RESUMEN
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.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , S-Adenosilmetionina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Catálisis , N-Metiltransferasa de Histona-Lisina/química , Histonas/química , Humanos , Cinética , Lisina/química , Lisina/metabolismo , Metilación , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional , S-Adenosilmetionina/química , Proteína p53 Supresora de Tumor/químicaRESUMEN
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.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/química , Isótopos/química , Unión Competitiva , Catálisis , Ciclo Celular , Simulación por Computador , Histonas/química , Humanos , Cinética , Lisina/química , Metilación , Modelos Moleculares , Modelos Teóricos , Metástasis de la Neoplasia , Péptidos/química , Estructura Secundaria de Proteína , S-Adenosilmetionina/química , Programas Informáticos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por SustratoRESUMEN
Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphoproteins in normal and diseased states is critical to fully characterize cell signaling. Towards phosphoprotein analysis tools, our lab reported kinase-catalyzed labeling where γ-phosphate modified ATP analogs are utilized by kinases to label peptides or protein substrates with a functional tag. In particular, the ATP-biotin analog was developed for kinase-catalyzed biotinylation. However, kinase-catalyzed labeling has been tested rigorously with only a few kinases, preventing use of ATP-biotin as a general tool. Here, biotinylation experiments, gel or HPLC-based quantification, and kinetic measurements indicated that twenty-five kinases throughout the kinome tree accepted ATP-biotin as a cosubstrate. With this rigorous characterization of ATP-biotin compatibility, kinase-catalyzed labeling is now immediately useful for studying phosphoproteins and characterizing the role of phosphorylation in various biological events.
Asunto(s)
Biotina/análogos & derivados , Proteínas Quinasas/química , Biotina/química , Biotinilación , Humanos , Cinética , Simulación del Acoplamiento Molecular , FosforilaciónRESUMEN
Kinase-catalyzed protein phosphorylation is an important post-translational modification that regulates a variety of cellular functions. Identification of the many substrates of a specific kinase is critical to fully characterize cell biology. Unfortunately, kinase-substrate interactions are often transient, which makes their identification challenging. Here, the transient kinase-substrate complex was stabilized by covalent crosslinking using γ-phosphate modified ATP analogs. Building upon prior use of an ATP-aryl azide photocrosslinking analog, we report here the creation of an ATP-benzophenone photocrosslinking analog. ATP-benzophenone displayed a higher conversion percentage but more diffuse crosslinking compared to the ATP-aryl azide analog. A docking study was also performed to rationalize the conversion and crosslinking data. In total, the photocrosslinking ATP analogs produced stable kinase-substrate complexes that are suitable for future applications characterizing cell signaling pathways.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Fosforilación/fisiología , Proteínas Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Humanos , Procesamiento Proteico-Postraduccional , Transducción de Señal , Especificidad por SustratoRESUMEN
Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphorylation is critical to understand cell biology. Our lab recently discovered kinase-catalyzed biotinylation, where ATP-biotin is utilized by kinases to label phosphopeptides or phosphoproteins with a biotin tag. To exploit kinase-catalyzed biotinylation for phosphoprotein purification and identification in a cellular context, the susceptibility of the biotin tag to phosphatases was characterized. We found that the phosphorylbiotin group on peptide and protein substrates was relatively insensitive to protein phosphatases. To understand how phosphatase stability would impact phosphoproteomics research applications, kinase-catalyzed biotinylation of cell lysates was performed in the presence of kinase or phosphatase inhibitors. We found that biotinylation with ATP-biotin was sensitive to inhibitors, although with variable effects compared to ATP phosphorylation. The results suggest that kinase-catalyzed biotinylation is well suited for phosphoproteomics studies, with particular utility towards monitoring low-abundance phosphoproteins or characterizing the influence of inhibitor drugs on protein phosphorylation.
Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , Proteómica , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Biocatálisis , Biotina/química , Biotina/metabolismo , Biotinilación , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas/química , Fosforilación/efectos de los fármacos , Proteínas Quinasas/química , Especificidad por SustratoRESUMEN
Kinase-catalyzed protein phosphorylation is an important biochemical process involved in cellular functions. We recently discovered that kinases promiscuously accept γ-modified ATP analogues as cosubstrates and used several ATP analogues as tools for studying protein phosphorylation. Herein, we explore the structural requirements of γ-modified ATP analogues for kinase compatibility. To understand the influence of linker length and composition, a series of ATP analogues was synthesized, and the efficiency of kinase-catalyzed labeling was determined by quantitative mass spectrometry. This study on factors influencing kinase cosubstrate promiscuity will enable design of ATP analogues for a variety of kinase-catalyzed labeling reactions.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Aminas/química , Proteínas Quinasas Dependientes de AMP Cíclico/química , Péptidos/química , Coloración y Etiquetado/métodos , Sitios de Unión , Catálisis , Cinética , Espectrometría de Masas , Simulación del Acoplamiento Molecular , Fosforilación , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Functional studies of the RNA N6-methyladenosine (m6A) modification have been limited by an inability to map individual m6A-modified sites in whole transcriptomes. To enable such studies, here, we introduce m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq), a method for quantitative, whole-transcriptome mapping of m6A at single-nucleotide resolution. The method requires only ~30 ng of poly(A) or rRNA-depleted RNA. We mapped m6A modification stoichiometries in RNA from cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We identified numerous cell-state-specific m6A sites whose methylation status was highly dynamic during cell differentiation. We observed changes of m6A stoichiometry as well as expression levels of transcripts encoding or regulated by key transcriptional factors (TFs) critical for HSPC differentiation. m6A-SAC-seq is a quantitative method to dissect the dynamics and functional roles of m6A sites in diverse biological processes using limited input RNA.
Asunto(s)
Procesamiento Postranscripcional del ARN , Transcriptoma , Animales , Humanos , Mamíferos/genética , Metilación , ARN/genética , ARN/metabolismo , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/genética , Transcriptoma/genéticaRESUMEN
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.
Asunto(s)
Diferenciación Celular , Fosfatasas de Especificidad Dual , Megacariocitos , Fosfatasas de la Proteína Quinasa Activada por Mitógenos , Adulto , Animales , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Arginina/metabolismo , Línea Celular , Fosfatasas de Especificidad Dual/metabolismo , Estabilidad de Enzimas , Células HEK293 , Sistema de Señalización de MAP Quinasas , Megacariocitos/citología , Megacariocitos/enzimología , Metilación , Ratones Endogámicos C57BL , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Síndromes Mielodisplásicos/enzimología , Síndromes Mielodisplásicos/patología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Poliubiquitina/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteolisis , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , UbiquitinaciónRESUMEN
Kinase-catalyzed protein phosphorylation plays an essential role in a variety of biological processes. Methods to detect phosphoproteins and phosphopeptides in cellular mixtures will aid in cell biological and signaling research. Our laboratory recently discovered the utility of γ-modified ATP analogues as tools for studying phosphorylation. Specifically, ATP-biotin can be used for labeling and visualizing phosphoproteins from cell lysates. Because the biotin tag is suitable for protein detection, the biotinylation reaction can be applied to multiple phosphoproteomics applications. Herein we report a general protocol for labeling phosphopeptides and phosphoproteins in biological samples using kinase-catalyzed biotinylation.