RESUMEN
DNA-protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription.
Asunto(s)
ADN Helicasas , Enzimas Reparadoras del ADN , Reparación del ADN , Proteínas de Unión a Poli-ADP-Ribosa , Proteolisis , ARN Polimerasa II , Transcripción Genética , Humanos , Proteínas Portadoras , ADN/metabolismo , ADN/genética , Daño del ADN , ADN Helicasas/metabolismo , ADN Helicasas/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Células HEK293 , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores de Interleucina-17 , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
CLEC16A is a membrane-associated C-type lectin protein that functions as a E3-ubiquitin ligase. CLEC16A regulates autophagy and mitophagy, and reportedly localizes to late endosomes. GWAS studies have associated CLEC16A SNPs to various auto-immune and neurological disorders, including multiple sclerosis and Parkinson disease. Studies in mouse models imply a role for CLEC16A in neurodegeneration. We identified bi-allelic CLEC16A truncating variants in siblings from unrelated families presenting with a severe neurodevelopmental disorder including microcephaly, brain atrophy, corpus callosum dysgenesis, and growth retardation. To understand the function of CLEC16A in neurodevelopment we used in vitro models and zebrafish embryos. We observed CLEC16A localization to early endosomes in HEK293T cells. Mass spectrometry of human CLEC16A showed interaction with endosomal retromer complex subunits and the endosomal ubiquitin ligase TRIM27. Expression of the human variant leading to C-terminal truncated CLEC16A, abolishes both its endosomal localization and interaction with TRIM27, suggesting a loss-of-function effect. CLEC16A knockdown increased TRIM27 adhesion to early endosomes and abnormal accumulation of endosomal F-actin, a sign of disrupted vesicle sorting. Mutagenesis of clec16a by CRISPR-Cas9 in zebrafish embryos resulted in accumulated acidic/phagolysosome compartments, in neurons and microglia, and dysregulated mitophagy. The autophagocytic phenotype was rescued by wild-type human CLEC16A but not the C-terminal truncated CLEC16A. Our results demonstrate that CLEC16A closely interacts with retromer components and regulates endosomal fate by fine-tuning levels of TRIM27 and polymerized F-actin on the endosome surface. Dysregulation of CLEC16A-mediated endosomal sorting is associated with neurodegeneration, but it also causes accumulation of autophagosomes and unhealthy mitochondria during brain development.
Asunto(s)
Actinas , Pez Cebra , Animales , Humanos , Proteínas de Unión al ADN/metabolismo , Endosomas/genética , Endosomas/metabolismo , Células HEK293 , Lectinas Tipo C/genética , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Nucleares/metabolismo , Transporte de Proteínas , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
TBR1 is a neuron-specific transcription factor involved in brain development and implicated in a neurodevelopmental disorder (NDD) combining features of autism spectrum disorder (ASD), intellectual disability (ID) and speech delay. TBR1 has been previously shown to interact with a small number of transcription factors and co-factors also involved in NDDs (including CASK, FOXP1/2/4 and BCL11A), suggesting that the wider TBR1 interactome may have a significant bearing on normal and abnormal brain development. Here, we have identified approximately 250 putative TBR1-interaction partners by affinity purification coupled to mass spectrometry. As well as known TBR1-interactors such as CASK, the identified partners include transcription factors and chromatin modifiers, along with ASD- and ID-related proteins. Five interaction candidates were independently validated using bioluminescence resonance energy transfer assays. We went on to test the interaction of these candidates with TBR1 protein variants implicated in cases of NDD. The assays uncovered disturbed interactions for NDD-associated variants and identified two distinct protein-binding domains of TBR1 that have essential roles in protein-protein interaction.
Asunto(s)
Trastornos del Neurodesarrollo , Proteínas de Dominio T Box , Humanos , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Unión Proteica/genética , Unión Proteica/fisiología , Proteínas/genética , Proteínas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Ubiquitin-specific protease 7 (USP7) has been implicated in cancer progression and neurodevelopment. However, its molecular targets remain poorly characterized. We combined quantitative proteomics, transcriptomics, and epigenomics to define the core USP7 network. Our multi-omics analysis reveals USP7 as a control hub that links genome regulation, tumor suppression, and histone H2A ubiquitylation (H2AK119ub1) by noncanonical Polycomb-repressive complexes (ncPRC1s). USP7 strongly stabilizes ncPRC1.6 and, to a lesser extent, ncPRC1.1. Moreover, USP7 represses expression of AUTS2, which suppresses H2A ubiquitylation by ncPRC1.3/5. Collectively, these USP7 activities promote the genomic deposition of H2AK119ub1 by ncPRC1, especially at transcriptionally repressed loci. Notably, USP7-dependent changes in H2AK119ub1 levels are uncoupled from H3K27me3. Even complete loss of the PRC1 catalytic core and H2AK119ub1 has only a limited effect on H3K27me3. Besides defining the USP7 regulome, our results reveal that H2AK119ub1 dosage is largely disconnected from H3K27me3.
RESUMEN
Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in structural and molecular diversity of dendritic spines. Active spines with large calcium ion (Ca2+ ) transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into spines via the actin-based motor myosin V. An increase in synaptic strength correlates with stable anchoring of the ER, followed by the formation of an organelle referred to as the spine apparatus. Here, we show that myosin V binds the Ca2+ sensor caldendrin, a brain-specific homolog of the well-known myosin V interactor calmodulin. While calmodulin is an essential activator of myosin V motor function, we found that caldendrin acts as an inhibitor of processive myosin V movement. In mouse and rat hippocampal neurons, caldendrin regulates spine apparatus localization to a subset of dendritic spines through a myosin V-dependent pathway. We propose that caldendrin transforms myosin into a stationary F-actin tether that enables the localization of ER tubules and formation of the spine apparatus in dendritic spines.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Espinas Dendríticas/metabolismo , Retículo Endoplásmico/metabolismo , Miosina Tipo V/metabolismo , Actinas/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Calmodulina/metabolismo , Retículo Endoplásmico Liso/metabolismo , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Espectrometría de Masas , Ratones Noqueados , Miosina Tipo V/genética , Dominios y Motivos de Interacción de Proteínas , Ratas WistarRESUMEN
The 10-subunit TFIIH complex is vital to transcription and nucleotide excision repair. Hereditary mutations in its smallest subunit, TTDA/GTF2H5, cause a photosensitive form of the rare developmental disorder trichothiodystrophy. Some trichothiodystrophy features are thought to be caused by subtle transcription or gene expression defects. TTDA/GTF2H5 knockout mice are not viable, making it difficult to investigate TTDA/GTF2H5 in vivo function. Here we show that deficiency of C. elegans TTDA ortholog GTF-2H5 is, however, compatible with life, in contrast to depletion of other TFIIH subunits. GTF-2H5 promotes TFIIH stability in multiple tissues and is indispensable for nucleotide excision repair, in which it facilitates recruitment of TFIIH to DNA damage. Strikingly, when transcription is challenged, gtf-2H5 embryos die due to the intrinsic TFIIH fragility in absence of GTF-2H5. These results support the idea that TTDA/GTF2H5 mutations cause transcription impairment underlying trichothiodystrophy and establish C. elegans as model for studying pathogenesis of this disease.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Reparación del ADN/genética , ADN de Helmintos/fisiología , Factores de Transcripción/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Alpha-2-antiplasmin (α2AP) is the main natural inhibitor of plasmin. The C-terminus of α2AP is crucial for the initial interaction with plasmin(ogen) and the rapid inhibitory mechanism. Approximately 35% of circulating α2AP has lost its C-terminus (non-plasminogen binding α2AP/NPB-α2AP) and thereby its rapid inhibitory capacity. The C-terminal cleavage site of α2AP is still unknown. A commercially available monoclonal antibody against α2AP (TC 3AP) detects intact but not NPB-α2AP, suggesting that the cleavage site is located N-terminally from the epitope of TC 3AP. OBJECTIVES: To determine the epitope of TC 3AP and then to localize the C-terminal cleavage site of α2AP. METHODS: For epitope mapping of TC 3AP, commercially available plasma purified α2AP was enzymatically digested with Asp-N, Glu-C, or Lys-N. The resulting peptides were immunoprecipitated using TC 3AP-loaded Dynabeads® Protein G. Bound peptides were eluted and analyzed by liquid chromatography-tandem mass spectometry (LC-MS/MS). To localize the C-terminal cleavage site precisely, α2AP (intact and NPB) was purified from plasma and analyzed by LC-MS/MS after enzymatic digestion with Arg-C. RESULTS: We localized the epitope of TC 3AP between amino acid residues Asp428 and Gly439. LC-MS/MS data from plasma purified α2AP showed that NPB-α2AP results from cleavage at Gln421-Asp422 as preferred site, but also after Leu417, Glu419, Gln420, or Asp422. CONCLUSIONS: The C-terminal cleavage site of human α2AP is located N-terminally from the TC 3AP epitope. Because C-terminal cleavage of α2AP can occur after multiple residues, different proteases may be responsible for the generation of NPB-α2AP.
Asunto(s)
Plasminógeno , alfa 2-Antiplasmina , Cromatografía Liquida , Fibrinolisina , Humanos , Espectrometría de Masas en TándemRESUMEN
The spine apparatus (SA) is an endoplasmic reticulum-related organelle that is present in a subset of dendritic spines in cortical and pyramidal neurons, and plays an important role in Ca2+ homeostasis and dendritic spine plasticity. The protein synaptopodin is essential for the formation of the SA and is widely used as a maker for this organelle. However, it is still unclear which factors contribute to its localization at selected synapses, and how it triggers local SA formation. In this study, we characterized development, localization and mobility of synaptopodin clusters in hippocampal primary neurons, as well as the molecular dynamics within these clusters. Interestingly, synaptopodin at the shaft-associated clusters is less dynamic than at spinous clusters. We identify the actin-based motor proteins myosin V (herein referring to both the myosin Va and Vb forms) and VI as novel interaction partners of synaptopodin, and demonstrate that myosin V is important for the formation and/or maintenance of the SA. We found no evidence of active microtubule-based transport of synaptopodin. Instead, new clusters emerge inside spines, which we interpret as the SA being assembled on-site.
Asunto(s)
Dendritas/metabolismo , Hipocampo/metabolismo , Proteínas de Microfilamentos/metabolismo , Miosina Tipo V/metabolismo , Animales , Dendritas/genética , Femenino , Hipocampo/citología , Ratones , Proteínas de Microfilamentos/genética , Miosina Tipo V/genética , Ratas , Ratas WistarRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The Mediator complex regulates transcription by connecting enhancers to promoters. High Mediator binding density defines super enhancers, which regulate cell-identity genes and oncogenes. Protein interactions of Mediator may explain its role in these processes but have not been identified comprehensively. Here, we purify Mediator from neural stem cells (NSCs) and identify 75 protein-protein interaction partners. We identify super enhancers in NSCs and show that Mediator-interacting chromatin modifiers colocalize with Mediator at enhancers and super enhancers. Transcription factor families with high affinity for Mediator dominate enhancers and super enhancers and can explain genome-wide Mediator localization. We identify E-box transcription factor Tcf4 as a key regulator of NSCs. Tcf4 interacts with Mediator, colocalizes with Mediator at super enhancers and regulates neurogenic transcription factor genes with super enhancers and broad H3K4me3 domains. Our data suggest that high binding-affinity for Mediator is an important organizing feature in the transcriptional network that determines NSC identity.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Redes Reguladoras de Genes/fisiología , Complejo Mediador/metabolismo , Células-Madre Neurales/fisiología , Neurogénesis/genética , Factor de Transcripción 4/metabolismo , Línea Celular , Elementos de Facilitación Genéticos/genética , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Regiones Promotoras Genéticas/genética , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Transcripción Genética/fisiologíaRESUMEN
The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability, and DNA interstrand crosslink repair in vertebrates. We identify HSF2BP, a protein previously described as testis specific and not characterized functionally, as an interactor of BRCA2 in mouse embryonic stem cells, where the 2 proteins form a constitutive complex. HSF2BP is transcribed in all cultured human cancer cell lines tested and elevated in some tumor samples. Inactivation of the mouse Hsf2bp gene results in male infertility due to a severe HR defect during spermatogenesis. The BRCA2-HSF2BP interaction is highly evolutionarily conserved and maps to armadillo repeats in HSF2BP and a 68-amino acid region between the BRC repeats and the DNA binding domain of human BRCA2 (Gly2270-Thr2337) encoded by exons 12 and 13. This region of BRCA2 does not harbor known cancer-associated missense mutations and may be involved in the reproductive rather than the tumor-suppressing function of BRCA2.
Asunto(s)
Proteína BRCA2/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Choque Térmico/metabolismo , Espermatogénesis , Animales , Proteína BRCA2/genética , Proteínas Portadoras/genética , Línea Celular Tumoral , Proteínas de Choque Térmico/genética , Humanos , Ratones , Mutación Missense , Dominios ProteicosRESUMEN
Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Adulto , Encéfalo/patología , Proteínas Portadoras/genética , Ciclo Celular/fisiología , Cilios/metabolismo , Femenino , Estudios de Asociación Genética/métodos , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Recién Nacido , Masculino , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/metabolismo , Microcefalia/genética , Mutación , Malformaciones del Sistema Nervioso/genética , Polimicrogiria/etiología , Polimicrogiria/patologíaRESUMEN
FOXP transcription factors play important roles in neurodevelopment, but little is known about how their transcriptional activity is regulated. FOXP proteins cooperatively regulate gene expression by forming homo- and hetero-dimers with each other. Physical associations with other transcription factors might also modulate the functions of FOXP proteins. However, few FOXP-interacting transcription factors have been identified so far. Therefore, we sought to discover additional transcription factors that interact with the brain-expressed FOXP proteins, FOXP1, FOXP2 and FOXP4, through affinity-purifications of protein complexes followed by mass spectrometry. We identified seven novel FOXP-interacting transcription factors (NR2F1, NR2F2, SATB1, SATB2, SOX5, YY1 and ZMYM2), five of which have well-estabslished roles in cortical development. Accordingly, we found that these transcription factors are co-expressed with FoxP2 in the deep layers of the cerebral cortex and also in the Purkinje cells of the cerebellum, suggesting that they may cooperate with the FoxPs to regulate neural gene expression in vivo. Moreover, we demonstrated that etiological mutations of FOXP1 and FOXP2, known to cause neurodevelopmental disorders, severely disrupted the interactions with FOXP-interacting transcription factors. Additionally, we pinpointed specific regions within FOXP2 sequence involved in mediating these interactions. Thus, by expanding the FOXP interactome we have uncovered part of a broader neural transcription factor network involved in cortical development, providing novel molecular insights into the transcriptional architecture underlying brain development and neurodevelopmental disorders.
Asunto(s)
Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Trastornos del Neurodesarrollo , Células de Purkinje/metabolismo , Proteínas Represoras , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Humanos , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/patología , Células de Purkinje/patología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Ubiquitylation is an important posttranslational protein modification that is involved in many cellular events. Immunopurification of peptides containing a K-ε-diglycine (diGly) remnant as a mark of ubiquitylation combined with mass spectrometric detection has resulted in an explosion of the number of identified ubiquitylation sites. Here, we present several significant improvements to this workflow, including fast, offline and crude high pH reverse-phase fractionation of tryptic peptides into only three fractions with simultaneous desalting prior to immunopurification and better control of the peptide fragmentation settings in the Orbitrap HCD cell. In addition, more efficient sample cleanup using a filter plug to retain the antibody beads results in a higher specificity for diGly peptides and less non-specific binding. These relatively simple modifications of the protocol result in the routine detection of over 23,000 diGly peptides from HeLa cells upon proteasome inhibition. The efficacy of this strategy is shown for lysates of both non-labeled and SILAC labeled cell lines. Furthermore, we demonstrate that this strategy is useful for the in-depth analysis of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. This study presents a valuable addition to the toolbox for ubiquitylation site analysis to uncover the deep ubiquitinome. SIGNIFICANCE: A K-ε-diglycine (diGly) mark on peptides after tryptic digestion of proteins indicates a site of ubiquitylation, a posttranslational modification involved in a wide range of cellular processes. Here, we report several improvements to methods for the isolation and detection of diGly peptides from complex biological mixtures such as cell lysates and brain tissue. This adapted method is robust, reproducible and outperforms previously published methods in terms of number of modified peptide identifications from a single sample. In-depth analysis of the ubiquitinome using mass spectrometry will lead to a better understanding of the roles of protein ubiquitylation in cellular events.
Asunto(s)
Espectrometría de Masas/métodos , Proteómica/métodos , Ubiquitinación , Sitios de Unión , Protocolos Clínicos , Glicilglicina/análisis , Células HeLa , Humanos , Métodos , Procesamiento Proteico-Postraduccional , Ubiquitina/metabolismoRESUMEN
The ubiquitin-proteasome system (UPS), a highly regulated mechanism including the active marking of proteins by ubiquitin to be degraded, is critical in regulating proteostasis. Dysfunctioning of the UPS has been implicated in diseases such as cancer and neurodegenerative disorders. Here we investigate the effects of proteasome malfunctioning on global proteome and ubiquitinome dynamics using SILAC proteomics in Drosophila S2 cells. dsRNA-mediated knockdown of specific proteasome target subunits is used to inactivate the proteasome. Upon this perturbation, both the global proteome and the ubiquitinome become modified to a great extent, with the overall impact on the ubiquitinome being the most dramatic. The abundances of â¼10% of all proteins are increased, while the abundances of the far majority of over 14â¯000 detected diGly peptides are increased, suggesting that the pool of ubiquitinated proteins is highly dynamic. Remarkably, several proteins show heterogeneous ubiquitination dynamics, with different lysine residues on the same protein showing either increased or decreased ubiquitination. This suggests the occurrence of simultaneous and functionally different ubiquitination events. This strategy offers a powerful tool to study the response of the ubiquitinome upon interruption of normal UPS activity by targeted interference and opens up new avenues for the dissection of the mode of action of individual components of the proteasome. Because this is to our knowledge the first comprehensive ubiquitinome screen upon proteasome malfunctioning in a fruit fly cell system, this data set will serve as a valuable repository for the Drosophila community.
Asunto(s)
Drosophila/química , Proteómica/métodos , Proteínas Ubiquitinadas/análisis , Animales , Técnicas de Silenciamiento del Gen , Complejo de la Endopetidasa Proteasomal/deficiencia , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/genética , ARN Bicatenario/genética , Ubiquitina/análisis , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Mutations in NIPBL are the most frequent cause of Cornelia de Lange syndrome (CdLS), a developmental disorder encompassing several neurological defects, including intellectual disability and seizures. How NIPBL mutations affect brain development is not understood. Here we identify Nipbl as a functional interaction partner of the neural transcription factor Zfp609 in brain development. Depletion of Zfp609 or Nipbl from cortical neural progenitors in vivo is detrimental to neuronal migration. Zfp609 and Nipbl overlap at genomic binding sites independently of cohesin and regulate genes that control cortical neuron migration. We find that Zfp609 and Nipbl interact with the Integrator complex, which functions in RNA polymerase 2 pause release. Indeed, Zfp609 and Nipbl co-localize at gene promoters containing paused RNA polymerase 2, and Integrator similarly regulates neuronal migration. Our data provide a rationale and mechanistic insights for the role of Nipbl in the neurological defects associated with CdLS.
Asunto(s)
Movimiento Celular/genética , Corteza Cerebral/crecimiento & desarrollo , Síndrome de Cornelia de Lange/genética , Regulación del Desarrollo de la Expresión Génica , Células-Madre Neurales/citología , Neuronas/citología , Transactivadores/genética , Factores de Transcripción/genética , Animales , Proteínas de Ciclo Celular/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Ratones , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , CohesinasRESUMEN
Heterozygous germline mutations in breast cancer 1 (BRCA1) strongly predispose women to breast cancer. BRCA1 plays an important role in DNA double-strand break (DSB) repair via homologous recombination (HR), which is important for tumor suppression. Although BRCA1-deficient cells are highly sensitive to treatment with DSB-inducing agents through their HR deficiency (HRD), BRCA1-associated tumors display heterogeneous responses to platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors in clinical trials. It is unclear whether all pathogenic BRCA1 mutations have similar effects on the response to therapy. Here, we have investigated mammary tumorigenesis and therapy sensitivity in mice carrying the Brca1185stop and Brca15382stop alleles, which respectively mimic the 2 most common BRCA1 founder mutations, BRCA1185delAG and BRCA15382insC. Both the Brca1185stop and Brca15382stop mutations predisposed animals to mammary tumors, but Brca1185stop tumors responded markedly worse to HRD-targeted therapy than did Brca15382stop tumors. Mice expressing Brca1185stop mutations also developed therapy resistance more rapidly than did mice expressing Brca15382stop. We determined that both murine Brca1185stop tumors and human BRCA1185delAG breast cancer cells expressed a really interesting new gene domain-less (RING-less) BRCA1 protein that mediated resistance to HRD-targeted therapies. Together, these results suggest that expression of RING-less BRCA1 may serve as a marker to predict poor response to DSB-inducing therapy in human cancer patients.
Asunto(s)
Proteína BRCA1/genética , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos , Eliminación de Gen , Neoplasias Mamarias Animales/genética , Alelos , Animales , Antineoplásicos/farmacología , Cisplatino/farmacología , Cruzamientos Genéticos , Daño del ADN , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Efecto Fundador , Mutación del Sistema de Lectura , Ingeniería Genética , Humanos , Masculino , Neoplasias Mamarias Animales/tratamiento farmacológico , Ratones , Mutación , Trasplante de Neoplasias , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Recombinación GenéticaRESUMEN
BACKGROUND: Elevated plasma cardiac troponin T (cTnT) levels in patients with neuromuscular disorders may erroneously lead to the diagnosis of acute myocardial infarction or myocardial injury. METHODS AND RESULTS: In 122 patients with Pompe disease, the relationship between cTnT, cardiac troponin I, creatine kinase (CK), CK-myocardial band levels, and skeletal muscle damage was assessed. ECG and echocardiography were used to evaluate possible cardiac disease. Patients were divided into classic infantile, childhood-onset, and adult-onset patients. cTnT levels were elevated in 82% of patients (median 27 ng/L, normal values <14 ng/L). Cardiac troponin I levels were normal in all patients, whereas CK-myocardial band levels were increased in 59% of patients. cTnT levels correlated with CK levels in all 3 subgroups (P<0.001). None of the abnormal ECGs recorded in 21 patients were indicative of acute myocardial infarction, and there were no differences in cTnT levels between patients with and without (n=90) abnormalities on ECG (median 28 ng/L in both groups). The median left ventricular mass index measured with echocardiography was normal in all the 3 subgroups. cTnT mRNA expression in skeletal muscle was not detectable in controls but was strongly induced in patients with Pompe disease. cTnT protein was identified by mass spectrometry in patient-derived skeletal muscle tissue. CONCLUSIONS: Elevated plasma cTnT levels in patients with Pompe disease are associated with skeletal muscle damage, rather than acute myocardial injury. Increased cTnT levels in Pompe disease and likely other neuromuscular disorders should be interpreted with caution to avoid unnecessary cardiac interventions.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II/sangre , Músculo Esquelético/metabolismo , Troponina T/sangre , Adolescente , Adulto , Niño , Preescolar , Creatina Quinasa/sangre , Electrocardiografía , Femenino , Regulación de la Expresión Génica , Enfermedad del Almacenamiento de Glucógeno Tipo II/patología , Enfermedad del Almacenamiento de Glucógeno Tipo II/fisiopatología , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Humanos , Lactante , Masculino , Persona de Mediana Edad , Músculo Esquelético/lesiones , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Troponina I/sangreRESUMEN
RATIONALE: The (pro)renin receptor ([P]RR) interacts with (pro)renin at concentrations that are >1000× higher than observed under (patho)physiological conditions. Recent studies have identified renin-angiotensin system-independent functions for (P)RR related to its association with the vacuolar H(+)-ATPase. OBJECTIVE: To uncover renin-angiotensin system-independent functions of the (P)RR. METHODS AND RESULTS: We used a proteomics-based approach to purify and identify (P)RR-interacting proteins. This resulted in identification of sortilin-1 (SORT1) as a high-confidence (P)RR-interacting protein, a finding which was confirmed by coimmunoprecipitation of endogenous (P)RR and SORT1. Functionally, silencing (P)RR expression in hepatocytes decreased SORT1 and low-density lipoprotein (LDL) receptor protein abundance and, as a consequence, resulted in severely attenuated cellular LDL uptake. In contrast to LDL, endocytosis of epidermal growth factor or transferrin remained unaffected by silencing of the (P)RR. Importantly, reduction of LDL receptor and SORT1 protein abundance occurred in the absence of changes in their corresponding transcript level. Consistent with a post-transcriptional event, degradation of the LDL receptor induced by (P)RR silencing could be reversed by lysosomotropic agents, such as bafilomycin A1. CONCLUSIONS: Our study identifies a renin-angiotensin system-independent function for the (P)RR in the regulation of LDL metabolism by controlling the levels of SORT1 and LDL receptor.