RESUMEN
Cancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103+ T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here, we show an unexpected and transient CD61 expression, which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling, improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically, the presence of CD61+ tumor-infiltrating T lymphocytes is associated with improved clinical outcomes, mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion, this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells, which potentiates a new target for immune-based cellular therapies.
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Antígenos CD , Apirasa , Cadenas alfa de Integrinas , Receptores de Antígenos de Linfocitos T , Transducción de Señal , Animales , Humanos , Ratones , Antígenos CD/metabolismo , Antígenos CD/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Cadenas alfa de Integrinas/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias/inmunología , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.
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Células Epiteliales/virología , Perfilación de la Expresión Génica/métodos , ARN Viral/genética , Proteínas de Unión al ARN/genética , Virus Sindbis/genética , Transcriptoma , Neoplasias del Cuello Uterino/virología , Regiones no Traducidas 5' , Sitios de Unión , Células Epiteliales/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Femenino , Regulación Viral de la Expresión Génica , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas del Complejo SMN , Virus Sindbis/crecimiento & desarrollo , Virus Sindbis/metabolismo , Virus Sindbis/patogenicidad , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Replicación ViralRESUMEN
BACKGROUND: Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac ß-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. METHODS: Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with ß-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. RESULTS: We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1 (histone deacetylase 1). Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. CONCLUSIONS: We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors.
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AMP Cíclico , Miocitos Cardíacos , Humanos , Proteómica , Hidrolasas Diéster Fosfóricas , Hipertrofia , AdrenérgicosRESUMEN
Marine phytoplankton can interchange trace metals in various biochemical functions, particularly under metal-limiting conditions. Here, we investigate the stimulating and toxicity effect of chromium (Cr) on a marine Chlorophyceae Osetreococcus tauri under Fe-replete and Fe-deficient conditions. We determined the growth, photosynthesis, and proteome expressions of Osetreococcus tauri cultured under different Cr and Fe concentrations. In Fe-replete conditions, the presence of Cr(VI) stimulated significantly the growth rate and the maximum yield of photochemistry of photosystem II (Fv /Fm ) of the phytoplankton, while the functional absorption cross-section of photosystem II (σPSII ) did not change. Minor additions of Cr(VI) partially rescued phytoplankton growth under Fe-limited conditions. Proteomic analysis of this alga grown in Fe-replete normal and Fe-replete with Cr addition media (10 µM Cr) showed that the presence of Cr significantly decreased the expression of phosphate-transporting proteins and photosynthetic proteins, while increasing the expression of proteins related to carbon assimilation. Cr can stimulate the growth and photosynthesis of O. tauri, but the effects are dependent on both the Cr(VI) concentration and the availability of Fe. The proteomic results further suggest that Cr(VI) addition might significantly increase starch production and carbon fixation.
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Complejo de Proteína del Fotosistema II , Proteómica , Complejo de Proteína del Fotosistema II/metabolismo , Cromo/toxicidad , Cromo/metabolismo , Fotosíntesis , Fitoplancton/metabolismo , Proteoma/metabolismoRESUMEN
CDK9 is a kinase critical for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. We also confirm the splicing factor SF3B1 as a target of CDK9 and show that SF3B1 in complex with polyadenylation factors is lost from chromatin after CDK9 inhibition. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.
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Factor B de Elongación Transcripcional Positiva , ARN Polimerasa II , Cromatina/genética , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , ARN , ARN Polimerasa II/metabolismo , Factores de Empalme de ARN/genética , Transcripción Genética , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Large RNA-binding complexes play a central role in gene expression and orchestrate production, function, and turnover of mRNAs. The accuracy and dynamics of RNA-protein interactions within these molecular machines are essential for their function and are mediated by RNA-binding proteins (RBPs). Here, we show that fission yeast whole-cell poly(A)+ RNA-protein crosslinking data provide information on the organization of RNA-protein complexes. To evaluate the relative enrichment of cellular RBPs on poly(A)+ RNA, we combine poly(A)+ RNA interactome capture with a whole-cell extract normalization procedure. This approach yields estimates of in vivo RNA-binding activities that identify subunits within multiprotein complexes that directly contact RNA. As validation, we trace RNA interactions of different functional modules of the 3' end processing machinery and reveal additional contacts. Extending our analysis to different mutants of the RNA exosome complex, we explore how substrate channeling through the complex is affected by mutation. Our data highlight the central role of the RNA helicase Mtl1 in regulation of the complex and provide insights into how different components contribute to engagement of the complex with substrate RNA. In addition, we characterize RNA-binding activities of novel RBPs that have been recurrently detected in the RNA interactomes of multiple species. We find that many of these, including cyclophilins and thioredoxins, are substoichiometric RNA interactors in vivo. Because RBPomes show very good overall agreement between species, we propose that the RNA-binding characteristics we observe in fission yeast are likely to apply to related proteins in higher eukaryotes as well.
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ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Ciclofilinas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Mutación , Subunidades de Proteína/metabolismo , Motivos de Unión al ARN , Proteínas de Unión al ARN/química , Ribosomas/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Transcripción Genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Alpha-synuclein pathology is associated with dopaminergic neuronal loss in the substantia nigra (SN) of Parkinson's patients. Working across human and mouse models, we investigated mechanisms by which the accumulation of soluble α-synuclein oligomers leads to neurodegeneration. Biochemical analysis of the midbrain of α-synuclein overexpressing BAC-transgenic male and female mice revealed age- and region-dependent mitochondrial dysfunction and accumulation of damaged proteins downstream of the RE1 Silencing Transcription Factor (REST). Vulnerable SN dopaminergic neurons displayed low REST levels compared with neighboring protected SN GABAergic neurons, which correlated with the accumulation of α-synuclein oligomers and disrupted mitochondrial morphology. Consistent with a protective role, REST levels were reduced in patient induced pluripotent stem cell-derived dopaminergic neurons carrying the SNCA-Triplication mutation, which accumulated α-synuclein oligomers and mitochondrial damage, and displayed REST target gene dysregulation. Furthermore, CRISPR-mediated REST KO induced mitochondrial dysfunction and impaired mitophagy in vitro Conversely, REST overexpression attenuated mitochondrial toxicity and mitochondrial morphology disruption through the transcription factor PGC-1α. Finally, decreased α-synuclein oligomer accumulation and mitochondrial dysfunction in mice correlated with nuclear REST and PGC-1α in protected SN GABAergic neurons compared with vulnerable dopaminergic neurons. Our findings show that increased levels of α-synuclein oligomers cause dopaminergic neuronal-specific dysfunction through mitochondrial toxicity, which can be attenuated by REST in an early model of Parkinsonian pathology. These findings highlight REST as a mediator of dopaminergic vulnerability in PD.SIGNIFICANCE STATEMENT Understanding early Parkinsonian pathophysiology through studies of advanced preclinical models is fundamental to the translation of disease-modifying therapies. Here we show disease-relevant levels of α-synuclein expression in mice leads to accumulation of α-synuclein oligomers in the absence of overt aggregation, and mitochondrial dysfunction in dopaminergic neurons lacking the RE1 Silencing Transcription Factor. Our findings identify the mechanism of action of RE1 Silencing Transcription Factor and PGC-1α as mediators of dopaminergic vulnerability in α-synuclein BAC-transgenic mice and induced pluripotent stem cell-derived dopaminergic cultures, highlighting their potential as therapeutic targets.
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Neuronas Dopaminérgicas/patología , Mitocondrias/patología , Proteínas Represoras/genética , Sinucleinopatías/genética , Sinucleinopatías/patología , alfa-Sinucleína/genética , Animales , Sistemas CRISPR-Cas , Cromosomas Artificiales Bacterianos , Femenino , Neuronas GABAérgicas/patología , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estrés Oxidativo , Enfermedad de Parkinson/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genéticaRESUMEN
Dissolved iron (Fe) is vanishingly low in the oceans, with ecological success conferred to microorganisms that can restructure their biochemistry to maintain high growth rates during Fe scarcity. Chemolithoautotrophic ammonia-oxidising archaea (AOA) are highly abundant in the oceans, constituting ~30% of cells below the photic zone. Here we examine the proteomic response of the AOA isolate Nitrosopumilus maritimus to growth-limiting Fe concentrations. Under Fe limitation, we observed a significant reduction in the intensity of Fe-dense ferredoxins associated with respiratory complex I whilst complex III and IV proteins with more central roles in the electron transport chain remain unchanged. We concomitantly observed an increase in the intensity of Fe-free functional alternatives such as flavodoxin and plastocyanin, thioredoxin and alkyl hydroperoxide which are known to mediate electron transport and reactive oxygen species detoxification, respectively. Under Fe limitation, we found a marked increase in the intensity of the ABC phosphonate transport system (Phn), highlighting an intriguing link between Fe and P cycling in N. maritimus. We hypothesise that an elevated uptake of exogenous phosphonates under Fe limitation may either supplement N. maritimus' endogenous methylphosphonate biosynthesis pathway - which requires Fe - or enhance the production of phosphonate-containing exopolysaccharides known to efficiently bind environmental Fe.
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Amoníaco , Archaea , Amoníaco/metabolismo , Archaea/metabolismo , Hierro/metabolismo , Nutrientes , Oxidación-Reducción , ProteómicaRESUMEN
The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome integrity and protection against cancer. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, recent studies provide evidence that PALB2 also associates with unperturbed chromatin. Here, we investigated the previously poorly described role of chromatin-associated PALB2 in undamaged cells. We found that PALB2 associates with active genes through its major binding partner, MRG15, which recognizes histone H3 trimethylated at lysine 36 (H3K36me3) by the SETD2 methyltransferase. Missense mutations that ablate PALB2 binding to MRG15 confer elevated sensitivity to the topoisomerase inhibitor camptothecin (CPT) and increased levels of aberrant metaphase chromosomes and DNA stress in gene bodies, which were suppressed by preventing DNA replication. Remarkably, the level of PALB2 at genic regions was frequently decreased, rather than increased, upon CPT treatment. We propose that the steady-state presence of PALB2 at active genes, mediated through the SETD2/H3K36me3/MRG15 axis, ensures an immediate response to DNA stress and therefore effective protection of these regions during DNA replication. This study provides a conceptual advance in demonstrating that the constitutive chromatin association of repair factors plays a key role in the maintenance of genome stability and furthers our understanding of why PALB2 defects lead to human genome instability syndromes.
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Cromatina/ultraestructura , Daño del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Factores de Transcripción/metabolismo , Proteína BRCA2/genética , Línea Celular Tumoral , Cromosomas/ultraestructura , Reparación del ADN , Replicación del ADN , Genoma Humano , Células HEK293 , Células HeLa , Humanos , Concentración 50 Inhibidora , Mutación , Unión Proteica , Proteómica , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Cancer precision medicine largely relies on knowledge about genetic aberrations in tumors and next-generation-sequencing studies have shown a high mutational complexity in many cancers. Although a large number of the observed mutations is believed to be not causally linked with cancer, the functional effects of many rare mutations but also of combinations of driver mutations are often unknown. Here, we perform a systems analysis of a model of EGFR-mutated nonsmall cell lung cancer resistant to targeted therapy that integrates whole exome sequencing, global time-course discovery phosphoproteomics and computational modeling to identify functionally relevant molecular alterations. Our approach allows for a complexity reduction from over 2,000 genetic events potentially involved in mediating resistance to only 44 phosphoproteins and 35 topologically close genetic alterations. We perform single- and combination-drug testing against the predicted phosphoproteins and discovered that targeting of HSPB1, DBNL and AKT1 showed potent antiproliferative effects overcoming resistance against EGFR-inhibitory therapy. Our approach may therefore be used to complement mutational profiling to identify functionally relevant molecular aberrations and propose combination therapies across cancers.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/genética , Fosforilación , Proteogenómica , Transducción de SeñalRESUMEN
Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins.
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Proteínas Bacterianas/metabolismo , Leptospira interrogans/metabolismo , Leptospirosis/microbiología , Animales , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos , Cobayas , Procesos Heterotróficos , Humanos , Leptospira interrogans/genética , Leptospira interrogans/crecimiento & desarrollo , Leptospira interrogans/patogenicidad , Masculino , Transporte de Proteínas , VirulenciaRESUMEN
Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm-Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract.
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Exosomas/inmunología , Inmunidad Innata , Sistema Urinario/inmunología , Adulto , Biomarcadores/orina , Exosomas/ultraestructura , Femenino , Humanos , Masculino , Microscopía Inmunoelectrónica , Proteoma/inmunología , Sistema Urinario/microbiología , Escherichia coli Uropatógena/crecimiento & desarrollo , Escherichia coli Uropatógena/inmunología , Adulto JovenRESUMEN
Protein phosphorylation is a common post-translational modification in eukaryotic cells and has a wide range of functional effects. Here, we used mass spectrometry to search for phosphorylated residues in all the proteins of influenza A and B viruses--to the best of our knowledge, the first time such a comprehensive approach has been applied to a virus. We identified 36 novel phosphorylation sites, as well as confirming 3 previously-identified sites. N-terminal processing and ubiquitination of viral proteins was also detected. Phosphorylation was detected in the polymerase proteins (PB2, PB1 and PA), glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M1), ion channel (M2), non-structural protein (NS1) and nuclear export protein (NEP). Many of the phosphorylation sites detected were conserved between influenza virus genera, indicating the fundamental importance of phosphorylation for all influenza viruses. Their structural context indicates roles for phosphorylation in regulating viral entry and exit (HA and NA); nuclear localisation (PB2, M1, NP, NS1 and, through NP and NEP, of the viral RNA genome); and protein multimerisation (NS1 dimers, M2 tetramers and NP oligomers). Using reverse genetics we show that for NP of influenza A viruses phosphorylation sites in the N-terminal NLS are important for viral growth, whereas mutating sites in the C-terminus has little or no effect. Mutating phosphorylation sites in the oligomerisation domains of NP inhibits viral growth and in some cases transcription and replication of the viral RNA genome. However, constitutive phosphorylation of these sites is not optimal. Taken together, the conservation, structural context and functional significance of phosphorylation sites implies a key role for phosphorylation in influenza biology. By identifying phosphorylation sites throughout the proteomes of influenza A and B viruses we provide a framework for further study of phosphorylation events in the viral life cycle and suggest a range of potential antiviral targets.
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Virus de la Influenza A/metabolismo , Virus de la Influenza B/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Proteoma/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Perros , Virus de la Influenza A/química , Virus de la Influenza B/química , Fosforilación , Proteoma/química , Proteínas Virales/químicaRESUMEN
KRAS is a proto-oncogene encoding a small GTPase. Mutations contribute to â¼30% of human solid tumours, including lung adenocarcinoma, pancreatic, and colorectal carcinomas. Most KRAS activating mutations interfere with GTP hydrolysis, essential for its role as a molecular switch, leading to alterations in their molecular environment and oncogenic signalling. However, the precise signalling cascades these mutations affect are poorly understood. Here, APEX2 proximity labelling was used to profile the molecular environment of WT, G12D, G13D, and Q61H-activating KRAS mutants under starvation and stimulation conditions. Through quantitative proteomics, we demonstrate the presence of known KRAS interactors, including ARAF and LZTR1, which are differentially captured by WT and KRAS mutants. Notably, the KRAS mutations G12D, G13D, and Q61H abrogate their association with LZTR1, thereby affecting turnover. Elucidating the implications of LZTR1-mediated regulation of KRAS protein levels in cancer may offer insights into therapeutic strategies targeting KRAS-driven malignancies.
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Neoplasias Colorrectales , Proteínas Proto-Oncogénicas p21(ras) , Ubiquitina-Proteína Ligasas , Humanos , Proteínas Cullin/genética , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal/genética , Factores de TranscripciónRESUMEN
The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.
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Factor de Transcripción E2F1 , Factor de Transcripción E2F4 , Células Madre Neoplásicas , Neoplasias Pancreáticas , Comunicación Paracrina , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Factor de Transcripción E2F1/metabolismo , Factor de Transcripción E2F1/genética , Línea Celular Tumoral , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Factor de Transcripción E2F4/metabolismo , Factor de Transcripción E2F4/genética , Animales , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Proteína de Retinoblastoma/metabolismo , Proteína de Retinoblastoma/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Factores de Transcripción p300-CBP/metabolismo , Factores de Transcripción p300-CBP/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Femenino , Proliferación Celular , Ratones , Transducción de Señal , Resistencia a Antineoplásicos/genéticaRESUMEN
BACKGROUND: Extracellular vesicles (EVs) are proposed to play a role in the development of cardiovascular diseases (CVDs) and are considered emerging markers of CVDs. n-3 PUFAs are abundant in oily fish and fish oil and are reported to reduce CVD risk, but there has been little research to date examining the effects of n-3 PUFAs on the generation and function of EVs. OBJECTIVES: We aimed to investigate the effects of fish oil supplementation on the number, generation, and function of EVs in subjects with moderate risk of CVDs. METHODS: A total of 40 participants with moderate risk of CVDs were supplemented with capsules containing either fish oil (1.9 g/d n-3 PUFAs) or control oil (high-oleic safflower oil) for 12 wk in a randomized, double-blind, placebo-controlled crossover intervention study. The effects of fish oil supplementation on conventional CVD and thrombogenic risk markers were measured, along with the number and fatty acid composition of circulating and platelet-derived EVs (PDEVs). PDEV proteome profiles were evaluated, and their impact on coagulation was assessed using assays including fibrin clot formation, thrombin generation, fibrinolysis, and ex vivo thrombus formation. RESULTS: n-3 PUFAs decreased the numbers of circulating EVs by 27%, doubled their n-3 PUFA content, and reduced their capacity to support thrombin generation by >20% in subjects at moderate risk of CVDs. EVs derived from n-3 PUFA-enriched platelets in vitro also resulted in lower thrombin generation, but did not alter thrombus formation in a whole blood ex vivo assay. CONCLUSIONS: Dietary n-3 PUFAs alter the number, composition, and function of EVs, reducing their coagulatory activity. This study provides clear evidence that EVs support thrombin generation and that this EV-dependent thrombin generation is reduced by n-3 PUFAs, which has implications for prevention and treatment of thrombosis. CLINICAL TRIAL REGISTRY: This trial was registered at clinicaltrials.gov as NCT03203512.
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Coagulación Sanguínea , Plaquetas , Estudios Cruzados , Vesículas Extracelulares , Ácidos Grasos Omega-3 , Humanos , Vesículas Extracelulares/metabolismo , Ácidos Grasos Omega-3/farmacología , Masculino , Femenino , Persona de Mediana Edad , Método Doble Ciego , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Suplementos Dietéticos , Enfermedades Cardiovasculares/prevención & control , Adulto , Aceites de Pescado/farmacología , Aceites de Pescado/administración & dosificación , Anciano , Ácidos Grasos/metabolismoRESUMEN
Patients with alcoholism and type 2 diabetes manifest altered metabolism, including elevated aldehyde levels and unusually low asparagine levels. We show that asparagine synthetase B (ASNS), the only human asparagine-forming enzyme, is inhibited by disease-relevant reactive aldehydes, including formaldehyde and acetaldehyde. Cellular studies show non-cytotoxic amounts of reactive aldehydes induce a decrease in asparagine levels. Biochemical analyses reveal inhibition results from reaction of the aldehydes with the catalytically important N-terminal cysteine of ASNS. The combined cellular and biochemical results suggest a possible mechanism underlying the low asparagine levels in alcoholism and diabetes. The results will stimulate research on the biological consequences of the reactions of aldehydes with nucleophilic residues.
RESUMEN
Acute myeloid leukemia (AML) is a heterogeneous disease characterized by genomic aberrations in oncogenes, cytogenetic abnormalities, and an aberrant epigenetic landscape. Nearly 50% of AML cases will relapse with current treatment. A major source of therapy resistance is the interaction of mesenchymal stroma with leukemic cells resulting in therapeutic protection. We aimed to determine pro-survival/anti-apoptotic protein networks involved in the stroma protection of leukemic cells. Proteomic profiling of cultured primary AML (n = 14) with Hs5 stroma cell line uncovered an up-regulation of energy-favorable metabolic proteins. Next, we modulated stroma-induced drug resistance with an epigenetic drug library, resulting in reduced apoptosis with histone deacetylase inhibitor (HDACi) treatment versus other epigenetic modifying compounds. Quantitative phosphoproteomic probing of this effect further revealed a metabolic-enriched phosphoproteome including significant up-regulation of acetyl-coenzyme A synthetase (ACSS2, S30) in leukemia-stroma HDACi treated cocultures compared with untreated monocultures. Validating these findings, we show ACSS2 substrate, acetate, promotes leukemic proliferation, ACSS2 knockout in leukemia cells inhibits leukemic proliferation and ACSS2 knockout in the stroma impairs leukemic metabolic fitness. Finally, we identify ACSS1/ACSS2-high expression AML subtype correlating with poor overall survival. Collectively, this study uncovers the leukemia-stroma phosphoproteome emphasizing a role for ACSS2 in mediating AML growth and drug resistance.
RESUMEN
BACKGROUND: Serratia sp. ATCC 39006 (S39006) is a Gram-negative enterobacterium that is virulent in plant and animal models. It produces a red-pigmented trypyrrole secondary metabolite, prodigiosin (Pig), and a carbapenem antibiotic (Car), as well as the exoenzymes, pectate lyase and cellulase. Secondary metabolite production in this strain is controlled by a complex regulatory network involving quorum sensing (QS). Hfq and RsmA (two RNA binding proteins and major post-transcriptional regulators of gene expression) play opposing roles in the regulation of several key phenotypes within S39006. Prodigiosin and carbapenem production was abolished, and virulence attenuated, in an S39006 ∆hfq mutant, while the converse was observed in an S39006 rsmA transposon insertion mutant. RESULTS: In order to define the complete regulon of Hfq and RsmA, deep sequencing of cDNA libraries (RNA-seq) was used to analyse the whole transcriptome of S39006 ∆hfq and rsmA::Tn mutants. Moreover, we investigated global changes in the proteome using an LC-MS/MS approach. Analysis of differential gene expression showed that Hfq and RsmA directly or indirectly regulate (at the level of RNA) 4% and 19% of the genome, respectively, with some correlation between RNA and protein expression. Pathways affected include those involved in antibiotic regulation, virulence, flagella synthesis, and surfactant production. Although Hfq and RsmA are reported to activate flagellum production in E. coli and an adherent-invasive E. coli hfq mutant was shown to have no flagella by electron microscopy, we found that flagellar production was increased in the S39006 rsmA and hfq mutants. Additionally, deletion of rsmA resulted in greater genomic flux with increased activity of two mobile genetic elements. This was confirmed by qPCR and analysis of rsmA culture supernatant revealed the presence of prophage DNA and phage particles. Finally, expression of a hypothetical protein containing DUF364 increased prodigiosin production and was controlled by a putative 5' cis-acting regulatory RNA element. CONCLUSION: Using a combination of transcriptomics and proteomics this study provides a systems-level understanding of Hfq and RsmA regulation and identifies similarities and differences in the regulons of two major regulators. Additionally our study indicates that RsmA regulates both core and variable genome regions and contributes to genome stability.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteína de Factor 1 del Huésped/genética , Proteína de Factor 1 del Huésped/metabolismo , Serratia/genética , Serratia/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Análisis por Conglomerados , Transporte de Electrón/genética , Flagelos/genética , Flagelos/metabolismo , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Mutación , Operón , Prodigiosina/metabolismo , Proteoma , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ARN , Serratia/patogenicidad , Serratia/virología , Transcriptoma , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Formaldehyde (HCHO) is a potent electrophile that is toxic above threshold levels, but which is also produced in the nuclei of eukaryotic cells by demethylases. We report studies with the four canonical human histones revealing that histone H2B reacts with HCHO, including as generated by a histone demethylase, to give a stable product. NMR studies show that HCHO reacts with the N-terminal proline and associated amide of H2B to give a 5,5-bicyclic aminal that is relatively stable to competition with HCHO scavengers. While the roles of histone modification by this reaction require further investigation, we demonstrated the potential of N-terminal aminal formation to modulate protein function by conducting biochemical and cellular studies on the effects of HCHO on catalysis by 4-oxalocrotonate tautomerase, which employs a nucleophilic N-terminal proline. The results suggest that reactions of N-terminal residues with HCHO and other aldehydes have potential to alter protein function.