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1.
Biochemistry ; 60(34): 2593-2609, 2021 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-34411482

RESUMEN

SHP2 is a protein tyrosine phosphatase that plays a critical role in the full activation of the Ras-MAPK pathway upon stimulation of receptor tyrosine kinases, which are frequently amplified or mutationally activated in human cancer. In addition, activating mutations in SHP2 result in developmental disorders and hematologic malignancies. Several allosteric inhibitors have been developed for SHP2 and are currently in clinical trials. Here, we report the development and evaluation of a SHP2 PROTAC created by conjugating RMC-4550 with pomalidomide using a PEG linker. This molecule is highly selective for SHP2, induces degradation of SHP2 in leukemic cells at submicromolar concentrations, inhibits MAPK signaling, and suppresses cancer cell growth. SHP2 PROTACs serve as an alternative strategy for targeting ERK-dependent cancers and are useful tools alongside allosteric inhibitors for dissecting the mechanisms by which SHP2 exerts its oncogenic activity.


Asunto(s)
Antineoplásicos/farmacología , Metanol/análogos & derivados , Neoplasias/tratamiento farmacológico , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirazinas/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Terapia Molecular Dirigida , Mutación , Neoplasias/metabolismo , Neoplasias/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteolisis , Transducción de Señal
2.
J Biol Chem ; 293(33): 12770-12780, 2018 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-29959229

RESUMEN

Set7/9 (also known as Set7, Set9, Setd7, and Kmt7) is a lysine methyltransferase that catalyzes the methylation of multiple substrates, including histone H3 and non-histone proteins. Although not essential for normal development and physiology, Set7/9-mediated methylation events play important roles in regulating cellular pathways involved in various human diseases, making Set7/9 a promising therapeutic target. Multiple Set7/9 inhibitors have been developed, which exhibit varying degrees of potency and selectivity in vitro However, validation of these compounds in vivo has been hampered by the lack of a reliable cellular biomarker for Set7/9 activity. Here, we report the identification of Rpl29, a ribosomal protein abundantly expressed in all cell types, as a major substrate of Set7/9. We show that Rpl29 lysine 5 (Rpl29K5) is methylated exclusively by Set7/9 and can be demethylated by Lsd1 (also known as Kdm1a). Rpl29 is not a core component of the ribosome translational machinery and plays a regulatory role in translation efficiency. Our results indicate that Rpl29 methylation has no effect on global protein synthesis but affects Rpl29 subcellular localization. Using an Rpl29 methylation-specific antibody, we demonstrate that Rpl29K5 methylation is present ubiquitously and validate that (R)-PFI-2, a Set7/9 inhibitor, efficiently reduces Rpl29K5 methylation in cell lines. Thus, Rpl29 methylation can serve as a specific cellular biomarker for measuring Set7/9 activity.


Asunto(s)
Factores de Coagulación Sanguínea/genética , Metilación de ADN , Regulación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Lisina/química , Proteínas Ribosómicas/fisiología , Animales , Factores de Coagulación Sanguínea/metabolismo , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Masculino , Ratones Noqueados , Procesamiento Proteico-Postraduccional , Proteínas de Unión al ARN , Transcripción Genética
3.
Bioorg Med Chem ; 25(24): 6479-6485, 2017 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-29089257

RESUMEN

The PTPN11 oncogene encodes the cytoplasmic protein tyrosine phosphatase SHP2, which, through its role in multiple signaling pathways, promotes the progression of hematological malignancies and other cancers. Here, we employ high-throughput screening to discover a lead chemical scaffold, the benzothiazolopyrimidones, that allosterically inhibits this oncogenic phosphatase by simultaneously engaging the C-SH2 and PTP domains. We improved our lead to generate an analogue that better suppresses SHP2 activity in vitro. Suppression of Erk phopsphorylation by the lead compound is also consistent with SHP2 inhibition in AML cells. Our findings provide an alternative starting point for therapeutic intervention and will catalyze investigations into the relationship between SHP2 conformational regulation, activity, and disease progression.


Asunto(s)
Benzotiazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Pirimidinonas/farmacología , Regulación Alostérica/efectos de los fármacos , Benzotiazoles/síntesis química , Benzotiazoles/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirimidinonas/síntesis química , Pirimidinonas/química , Relación Estructura-Actividad
4.
Mol Cell Proteomics ; 13(1): 372-87, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24129315

RESUMEN

Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine; asymmetric dimethyl arginine; and monomethyl, dimethyl, and trimethyl lysine motifs. These antibodies were used to perform immunoaffinity purification of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell line and mouse tissues, and ∼160 lysine methylation sites in human cell line HCT116. The number of methylation sites identified in this study exceeds those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared with those found in mouse embryo shows a tissue-specific distribution of arginine methylation, and extends the types of proteins that are known to be arginine methylated to include many new protein types. Many arginine-methylated proteins that we identified from the brain, including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, whereas the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.


Asunto(s)
Arginina/metabolismo , Encéfalo/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Secuencias de Aminoácidos/genética , Animales , Arginina/genética , Cromatografía Liquida , Células HCT116 , Humanos , Lisina/genética , Metilación , Ratones , Espectrometría de Masas en Tándem
5.
J Biol Chem ; 289(52): 36048-58, 2014 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-25381250

RESUMEN

The non-receptor isoform of protein-tyrosine phosphatase ϵ (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas the Src SH2 domain did not bind at all, suggesting that GRB2 may link PTPe with downstream molecules. Further studies indicated that GRB2 is required for activation of Src by cyt-PTPe in osteoclast-like cells (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that GRB2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs.


Asunto(s)
Proteína Adaptadora GRB2/fisiología , Osteoclastos/enzimología , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/fisiología , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Células HEK293 , Humanos , Ratones de la Cepa 129 , Ratones Noqueados , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional
6.
J Biol Chem ; 287(1): 429-437, 2012 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-22072714

RESUMEN

In epigenetic signaling pathways, histone tails are heavily modified, resulting in the recruitment of effector molecules that can influence transcription. One such molecule, plant homeodomain finger protein 20 (PHF20), uses a Tudor domain to read dimethyl lysine residues and is a known component of the MOF (male absent on the first) histone acetyltransferase protein complex, suggesting it plays a role in the cross-talk between lysine methylation and histone acetylation. We sought to investigate the biological role of PHF20 by generating a knockout mouse. Without PHF20, mice die shortly after birth and display a wide variety of phenotypes within the skeletal and hematopoietic systems. Mechanistically, PHF20 is not required for maintaining the global H4K16 acetylation levels or locus specific histone acetylation but instead works downstream in transcriptional regulation of MOF target genes.


Asunto(s)
Regulación de la Expresión Génica/genética , Histona Acetiltransferasas/metabolismo , Proteínas de Homeodominio/genética , Lisina/metabolismo , Animales , Proteínas de Unión al ADN , Femenino , Técnicas de Inactivación de Genes , Histonas/química , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Factores de Transcripción , Transcripción Genética/genética
7.
Science ; 381(6659): 794-799, 2023 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-37590355

RESUMEN

The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product-inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRASG12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRASG12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRASG12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).


Asunto(s)
Productos Biológicos , Ciclofilina A , Inmunofilinas , Chaperonas Moleculares , Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/uso terapéutico , Cisteína/química , Cisteína/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Ciclofilina A/química , Ciclofilina A/metabolismo , Inmunofilinas/química , Inmunofilinas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética
8.
Bio Protoc ; 12(3): e4311, 2022 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-35284598

RESUMEN

Cells sense and respond to mitogens by activating a cascade of signaling events, primarily mediated by tyrosine phosphorylation (pY). Because of its key roles in cellular homeostasis, deregulation of this signaling is often linked to oncogenesis. To understand the mechanisms underlying these signaling pathway aberrations, it is necessary to quantify tyrosine phosphorylation on a global scale in cancer cell models. However, the majority of the protein phosphorylation events occur on serine (86%) and threonine (12%) residues, whereas only 2% of phosphorylation events occur on tyrosine residues ( Olsen et al., 2006 ). The low stoichiometry of tyrosine phosphorylation renders it difficult to quantify cellular pY events comprehensively with high mass accuracy and reproducibility. Here, we describe a detailed protocol for isolating and quantifying tyrosine phosphorylated peptides from drug-perturbed, growth factor-stimulated cancer cells, using immunoaffinity purification and tandem mass tags (TMT) coupled with mass spectrometry.

9.
Cancer Res ; 82(11): 2141-2155, 2022 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-35311954

RESUMEN

The protein tyrosine phosphatase SHP2 is crucial for oncogenic transformation of acute myeloid leukemia (AML) cells expressing mutated receptor tyrosine kinases. SHP2 is required for full RAS-ERK activation to promote cell proliferation and survival programs. Allosteric SHP2 inhibitors act by stabilizing SHP2 in its autoinhibited conformation and are currently being tested in clinical trials for tumors with overactivation of the RAS/ERK pathway, alone and in various drug combinations. In this study, we established cells with acquired resistance to the allosteric SHP2 inhibitor SHP099 from two FLT3-ITD (internal tandem duplication)-positive AML cell lines. Label-free and isobaric labeling quantitative mass spectrometry-based phosphoproteomics of these resistant models demonstrated that AML cells can restore phosphorylated ERK (pERK) in the presence of SHP099, thus developing adaptive resistance. Mechanistically, SHP2 inhibition induced tyrosine phosphorylation and feedback-driven activation of the FLT3 receptor, which in turn phosphorylated SHP2 on tyrosine 62. This phosphorylation stabilized SHP2 in its open conformation, preventing SHP099 binding and conferring resistance. Combinatorial inhibition of SHP2 and MEK or FLT3 prevented pERK rebound and resistant cell growth. The same mechanism was observed in a FLT3-mutated B-cell acute lymphoblastic leukemia cell line and in the inv(16)/KitD816Y AML mouse model, but allosteric inhibition of Shp2 did not impair the clonogenic ability of normal bone marrow progenitors. Together, these results support the future use of SHP2 inhibitor combinations for clinical applications. SIGNIFICANCE: These findings suggest that combined inhibition of SHP2 and FLT3 effectively treat FLT3-ITD-positive AML, highlighting the need for development of more potent SHP2 inhibitors and combination therapies for clinical applications.


Asunto(s)
Apoptosis , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Piperidinas , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Pirimidinas , Regulación Alostérica , Animales , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Ratones , Mutación , Fosforilación , Piperidinas/farmacología , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Tirosina/metabolismo , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo
10.
Elife ; 102021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33755016

RESUMEN

SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.


Asunto(s)
Receptores ErbB/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteómica/métodos , Catálisis , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Ocludina/metabolismo , Fosfolipasa C gamma/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Piperidinas/metabolismo , Piperidinas/farmacología , Unión Proteica , Pirimidinas/metabolismo , Pirimidinas/farmacología , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Dominios Homologos src
11.
Nat Commun ; 9(1): 4508, 2018 10 30.
Artículo en Inglés | MEDLINE | ID: mdl-30375388

RESUMEN

Activating mutations in PTPN11, encoding the cytosolic protein tyrosine phosphatase SHP2, result in developmental disorders and act as oncogenic drivers in patients with hematologic cancers. The allosteric inhibitor SHP099 stabilizes the wild-type SHP2 enzyme in an autoinhibited conformation that is itself destabilized by oncogenic mutations. Here, we report the impact of the highly activated and most frequently observed mutation, E76K, on the structure of SHP2, and investigate the effect of E76K and other oncogenic mutations on allosteric inhibition by SHP099. SHP2E76K adopts an open conformation but can be restored to the closed, autoinhibited conformation, near-identical to the unoccupied wild-type enzyme, when complexed with SHP099. SHP099 inhibitory activity against oncogenic SHP2 variants in vitro and in cells scales inversely with the activating strength of the mutation, indicating that either oncoselective or vastly more potent inhibitors will be necessary to suppress oncogenic signaling by the most strongly activating SHP2 mutations in cancer.


Asunto(s)
Regulación Alostérica/genética , Piperidinas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Pirimidinas/metabolismo , Humanos , Mutación , Proteínas Oncogénicas , Piperidinas/farmacología , Conformación Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/ultraestructura , Pirimidinas/farmacología
12.
Life Sci Alliance ; 1(5): e201800117, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-30456381

RESUMEN

The coactivator-associated arginine methyltransferase (CARM1) functions as a regulator of transcription by methylating a diverse array of substrates. To broaden our understanding of CARM1's mechanistic actions, we sought to identify additional substrates for this enzyme. To do this, we generated CARM1 substrate motif antibodies, and used immunoprecipitation coupled with mass spectrometry to identify cellular targets of CARM1, including mediator complex subunit 12 (MED12) and the lysine methyltransferase KMT2D. Both of these proteins are implicated in enhancer function. We identified the major CARM1-mediated MED12 methylation site as arginine 1899 (R1899), which interacts with the Tudor domain-containing effector molecule, TDRD3. Chromatin immunoprecipitation-seq studies revealed that CARM1 and the methyl mark it deposits are tightly associated with ERα-specific enhancers and positively modulate transcription of estrogen-regulated genes. In addition, we showed that the methylation of MED12, at the R1899 site, and the recruitment of TDRD3 by this methylated motif are critical for the ability of MED12 to interact with activating noncoding RNAs.

13.
Sci Rep ; 6: 28718, 2016 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-27338245

RESUMEN

Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes - PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies.


Asunto(s)
Anticuerpos/química , Arginina/química , Biblioteca de Péptidos , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/química , Secuencias de Aminoácidos , Animales , Línea Celular , Mapeo Epitopo , Proteínas F-Box/química , Humanos , Metilación , Ratones , Proteínas Recombinantes/química , Proteínas Represoras/química , Transducción de Señal , Especificidad por Sustrato
14.
Sci Rep ; 3: 1311, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23419748

RESUMEN

Arginine methylation is a common posttranslational modification that is found on both histone and non-histone proteins. Three types of arginine methylation exist in mammalian cells: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). PRMT1 is the primary methyltransferase that deposits the ADMA mark, and it accounts for over 90% of this type of methylation. Here, we show that with the loss of PRMT1 activity, there are major increases in global MMA and SDMA levels, as detected by type-specific antibodies. Amino acid analysis confirms that MMA and SDMA levels accumulate when ADMA levels are reduced. These findings reveal the dynamic interplay between different arginine methylation types in the cells, and that the pre-existence of the dominant ADMA mark can block the occurrence of SDMA and MMA marks on the same substrate. This study provides clear evidence of competition for different arginine methylation types on the same substrates.


Asunto(s)
Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Arginina/análogos & derivados , Arginina/química , Arginina/metabolismo , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Metilación , Proteína-Arginina N-Metiltransferasas/deficiencia , Proteína-Arginina N-Metiltransferasas/genética , Proteolisis , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Especificidad por Sustrato
15.
Methods Enzymol ; 512: 71-92, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22910203

RESUMEN

Arginine methylation was discovered in the mid-1960s. About 15 years ago, the first protein arginine N-methyltransferase (PRMT) enzyme was described. The PRMT family now stands at nine members, and these enzymes play a key role in regulating a multitude of cellular events. The majority of the PRMTs have been deleted in mice, thus providing genetically tractable systems for in vivo and cell-based studies. These studies have implicated this posttranslational modification in chromatin remodeling, transcriptional regulation, RNA processing, protein/RNA trafficking, signal transduction, and DNA repair. In this chapter, we introduce different approaches that have been developed to assess protein arginine methylation levels and characterize PRMT substrates.


Asunto(s)
Arginina/química , Pruebas de Enzimas , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/química , Proteínas Recombinantes de Fusión/química , Animales , Anticuerpos Monoclonales de Origen Murino/química , Extractos Celulares/química , Proteínas de Unión al ADN/química , Inhibidores Enzimáticos/química , Escherichia coli , Fibroblastos/enzimología , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Metilación , Ratones , Fragmentos de Péptidos/química , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/biosíntesis , Proteína-Arginina N-Metiltransferasas/genética , Conejos , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/biosíntesis , Reticulocitos/enzimología
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