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1.
Mol Cell ; 82(8): 1557-1572.e7, 2022 04 21.
Artículo en Inglés | MEDLINE | ID: mdl-35180429

RESUMEN

During the translation surveillance mechanism known as ribosome-associated quality control, the ASC-1 complex (ASCC) disassembles ribosomes stalled on the mRNA. Here, we show that there are two distinct classes of stalled ribosome. Ribosomes stalled by translation elongation inhibitors or methylated mRNA are short lived in human cells because they are split by the ASCC. In contrast, although ultraviolet light and 4-nitroquinoline 1-oxide induce ribosome stalling by damaging mRNA, and the ASCC is recruited to these stalled ribosomes, we found that they are refractory to the ASCC. Consequently, unresolved UV- and 4NQO-stalled ribosomes persist in human cells. We show that ribosome stalling activates cell-cycle arrest, partly through ZAK-p38MAPK signaling, and that this cell-cycle delay is prolonged when the ASCC cannot resolve stalled ribosomes. Thus, we propose that the sensitivity of stalled ribosomes to the ASCC influences the kinetics of stall resolution, which in turn controls the adaptive stress response.


Asunto(s)
Daño del ADN , Ribosomas , Humanos , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismo
2.
Clin Chem Lab Med ; 62(6): 1206-1216, 2024 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-38253336

RESUMEN

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.


Asunto(s)
COVID-19 , Espectrometría de Masas , Técnicas de Diagnóstico Molecular , ARN Viral , SARS-CoV-2 , Saliva , Humanos , Saliva/virología , Saliva/química , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/inmunología , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/virología , ARN Viral/análisis , Espectrometría de Masas/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Masculino , Sensibilidad y Especificidad , Femenino , Persona de Mediana Edad , Fosfoproteínas/análisis , Fosfoproteínas/inmunología , Proteínas de la Nucleocápside de Coronavirus/análisis , Proteínas de la Nucleocápside de Coronavirus/inmunología , Antígenos Virales/análisis , Antígenos Virales/inmunología , Adulto , Cromatografía Liquida/métodos
3.
Mol Cell ; 61(6): 834-49, 2016 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-26990987

RESUMEN

The death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIPL only) or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate.


Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Caspasa 8/genética , Linaje de la Célula/genética , Isoformas de Proteínas/genética , Apoptosis/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Humanos , Mutagénesis , Unión Proteica , Isoformas de Proteínas/metabolismo , Espectrometría de Masas en Tándem
4.
Mol Psychiatry ; 27(3): 1455-1468, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34937870

RESUMEN

Schizophrenia (SCZ) and bipolar disorder are debilitating neuropsychiatric disorders arising from a combination of environmental and genetic factors. Novel open reading frames (nORFs) are genomic loci that give rise to previously uncharacterized transcripts and protein products. In our previous work, we have shown that nORFs can be biologically regulated and that they may play a role in cancer and rare diseases. More importantly, we have shown that nORFs may emerge in accelerated regions of the genome giving rise to species-specific functions. We hypothesize that nORFs represent a potentially important group of biological factors that may contribute to SCZ and bipolar disorder pathophysiology. Human accelerated regions (HARs) are genomic features showing human-lineage-specific rapid evolution that may be involved in biological regulation and have additionally been found to associate with SCZ genes. Transposable elements (TEs) are another set of genomic features that have been shown to regulate gene expression. As with HARs, their relevance to SCZ has also been suggested. Here, nORFs are investigated in the context of HARs and TEs. This work shows that nORFs whose expression is disrupted in SCZ and bipolar disorder are in close proximity to HARs and TEs and that some of them are significantly associated with SCZ and bipolar disorder genomic hotspots. We also show that nORF encoded proteins can form structures and potentially constitute novel drug targets.


Asunto(s)
Trastorno Bipolar , Esquizofrenia , Trastorno Bipolar/genética , Elementos Transponibles de ADN/genética , Estudio de Asociación del Genoma Completo , Humanos , Sistemas de Lectura Abierta/genética , Esquizofrenia/genética , Esquizofrenia/metabolismo
5.
Clin Chem Lab Med ; 61(2): 302-310, 2023 01 27.
Artículo en Inglés | MEDLINE | ID: mdl-36395058

RESUMEN

OBJECTIVES: During 2020, the UK's Department of Health and Social Care (DHSC) established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity. METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK (ADE) and AYNVTQAFGR (AYN), derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study. RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen's Kappa 0.90). Excluding Cq>32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen's Kappa 0.95. CONCLUSIONS: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Pandemias , COVID-19/diagnóstico , Prueba de COVID-19 , Espectrometría de Masas en Tándem/métodos , Cromatografía Liquida , Estudios Prospectivos , Técnicas de Laboratorio Clínico/métodos , Sensibilidad y Especificidad , Péptidos
6.
Nucleic Acids Res ; 49(1): 458-478, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33332560

RESUMEN

The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5'ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.


Asunto(s)
Autoantígenos/fisiología , Proteína I de Unión a Poli(A)/fisiología , Polirribosomas/metabolismo , Biosíntesis de Proteínas/fisiología , ARN Mensajero/metabolismo , Ribonucleoproteínas/fisiología , Serina-Treonina Quinasas TOR/fisiología , Regiones no Traducidas 5'/genética , Autoantígenos/genética , Regulación de la Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 2 de la Rapamicina/antagonistas & inhibidores , Mutagénesis Sitio-Dirigida , Mutación Missense , Naftiridinas/farmacología , Mutación Puntual , Biosíntesis de Proteínas/genética , Interferencia de ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/genética , Antígeno SS-B
7.
J Biol Chem ; 295(38): 13277-13286, 2020 09 18.
Artículo en Inglés | MEDLINE | ID: mdl-32723862

RESUMEN

The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per-ARNT-Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.


Asunto(s)
Corteza Cerebral/química , Canales de Potasio Éter-A-Go-Go/química , Hemo/química , Neuronas/química , Corteza Cerebral/metabolismo , Canales de Potasio Éter-A-Go-Go/metabolismo , Hemo/metabolismo , Humanos , Neuronas/metabolismo , Unión Proteica , Dominios Proteicos
8.
Biochem J ; 477(4): 773-786, 2020 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-32011671

RESUMEN

NF-κB repressing factor (NKRF) was recently identified as an RNA binding protein that together with its associated proteins, the 5'-3' exonuclease XRN2 and the helicase DHX15, is required to process the precursor ribosomal RNA. XRN2 is a multi-functional ribonuclease that is also involved in processing mRNAs, tRNAs and lncRNAs. The activity and stability of XRN2 are controlled by its binding partners, PAXT-1, CDKN2AIP and CDKN2AIPNL. In each case, these proteins interact with XRN2 via an XRN2 binding domain (XTBD), the structure and mode of action of which is highly conserved. Rather surprisingly, although NKRF interacts directly with XRN2, it was not predicted to contain such a domain, and NKRF's interaction with XRN2 was therefore unexplained. We have identified an alternative upstream AUG start codon within the transcript that encodes NKRF and demonstrate that the full-length form of NKRF contains an XTBD that is conserved across species. Our data suggest that NKRF is tethered in the nucleolus by binding directly to rRNA and that the XTBD in the N-terminal extension of NKRF is essential for the retention of XRN2 in this sub-organelle. Thus, we propose NKRF regulates the early steps of pre-rRNA processing during ribosome biogenesis by controlling the spatial distribution of XRN2 and our data provide further support for the XTBD as an XRN2 interacting motif.


Asunto(s)
Nucléolo Celular/metabolismo , Exorribonucleasas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Exorribonucleasas/genética , Células HeLa , Humanos , Unión Proteica , Proteínas Represoras/genética , Homología de Secuencia
9.
Nucleic Acids Res ; 47(15): 8224-8238, 2019 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-31180491

RESUMEN

The CCR4-NOT complex plays an important role in the translational repression and deadenylation of mRNAs. However, little is known about the specific roles of interacting factors. We demonstrate that the DEAD-box helicases eIF4A2 and DDX6 interact directly with the MA3 and MIF domains of CNOT1 and compete for binding. Furthermore, we now show that incorporation of eIF4A2 into the CCR4-NOT complex inhibits CNOT7 deadenylation activity in contrast to DDX6 which enhances CNOT7 activity. Polyadenylation tests (PAT) on endogenous mRNAs determined that eIF4A2 bound mRNAs have longer poly(A) tails than DDX6 bound mRNAs. Immunoprecipitation experiments show that eIF4A2 does not inhibit CNOT7 association with the CCR4-NOT complex but instead inhibits CNOT7 activity. We identified a CCR4-NOT interacting factor, TAB182, that modulates helicase recruitment into the CCR4-NOT complex, potentially affecting the outcome for the targeted mRNA. Together, these data show that the fate of an mRNA is dependent on the specific recruitment of either eIF4A2 or DDX6 to the CCR4-NOT complex which results in different pathways for translational repression and mRNA deadenylation.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Exorribonucleasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión/genética , Unión Competitiva , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , Exorribonucleasas/genética , Células HEK293 , Células HeLa , Humanos , Modelos Genéticos , Unión Proteica , Dominios Proteicos , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Proteínas Represoras/genética , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Factores de Transcripción/genética
10.
Mol Cell ; 47(2): 291-305, 2012 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-22683266

RESUMEN

Formation of the death-inducing signaling complex (DISC) is a critical step in death receptor-mediated apoptosis, yet the mechanisms underlying assembly of this key multiprotein complex remain unclear. Using quantitative mass spectrometry, we have delineated the stoichiometry of the native TRAIL DISC. While current models suggest that core DISC components are present at a ratio of 1:1, our data indicate that FADD is substoichiometric relative to TRAIL-Rs or DED-only proteins; strikingly, there is up to 9-fold more caspase-8 than FADD in the DISC. Using structural modeling, we propose an alternative DISC model in which procaspase-8 molecules interact sequentially, via their DED domains, to form a caspase-activating chain. Mutating key interacting residues in procaspase-8 DED2 abrogates DED chain formation in cells and disrupts TRAIL/CD95 DISC-mediated procaspase-8 activation in a functional DISC reconstitution model. This provides direct experimental evidence for a DISC model in which DED chain assembly drives caspase-8 dimerization/activation, thereby triggering cell death.


Asunto(s)
Apoptosis , Caspasa 8/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Línea Celular Tumoral , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Células Jurkat , Espectrometría de Masas/métodos , Modelos Biológicos , Modelos Moleculares , Conformación Molecular , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/química , Receptor fas/química
12.
J Proteome Res ; 13(7): 3360-9, 2014 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-24854137

RESUMEN

Despite the increasing popularity of data-independent acquisition workflows, data-dependent acquisition (DDA) is still the prevalent method of LC-MS-based proteomics. DDA is the basis of isobaric mass tagging technique, a powerful MS2 quantification strategy that allows coanalysis of up to 10 proteomics samples. A well-documented limitation of DDA, however, is precursor coselection, whereby a target peptide is coisolated with other ions for fragmentation. Here, we investigated if additional peptide purification by traveling wave ion mobility separation (TWIMS) can reduce precursor contamination using a mixture of Saccharomyces cerevisiae and HeLa proteomes. In accordance with previous reports on FAIMS-Orbitrap instruments, we find that TWIMS provides a remarkable improvement (on average 2.85 times) in the signal-to-noise ratio for sequence ions. We also report that TWIMS reduces reporter ions contamination by around one-third (to 14-15% contamination) and even further (to 6-9%) when combined with a narrowed quadrupole isolation window. We discuss challenges associated with applying TWIMS purification to isobaric mass tagging experiments, including correlation between ion m/z and drift time, which means that coselected peptides are expected to have similar mobility. We also demonstrate that labeling results in peptides having more uniform m/z and drift time distributions than observed for unlabeled peptides. Data are available via ProteomeXchange with identifier PXD001047.


Asunto(s)
Proteoma/química , Cromatografía Liquida , Células HeLa , Humanos , Peso Molecular , Proteoma/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Relación Señal-Ruido , Espectrometría de Masas en Tándem
13.
Mol Pharmacol ; 79(5): 844-54, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21325018

RESUMEN

An initial stage of many neurodegenerative processes is associated with compromised synaptic function and precedes synapse loss, neurite fragmentation, and neuronal death. We showed previously that deficiency of heme, regulating many proteins of pharmacological importance, causes neurodegeneration of primary cortical neurons via N-methyl-d-aspartate receptor (NMDAR)-dependent suppression of the extracellular signal-regulated kinase 1/2 pathway. Here, we asked whether the reduction of heme causes synaptic perturbation before neurite fragmentation in neuronal cultures and investigated molecular mechanisms of synaptic dysfunction in these cells. We showed the change in the NR2B subunit phosphorylation that correlates with compromised NMDAR function after the reduction of regulatory heme and a rapid rescue of NR2B phosphorylation and NMDAR function by exogenous heme. Electrophysiological recordings demonstrated diminished NMDAR currents and NMDAR-mediated calcium influx after 24 h of inhibition of heme synthesis. These effects were reversed by treatment with heme; however, inhibition of the Src family kinases abolished the rescue effect of heme on NMDA-evoked currents. Diminished NMDAR current and Ca(2+) influx resulted in suppressed cGMP production and impairment of spine formation. Exogenous heme exerted rescue effects on NR2B tyrosine phosphorylation and NMDA-evoked currents within minutes, suggesting direct interactions within the NMDAR complex. These synaptic changes after inhibition of heme synthesis occurred at this stage without apparent dysfunction of major hemoproteins. We conclude that regulatory heme is necessary in maintaining NR2B phosphorylation and NMDAR function. NMDAR failure occurs before neurite fragmentation and may be a causal factor in neurodegeneration; this could suggest a route for an early pharmacological intervention.


Asunto(s)
Hemo/metabolismo , Neuronas/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , Adenosina Trifosfato/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Femenino , Hemo/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Oxidación-Reducción , Fosforilación , Reacción en Cadena de la Polimerasa , Receptores de N-Metil-D-Aspartato/metabolismo , Tirosina/metabolismo
14.
Mol Cell Proteomics ; 8(7): 1501-15, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19346216

RESUMEN

We used shotgun proteomics to identify plasma membrane and lipid raft proteins purified from B cells obtained from mantle cell lymphoma (MCL) patients in leukemic phase. Bioinformatics identified 111 transmembrane proteins, some of which were profiled in primary MCL cases, MCL-derived cell lines, and normal B cells using RT-PCR and Western blotting. Several transmembrane proteins, including CD27, CD70, and CD31 (PECAM-1), were overexpressed when compared with normal B cells. CD70 was up-regulated (>10-fold) in three of five MCL patients along with its cognate receptor CD27, which was up-regulated (4-9-fold) in five of five patients, suggesting that MCL cells may undergo autocrine stimulation via this signaling pathway. Activated calpain I and protein kinase C betaII were also detected in the plasma membranes, suggesting that these proteins are constitutively active in MCL. Protein kinase C betaII has been associated with lipid rafts, and shotgun proteomics/protein profiling revealed that key lipid raft proteins, raftlin (four of five patients) and CSK (C-terminal Src kinase)-binding protein (Cbp)/phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG) (four of four patients) were down-regulated in MCL. Levels of other known lipid raft proteins, such as Lyn kinase and flotillin 1, were similar to normal B cells. However, 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, was associated with lipid rafts and was up-regulated approximately 7-fold in MCL compared with normal B cells. Significantly inhibitors of 5-LO activity (AA861) and 5-LO-activating protein (FLAP) (MK886, its activating enzyme) induced apoptosis in MCL cell lines and primary chronic lymphocytic leukemia cells, indicating an important role for the leukotriene biosynthetic pathway in MCL and other B cell malignancies. Thus, using shotgun proteomics and mRNA and protein expression profiling we identified a subset of known and unknown transmembrane proteins with aberrant expression in MCL plasma membranes. These proteins may play a role in the pathology of the disease and are potential therapeutic targets in MCL.


Asunto(s)
Membrana Celular/química , Linfoma de Células del Manto/química , Linfoma de Células del Manto/fisiopatología , Proteínas de la Membrana/análisis , Proteínas de Neoplasias/análisis , Análisis por Matrices de Proteínas/métodos , Transducción de Señal/fisiología , Adulto , Animales , Apoptosis/fisiología , Araquidonato 5-Lipooxigenasa/metabolismo , Membrana Celular/metabolismo , Biología Computacional , Humanos , Isoenzimas/análisis , Inhibidores de la Lipooxigenasa , Masculino , Microdominios de Membrana/química , Proteína Quinasa C/análisis , Proteína Quinasa C beta , Proteómica/métodos , Células Tumorales Cultivadas
15.
Nat Commun ; 12(1): 819, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547302

RESUMEN

Regulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.


Asunto(s)
Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/química , Caspasa 8/química , Proteína de Dominio de Muerte Asociada a Fas/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Dominio Catalítico , Clonación Molecular , Microscopía por Crioelectrón , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/química , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/genética , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Muerte Celular Regulada/genética , Factor de Necrosis Tumoral alfa/química , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo
16.
Cell Rep ; 26(8): 2028-2036.e6, 2019 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-30784586

RESUMEN

Although poly-ADP-ribosylation (PARylation) of DNA repair factors had been well documented, its role in the repair of DNA double-strand breaks (DSBs) is poorly understood. NR4A nuclear orphan receptors were previously linked to DSB repair; however, their function in the process remains elusive. Classically, NR4As function as transcription factors using a specialized tandem zinc-finger DNA-binding domain (DBD) for target gene induction. Here, we show that NR4A DBD is bi-functional and can bind poly-ADP-ribose (PAR) through a pocket localized in the second zinc finger. Separation-of-function mutants demonstrate that NR4A PAR binding, while dispensable for transcriptional activity, facilitates repair of radiation-induced DNA double-strand breaks in G1. Moreover, we define DNA-PKcs protein as a prominent target of ionizing radiation-induced PARylation. Mechanistically, NR4As function by directly targeting poly-ADP-ribosylated DNA-PKcs to facilitate its autophosphorylation-promoting DNA-PK kinase assembly at DNA lesions. Selective targeting of the PAR-binding pocket of NR4A presents an opportunity for cancer therapy.


Asunto(s)
Reparación del ADN , Proteína Quinasa Activada por ADN/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Sitios de Unión , Línea Celular Tumoral , Proteína Quinasa Activada por ADN/química , Células HEK293 , Humanos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/química , Poli ADP Ribosilación , Poli Adenosina Difosfato Ribosa/química , Poli Adenosina Difosfato Ribosa/metabolismo , Unión Proteica , Dedos de Zinc
17.
Genome Biol ; 20(1): 262, 2019 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-31791371

RESUMEN

BACKGROUND: Regulation of the mRNA life cycle is central to gene expression control and determination of cell fate. miRNAs represent a critical mRNA regulatory mechanism, but despite decades of research, their mode of action is still not fully understood. RESULTS: Here, we show that eIF4A2 is a major effector of the repressive miRNA pathway functioning via the Ccr4-Not complex. We demonstrate that while DDX6 interacts with Ccr4-Not, its effects in the mechanism are not as pronounced. Through its interaction with the Ccr4-Not complex, eIF4A2 represses mRNAs at translation initiation. We show evidence that native eIF4A2 has similar RNA selectivity to chemically inhibited eIF4A1. eIF4A2 exerts its repressive effect by binding purine-rich motifs which are enriched in the 5'UTR of target mRNAs directly upstream of the AUG start codon. CONCLUSIONS: Our data support a model whereby purine motifs towards the 3' end of the 5'UTR are associated with increased ribosome occupancy and possible uORF activation upon eIF4A2 binding.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Regulación de la Expresión Génica , MicroARNs/fisiología , Receptores CCR4/metabolismo , Factores de Transcripción/metabolismo , Regiones no Traducidas 5' , Humanos
18.
Nat Commun ; 9(1): 3877, 2018 09 24.
Artículo en Inglés | MEDLINE | ID: mdl-30250067

RESUMEN

PAXX is a recently identified component of the nonhomologous end joining (NHEJ) DNA repair pathway. The molecular mechanisms of PAXX action remain largely unclear. Here we characterise the interactomes of PAXX and its paralogs, XLF and XRCC4, to show that these factors share the ability to interact with DNA polymerase λ (Pol λ), stimulate its activity and are required for recruitment of Pol λ to laser-induced DNA damage sites. Stimulation of Pol λ activity by XRCC4 paralogs requires a direct interaction between the SP/8 kDa domain of Pol λ and their N-terminal head domains to facilitate recognition of the 5' end of substrate gaps. Furthermore, PAXX and XLF collaborate with Pol λ to promote joining of incompatible DNA ends and are redundant in supporting Pol λ function in vivo. Our findings identify Pol λ as a novel downstream effector of PAXX function and show XRCC4 paralogs act in synergy to regulate polymerase activity in NHEJ.


Asunto(s)
Reparación del ADN por Unión de Extremidades/fisiología , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Roturas del ADN de Doble Cadena/efectos de la radiación , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/aislamiento & purificación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Células HEK293 , Humanos , Rayos Láser/efectos adversos , Mutagénesis Sitio-Dirigida , Unión Proteica/fisiología , Dominios Proteicos/fisiología , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en Tándem/métodos
19.
Bioorg Med Chem ; 13(24): 6727-31, 2005 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-16105739

RESUMEN

To study the biological effects of quercetin, authentic products of quercetin metabolism are required as standards. The synthesis of quercetin sulfate standards is thus described. Quercetin was reacted with a 10-fold molar excess of sulfur trioxide-N-triethylamine, and the products were analyzed by HPLC and mass spectrometry. Four monosulfates and three disulfates were identified, and structural inferences were drawn by 1H NMR spectrometry of HPLC peak isolates. Analysis of the urine of rats that had received quercetin (1.9 g/kg po) yielded a single peak, which by comparison with the products of the reaction between quercetin and sulfur trioxide-N-triethylamine was identified as quercetin 3'-O-sulfate.


Asunto(s)
Quercetina/química , Quercetina/orina , Sulfatos/síntesis química , Sulfatos/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética , Estructura Molecular , Quercetina/metabolismo , Quercetina/farmacología , Ratas , Ratas Endogámicas F344 , Sulfatos/química
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