RESUMEN
Thalidomide has a dark history as a teratogen, but in recent years, its derivates have been shown to function as potent chemotherapeutic agents. These drugs bind cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex, and modify its degradation targets. Despite these insights, remarkably little is known about the normal function of cereblon in development. Here, we employ Ciona, a simple invertebrate chordate, to identify endogenous Crbn targets. In Ciona, Crbn is specifically expressed in developing muscles during tail elongation before they acquire contractile activity. Crbn expression is activated by Mrf, the ortholog of MYOD1, a transcription factor important for muscle differentiation. CRISPR/Cas9-mediated mutations of Crbn lead to precocious onset of muscle contractions. By contrast, overexpression of Crbn delays contractions and is associated with decreased expression of contractile protein genes such as troponin. This reduction is possibly due to reduced Mrf protein levels without altering Mrf mRNA levels. Our findings suggest that Mrf and Crbn form a negative feedback loop to control the precision of muscle differentiation during tail elongation.
Asunto(s)
Ciona intestinalis , Músculos , Péptido Hidrolasas , Animales , Proteínas Portadoras , Ciona intestinalis/genética , Ciona intestinalis/metabolismo , Músculos/metabolismo , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Talidomida/efectos adversos , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Larva/genética , Larva/metabolismoRESUMEN
Hypoxia-induced angiogenesis maintains tissue oxygen supply and protects against ischemia but also enhances tumor progression and malignancy. This is mediated through activation of transcription factors like hypoxia-inducible factor 1 (HIF-1) and c-Myc, yet the impact of hypoxia on negative regulators of angiogenesis is unknown. During vascular development, seryl-tRNA synthetase (SerRS) regulates angiogenesis through a novel mechanism by counteracting c-Myc and transcriptionally repressing vascular endothelial growth factor A (VEGFA) expression. Here, we reveal that the transcriptional repressor role of SerRS is inactivated under hypoxia through phosphorylation by ataxia telangiectasia mutated (ATM) and ataxia telangiectasia mutated and RAD3-related (ATR) at Ser101 and Ser241 to attenuate its DNA binding capacity. In zebrafish, SerRSS101D/S241D, a phosphorylation-mimicry mutant, cannot suppress VEGFA expression to support normal vascular development. Moreover, expression of SerRSS101A/S241A, a phosphorylation-deficient and constitutively active mutant, prevents hypoxia-induced binding of c-Myc and HIF-1 to the VEGFA promoter, and activation of VEGFA expression. Consistently, SerRSS101A/S241A strongly inhibits normal and tumor-derived angiogenesis in mice. Therefore, we reveal a key step regulating hypoxic angiogenesis and highlight the importance of nuclear SerRS in post-developmental angiogenesis regulation in addition to vascular development. The role of nuclear SerRS in inhibiting both c-Myc and HIF-1 may provide therapeutic opportunities to correct dysregulation of angiogenesis in pathological settings.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neovascularización Patológica/genética , Serina-ARNt Ligasa/metabolismo , Inductores de la Angiogénesis , Animales , Animales Modificados Genéticamente , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Línea Celular , Femenino , Células HEK293 , Humanos , Hipoxia/metabolismo , Hipoxia/fisiopatología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Ratones Desnudos , Fosforilación , Serina-ARNt Ligasa/fisiología , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
CC-122 is a next-generation cereblon E3 ligase-modulating agent that has demonstrated promising clinical efficacy in patients with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Mechanistically, CC-122 induces the degradation of IKZF1/3, leading to T-cell activation and robust cell-autonomous killing in DLBCL. We report a genome-wide CRISPR/Cas9 screening for CC-122 in a DLBCL cell line SU-DHL-4 with follow-up mechanistic characterization in 6 DLBCL cell lines to identify genes regulating the response to CC-122. Top-ranked CC-122 resistance genes encode, not only well-defined members or regulators of the CUL4/DDB1/RBX1/CRBN E3 ubiquitin ligase complex, but also key components of signaling and transcriptional networks that have not been shown to modulate the response to cereblon modulators. Ablation of CYLD, NFKBIA, TRAF2, or TRAF3 induces hyperactivation of the canonical and/or noncanonical NF-κB pathways and subsequently diminishes CC-122-induced apoptosis in 5 of 6 DLBCL cell lines. Depletion of KCTD5, the substrate adaptor of the CUL3/RBX1/KCTD5 ubiquitin ligase complex, promotes the stabilization of its cognate substrate, GNG5, resulting in CC-122 resistance in HT, SU-DHL-4, and WSU-DLCL2. Furthermore, knockout of AMBRA1 renders resistance to CC-122 in SU-DHL-4 and U-2932, whereas knockout of RFX7 leads to resistance specifically in SU-DHL-4. The ubiquitous and cell line-specific mechanisms of CC-122 resistance in DLBCL cell lines revealed in this work pinpoint genetic alternations that are potentially associated with clinical resistance in patients and facilitate the development of biomarker strategies for patient stratification, which may improve clinical outcomes of patients with R/R DLBCL.
Asunto(s)
Linfoma de Células B Grandes Difuso , Piperidonas , Proteínas Adaptadoras Transductoras de Señales , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Canales de Potasio , Quinazolinonas , Ubiquitina-Proteína LigasasRESUMEN
A hallmark of a group of neurodegenerative diseases such as Alzheimer disease is the formation of neurofibrillary tangles, which are principally composed of bundles of filaments formed by microtubule-associated protein Tau. Clarifying how natively unstructured Tau protein forms abnormal aggregates is of central importance for elucidating the etiology of these diseases. There is considerable evidence showing that zinc, as an essential element that is highly concentrated in brain, is linked to the development or progression of these diseases. Herein, by using recombinant human Tau fragment Tau(244-372) and its mutants, we have investigated the effect of zinc on the aggregation of Tau. Low micromolar concentrations of Zn(2+) dramatically accelerate fibril formation of wild-type Tau(244-372) under reducing conditions, compared with no Zn(2+). Higher concentrations of Zn(2+), however, induce wild-type Tau(244-372) to form granular aggregates in reducing conditions. Moreover, these non-fibrillar aggregates assemble into mature Tau filaments when Zn(2+) has been chelated by EDTA. Unlike wild-type Tau(244-372), low micromolar concentrations of Zn(2+) have no obvious effects on fibrillization kinetics of single mutants C291A and C322A and double mutant C291A/C322A under reducing conditions. The results from isothermal titration calorimetry show that one Zn(2+) binds to one Tau molecule via tetrahedral coordination to Cys-291 and Cys-322 as well as two histidines, with moderate, micromolar affinity. Our data demonstrate that low micromolar zinc accelerates the fibrillization of human Tau protein via bridging Cys-291 and Cys-322 in physiological reducing conditions, providing clues to understanding the relationship between zinc dyshomeostasis and the etiology of neurodegenerative diseases.
Asunto(s)
Cisteína/metabolismo , Ovillos Neurofibrilares , Fragmentos de Péptidos , Zinc/metabolismo , Proteínas tau , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Disulfuros/metabolismo , Humanos , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Ovillos Neurofibrilares/química , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Oxidación-Reducción , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinámica , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) are key enzymes in the mRNA translation machinery, yet they possess numerous non-canonical functions developed during the evolution of complex organisms. The aaRSs and aaRS-interacting multi-functional proteins (AIMPs) are continually being implicated in tumorigenesis, but these connections are often limited in scope, focusing on specific aaRSs in distinct cancer subtypes. Here, we analyze publicly available genomic and transcriptomic data on human cytoplasmic and mitochondrial aaRSs across many cancer types. As high-throughput technologies have improved exponentially, large-scale projects have systematically quantified genetic alteration and expression from thousands of cancer patient samples. One such project is the Cancer Genome Atlas (TCGA), which processed over 20,000 primary cancer and matched normal samples from 33 cancer types. The wealth of knowledge provided from this undertaking has streamlined the identification of cancer drivers and suppressors. We examined aaRS expression data produced by the TCGA project and combined this with patient survival data to recognize trends in aaRSs' impact on cancer both molecularly and prognostically. We further compared these trends to an established tumor suppressor and a proto-oncogene. We observed apparent upregulation of many tRNA synthetase genes with aggressive cancer types, yet, at the individual gene level, some aaRSs resemble a tumor suppressor while others show similarities to an oncogene. This study provides an unbiased, overarching perspective on the relationship of aaRSs with cancers and identifies certain aaRS family members as promising therapeutic targets or potential leads for developing biological therapy for cancer.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Mutación , Neoplasias/enzimología , Neoplasias/mortalidad , Aminoacil-ARNt Sintetasas/metabolismo , Bases de Datos de Proteínas , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Variación Genética , Humanos , Neoplasias/genética , Proteómica/métodos , Proto-Oncogenes Mas , Análisis de SupervivenciaRESUMEN
Both Ras protein and calcium play significant roles in various cellular processes via complex signaling transduction networks. However, it is not well understood whether and how Ca(2+) can directly regulate Ras function. Here we demonstrate by isothermal titration calorimetry that Ca(2+) directly binds to the H-Ras.GDP.Mg(2+) complex with moderate affinity at the first binding site followed by two weak binding events. The results from limited proteinase degradation show that Ca(2+) protects the fragments of H-Ras from being further degraded by trypsin and by proteinase K. HPLC studies together with fluorescence spectroscopic measurements indicate that binding of Ca(2+) to the H-Ras.GDP.Mg(2+) complex remarkably promotes guanine nucleotide exchange on H-Ras under emulated physiological Ca(2+) concentration conditions. Addition of high concentrations of either of two macromolecular crowding agents, Ficoll 70 and dextran 70, dramatically enhances H-Ras guanine nucleotide exchange extent in the presence of Ca(2+) at emulated physiological concentrations, and the nucleotide exchange extent increases significantly with the concentrations of crowding agents. Together, these results indicate that binding of calcium ions to H-Ras remarkably promotes H-Ras guanine nucleotide exchange under emulated physiological conditions. We thus propose that Ca(2+) may activate Ras signaling pathway by interaction with Ras, providing clues to understand the role of calcium in regulating Ras function in physiological environments.
Asunto(s)
Calcio/metabolismo , Nucleótidos de Guanina/metabolismo , Proteínas ras/metabolismo , Proteínas ras/fisiología , Secuencia de Aminoácidos , Sitios de Unión , Calcio/farmacología , Simulación por Computador , Relación Dosis-Respuesta a Droga , Humanos , Iones/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Concentración Osmolar , Unión Proteica/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Dominant mutations in glycyl-tRNA synthetase (GlyRS) cause a subtype of Charcot-Marie-Tooth neuropathy (CMT2D). Although previous studies have shown that GlyRS mutants aberrantly interact with Nrp1, giving insight into the disease's specific effects on motor neurons, these cannot explain length-dependent axonal degeneration. Here, we report that GlyRS mutants interact aberrantly with HDAC6 and stimulate its deacetylase activity on α-tubulin. A decrease in α-tubulin acetylation and deficits in axonal transport are observed in mice peripheral nerves prior to disease onset. An HDAC6 inhibitor used to restore α-tubulin acetylation rescues axonal transport deficits and improves motor functions of CMT2D mice. These results link the aberrant GlyRS-HDAC6 interaction to CMT2D pathology and suggest HDAC6 as an effective therapeutic target. Moreover, the HDAC6 interaction differs from Nrp1 interaction among GlyRS mutants and correlates with divergent clinical presentations, indicating the existence of multiple and different mechanisms in CMT2D.
Asunto(s)
Transporte Axonal/genética , Axones/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Glicina-ARNt Ligasa/metabolismo , Histona Desacetilasa 6/metabolismo , Neuronas Motoras/metabolismo , Acetilación , Animales , Transporte Axonal/efectos de los fármacos , Enfermedad de Charcot-Marie-Tooth/genética , Modelos Animales de Enfermedad , Femenino , Glicina-ARNt Ligasa/genética , Células HEK293 , Histona Desacetilasa 6/antagonistas & inhibidores , Humanos , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas del Tejido Nervioso/metabolismo , Neuropilina-1/metabolismo , Nervios Periféricos/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
The effects of four single macromolecular crowding agents, Ficoll 70, dextran 70, polyethylene glycol (PEG) 2000, and calf thymus DNA (CT DNA), and three mixed crowding agents containing both CT DNA and polysaccharide (or PEG 2000) on the refolding of guanidine hydrochloride-denatured rabbit muscle creatine kinase (MM-CK) have been examined by activity assay. When the total concentration of the mixed crowding agent is 100 g/l, in which the weight ratio of CT DNA to Ficoll 70 is 1:9, the refolding yield of MM-CK after refolding for 3 h under these conditions increases 23% compared with that in the presence of 10 g/l CT DNA, 18% compared with 100 g/l Ficoll 70, and 19% compared with that in the absence of crowding agents. A remarkable increase in the refolding yield of MM-CK by a mixed crowding agent containing CT DNA and dextran 70 (or PEG 2000) is also observed. Further folding kinetics analyses show that these three mixed crowding agents remarkably accelerate the refolding of MM-CK, compared with single crowding agents. Aggregation of MM-CK in the presence of any of the three mixed crowding agents is less serious than that in the presence of a single crowding agent at the same concentration but more serious than that in the absence of crowding agents. Both the refolding yield and the refolding rate of MM-CK in mixtures of these agents are increased relative to the individual agents by themselves, indicating that mixed macromolecular crowding agents are more favorable to MM-CK folding and can be used to reflect the physiological environment more accurately than single crowding agents.
Asunto(s)
Forma MM de la Creatina-Quinasa/química , Pliegue de Proteína , Animales , ADN/química , Dextranos/química , Dimerización , Ficoll/química , Cinética , Polietilenglicoles/química , Renaturación de Proteína , ConejosRESUMEN
Neddylation is a post-translational modification that controls the cell cycle and proliferation by conjugating the ubiquitin-like protein NEDD8 to specific targets. Here we report that glycyl-tRNA synthetase (GlyRS), an essential enzyme in protein synthesis, also plays a critical role in neddylation. In human cells, knockdown of GlyRS, but not knockdown of a different tRNA synthetase, decreased the global level of neddylation and caused cell-cycle abnormality. This function of GlyRS is achieved through direct interactions with multiple components of the neddylation pathway, including NEDD8, E1, and E2 (Ubc12). Using various structural and functional approaches, we show that GlyRS binds the APPBP1 subunit of E1 and captures and protects activated E2 (NEDD8-conjugated Ubc12) before the activated E2 reaches a downstream target. Therefore, GlyRS functions as a chaperone that critically supports neddylation. This function is probably conserved in all eukaryotic GlyRS enzymes and may contribute to the strong association of GlyRS with cancer progression.
Asunto(s)
Glicina-ARNt Ligasa/fisiología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinas/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/mortalidad , Dominio Catalítico , Ciclo Celular , Glicina-ARNt Ligasa/química , Células HEK293 , Células HeLa , Humanos , Estimación de Kaplan-Meier , Cinética , Proteína NEDD8 , Modelos de Riesgos Proporcionales , Unión Proteica , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteolisis , Enzimas Activadoras de Ubiquitina/química , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitinas/químicaRESUMEN
Recent studies suggested an essential role for seryl-tRNA synthetase (SerRS) in vascular development. This role is specific to SerRS among all tRNA synthetases and is independent of its well-known aminoacylation function in protein synthesis. A unique nucleus-directing domain, added at the invertebrate-to-vertebrate transition, confers this novel non-translational activity of SerRS. Previous studies showed that SerRS, in some unknown way, controls VEGFA expression to prevent vascular over-expansion. Using in vitro, cell and animal experiments, we show here that SerRS intervenes by antagonizing c-Myc, the major transcription factor promoting VEGFA expression, through a tandem mechanism. First, by direct head-to-head competition, nuclear-localized SerRS blocks c-Myc from binding to the VEGFA promoter. Second, DNA-bound SerRS recruits the SIRT2 histone deacetylase to erase prior c-Myc-promoted histone acetylation. Thus, vertebrate SerRS and c-Myc is a pair of 'Yin-Yang' transcriptional regulator for proper development of a functional vasculature. Our results also discover an anti-angiogenic activity for SIRT2.DOI: http://dx.doi.org/10.7554/eLife.02349.001.
Asunto(s)
Proteínas Proto-Oncogénicas c-myc/genética , Serina-ARNt Ligasa/genética , Secuencia de Aminoácidos , Inductores de la Angiogénesis/farmacología , Animales , Línea Celular , Epigénesis Genética , Femenino , Silenciador del Gen , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Conformación Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Serina-ARNt Ligasa/farmacología , Sirtuina 2/genética , Sirtuina 2/farmacología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pez CebraRESUMEN
A polyphasic taxonomic approach was used to study strain 12116T, which was isolated from desert sand in Xinjiang, China. Cells of the isolate were Gram-negative, non-motile, rod-shaped and aerobic. MK-7 was the predominant respiratory menaquinone. The DNA G+C content was 46.3 mol%. A phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate was most closely related to the members of the genus Dyadobacter, with similarities ranging from 94.7 to 96.6 %. Therefore, on the basis of genotypic and phenotypic data, strain 12116T represents a novel species of the genus Dyadobacter, for which the name Dyadobacter alkalitolerans sp. nov. is proposed. The type strain is 12116T (=CCTCC AB 207176T =NRRL B-51268T).