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1.
Annu Rev Biochem ; 89: 135-158, 2020 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-31815535

RESUMEN

DNA methylation at the 5-position of cytosine (5mC) plays vital roles in mammalian development. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), and the two DNMT families, DNMT3 and DNMT1, are responsible for methylation establishment and maintenance, respectively. Since their discovery, biochemical and structural studies have revealed the key mechanisms underlying how DNMTs catalyze de novo and maintenance DNA methylation. In particular, recent development of low-input genomic and epigenomic technologies has deepened our understanding of DNA methylation regulation in germ lines and early stage embryos. In this review, we first describe the methylation machinery including the DNMTs and their essential cofactors. We then discuss how DNMTs are recruited to or excluded from certain genomic elements. Lastly, we summarize recent understanding of the regulation of DNA methylation dynamics in mammalian germ lines and early embryos with a focus on both mice and humans.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN/genética , Regulación del Desarrollo de la Expresión Génica , Genoma , Animales , Coenzimas/química , Coenzimas/metabolismo , Islas de CpG , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN Metiltransferasa 3A , Embrión de Mamíferos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Oocitos/citología , Oocitos/enzimología , Oocitos/crecimiento & desarrollo , Transducción de Señal , Espermatozoides/citología , Espermatozoides/enzimología , Espermatozoides/crecimiento & desarrollo
2.
Annu Rev Biochem ; 88: 191-220, 2019 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-30883196

RESUMEN

Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.


Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica/métodos , Ingeniería de Proteínas/métodos , ARN Guía de Kinetoplastida/genética , Desaminasas APOBEC/genética , Desaminasas APOBEC/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Artefactos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Transferencia de Gen , Genoma Humano , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Programas Informáticos
3.
Annu Rev Biochem ; 86: 749-775, 2017 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-28226215

RESUMEN

Peroxiredoxins (Prxs) constitute a major family of peroxidases, with mammalian cells expressing six Prx isoforms (PrxI to PrxVI). Cells produce hydrogen peroxide (H2O2) at various intracellular locations where it can serve as a signaling molecule. Given that Prxs are abundant and possess a structure that renders the cysteine (Cys) residue at the active site highly sensitive to oxidation by H2O2, the signaling function of this oxidant requires extensive and highly localized regulation. Recent findings on the reversible regulation of PrxI through phosphorylation at the centrosome and on the hyperoxidation of the Cys at the active site of PrxIII in mitochondria are described in this review as examples of such local regulation of H2O2 signaling. Moreover, their high affinity for and sensitivity to oxidation by H2O2 confer on Prxs the ability to serve as sensors and transducers of H2O2 signaling through transfer of their oxidation state to bound effector proteins.


Asunto(s)
Ritmo Circadiano/genética , Regulación de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Peroxirredoxinas/metabolismo , Animales , Dominio Catalítico , Centrosoma/metabolismo , Centrosoma/ultraestructura , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Mitocondrias/ultraestructura , Mitosis , Oxidación-Reducción , Peroxirredoxinas/genética , Fosforilación , Transducción de Señal
4.
Annu Rev Biochem ; 86: 685-714, 2017 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-28301740

RESUMEN

Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized as precursors in the cytosol and are imported into mitochondria by five transport pathways. The protein import machineries of the mitochondrial membranes and aqueous compartments reveal a remarkable variability of mechanisms for protein recognition, translocation, and sorting. The protein translocases do not operate as separate entities but are connected to each other and to machineries with functions in energetics, membrane organization, and quality control. Here, we discuss the versatility and dynamic organization of the mitochondrial protein import machineries. Elucidating the molecular mechanisms of mitochondrial protein translocation is crucial for understanding the integration of protein translocases into a large network that controls organelle biogenesis, function, and dynamics.


Asunto(s)
Proteínas Portadoras/metabolismo , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Células Eucariotas/metabolismo , Células Eucariotas/ultraestructura , Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Mitocondrias/ultraestructura , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Membranas Mitocondriales/ultraestructura , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Biogénesis de Organelos , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Precursores de Proteínas/química , Precursores de Proteínas/genética , Transporte de Proteínas
5.
Annu Rev Biochem ; 86: 305-331, 2017 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-28375741

RESUMEN

The identification of heterozygous mutations in the metabolic enzyme isocitrate dehydrogenase (IDH) in subsets of cancers, including secondary glioblastoma, acute myeloid leukemia, intrahepatic cholangiocarcinoma, and chondrosarcomas, led to intense discovery efforts to delineate the mutations' involvement in carcinogenesis and to develop therapeutics, which we review here. The three IDH isoforms (nicotinamide adenine dinucleotide phosphate-dependent IDH1 and IDH2, and nicotinamide adenine dinucleotide-dependent IDH3) contribute to regulating the circuitry of central metabolism. Several biochemical and genetic observations led to the discovery of the neomorphic production of the oncometabolite (R)-2-hydroxyglutarate (2-HG) by mutant IDH1 and IDH2 (mIDH). Heterozygous mutation of IDH1/2 and accumulation of 2-HG cause profound metabolic and epigenetic dysregulation, including inhibition of normal cellular differentiation, leading to disease. Crystallographic structural studies during the development of compounds targeting mIDH demonstrated common allosteric inhibition by distinct chemotypes. Ongoing clinical trials in patients with mIDH advanced hematologic malignancies have demonstrated compelling clinical proof-of-concept, validating the biology and drug discovery approach.


Asunto(s)
Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Glutaratos/metabolismo , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Acetamidas/síntesis química , Acetamidas/uso terapéutico , Antineoplásicos/síntesis química , Bencenoacetamidas/síntesis química , Bencenoacetamidas/uso terapéutico , Bencimidazoles/síntesis química , Bencimidazoles/uso terapéutico , Biomarcadores de Tumor/análisis , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/uso terapéutico , Expresión Génica , Glutaratos/análisis , Humanos , Imidazoles/síntesis química , Imidazoles/uso terapéutico , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Modelos Moleculares , Mutación , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Investigación Biomédica Traslacional
6.
Cell ; 169(3): 381-405, 2017 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-28431241

RESUMEN

The Ser and Thr kinase AKT, also known as protein kinase B (PKB), was discovered 25 years ago and has been the focus of tens of thousands of studies in diverse fields of biology and medicine. There have been many advances in our knowledge of the upstream regulatory inputs into AKT, key multifunctional downstream signaling nodes (GSK3, FoxO, mTORC1), which greatly expand the functional repertoire of AKT, and the complex circuitry of this dynamically branching and looping signaling network that is ubiquitous to nearly every cell in our body. Mouse and human genetic studies have also revealed physiological roles for the AKT network in nearly every organ system. Our comprehension of AKT regulation and functions is particularly important given the consequences of AKT dysfunction in diverse pathological settings, including developmental and overgrowth syndromes, cancer, cardiovascular disease, insulin resistance and type 2 diabetes, inflammatory and autoimmune disorders, and neurological disorders. There has also been much progress in developing AKT-selective small molecule inhibitors. Improved understanding of the molecular wiring of the AKT signaling network continues to make an impact that cuts across most disciplines of the biomedical sciences.


Asunto(s)
Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Quimioterapia , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Terapia Molecular Dirigida , Fosforilación , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores
7.
Cell ; 171(3): 628-641.e26, 2017 Oct 19.
Artículo en Inglés | MEDLINE | ID: mdl-29053969

RESUMEN

Ferroptosis is a form of programmed cell death that is pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure, and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.


Asunto(s)
Lesión Renal Aguda/patología , Asma/patología , Lesiones Traumáticas del Encéfalo/patología , Muerte Celular , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Lesión Renal Aguda/metabolismo , Animales , Apoptosis , Asma/metabolismo , Lesiones Traumáticas del Encéfalo/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Humanos , Isoenzimas/metabolismo , Lipooxigenasa/química , Lipooxigenasa/metabolismo , Ratones , Modelos Moleculares , Oxazolidinonas/farmacología , Oxidación-Reducción , Proteínas de Unión a Fosfatidiletanolamina/química
8.
Annu Rev Biochem ; 85: 431-54, 2016 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-26844395

RESUMEN

Recent developments indicate that macrodomains, an ancient and diverse protein domain family, are key players in the recognition, interpretation, and turnover of ADP-ribose (ADPr) signaling. Crucial to this is the ability of macrodomains to recognize ADPr either directly, in the form of a metabolic derivative, or as a modification covalently bound to proteins. Thus, macrodomains regulate a wide variety of cellular and organismal processes, including DNA damage repair, signal transduction, and immune response. Their importance is further indicated by the fact that dysregulation or mutation of a macrodomain is associated with several diseases, including cancer, developmental defects, and neurodegeneration. In this review, we summarize the current insights into macrodomain evolution and how this evolution influenced their structural and functional diversification. We highlight some aspects of macrodomain roles in pathobiology as well as their emerging potential as therapeutic targets.


Asunto(s)
Reparación del ADN , Proteínas de Escherichia coli/química , Neoplasias/enzimología , Poli(ADP-Ribosa) Polimerasas/química , Procesamiento Proteico-Postraduccional , Proteínas Represoras/química , Virosis/enzimología , Adenosina Difosfato Ribosa/química , Adenosina Difosfato Ribosa/metabolismo , Animales , Daño del ADN , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolución Molecular , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Familia de Multigenes , Neoplasias/química , Neoplasias/genética , Neoplasias/patología , Filogenia , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Dominios Proteicos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Homología Estructural de Proteína , Virosis/genética , Virosis/patología , Virosis/virología
9.
Nat Rev Mol Cell Biol ; 20(9): 515-534, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31110302

RESUMEN

PI3Ks are a family of lipid kinases that phosphorylate intracellular inositol lipids to regulate signalling and intracellular vesicular traffic. Mammals have eight isoforms of PI3K, divided into three classes. The class I PI3Ks generate 3-phosphoinositide lipids, which directly activate signal transduction pathways. In addition to being frequently genetically activated in cancer, similar mutations in class I PI3Ks have now also been found in a human non-malignant overgrowth syndrome and a primary immune disorder that predisposes to lymphoma. The class II and class III PI3Ks are regulators of membrane traffic along the endocytic route, in endosomal recycling and autophagy, with an often indirect effect on cell signalling. Here, we summarize current knowledge of the different PI3K classes and isoforms, focusing on recently uncovered biological functions and the mechanisms by which these kinases are activated. Deeper insight into the PI3K isoforms will undoubtedly continue to contribute to a better understanding of fundamental cell biological processes and, ultimately, of human disease.


Asunto(s)
Endosomas/metabolismo , Linfoma/enzimología , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal , Animales , Transporte Biológico Activo , Endocitosis , Humanos , Isoenzimas/metabolismo , Linfoma/patología
10.
Cell ; 160(3): 489-502, 2015 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-25619690

RESUMEN

Protein kinase C (PKC) isozymes have remained elusive cancer targets despite the unambiguous tumor promoting function of their potent ligands, phorbol esters, and the prevalence of their mutations. We analyzed 8% of PKC mutations identified in human cancers and found that, surprisingly, most were loss of function and none were activating. Loss-of-function mutations occurred in all PKC subgroups and impeded second-messenger binding, phosphorylation, or catalysis. Correction of a loss-of-function PKCß mutation by CRISPR-mediated genome editing in a patient-derived colon cancer cell line suppressed anchorage-independent growth and reduced tumor growth in a xenograft model. Hemizygous deletion promoted anchorage-independent growth, revealing that PKCß is haploinsufficient for tumor suppression. Several mutations were dominant negative, suppressing global PKC signaling output, and bioinformatic analysis suggested that PKC mutations cooperate with co-occurring mutations in cancer drivers. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapies should focus on restoring, not inhibiting, PKC activity.


Asunto(s)
Proteína Quinasa C/química , Proteína Quinasa C/genética , Animales , Línea Celular Tumoral , Transferencia Resonante de Energía de Fluorescencia , Genes Supresores de Tumor , Xenoinjertos , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones Desnudos , Modelos Moleculares , Mutación , Trasplante de Neoplasias , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína
11.
Cell ; 158(6): 1309-1323, 2014 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-25215489

RESUMEN

The balance between oxidative and nonoxidative glucose metabolism is essential for a number of pathophysiological processes. By deleting enzymes that affect aerobic glycolysis with different potencies, we examine how modulating glucose metabolism specifically affects hematopoietic and leukemic cell populations. We find that a deficiency in the M2 pyruvate kinase isoform (PKM2) reduces the levels of metabolic intermediates important for biosynthesis and impairs progenitor function without perturbing hematopoietic stem cells (HSCs), whereas lactate dehydrogenase A (LDHA) deletion significantly inhibits the function of both HSCs and progenitors during hematopoiesis. In contrast, leukemia initiation by transforming alleles putatively affecting either HSCs or progenitors is inhibited in the absence of either PKM2 or LDHA, indicating that the cell-state-specific responses to metabolic manipulation in hematopoiesis do not apply to the setting of leukemia. This finding suggests that fine-tuning the level of glycolysis may be explored therapeutically for treating leukemia while preserving HSC function.


Asunto(s)
Glucólisis , Hematopoyesis , Leucemia/metabolismo , Animales , Eliminación de Gen , Células Madre Hematopoyéticas/metabolismo , Humanos , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Lactato Deshidrogenasa 5 , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo
12.
Cell ; 157(5): 1088-103, 2014 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-24855946

RESUMEN

Cancer cells must integrate multiple biosynthetic demands to drive indefinite proliferation. How these key cellular processes, such as metabolism and protein synthesis, crosstalk to fuel cancer cell growth is unknown. Here, we uncover the mechanism by which the Myc oncogene coordinates the production of the two most abundant classes of cellular macromolecules, proteins, and nucleic acids in cancer cells. We find that a single rate-limiting enzyme, phosphoribosyl-pyrophosphate synthetase 2 (PRPS2), promotes increased nucleotide biosynthesis in Myc-transformed cells. Remarkably, Prps2 couples protein and nucleotide biosynthesis through a specialized cis-regulatory element within the Prps2 5' UTR, which is controlled by the oncogene and translation initiation factor eIF4E downstream Myc activation. We demonstrate with a Prps2 knockout mouse that the nexus between protein and nucleotide biosynthesis controlled by PRPS2 is crucial for Myc-driven tumorigenesis. Together, these studies identify a translationally anchored anabolic circuit critical for cancer cell survival and an unexpected vulnerability for "undruggable" oncogenes, such as Myc. PAPERFLICK:


Asunto(s)
Carcinogénesis , Nucleótidos/biosíntesis , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ribosa-Fosfato Pirofosfoquinasa/genética , Regiones no Traducidas 5' , Animales , Linfocitos B/metabolismo , Secuencia de Bases , Linfoma de Burkitt/metabolismo , Línea Celular Tumoral , Células Cultivadas , Células Madre Embrionarias , Factor 4E Eucariótico de Iniciación/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Células 3T3 NIH , Ribosa-Fosfato Pirofosfoquinasa/metabolismo
13.
Mol Cell ; 81(21): 4509-4526.e10, 2021 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-34560002

RESUMEN

The interferon (IFN) pathway is critical for cytotoxic T cell activation, which is central to tumor immunosurveillance and successful immunotherapy. We demonstrate here that PKCλ/ι inactivation results in the hyper-stimulation of the IFN cascade and the enhanced recruitment of CD8+ T cells that impaired the growth of intestinal tumors. PKCλ/ι directly phosphorylates and represses the activity of ULK2, promoting its degradation through an endosomal microautophagy-driven ubiquitin-dependent mechanism. Loss of PKCλ/ι results in increased levels of enzymatically active ULK2, which, by direct phosphorylation, activates TBK1 to foster the activation of the STING-mediated IFN response. PKCλ/ι inactivation also triggers autophagy, which prevents STING degradation by chaperone-mediated autophagy. Thus, PKCλ/ι is a hub regulating the IFN pathway and three autophagic mechanisms that serve to maintain its homeostatic control. Importantly, single-cell multiplex imaging and bioinformatics analysis demonstrated that low PKCλ/ι levels correlate with enhanced IFN signaling and good prognosis in colorectal cancer patients.


Asunto(s)
Neoplasias Colorrectales/metabolismo , Interferones/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal , Adulto , Anciano , Anciano de 80 o más Años , Animales , Autofagia , Linfocitos T CD8-positivos/metabolismo , Carcinogénesis , Transformación Celular Neoplásica , Neoplasias Colorrectales/mortalidad , Cicloheximida/química , Femenino , Células HEK293 , Humanos , Inmunofenotipificación , Factor 3 Regulador del Interferón/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Persona de Mediana Edad , Trasplante de Neoplasias , Fosforilación , Pronóstico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción , Regulación hacia Arriba
14.
Mol Cell ; 81(2): 398-407.e4, 2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33340489

RESUMEN

Mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and proliferation by sensing fluctuations in environmental cues such as nutrients, growth factors, and energy levels. The Rag GTPases (Rags) serve as a critical module that signals amino acid (AA) availability to modulate mTORC1 localization and activity. Recent studies have demonstrated how AAs regulate mTORC1 activity through Rags. Here, we uncover an unconventional pathway that activates mTORC1 in response to variations in threonine (Thr) levels via mitochondrial threonyl-tRNA synthetase TARS2. TARS2 interacts with inactive Rags, particularly GTP-RagC, leading to increased GTP loading of RagA. mTORC1 activity in cells lacking TARS2 is resistant to Thr repletion, showing that TARS2 is necessary for Thr-dependent mTORC1 activation. The requirement of TARS2, but not cytoplasmic threonyl-tRNA synthetase TARS, for this effect demonstrates an additional layer of complexity in the regulation of mTORC1 activity.


Asunto(s)
Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Mitocondrias/metabolismo , Proteínas de Unión al GTP Monoméricas/genética , Treonina-ARNt Ligasa/genética , Treonina/metabolismo , Regulación de la Expresión Génica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Unión Proteica , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína Reguladora Asociada a mTOR/genética , Proteína Reguladora Asociada a mTOR/metabolismo , Transducción de Señal , Treonina-ARNt Ligasa/antagonistas & inhibidores , Treonina-ARNt Ligasa/metabolismo
15.
Nat Rev Mol Cell Biol ; 17(2): 83-96, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26648264

RESUMEN

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference.


Asunto(s)
Adenosina Desaminasa/genética , Adenosina/metabolismo , Genoma , Inosina/metabolismo , Edición de ARN , ARN Mensajero/genética , Adenosina Desaminasa/metabolismo , Elementos Alu , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Transducción de Señal
16.
Cell ; 153(5): 1050-63, 2013 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-23706742

RESUMEN

RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110ß isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110ß, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110ß via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110ß. Cells from mice carrying mutations in the p110ß RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110ß downstream of GPCRs.


Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Fibroblastos/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Animales , Quimiotaxis , Fosfatidilinositol 3-Quinasa Clase I/química , Fibrosis/inducido químicamente , Fibrosis/prevención & control , Reguladores de Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Isoenzimas/metabolismo , Pulmón/patología , Ratones , Dominios y Motivos de Interacción de Proteínas , Proteína de Unión al GTP rac1/metabolismo , Proteínas ras/química
17.
Cell ; 155(2): 397-409, 2013 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-24120138

RESUMEN

The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.


Asunto(s)
Neoplasias de la Mama/metabolismo , Eliminación de Gen , Neoplasias Mamarias Experimentales/metabolismo , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Animales , Secuencia de Bases , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Exones , Femenino , Técnicas de Inactivación de Genes , Xenoinjertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Mutación , Metástasis de la Neoplasia , Trasplante de Neoplasias , Empalme del ARN
18.
Mol Cell ; 80(1): 87-101.e5, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32931746

RESUMEN

Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.


Asunto(s)
Neoplasias Mamarias Animales/enzimología , Neoplasias Mamarias Animales/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Carcinogénesis/patología , Femenino , Eliminación de Gen , Humanos , Insulina/metabolismo , Isoenzimas/metabolismo , Metástasis de la Neoplasia , Neutrófilos/metabolismo , Receptor ErbB-2/metabolismo
19.
Mol Cell ; 80(2): 279-295.e8, 2020 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-33065020

RESUMEN

The PTEN tumor suppressor controls cell death and survival by regulating functions of various molecular targets. While the role of PTEN lipid-phosphatase activity on PtdIns(3,4,5)P3 and inhibition of PI3K pathway is well characterized, the biological relevance of PTEN protein-phosphatase activity remains undefined. Here, using knockin (KI) mice harboring cancer-associated and functionally relevant missense mutations, we show that although loss of PTEN lipid-phosphatase function cooperates with oncogenic PI3K to promote rapid mammary tumorigenesis, the additional loss of PTEN protein-phosphatase activity triggered an extensive cell death response evident in early and advanced mammary tumors. Omics and drug-targeting studies revealed that PI3Ks act to reduce glucocorticoid receptor (GR) levels, which are rescued by loss of PTEN protein-phosphatase activity to restrain cell survival. Thus, we find that the dual regulation of GR by PI3K and PTEN functions as a rheostat that can be exploited for the treatment of PTEN loss-driven cancers.


Asunto(s)
Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Fosfohidrolasa PTEN/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Carcinogénesis , Muerte Celular , Línea Celular Tumoral , Proliferación Celular , Dexametasona/farmacología , Femenino , Humanos , Isoenzimas/metabolismo , Ratones , Modelos Biológicos , Mutación/genética , Organoides/patología , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Estabilidad Proteica , Proteoma/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo
20.
Mol Cell ; 79(3): 376-389.e8, 2020 08 06.
Artículo en Inglés | MEDLINE | ID: mdl-32640193

RESUMEN

Activation of dual-specificity tyrosine-phosphorylation-regulated kinases 1A and 1B (DYRK1A and DYRK1B) requires prolyl hydroxylation by PHD1 prolyl hydroxylase. Prolyl hydroxylation of DYRK1 initiates a cascade of events leading to the release of molecular constraints on von Hippel-Lindau (VHL) ubiquitin ligase tumor suppressor function. However, the proline residue of DYRK1 targeted by hydroxylation and the role of prolyl hydroxylation in tyrosine autophosphorylation of DYRK1 are unknown. We found that a highly conserved proline in the CMGC insert of the DYRK1 kinase domain is hydroxylated by PHD1, and this event precedes tyrosine autophosphorylation. Mutation of the hydroxylation acceptor proline precludes tyrosine autophosphorylation and folding of DYRK1, resulting in a kinase unable to preserve VHL function and lacking glioma suppression activity. The consensus proline sequence is shared by most CMGC kinases, and prolyl hydroxylation is essential for catalytic activation. Thus, formation of prolyl-hydroxylated intermediates is a novel mechanism of kinase maturation and likely a general mechanism of regulation of CMGC kinases in eukaryotes.


Asunto(s)
Neoplasias Encefálicas/genética , Glioma/genética , Isoenzimas/genética , Prolina/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Cristalografía por Rayos X , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Glioma/patología , Células HEK293 , Xenoinjertos , Humanos , Hidroxilación , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Ratones , Ratones Desnudos , Proteína Quinasa 14 Activada por Mitógenos/química , Proteína Quinasa 14 Activada por Mitógenos/genética , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Modelos Moleculares , Mutación , Neuroglía/metabolismo , Neuroglía/patología , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Secundaria de Proteína , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Quinasas DyrK
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