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1.
EMBO Rep ; 25(3): 1589-1622, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38297188

RESUMEN

Embryonic genome activation (EGA) occurs during preimplantation development and is characterized by the initiation of de novo transcription from the embryonic genome. Despite its importance, the regulation of EGA and the transcription factors involved in this process are poorly understood. Paired-like homeobox (PRDL) family proteins are implicated as potential transcriptional regulators of EGA, yet the PRDL-mediated gene regulatory networks remain uncharacterized. To investigate the function of PRDL proteins, we are identifying the molecular interactions and the functions of a subset family of the Eutherian Totipotent Cell Homeobox (ETCHbox) proteins, seven PRDL family proteins and six other transcription factors (TFs), all suggested to participate in transcriptional regulation during preimplantation. Using mass spectrometry-based interactomics methods, AP-MS and proximity-dependent biotin labeling, and chromatin immunoprecipitation sequencing we derive the comprehensive regulatory networks of these preimplantation TFs. By these interactomics tools we identify more than a thousand high-confidence interactions for the 21 studied bait proteins with more than 300 interacting proteins. We also establish that TPRX2, currently assigned as pseudogene, is a transcriptional activator.


Asunto(s)
Proteínas de Homeodominio , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Proteínas de Homeodominio/genética , Genes Homeobox , Genoma
2.
EMBO Rep ; 23(6): e54041, 2022 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-35384245

RESUMEN

Much cell-to-cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we apply three complementary methods to map the molecular context and substrate profiles of RTKs. We use affinity purification coupled to mass spectrometry (AP-MS) to characterize stable binding partners and RTK-protein complexes, proximity-dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also use kinase-deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well-studied RTKs, offers insights into the functions of less well-studied RTKs, and highlights RTK-RTK interactions and shared signaling pathways.


Asunto(s)
Proteínas Tirosina Quinasas Receptoras , Transducción de Señal , Membrana Celular/metabolismo , Humanos , Fosforilación , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Tirosina/metabolismo
3.
Nucleic Acids Res ; 49(19): 10895-10910, 2021 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-34634806

RESUMEN

N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two abundant modifications found in mRNAs and ncRNAs that can regulate multiple aspects of RNA biology. They function mainly by regulating interactions with specific RNA-binding proteins. Both modifications are linked to development, disease and stress response. To date, three methyltransferases and two demethylases have been identified that modify adenosines in mammalian mRNAs. Here, we present a comprehensive analysis of the interactomes of these enzymes. PCIF1 protein network comprises mostly factors involved in nascent RNA synthesis by RNA polymerase II, whereas ALKBH5 is closely linked with most aspects of pre-mRNA processing and mRNA export to the cytoplasm. METTL16 resides in subcellular compartments co-inhabited by several other RNA modifiers and processing factors. FTO interactome positions this demethylase at a crossroad between RNA transcription, RNA processing and DNA replication and repair. Altogether, these enzymes share limited spatial interactomes, pointing to specific molecular mechanisms of their regulation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Adenosina/análogos & derivados , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Metiltransferasas/genética , Proteínas Nucleares/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Reparación del ADN , Replicación del ADN , Ontología de Genes , Células HEK293 , Humanos , Metiltransferasas/metabolismo , Anotación de Secuencia Molecular , Proteínas Nucleares/metabolismo , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Transcripción Genética
4.
Mol Syst Biol ; 17(11): e10396, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34709727

RESUMEN

Treatment options for COVID-19, caused by SARS-CoV-2, remain limited. Understanding viral pathogenesis at the molecular level is critical to develop effective therapy. Some recent studies have explored SARS-CoV-2-host interactomes and provided great resources for understanding viral replication. However, host proteins that functionally associate with SARS-CoV-2 are localized in the corresponding subnetwork within the comprehensive human interactome. Therefore, constructing a downstream network including all potential viral receptors, host cell proteases, and cofactors is necessary and should be used as an additional criterion for the validation of critical host machineries used for viral processing. This study applied both affinity purification mass spectrometry (AP-MS) and the complementary proximity-based labeling MS method (BioID-MS) on 29 viral ORFs and 18 host proteins with potential roles in viral replication to map the interactions relevant to viral processing. The analysis yields a list of 693 hub proteins sharing interactions with both viral baits and host baits and revealed their biological significance for SARS-CoV-2. Those hub proteins then served as a rational resource for drug repurposing via a virtual screening approach. The overall process resulted in the suggested repurposing of 59 compounds for 15 protein targets. Furthermore, antiviral effects of some candidate drugs were observed in vitro validation using image-based drug screen with infectious SARS-CoV-2. In addition, our results suggest that the antiviral activity of methotrexate could be associated with its inhibitory effect on specific protein-protein interactions.


Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Descubrimiento de Drogas , Interacciones Huésped-Patógeno/efectos de los fármacos , Proteoma/efectos de los fármacos , SARS-CoV-2/fisiología , COVID-19/virología , Reposicionamiento de Medicamentos , Humanos , Espectrometría de Masas , Metotrexato/farmacología , Proteómica , Replicación Viral/efectos de los fármacos
5.
Redox Biol ; 69: 103031, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38184997

RESUMEN

The Kelch-like ECH-associated protein 1 (KEAP1) - Nuclear factor erythroid 2 -related factor 2 (NRF2) pathway is the major transcriptional stress response system in cells against oxidative and electrophilic stress. NRF2 is frequently constitutively active in many cancers, rendering the cells resistant to chemo- and radiotherapy. Loss-of-function (LOF) mutations in the repressor protein KEAP1 are common in non-small cell lung cancer, particularly adenocarcinoma. While the mutations can occur throughout the gene, they are enriched in certain areas, indicating that these may have unique functional importance. In this study, we show that in the GSEA analysis of TCGA lung adenocarcinoma RNA-seq data, the KEAP1 mutations in R320 and R470 were associated with enhanced Tumor Necrosis Factor alpha (TNFα) - Nuclear Factor kappa subunit B (NFκB) signaling as well as MYC and MTORC1 pathways. To address the functional role of these hotspot mutations, affinity purification and mass spectrometry (AP-MS) analysis of wild type (wt) KEAP1 and its mutation forms, R320Q and R470C were employed to interrogate differences in the protein interactome. We identified TNF receptor associated factor 2 (TRAF2) as a putative protein interaction partner. Both mutant KEAP1 forms showed increased interaction with TRAF2 and other anti-apoptotic proteins, suggesting that apoptosis signalling could be affected by the protein interactions. A549 lung adenocarcinoma cells overexpressing mutant KEAP1 showed high TRAF2-mediated NFκB activity and increased protection against apoptosis, XIAP being one of the key proteins involved in anti-apoptotic signalling. To conclude, KEAP1 R320Q and R470C and its interaction with TRAF2 leads to activation of NFκB pathway, thereby protecting against apoptosis.


Asunto(s)
Adenocarcinoma del Pulmón , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Línea Celular Tumoral , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Adenocarcinoma del Pulmón/genética , Apoptosis/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Mutación
6.
Cancers (Basel) ; 15(17)2023 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-37686522

RESUMEN

Chromosomal translocations creating fusion genes are common cancer drivers. The oncogenic ETV6-NTRK3 (EN) gene fusion joins the sterile alpha domain of the ETV6 transcription factor with the tyrosine kinase domain of the neurotrophin-3 receptor NTRK3. Four EN variants with alternating break points have since been detected in a wide range of human cancers. To provide molecular level insight into EN oncogenesis, we employed a proximity labeling mass spectrometry approach to define the molecular context of the fusions. We identify in total 237 high-confidence interactors, which link EN fusions to several key signaling pathways, including ERBB, insulin and JAK/STAT. We then assessed the effects of EN variants on these pathways, and showed that the pan NTRK inhibitor Selitrectinib (LOXO-195) inhibits the oncogenic activity of EN2, the most common variant. This systems-level analysis defines the molecular framework in which EN oncofusions operate to promote cancer and provides some mechanisms for therapeutics.

7.
iScience ; 26(3): 106172, 2023 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-36876139

RESUMEN

The paired-like homeobox transcription factor LEUTX is expressed in human preimplantation embryos between the 4- and 8-cell stages, and then silenced in somatic tissues. To characterize the function of LEUTX, we performed a multiomic characterization of LEUTX using two proteomics methods and three genome-wide sequencing approaches. Our results show that LEUTX stably interacts with the EP300 and CBP histone acetyltransferases through its 9 amino acid transactivation domain (9aaTAD), as mutation of this domain abolishes the interactions. LEUTX targets genomic cis-regulatory sequences that overlap with repetitive elements, and through these elements it is suggested to regulate the expression of its downstream genes. We find LEUTX to be a transcriptional activator, upregulating several genes linked to preimplantation development as well as 8-cell-like markers, such as DPPA3 and ZNF280A. Our results support a role for LEUTX in preimplantation development as an enhancer binding protein and as a potent transcriptional activator.

8.
Stem Cell Reports ; 17(7): 1743-1756, 2022 07 12.
Artículo en Inglés | MEDLINE | ID: mdl-35777358

RESUMEN

Embryonic genome activation (EGA) is critical for embryonic development. However, our understanding of the regulatory mechanisms of human EGA is still incomplete. Human embryonic stem cells (hESCs) are an established model for studying developmental processes, but they resemble epiblast and are sub-optimal for modeling EGA. DUX4 regulates human EGA by inducing cleavage-stage-specific genes, while it also induces cell death. We report here that a short-pulsed expression of DUX4 in primed hESCs activates an EGA-like gene expression program in up to 17% of the cells, retaining cell viability. These DUX4-induced cells resembled eight-cell stage blastomeres and were named induced blastomere-like (iBM) cells. The iBM cells showed marked reduction of POU5F1 protein, as previously observed in mouse two-cell-like cells. Finally, the iBM cells were successfully enriched using an antibody against NaPi2b (SLC34A2), which is expressed in human blastomeres. The iBM cells provide an improved model system to study human EGA transcriptome.


Asunto(s)
Blastómeros , Proteínas de Homeodominio/metabolismo , Células Madre Embrionarias Humanas , Animales , Blastómeros/metabolismo , Desarrollo Embrionario/genética , Femenino , Genes Homeobox , Genoma Humano , Proteínas de Homeodominio/genética , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ratones , Embarazo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/metabolismo
9.
iScience ; 25(4): 104137, 2022 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-35402882

RESUMEN

Double homeobox 4 (DUX4) is expressed at the early pre-implantation stage in human embryos. Here we show that induced human DUX4 expression substantially alters the chromatin accessibility of non-coding DNA and activates thousands of newly identified transcribed enhancer-like regions, preferentially located within ERVL-MaLR repeat elements. CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs results in the increased expression of target embryonic genome activation (EGA) genes ZSCAN4 and KHDC1P1. We show that DUX4 is markedly enriched in human zygotes, followed by intense nuclear DUX4 localization preceding and coinciding with minor EGA. DUX4 knockdown in human zygotes led to changes in the EGA transcriptome but did not terminate the embryos. We also show that the DUX4 protein interacts with the Mediator complex via the C-terminal KIX binding motif. Our findings contribute to the understanding of DUX4 as a regulator of the non-coding genome.

10.
Blood Adv ; 6(7): 2444-2451, 2022 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-34920454

RESUMEN

Helios, encoded by IKZF2, is a member of the Ikaros family of transcription factors with pivotal roles in T-follicular helper, NK- and T-regulatory cell physiology. Somatic IKZF2 mutations are frequently found in lymphoid malignancies. Although germline mutations in IKZF1 and IKZF3 encoding Ikaros and Aiolos have recently been identified in patients with phenotypically similar immunodeficiency syndromes, the effect of germline mutations in IKZF2 on human hematopoiesis and immunity remains enigmatic. We identified germline IKZF2 mutations (one nonsense (p.R291X)- and 4 distinct missense variants) in six patients with systemic lupus erythematosus, immune thrombocytopenia or EBV-associated hemophagocytic lymphohistiocytosis. Patients exhibited hypogammaglobulinemia, decreased number of T-follicular helper and NK cells. Single-cell RNA sequencing of PBMCs from the patient carrying the R291X variant revealed upregulation of proinflammatory genes associated with T-cell receptor activation and T-cell exhaustion. Functional assays revealed the inability of HeliosR291X to homodimerize and bind target DNA as dimers. Moreover, proteomic analysis by proximity-dependent Biotin Identification revealed aberrant interaction of 3/5 Helios mutants with core components of the NuRD complex conveying HELIOS-mediated epigenetic and transcriptional dysregulation.


Asunto(s)
Mutación de Línea Germinal , Proteómica , Células Germinativas , Humanos , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo , Linfocitos T Reguladores/metabolismo
11.
Sci Rep ; 11(1): 966, 2021 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-33441730

RESUMEN

Rhegmatogenous retinal detachment (RRD) is an ophthalmic emergency, which usually requires prompt surgery to prevent further detachment and restore sensory function. Although several individual factors have been suggested, a systems level understanding of molecular pathomechanisms underlying this severe eye disorder is lacking. To address this gap in knowledge we performed the molecular level systems pathology analysis of the vitreous from 127 patients with RRD using state-of-the art quantitative mass spectrometry to identify the individual key proteins, as well as the biochemical pathways contributing to the development of the disease. RRD patients have specific vitreous proteome profiles compared to other diseases such as macular hole, pucker, or proliferative diabetic retinopathy eyes. Our data indicate that various mechanisms, including glycolysis, photoreceptor death, and Wnt and MAPK signaling, are activated during or after the RRD to promote retinal cell survival. In addition, platelet-mediated wound healing processes, cell adhesion molecules reorganization and apoptotic processes were detected during RRD progression or proliferative vitreoretinopathy formation. These findings improve the understanding of RRD pathogenesis, identify novel targets for treatment of this ophthalmic disease, and possibly affect the prognosis of eyes treated or operated upon due to RRD.


Asunto(s)
Retina/patología , Desprendimiento de Retina/patología , Apoptosis/fisiología , Plaquetas/metabolismo , Plaquetas/patología , Adhesión Celular/fisiología , Proliferación Celular/fisiología , Supervivencia Celular/fisiología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteoma/metabolismo , Retina/metabolismo , Desprendimiento de Retina/metabolismo , Perforaciones de la Retina/metabolismo , Perforaciones de la Retina/patología , Transducción de Señal/fisiología , Vitreorretinopatía Proliferativa/metabolismo , Vitreorretinopatía Proliferativa/patología , Cuerpo Vítreo/metabolismo , Cuerpo Vítreo/patología , Cicatrización de Heridas/fisiología
12.
Sci Immunol ; 6(65): eabe3981, 2021 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-34826259

RESUMEN

Helios, a member of the Ikaros family of transcription factors, is predominantly expressed in developing thymocytes, activated T cells, and regulatory T cells (Tregs). Studies in mice have emphasized its role in maintenance of Treg immunosuppressive functions by stabilizing Foxp3 expression and silencing the Il2 locus. However, its contribution to human immune homeostasis and the precise mechanisms by which Helios regulates other T cell subsets remain unresolved. Here, we investigated a patient with recurrent respiratory infections and hypogammaglobulinemia and identified a germline homozygous missense mutation in IKZF2 encoding Helios (p.Ile325Val). We found that HeliosI325V retains DNA binding and dimerization properties but loses interaction with several partners, including epigenetic remodelers. Whereas patient Tregs showed increased IL-2 production, patient conventional T cells had decreased accessibility of the IL2 locus and consequently reduced IL-2 production. Reduced chromatin accessibility was not exclusive to the IL2 locus but involved a variety of genes associated with T cell activation. Single-cell RNA sequencing of peripheral blood mononuclear cells revealed gene expression signatures indicative of a shift toward a proinflammatory, effector-like status in patient CD8+ T cells. Moreover, patient CD4+ T cells exhibited a pronounced defect in proliferation with delayed expression of surface checkpoint inhibitors, suggesting an impaired onset of the T cell activation program. Collectively, we identified a previously uncharacterized, germline-encoded inborn error of immunity and uncovered a cell-specific defect in Helios-dependent epigenetic regulation. Binding of Helios with specific partners mediates this regulation, which is ultimately necessary for the transcriptional programs that enable T cell homeostasis in health and disease.


Asunto(s)
Células Germinativas/inmunología , Factor de Transcripción Ikaros/inmunología , Adolescente , Epigénesis Genética/genética , Epigénesis Genética/inmunología , Humanos , Factor de Transcripción Ikaros/genética , Interleucina-2/biosíntesis , Masculino , Mutación Missense , Linfocitos T Reguladores/inmunología
13.
Nat Protoc ; 15(10): 3182-3211, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32778839

RESUMEN

Affinity purification coupled with mass spectrometry (AP-MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics studies. Whereas AP-MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. Integration of AP-MS and BioID data has been shown to comprehensively characterize a protein's molecular context, but interactome analysis using both methods in parallel is still labor and resource intense with respect to cell line generation and protein purification. Therefore, we developed the Multiple Approaches Combined (MAC)-tag workflow, which allows for both AP-MS and BioID analysis with a single construct and with almost identical protein purification and mass spectrometry (MS) identification procedures. We have applied the MAC-tag workflow to a selection of subcellular markers to provide a global view of the cellular protein interactome landscape. This localization database is accessible via our online platform ( http://proteomics.fi ) to predict the cellular localization of a protein of interest (POI) depending on its identified interactors. In this protocol, we present the detailed three-stage procedure for the MAC-tag workflow: (1) cell line generation for the MAC-tagged POI; (2) parallel AP-MS and BioID protein purification followed by MS analysis; and (3) protein interaction data analysis, data filtration and visualization with our localization visualization platform. The entire procedure can be completed within 25 d.


Asunto(s)
Espectrometría de Masas/métodos , Mapeo de Interacción de Proteínas/métodos , Purificación por Afinidad en Tándem/métodos , Biotinilación , Línea Celular , Cromatografía de Afinidad/métodos , Humanos , Mapas de Interacción de Proteínas/fisiología , Proteínas/metabolismo , Proteómica/métodos , Flujo de Trabajo
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