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2.
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
3.
SLAS Discov ; 26(9): 1200-1211, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34192965

RESUMO

The COVID-19 pandemic has clearly brought the healthcare systems worldwide to a breaking point, along with devastating socioeconomic consequences. The SARS-CoV-2 virus, which causes the disease, uses RNA capping to evade the human immune system. Nonstructural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small-molecule inhibitors of nsp14 methyltransferase (MTase) activity, we developed and employed a radiometric MTase assay to screen a library of 161 in-house synthesized S-adenosylmethionine (SAM) competitive MTase inhibitors and SAM analogs. Among six identified screening hits, SS148 inhibited nsp14 MTase activity with an IC50 value of 70 ± 6 nM and was selective against 20 human protein lysine MTases, indicating significant differences in SAM binding sites. Interestingly, DS0464 with an IC50 value of 1.1 ± 0.2 µM showed a bisubstrate competitive inhibitor mechanism of action. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein MTases. The structure-activity relationship provided by these compounds should guide the optimization of selective bisubstrate nsp14 inhibitors and may provide a path toward a novel class of antivirals against COVID-19, and possibly other coronaviruses.


Assuntos
COVID-19/genética , Exorribonucleases/genética , Ligação Proteica/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , Antivirais/farmacologia , Sítios de Ligação/genética , COVID-19/virologia , Humanos , Metilação , Pandemias , RNA Viral/genética , SARS-CoV-2/patogenicidade , Replicação Viral/genética
4.
Cell Rep ; 36(4): 109421, 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34320342

RESUMO

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

5.
bioRxiv ; 2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33619486

RESUMO

The COVID-19 pandemic has clearly brought the healthcare systems world-wide to a breaking point along with devastating socioeconomic consequences. The SARS-CoV-2 virus which causes the disease uses RNA capping to evade the human immune system. Non-structural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small molecule inhibitors of nsp14 methyltransferase (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC 50 value of 70 ± 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC 50 value of 1.1 ± 0.2 µM showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses.

6.
Nat Chem Biol ; 17(2): 129-137, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33414556

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 , Nanopartículas
7.
Biochimie ; 183: 100-107, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33476699

RESUMO

The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme's active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.


Assuntos
Inibidores Enzimáticos/química , Metilenotetra-Hidrofolato Redutase (NADPH2)/antagonistas & inibidores , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , S-Adenosilmetionina/química , Regulação Alostérica , Humanos , Domínios Proteicos
8.
Nucleic Acids Res ; 49(1): 177-189, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33313896

RESUMO

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-CBP
9.
Nano Lett ; 20(11): 7819-7827, 2020 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-33119310

RESUMO

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 , Nanotecnologia
11.
Cell Chem Biol ; 27(8): 1073-1083.e12, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32521230

RESUMO

ATP is an important energy metabolite and allosteric signal in health and disease. ATP-interacting proteins, such as P2 receptors, control inflammation, cell death, migration, and wound healing. However, identification of allosteric ATP sites remains challenging, and our current inventory of ATP-controlled pathways is likely incomplete. Here, we develop and verify mipATP as a minimally invasive photoaffinity probe for ATP-interacting proteins. Its N6 functionalization allows target enrichment by UV crosslinking and conjugation to reporter tags by "click" chemistry. The additions are compact, allowing mipATP to completely retain the calcium signaling responses of native ATP in vitro and in vivo. mipATP specifically enriched for known nucleotide binders in A549 cell lysates and membrane fractions. In addition, it retrieved unannotated ATP interactors, such as the FAS receptor, CD44, and various SLC transporters. Thus, mipATP is a promising tool to identify allosteric ATP sites in the proteome.


Assuntos
Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo , Proteoma/análise , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/síntese química , Aminoácidos/química , Aminoácidos/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Sinalização do Cálcio , Calmodulina/genética , Calmodulina/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Cromatografia Líquida de Alta Pressão , Química Click , Corantes Fluorescentes/química , Humanos , Marcação por Isótopo , Larva/metabolismo , Imagem Óptica , Proteoma/metabolismo , Espectrometria de Massas em Tandem , Raios Ultravioleta , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo
12.
Elife ; 82019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31657716

RESUMO

CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription. Its pharmacological inhibition is a promising anti-cancer strategy. Here SKI-73 (6a in this work) is presented as a CARM1 chemical probe with pro-drug properties. SKI-73 (6a) can rapidly penetrate cell membranes and then be processed into active inhibitors, which are retained intracellularly with 10-fold enrichment for several days. These compounds were characterized for their potency, selectivity, modes of action, and on-target engagement. SKI-73 (6a) recapitulates the effect of CARM1 knockout against breast cancer cell invasion. Single-cell RNA-seq analysis revealed that the SKI-73(6a)-associated reduction of invasiveness acts by altering epigenetic plasticity and suppressing the invasion-prone subpopulation. Interestingly, SKI-73 (6a) and CARM1 knockout alter the epigenetic plasticity with remarkable difference, suggesting distinct modes of action for small-molecule and genetic perturbations. We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process.


Assuntos
Neoplasias da Mama/genética , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Epigenômica/métodos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Algoritmos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Feminino , Humanos , Cinética , Células MCF-7 , Modelos Químicos , Estrutura Molecular , Ligação Proteica , Proteína-Arginina N-Metiltransferases/metabolismo
13.
Elife ; 82019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31081496

RESUMO

Elucidating the conformational heterogeneity of proteins is essential for understanding protein function and developing exogenous ligands. With the rapid development of experimental and computational methods, it is of great interest to integrate these approaches to illuminate the conformational landscapes of target proteins. SETD8 is a protein lysine methyltransferase (PKMT), which functions in vivo via the methylation of histone and nonhistone targets. Utilizing covalent inhibitors and depleting native ligands to trap hidden conformational states, we obtained diverse X-ray structures of SETD8. These structures were used to seed distributed atomistic molecular dynamics simulations that generated a total of six milliseconds of trajectory data. Markov state models, built via an automated machine learning approach and corroborated experimentally, reveal how slow conformational motions and conformational states are relevant to catalysis. These findings provide molecular insight on enzymatic catalysis and allosteric mechanisms of a PKMT via its detailed conformational landscape.


Assuntos
Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Conformação Proteica
14.
J Am Chem Soc ; 141(20): 8064-8067, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31034218

RESUMO

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/química
15.
Nat Commun ; 10(1): 19, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30604761

RESUMO

Protein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4+ T cell differentiation assays revealing the potential of individual probes to alter multiple T cell subpopulations which may have implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity. This chemical probe collection and associated data form a resource for the study of methylation-mediated signaling in epigenetics, inflammation and beyond.


Assuntos
Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Histonas/metabolismo , Proteínas Metiltransferases/antagonistas & inibidores , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Ensaios Enzimáticos/métodos , Epigenômica/métodos , Células HEK293 , Histona-Lisina N-Metiltransferase , Humanos , Células Jurkat , Metilação/efeitos dos fármacos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional/genética , Células Th1/efeitos dos fármacos , Células Th1/fisiologia
16.
J Mol Biol ; 430(21): 4168-4182, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30153436

RESUMO

Activation of the retinoic acid (RA) signaling pathway is important for controlling embryonic stem cell differentiation and development. Modulation of this pathway occurs through the recruitment of different epigenetic regulators at the retinoic acid receptors (RARs) located at RA-responsive elements and/or RA-responsive regions of RA-regulated genes. Coactivator-associated arginine methyltransferase 1 (CARM1, PRMT4) is a protein arginine methyltransferase that also functions as a transcriptional coactivator. Previous studies highlight CARM1's importance in the differentiation of different cell types. We address CARM1 function during RA-induced differentiation of murine embryonic stem cells (mESCs) using shRNA lentiviral transduction and CRISPR/Cas9 technology to deplete CARM1 in mESCs. We identify CARM1 as a novel transcriptional coactivator required for the RA-associated decrease in Rex1 (Zfp42) and for the RA induction of a subset of RA-regulated genes, including CRABP2 and NR2F1 (Coup-TF1). Furthermore, CARM1 is required for mESCs to differentiate into extraembryonic endoderm in response to RA. We next characterize the epigenetic mechanisms that contribute to RA-induced transcriptional activation of CRABP2 and NR2F1 in mESCs and show for the first time that CARM1 is required for this activation. Collectively, our data demonstrate that CARM1 is required for transcriptional activation of a subset of RA target genes, and we uncover changes in the recruitment of Suz12 and the epigenetic H3K27me3 and H3K27ac marks at gene regulatory regions for CRABP2 and NR2F1 during RA-induced differentiation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , Animais , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/citologia , Camundongos , Camundongos Knockout , Proteína-Arginina N-Metiltransferases/genética , RNA Interferente Pequeno/genética
17.
Chem Rev ; 118(14): 6656-6705, 2018 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-29927582

RESUMO

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 Substrato
18.
J Am Chem Soc ; 139(48): 17213-17216, 2017 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-29116772

RESUMO

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/métodos
20.
Proc Natl Acad Sci U S A ; 113(52): E8369-E8378, 2016 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-27940912

RESUMO

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 Substrato
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