Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 268
Filtrar
2.
Mol Syst Biol ; 17(9): e10156, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34569154

RESUMO

Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho-signaling in drug-treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.

3.
J Clin Oncol ; : JCO2101691, 2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34591593

RESUMO

PURPOSE: Approximately 10%-40% of patients with lung cancer report no history of tobacco smoking (never-smokers). We analyzed whole-exome and RNA-sequencing data of 160 tumor and normal lung adenocarcinoma (LUAD) samples from never-smokers to identify clinically actionable alterations and gain insight into the environmental and hereditary risk factors for LUAD among never-smokers. METHODS: We performed whole-exome and RNA-sequencing of 88 and 69 never-smoker LUADs. We analyzed these data in conjunction with data from 76 never-smoker and 299 smoker LUAD samples sequenced by The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium. RESULTS: We observed a high prevalence of clinically actionable driver alterations in never-smoker LUADs compared with smoker LUADs (78%-92% v 49.5%; P < .0001). Although a subset of never-smoker samples demonstrated germline alterations in DNA repair genes, the frequency of samples showing germline variants in cancer predisposing genes was comparable between smokers and never-smokers (6.4% v 6.9%; P = .82). A subset of never-smoker samples (5.9%) showed mutation signatures that were suggestive of passive exposure to cigarette smoke. Finally, analysis of RNA-sequencing data showed distinct immune transcriptional subtypes of never-smoker LUADs that varied in their expression of clinically relevant immune checkpoint molecules and immune cell composition. CONCLUSION: In this comprehensive genomic and transcriptome analysis of never-smoker LUADs, we observed a potential role for germline variants in DNA repair genes and passive exposure to cigarette smoke in the pathogenesis of a subset of never-smoker LUADs. Our findings also show that clinically actionable driver alterations are highly prevalent in never-smoker LUADs, highlighting the need for obtaining biopsies with adequate cellularity for clinical genomic testing in these patients.

4.
Mol Cell Proteomics ; : 100154, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34592423

RESUMO

Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites are necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-ɛ-GG peptides are enriched with anti-K-ε-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-ε-GG antibody (mK-ε-GG) and a magnetic particle processor. We report the identification of ∼20,000 ubiquitylation sites from a TMT10-plex with 500 µg input per sample processed in ∼2 hours. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility and significantly reduced processing time. The automated method also significantly reduced variability across process replicates compared to the manual method. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of the method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples.

5.
Nat Commun ; 12(1): 4980, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404792

RESUMO

Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, we show that it is possible to map specific protein subclasses within distinct compartments of living cells. In particular, we develop a method to enrich subcompartment-specific RNA binding proteins (RBPs) by combining peroxidase-catalyzed PL with organic-aqueous phase separation of crosslinked protein-RNA complexes ("APEX-PS"). We use APEX-PS to generate datasets of nuclear, nucleolar, and outer mitochondrial membrane (OMM) RBPs, which can be mined for novel functions. For example, we find that the OMM RBP SYNJ2BP retains specific nuclear-encoded mitochondrial mRNAs at the OMM during translation stress, facilitating their local translation and import of protein products into the mitochondrion during stress recovery. Functional PL in general, and APEX-PS in particular, represent versatile approaches for the discovery of proteins with novel function in specific subcellular compartments.


Assuntos
RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fosforilação , Proteoma/metabolismo , Proteômica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética
6.
Elife ; 102021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34414886

RESUMO

The trafficking of specific protein cohorts to correct subcellular locations at correct times is essential for every signaling and regulatory process in biology. Gene perturbation screens could provide a powerful approach to probe the molecular mechanisms of protein trafficking, but only if protein localization or mislocalization can be tied to a simple and robust phenotype for cell selection, such as cell proliferation or fluorescence-activated cell sorting (FACS). To empower the study of protein trafficking processes with gene perturbation, we developed a genetically encoded molecular tool named HiLITR (High-throughput Localization Indicator with Transcriptional Readout). HiLITR converts protein colocalization into proteolytic release of a membrane-anchored transcription factor, which drives the expression of a chosen reporter gene. Using HiLITR in combination with FACS-based CRISPRi screening in human cell lines, we identified genes that influence the trafficking of mitochondrial and ER tail-anchored proteins. We show that loss of the SUMO E1 component SAE1 results in mislocalization and destabilization of many mitochondrial tail-anchored proteins. We also demonstrate a distinct regulatory role for EMC10 in the ER membrane complex, opposing the transmembrane-domain insertion activity of the complex. Through transcriptional integration of complex cellular functions, HiLITR expands the scope of biological processes that can be studied by genetic perturbation screening technologies.

8.
Mol Cell Proteomics ; 20: 100133, 2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34391888

RESUMO

MS is the most effective method to directly identify peptides presented on human leukocyte antigen (HLA) molecules. However, current standard approaches often use 500 million or more cells as input to achieve high coverage of the immunopeptidome, and therefore, these methods are not compatible with the often limited amounts of tissue available from clinical tumor samples. Here, we evaluated microscaled basic reversed-phase fractionation to separate HLA peptide samples offline followed by ion mobility coupled to LC-MS/MS for analysis. The combination of these two separation methods enabled identification of 20% to 50% more peptides compared with samples analyzed without either prior fractionation or use of ion mobility alone. We demonstrate coverage of HLA immunopeptidomes with up to 8107 distinct peptides starting with as few as 100 million cells. The increased sensitivity obtained using our methods can provide data useful to improve HLA-binding prediction algorithms as well as to enable detection of clinically relevant epitopes such as neoantigens.

9.
Sci Data ; 8(1): 226, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34433823

RESUMO

While gene expression profiling has traditionally been the method of choice for large-scale perturbational profiling studies, proteomics has emerged as an effective tool in this context for directly monitoring cellular responses to perturbations. We previously reported a pilot library containing 3400 profiles of multiple perturbations across diverse cellular backgrounds in the reduced-representation phosphoproteome (P100) and chromatin space (Global Chromatin Profiling, GCP). Here, we expand our original dataset to include profiles from a new set of cardiotoxic compounds and from astrocytes, an additional neural cell model, totaling 5300 proteomic signatures. We describe filtering criteria and quality control metrics used to assess and validate the technical quality and reproducibility of our data. To demonstrate the power of the library, we present two case studies where data is queried using the concept of "connectivity" to obtain biological insight. All data presented in this study have been deposited to the ProteomeXchange Consortium with identifiers PXD017458 (P100) and PXD017459 (GCP) and can be queried at https://clue.io/proteomics .


Assuntos
Antineoplásicos/toxicidade , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cardiotoxinas/toxicidade , Inibidores de Proteínas Quinases/toxicidade , Proteômica , Linhagem Celular Tumoral , Humanos , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoma
10.
Nature ; 596(7870): 119-125, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34290406

RESUMO

Interactions between T cell receptors (TCRs) and their cognate tumour antigens are central to antitumour immune responses1-3; however, the relationship between phenotypic characteristics and TCR properties is not well elucidated. Here we show, by linking the antigenic specificity of TCRs and the cellular phenotype of melanoma-infiltrating lymphocytes at single-cell resolution, that tumour specificity shapes the expression state of intratumoural CD8+ T cells. Non-tumour-reactive T cells were enriched for viral specificities and exhibited a non-exhausted memory phenotype, whereas melanoma-reactive lymphocytes predominantly displayed an exhausted state that encompassed diverse levels of differentiation but rarely acquired memory properties. These exhausted phenotypes were observed both among clonotypes specific for public overexpressed melanoma antigens (shared across different tumours) or personal neoantigens (specific for each tumour). The recognition of such tumour antigens was provided by TCRs with avidities inversely related to the abundance of cognate targets in melanoma cells and proportional to the binding affinity of peptide-human leukocyte antigen (HLA) complexes. The persistence of TCR clonotypes in peripheral blood was negatively affected by the level of intratumoural exhaustion, and increased in patients with a poor response to immune checkpoint blockade, consistent with chronic stimulation mediated by residual tumour antigens. By revealing how the quality and quantity of tumour antigens drive the features of T cell responses within the tumour microenvironment, we gain insights into the properties of the anti-melanoma TCR repertoire.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma/imunologia , Especificidade por Substrato/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Regulação da Expressão Gênica , Humanos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/sangue , Fenótipo , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Célula Única , Transcriptoma/genética , Microambiente Tumoral
11.
Nat Commun ; 12(1): 4375, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272366

RESUMO

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Piridazinas/química , Monofosfato de Adenosina/química , Varredura Diferencial de Calorimetria , Domínio Catalítico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Microscopia Crioeletrônica , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Endorribonucleases/química , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Espectrometria de Massas , Complexos Multienzimáticos/ultraestrutura , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Piridazinas/farmacologia , Proteínas Recombinantes , Tetra-Hidroisoquinolinas/química
12.
Cancer Cell ; 39(6): 827-844.e10, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34129824

RESUMO

The core cohesin subunit STAG2 is recurrently mutated in Ewing sarcoma but its biological role is less clear. Here, we demonstrate that cohesin complexes containing STAG2 occupy enhancer and polycomb repressive complex (PRC2)-marked regulatory regions. Genetic suppression of STAG2 leads to a compensatory increase in cohesin-STAG1 complexes, but not in enhancer-rich regions, and results in reprogramming of cis-chromatin interactions. Strikingly, in STAG2 knockout cells the oncogenic genetic program driven by the fusion transcription factor EWS/FLI1 was highly perturbed, in part due to altered enhancer-promoter contacts. Moreover, loss of STAG2 also disrupted PRC2-mediated regulation of gene expression. Combined, these transcriptional changes converged to modulate EWS/FLI1, migratory, and neurodevelopmental programs. Finally, consistent with clinical observations, functional studies revealed that loss of STAG2 enhances the metastatic potential of Ewing sarcoma xenografts. Our findings demonstrate that STAG2 mutations can alter chromatin architecture and transcriptional programs to promote an aggressive cancer phenotype.

13.
Cell ; 184(15): 3962-3980.e17, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34171305

RESUMO

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.


Assuntos
Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Fases de Leitura Aberta/genética , Peptídeos/imunologia , Proteoma/imunologia , SARS-CoV-2/imunologia , Células A549 , Alelos , Sequência de Aminoácidos , Animais , Apresentação do Antígeno/imunologia , COVID-19/imunologia , COVID-19/virologia , Feminino , Células HEK293 , Humanos , Cinética , Masculino , Camundongos , Peptídeos/química , Linfócitos T/imunologia
14.
Mol Cell Proteomics ; 20: 100116, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34146720

RESUMO

Immunotherapies have emerged to treat diseases by selectively modulating a patient's immune response. Although the roles of T and B cells in adaptive immunity have been well studied, it remains difficult to select targets for immunotherapeutic strategies. Because human leukocyte antigen class II (HLA-II) peptides activate CD4+ T cells and regulate B cell activation, proliferation, and differentiation, these peptide antigens represent a class of potential immunotherapy targets and biomarkers. To better understand the molecular basis of how HLA-II antigen presentation is involved in disease progression and treatment, systematic HLA-II peptidomics combined with multiomic analyses of diverse cell types in healthy and diseased states is required. For this reason, MS-based innovations that facilitate investigations into the interplay between disease pathologies and the presentation of HLA-II peptides to CD4+ T cells will aid in the development of patient-focused immunotherapies.

15.
Nat Commun ; 12(1): 3346, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099720

RESUMO

Characterizing the human leukocyte antigen (HLA) bound ligandome by mass spectrometry (MS) holds great promise for developing vaccines and drugs for immune-oncology. Still, the identification of non-tryptic peptides presents substantial computational challenges. To address these, we synthesized and analyzed >300,000 peptides by multi-modal LC-MS/MS within the ProteomeTools project representing HLA class I & II ligands and products of the proteases AspN and LysN. The resulting data enabled training of a single model using the deep learning framework Prosit, allowing the accurate prediction of fragment ion spectra for tryptic and non-tryptic peptides. Applying Prosit demonstrates that the identification of HLA peptides can be improved up to 7-fold, that 87% of the proposed proteasomally spliced HLA peptides may be incorrect and that dozens of additional immunogenic neo-epitopes can be identified from patient tumors in published data. Together, the provided peptides, spectra and computational tools substantially expand the analytical depth of immunopeptidomics workflows.


Assuntos
Aprendizado Profundo , Peptídeos/imunologia , Espectrometria de Massas em Tandem/métodos , Linhagem Celular , Epitopos , Proteínas da Matriz Extracelular/metabolismo , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Ligantes , Espectrometria de Massas , Medicina Molecular , Peptídeos/metabolismo , Proteômica
16.
mSphere ; 6(3): e0021121, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34047655

RESUMO

Clostridioides difficile is a leading cause of health care-associated infections worldwide. These infections are transmitted by C. difficile's metabolically dormant, aerotolerant spore form. Functional spore formation depends on the assembly of two protective layers, a thick layer of modified peptidoglycan known as the cortex layer and a multilayered proteinaceous meshwork known as the coat. We previously identified two spore morphogenetic proteins, SpoIVA and SipL, that are essential for recruiting coat proteins to the developing forespore and making functional spores. While SpoIVA and SipL directly interact, the identities of the proteins they recruit to the forespore remained unknown. Here, we used mass spectrometry-based affinity proteomics to identify proteins that interact with the SpoIVA-SipL complex. These analyses identified the Peptostreptococcaceae family-specific, sporulation-induced bitopic membrane protein CD3457 (renamed SpoVQ) as a protein that interacts with SipL and SpoIVA. Loss of SpoVQ decreased heat-resistant spore formation by ∼5-fold and reduced cortex thickness ∼2-fold; the thinner cortex layer of ΔspoVQ spores correlated with higher levels of spontaneous germination (i.e., in the absence of germinant). Notably, loss of SpoVQ in either spoIVA or sipL mutants prevented cortex synthesis altogether and greatly impaired the localization of a SipL-mCherry fusion protein around the forespore. Thus, SpoVQ is a novel regulator of C. difficile cortex synthesis that appears to link cortex and coat formation. The identification of SpoVQ as a spore morphogenetic protein further highlights how Peptostreptococcaceae family-specific mechanisms control spore formation in C. difficile. IMPORTANCE The Centers for Disease Control has designated Clostridioides difficile as an urgent threat because of its intrinsic antibiotic resistance. C. difficile persists in the presence of antibiotics in part because it makes metabolically dormant spores. While recent work has shown that preventing the formation of infectious spores can reduce C. difficile disease recurrence, more selective antisporulation therapies are needed. The identification of spore morphogenetic factors specific to C. difficile would facilitate the development of such therapies. In this study, we identified SpoVQ (CD3457) as a spore morphogenetic protein specific to the Peptostreptococcaceae family that regulates the formation of C. difficile's protective spore cortex layer. SpoVQ acts in concert with the known spore coat morphogenetic factors, SpoIVA and SipL, to link formation of the protective coat and cortex layers. These data reveal a novel pathway that could be targeted to prevent the formation of infectious C. difficile spores.

18.
Nature ; 595(7866): 309-314, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33953401

RESUMO

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape1,2. Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR-Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape3-5. We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution6. SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy.


Assuntos
Inativação Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias/genética , Neoplasias/imunologia , Animais , Antígenos Virais/imunologia , Sistemas CRISPR-Cas/genética , Cromatina/genética , Cromatina/metabolismo , Elementos de DNA Transponíveis/genética , Modelos Animais de Doenças , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia
19.
Nat Metab ; 3(6): 786-797, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34045743

RESUMO

Maximal oxygen uptake (VO2max) is a direct measure of human cardiorespiratory fitness and is associated with health. However, the molecular determinants of interindividual differences in baseline (intrinsic) VO2max, and of increases of VO2max in response to exercise training (ΔVO2max), are largely unknown. Here, we measure ~5,000 plasma proteins using an affinity-based platform in over 650 sedentary adults before and after a 20-week endurance-exercise intervention and identify 147 proteins and 102 proteins whose plasma levels are associated with baseline VO2max and ΔVO2max, respectively. Addition of a protein biomarker score derived from these proteins to a score based on clinical traits improves the prediction of an individual's ΔVO2max. We validate findings in a separate exercise cohort, further link 21 proteins to incident all-cause mortality in a community-based cohort and reproduce the specificity of ~75% of our key findings using antibody-based assays. Taken together, our data shed light on biological pathways relevant to cardiorespiratory fitness and highlight the potential additive value of protein biomarkers in identifying exercise responsiveness in humans.


Assuntos
Proteínas Sanguíneas , Aptidão Cardiorrespiratória , Proteoma , Proteômica , Biomarcadores , Exercício Físico , Humanos , Estilo de Vida , Redes e Vias Metabólicas , Consumo de Oxigênio , Proteômica/métodos
20.
Science ; 372(6543): 716-721, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33986176

RESUMO

Transcription and metabolism both influence cell function, but dedicated transcriptional control of metabolic pathways that regulate cell fate has rarely been defined. We discovered, using a chemical suppressor screen, that inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) rescues erythroid differentiation in bloodless zebrafish moonshine (mon) mutant embryos defective for transcriptional intermediary factor 1 gamma (tif1γ). This rescue depends on the functional link of DHODH to mitochondrial respiration. The transcription elongation factor TIF1γ directly controls coenzyme Q (CoQ) synthesis gene expression. Upon tif1γ loss, CoQ levels are reduced, and a high succinate/α-ketoglutarate ratio leads to increased histone methylation. A CoQ analog rescues mon's bloodless phenotype. These results demonstrate that mitochondrial metabolism is a key output of a lineage transcription factor that drives cell fate decisions in the early blood lineage.


Assuntos
Eritropoese , Mitocôndrias/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética , Proteínas de Peixe-Zebra/metabolismo , Animais , Ciclo do Ácido Cítrico , Metilação de DNA , Transporte de Elétrons , Embrião não Mamífero/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Histonas/metabolismo , Leflunomida/farmacologia , Redes e Vias Metabólicas , Metilação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Consumo de Oxigênio , Fatores de Transcrição/genética , Ubiquinona/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...