resveratrol. Further studies are needed to understand the anticancer mechanisms of these derivatives.
Assuntos
Antineoplásicos/farmacologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Salicilatos/farmacologia , Estilbenos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HT29 , Células Hep G2 , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Resveratrol , Salicilatos/química , Estilbenos/química , DNA Metiltransferase 3BRESUMO
Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. With two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme-cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-ß-sp(2) carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.
Assuntos
Antígenos de Histocompatibilidade , Histona-Lisina N-Metiltransferase , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Neoplasias , Neoplasias , Proteína-Arginina N-Metiltransferases , S-Adenosilmetionina , Células HEK293 , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metilação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/química , Neoplasias/genética , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/genética , S-Adenosilmetionina/metabolismo , Especificidade por SubstratoRESUMO
Methionine adenosyltransferases (MATs) catalyze the formation of S-adenosyl-l-methionine (SAM) inside living cells. Recently, S-alkyl analogues of SAM have been documented as cofactor surrogates to label novel targets of methyltransferases. However, these chemically synthesized SAM analogues are not suitable for cell-based studies because of their poor membrane permeability. This issue was recently addressed under a cellular setting through a chemoenzymatic strategy to process membrane-permeable S-alkyl analogues of methionine (SAAMs) into the SAM analogues with engineered MATs. Here we describe a general sensitive activity assay for engineered MATs by converting the reaction products into S-alkylthioadenosines, followed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) quantification. With this assay, 40 human MAT mutants were evaluated against 7 SAAMs as potential substrates. The structure-activity relationship revealed that, besides better engaged SAAM binding by the MAT mutants (lower Km value in contrast to native MATs), the gained activity toward the bulky SAAMs stems from their ability to maintain the desired linear SN2 transition state (reflected by higher kcat value). Here the I117A mutant of human MATI was identified as the most active variant for biochemical production of SAM analogues from diverse SAAMs.
Assuntos
Ensaios Enzimáticos/métodos , Metionina Adenosiltransferase/metabolismo , Engenharia de Proteínas , S-Adenosilmetionina/análogos & derivados , S-Adenosilmetionina/metabolismo , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Cinética , Metionina Adenosiltransferase/química , Metionina Adenosiltransferase/genética , Dados de Sequência Molecular , Mutação , Ratos , Especificidade por SubstratoRESUMO
The identification of specific substrates of glutathione S-transferases (GSTs) is important for understanding drug metabolism. A method termed bioorthogonal identification of GST substrates (BIGS) was developed, in which a reduced glutathione (GSH) analogue was developed for recognition by a rationally engineered GST to label the substrates of the corresponding native GST. A K44G-W40A-R41A mutant (GST-KWR) of the mu-class glutathione S-transferases GSTM1 was shown to be active with a clickable GSH analogue (GSH-R1) as the cosubstrate. The GSH-R1 conjugation products can react with an azido-based biotin probe for ready enrichment and MS identification. Proof-of-principle studies were carried to detect the products of GSH-R1 conjugation to 1-chloro-2,4-dinitrobenzene (CDNB) and dopamine quinone. The BIGS technology was then used to identify GSTM1 substrates in the Chinese herbal medicine Ganmaocongji.
Assuntos
Glutationa Transferase/metabolismo , Química Click , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/metabolismo , Glutationa Transferase/química , Glutationa Transferase/genética , Espectrometria de Massas , Modelos Moleculares , Análise Serial de Proteínas , Engenharia de Proteínas , Especificidade por SubstratoRESUMO
Protein arginine methylation has emerged as a key post-translational modification responsible for many facets of eukaryotic gene expression. To better understand the extent of this modification in cellular pathways, we carried out bioorthogonal methylation profiling in Saccharomyces cerevisiae to comprehensively identify the in vivo substrates of the major yeast protein arginine methyltransferase Hmt1. Gene ontology analysis of candidate substrates revealed an enrichment of proteins involved in the process of translation. We verified one such factor, eIF1A, by in vitro methylation. Three sites on eIF1A were found to be responsible for its methylation: R13, R14, and R62, with varied capacity by which each site contributed to the overall methylation capacity in vitro . To determine the role of methylation in eIF1A function, we used a battery of arginine-to-alanine substitution mutants to evaluate translation fidelity in these mutants. Our data show that substitution mutants at R13 and R14 in the N-terminal tail improved the fidelity of start codon recognition in an initiation fidelity assay. Overall, our data suggest that Hmt1-mediated methylation of eIF1A fine-tunes the fidelity of start codon recognition for proper translation initiation.
RESUMO
Activating mutations in PIK3CA are frequently found in estrogen-receptor-positive (ER+) breast cancer, and the combination of the phosphatidylinositol 3-kinase (PI3K) inhibitor alpelisib with anti-ER inhibitors is approved for therapy. We have previously demonstrated that the PI3K pathway regulates ER activity through phosphorylation of the chromatin modifier KMT2D. Here, we discovered a methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. SMYD2 loss attenuates alpelisib-induced KMT2D chromatin binding and alpelisib-mediated changes in gene expression, including ER-dependent transcription. Knockdown or pharmacological inhibition of SMYD2 sensitizes breast cancer cells, patient-derived organoids, and tumors to PI3K/AKT inhibition and endocrine therapy in part through KMT2D K1330 methylation. Together, our findings uncover a regulatory crosstalk between post-translational modifications that fine-tunes KMT2D function at the chromatin. This provides a rationale for the use of SMYD2 inhibitors in combination with PI3Kα/AKT inhibitors in the treatment of ER+/PIK3CA mutant breast cancer.
Assuntos
Neoplasias da Mama , Cromatina , Histona-Lisina N-Metiltransferase , Humanos , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Neoplasias da Mama/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Metilação/efeitos dos fármacos , Linhagem Celular Tumoral , Animais , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Receptores de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacosRESUMO
Protein methyltransferases (PMTs) have emerged as important epigenetic regulators in myriad biological processes in both normal physiology and disease conditions. However, elucidating PMT-regulated epigenetic processes has been hampered by ambiguous knowledge about in vivo activities of individual PMTs particularly because of their overlapping but nonredundant functions. To address limitations of conventional approaches in mapping chromatin modification of specific PMTs, we have engineered the chromatin-modifying apparatus and formulated a novel technology, termed clickable chromatin enrichment with parallel DNA sequencing (CliEn-seq), to probe genome-wide chromatin modification within living cells. The three-step approach of CliEn-seq involves in vivo synthesis of S-adenosyl-L-methionine (SAM) analogues from cell-permeable methionine analogues by engineered SAM synthetase (methionine adenosyltransferase or MAT), in situ chromatin modification by engineered PMTs, subsequent enrichment and sequencing of the uniquely modified chromatins. Given critical roles of the chromatin-modifying enzymes in epigenetics and structural similarity among many PMTs, we envision that the CliEn-seq technology is generally applicable in deciphering chromatin methylation events of individual PMTs in diverse biological settings.
Assuntos
Cromatina/metabolismo , Metilação de DNA , Engenharia Genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Processamento de Proteína Pós-Traducional , Cromatina/genética , Epigenômica , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/isolamento & purificação , Células HEK293 , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/isolamento & purificação , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/isolamento & purificação , Humanos , Modelos Moleculares , S-Adenosilmetionina/biossíntese , S-Adenosilmetionina/químicaRESUMO
Posttranslational methylation by S-adenosyl-L-methionine(SAM)-dependent methyltransferases plays essential roles in modulating protein function in both normal and disease states. As such, there is a growing need to develop chemical reporters to examine the physiological and pathological roles of protein methyltransferases. Several sterically bulky SAM analogues have previously been used to label substrates of specific protein methyltransferases. However, broad application of these compounds has been limited by their general incompatibility with native enzymes. Here we report a SAM surrogate, ProSeAM (propargylic Se-adenosyl-l-selenomethionine), as a reporter of methyltransferases. ProSeAM can be processed by multiple protein methyltransferases for substrate labeling. In contrast, sulfur-based propargylic SAM undergoes rapid decomposition at physiological pH, likely via an allene intermediate. In conjunction with fluorescent/affinity-based azide probes, copper-catalyzed azide-alkyne cycloaddition chemistry, in-gel fluorescence visualization and proteomic analysis, we further demonstrated ProSeAM's utility to profile substrates of endogenous methyltransferases in diverse cellular contexts. These results thus feature ProSeAM as a convenient probe to study the activities of endogenous protein methyltransferases.
Assuntos
Metiltransferases/metabolismo , Selenometionina/análogos & derivados , Linhagem Celular Tumoral , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Metilação , Metiltransferases/química , Modelos Moleculares , Estrutura Molecular , Selenometionina/síntese química , Selenometionina/química , Selenometionina/metabolismoRESUMO
Protein methyltransferases (PMTs) play critical roles in multiple biological processes. Because PMTs often function in vivo through forming multimeric protein complexes, dissecting their activities in the native contexts is challenging but relevant. To address such a need, we envisioned a Bioorthogonal Profiling of Protein Methylation (BPPM) technology, in which a SAM analogue cofactor can be utilized by multiple rationally engineered PMTs to label substrates of the corresponding native PMTs. Here, 4-azidobut-2-enyl derivative of S-adenosyl-L-methionine (Ab-SAM) was reported as a suitable BPPM cofactor. The resultant cofactor-enzyme pairs were implemented to label specifically the substrates of closely related PMTs (e.g., EuHMT1 and EuHMT2) in a complex cellular mixture. The BPPM approach, coupled with mass spectrometric analysis, enables the identification of the nonhistone targets of EuHMT1/2. Comparison of EuHMT1/2's methylomes indicates that the two human PMTs, although similar in terms of their primary sequences, can act on the distinct sets of nonhistone targets. Given the conserved active sites of PMTs, Ab-SAM and its use in BPPM are expected to be transferable to other PMTs for target identification.
Assuntos
Azidas/química , Proteínas Metiltransferases/química , Proteínas Metiltransferases/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Homologia de Sequência de Aminoácidos , Química Click , Coenzimas/metabolismo , Células HEK293 , Humanos , Metilação , Proteoma/metabolismo , Reprodutibilidade dos TestesRESUMO
Epigenetic regulation is involved in numerous physiological and pathogenic processes. Among the key regulators that orchestrate epigenetic signaling are over 50 human protein lysine methyltransferases (PKMTs). Interrogation of the functions of individual PKMTs can be facilitated by target-specific PKMT inhibitors. Given the emerging need for such small molecules, we envisioned an approach to identify target-specific methyltransferase inhibitors by screening privileged small-molecule scaffolds against diverse methyltransferases. In this work, we demonstrated the feasibility of such an approach by identifying the inhibitors of SETD2. N-propyl sinefungin (Pr-SNF) was shown to interact preferentially with SETD2 by matching the distinct transition-state features of SETD2's catalytically active conformer. With Pr-SNF as a structure probe, we further revealed the dual roles of SETD2's post-SET loop in regulating substrate access through a distinct topological reconfiguration. Privileged sinefungin scaffolds are expected to have broad use as structure and chemical probes of methyltransferases.