RESUMEN
CD4+ T helper 17 (TH17) cells protect barrier tissues but also trigger autoimmunity. The mechanisms behind these opposing processes remain unclear. Here, we found that the transcription factor EGR2 controlled the transcriptional program of pathogenic TH17 cells in the central nervous system (CNS) but not that of protective TH17 cells at barrier sites. EGR2 was significantly elevated in myelin-reactive CD4+ T cells from patients with multiple sclerosis and mice with autoimmune neuroinflammation. The EGR2 transcriptional program was intricately woven within the TH17 cell transcriptional regulatory network and showed high interconnectivity with core TH17 cell-specific transcription factors. Mechanistically, EGR2 enhanced TH17 cell differentiation and myeloid cell recruitment to the CNS by upregulating pathogenesis-associated genes and myelomonocytic chemokines. T cell-specific deletion of Egr2 attenuated neuroinflammation without compromising the host's ability to control infections. Our study shows that EGR2 regulates tissue-specific and disease-specific functions in pathogenic TH17 cells in the CNS.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Ratones , Diferenciación Celular , Sistema Nervioso Central , Ratones Endogámicos C57BL , Enfermedades Neuroinflamatorias , Células TH1 , Células Th17 , Factores de Transcripción , Virulencia , HumanosRESUMEN
CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA-targeting modules. TniQ residues, proximal to CRISPR RNA (crRNA), are required for recognizing different crRNA categories, revealing an unappreciated role of TniQ to direct transposition into different classes of crRNA targets. To investigate adaptations allowing CAST elements to utilize attachment sites inaccessible to CRISPR-Cas surveillance complexes, we compared and contrasted PAM sequence requirements in both I-F3b CAST and I-F1 CRISPR-Cas systems. We identify specific amino acids that enable a wider range of PAM sequences to be accommodated in I-F3b CAST elements compared with I-F1 CRISPR-Cas, enabling CAST elements to access attachment sites as sequences drift and evade host surveillance. Together, this evidence points to the central role of TniQ in facilitating the acquisition of CRISPR effector complexes for RNA-guided DNA transposition.
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Proteínas Asociadas a CRISPR , ARN , ADN/genética , Sistemas CRISPR-Cas , Proteínas Asociadas a CRISPR/genéticaRESUMEN
CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism1-3. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA-TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC-TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein-DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.
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Sistemas CRISPR-Cas , Elementos Transponibles de ADN , Edición Génica , Holoenzimas , Complejos Multiproteicos , ARN Guía de Sistemas CRISPR-Cas , Transposasas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/ultraestructura , Edición Génica/métodos , Transposasas/química , Transposasas/metabolismo , Transposasas/ultraestructura , ARN Guía de Sistemas CRISPR-Cas/genética , Holoenzimas/química , Holoenzimas/metabolismo , Holoenzimas/ultraestructura , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Microscopía por Crioelectrón , Subunidades Ribosómicas/química , Subunidades Ribosómicas/metabolismo , Subunidades Ribosómicas/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructuraRESUMEN
Inhibition of the tumour suppressive function of p53 (encoded by TP53) is paramount for cancer development in humans. However, p53 remains unmutated in the majority of cases of glioblastoma (GBM)-the most common and deadly adult brain malignancy1,2. Thus, how p53-mediated tumour suppression is countered in TP53 wild-type (TP53WT) GBM is unknown. Here we describe a GBM-specific epigenetic mechanism in which the chromatin regulator bromodomain-containing protein 8 (BRD8) maintains H2AZ occupancy at p53 target loci through the EP400 histone acetyltransferase complex. This mechanism causes a repressive chromatin state that prevents transactivation by p53 and sustains proliferation. Notably, targeting the bromodomain of BRD8 displaces H2AZ, enhances chromatin accessibility and engages p53 transactivation. This in turn enforces cell cycle arrest and tumour suppression in TP53WT GBM. In line with these findings, BRD8 is highly expressed with H2AZ in proliferating single cells of patient-derived GBM, and is inversely correlated with CDKN1A, a canonical p53 target that encodes p21 (refs. 3,4). This work identifies BRD8 as a selective epigenetic vulnerability for a malignancy for which treatment has not improved for decades. Moreover, targeting the bromodomain of BRD8 may be a promising therapeutic strategy for patients with TP53WT GBM.
Asunto(s)
Epigénesis Genética , Glioblastoma , Factores de Transcripción , Proteína p53 Supresora de Tumor , Adulto , Humanos , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Histonas/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proliferación CelularRESUMEN
Throughout an individual's lifetime, genomic alterations accumulate in somatic cells1-11. However, the mutational landscape induced by retrotransposition of long interspersed nuclear element-1 (L1), a widespread mobile element in the human genome12-14, is poorly understood in normal cells. Here we explored the whole-genome sequences of 899 single-cell clones established from three different cell types collected from 28 individuals. We identified 1,708 somatic L1 retrotransposition events that were enriched in colorectal epithelium and showed a positive relationship with age. Fingerprinting of source elements showed 34 retrotransposition-competent L1s. Multidimensional analysis demonstrated that (1) somatic L1 retrotranspositions occur from early embryogenesis at a substantial rate, (2) epigenetic on/off of a source element is preferentially determined in the early organogenesis stage, (3) retrotransposition-competent L1s with a lower population allele frequency have higher retrotransposition activity and (4) only a small fraction of L1 transcripts in the cytoplasm are finally retrotransposed in somatic cells. Analysis of matched cancers further suggested that somatic L1 retrotransposition rate is substantially increased during colorectal tumourigenesis. In summary, this study illustrates L1 retrotransposition-induced somatic mosaicism in normal cells and provides insights into the genomic and epigenomic regulation of transposable elements over the human lifetime.
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Colon , Elementos Transponibles de ADN , Mucosa Intestinal , Retroelementos , Humanos , Carcinogénesis/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Elementos Transponibles de ADN/genética , Genómica , Elementos de Nucleótido Esparcido Largo/genética , Retroelementos/genética , Envejecimiento/genética , Frecuencia de los Genes , Mosaicismo , Epigenómica , Genoma Humano/genética , Colon/metabolismo , Mucosa Intestinal/metabolismo , Desarrollo Embrionario/genéticaAsunto(s)
Investigación Biomédica , COVID-19/inmunología , Síndrome de Liberación de Citoquinas/inmunología , Citocinas/inmunología , Sistema Inmunológico/inmunología , SARS-CoV-2/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Factores de Edad , Animales , Autoinmunidad , COVID-19/metabolismo , COVID-19/terapia , COVID-19/virología , Síndrome de Liberación de Citoquinas/metabolismo , Síndrome de Liberación de Citoquinas/virología , Citocinas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Sistema Inmunológico/metabolismo , Sistema Inmunológico/virología , Pronóstico , Factores de Riesgo , SARS-CoV-2/patogenicidad , Síndrome de Respuesta Inflamatoria Sistémica/metabolismo , Síndrome de Respuesta Inflamatoria Sistémica/terapia , Síndrome de Respuesta Inflamatoria Sistémica/virologíaRESUMEN
Alternative polyadenylation (APA) contributes to transcriptome complexity by generating mRNA isoforms with varying 3' UTR lengths. APA leading to 3' UTR shortening (3' US) is a common feature of most cancer cells; however, the molecular mechanisms are not understood. Here, we describe a widespread mechanism promoting 3' US in cancer through ubiquitination of the mRNA 3' end processing complex protein, PCF11, by the cancer-specific MAGE-A11-HUWE1 ubiquitin ligase. MAGE-A11 is normally expressed only in the male germline but is frequently re-activated in cancers. MAGE-A11 is necessary for cancer cell viability and is sufficient to drive tumorigenesis. Screening for targets of MAGE-A11 revealed that it ubiquitinates PCF11, resulting in loss of CFIm25 from the mRNA 3' end processing complex. This leads to APA of many transcripts affecting core oncogenic and tumor suppressors, including cyclin D2 and PTEN. These findings provide insights into the molecular mechanisms driving APA in cancer and suggest therapeutic strategies.
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Regiones no Traducidas 3'/genética , Antígenos de Neoplasias/metabolismo , Neoplasias Pulmonares/patología , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/patología , ARN Mensajero/metabolismo , Ubiquitina/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Animales , Antígenos de Neoplasias/genética , Apoptosis , Biomarcadores de Tumor , Carcinogénesis , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Proliferación Celular , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Poliadenilación , Empalme del ARN , ARN Mensajero/genética , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Ensayos Antitumor por Modelo de Xenoinjerto , Factores de Escisión y Poliadenilación de ARNm/genéticaAsunto(s)
Angioedema/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Síndrome de Liberación de Citoquinas/inmunología , Citocinas/metabolismo , Neumonía Viral/inmunología , Angioedema/sangre , Angioedema/patología , Angioedema/virología , Enzima Convertidora de Angiotensina 2 , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Antivirales/uso terapéutico , Biomarcadores/sangre , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico/métodos , Congresos como Asunto , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/epidemiología , Síndrome de Liberación de Citoquinas/sangre , Síndrome de Liberación de Citoquinas/virología , Citocinas/antagonistas & inhibidores , Citocinas/sangre , Citocinas/inmunología , Humanos , Internet , Sistema Calicreína-Quinina/efectos de los fármacos , Sistema Calicreína-Quinina/inmunología , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/sangre , Neumonía Viral/diagnóstico , Neumonía Viral/epidemiología , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/patología , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Factores de Tiempo , Tiempo de Tratamiento , Tratamiento Farmacológico de COVID-19RESUMEN
The mammalian small intestine epithelium harbors a peculiar population of CD4+CD8αα+ T cells that are derived from mature CD4+ T cells through reprogramming of lineage-specific transcription factors. CD4+CD8αα+ T cells occupy a unique niche in T cell biology because they exhibit mixed phenotypes and functional characteristics of both CD4+ helper and CD8+ cytotoxic T cells. The molecular pathways driving their generation are not fully mapped. However, recent studies demonstrate the unique role of the commensal gut microbiota as well as distinct cytokine and chemokine requirements in the differentiation and survival of these cells. We review the established and newly identified factors involved in the generation of CD4+CD8αα+ intraepithelial lymphocytes (IELs) and place them in the context of the molecular machinery that drives their phenotypic and functional differentiation.
Asunto(s)
Linfocitos Intraepiteliales , Humanos , Animales , Diferenciación Celular , Factores de Transcripción/metabolismo , Linfocitos T Citotóxicos , Linfocitos T CD8-positivos , Mucosa Intestinal/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , MamíferosRESUMEN
The cognitive abilities that characterize humans are thought to emerge from unique features of the cortical circuit architecture of the human brain, which include increased cortico-cortical connectivity. However, the evolutionary origin of these changes in connectivity and how they affected cortical circuit function and behaviour are currently unknown. The human-specific gene duplication SRGAP2C emerged in the ancestral genome of the Homo lineage before the major phase of increase in brain size1,2. SRGAP2C expression in mice increases the density of excitatory and inhibitory synapses received by layer 2/3 pyramidal neurons (PNs)3-5. Here we show that the increased number of excitatory synapses received by layer 2/3 PNs induced by SRGAP2C expression originates from a specific increase in local and long-range cortico-cortical connections. Mice humanized for SRGAP2C expression in all cortical PNs displayed a shift in the fraction of layer 2/3 PNs activated by sensory stimulation and an enhanced ability to learn a cortex-dependent sensory-discrimination task. Computational modelling revealed that the increased layer 4 to layer 2/3 connectivity induced by SRGAP2C expression explains some of the key changes in sensory coding properties. These results suggest that the emergence of SRGAP2C at the birth of the Homo lineage contributed to the evolution of specific structural and functional features of cortical circuits in the human cortex.
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Corteza Cerebral , Vías Nerviosas , Animales , Femenino , Humanos , Masculino , Ratones , Señalización del Calcio , Corteza Cerebral/anatomía & histología , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Discriminación en Psicología , Ratones Transgénicos , Vías Nerviosas/fisiología , Tamaño de los Órganos , Células Piramidales/fisiología , Sinapsis/metabolismoRESUMEN
Cellular dynamics and fate decision in early human embryogenesis remain largely unknown owing to the challenges of performing studies in human embryos1. Here, we explored whole-genomes of 334 single-cell colonies and targeted deep sequences of 379 bulk tissues obtained from various anatomical locations of seven recently deceased adult human donors. Using somatic mutations as an intrinsic barcode, we reconstructed early cellular phylogenies that demonstrate (1) an endogenous mutational rate that is higher in the first cell division but decreases to approximately one per cell per cell division later in life; (2) universal unequal contribution of early cells to embryo proper, resulting from early cellular bottlenecks that stochastically set aside epiblast cells within the embryo; (3) examples of varying degrees of early clonal imbalances between tissues on the left and right sides of the body, different germ layers and specific anatomical parts and organs; (4) emergence of a few ancestral cells that will substantially contribute to adult cell pools in blood and liver; and (5) presence of mitochondrial DNA heteroplasmy in the fertilized egg. Our approach also provides insights into the age-related mutational processes and loss of sex chromosomes in normal somatic cells. In sum, this study provides a foundation for future studies to complete cellular phylogenies in human embryogenesis.
Asunto(s)
Linaje de la Célula/genética , Células Clonales/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Mutación , ADN Mitocondrial/genética , Embrión de Mamíferos/embriología , Femenino , Humanos , Masculino , Tasa de MutaciónRESUMEN
The bacterium Vibrio vulnificus causes fatal septicemia in humans. Previously, we reported that an extracellular metalloprotease, vEP-45, secreted by V. vulnificus, undergoes self-proteolysis to generate a 34 kDa protease (vEP-34) by losing its C-terminal domain to produce the C-ter100 peptide. Moreover, we revealed that vEP-45 and vEP-34 proteases induce blood coagulation and activate the kallikrein/kinin system. However, the role of the C-ter100 peptide fragment released from vEP-45 in inducing inflammation is still unclear. Here, we elucidate, for the first time, the effects of C-ter100 on inducing inflammation and activating host innate immunity. Our results showed that C-ter100 could activate NF-κB by binding to the receptor TLR4, thereby promoting the secretion of inflammatory cytokines and molecules, such as TNF-α and nitric oxide (NO). Furthermore, C-ter100 could prime and activate the NLRP3 inflammasome (NLRP3, ASC, and caspase 1), causing IL-1ß secretion. In mice, C-ter100 induced the recruitment of immune cells, such as neutrophils and monocytes, along with histamine release into the plasma. Furthermore, the inflammatory response induced by C-ter100 could be effectively neutralized by an anti-C-ter100 monoclonal antibody (C-ter100Mab). These results demonstrate that C-ter100 can be a pathogen-associated molecular pattern (PAMP) that activates an innate immune response during Vibrio infection and could be a target for the development of antibiotics.
RESUMEN
Lineage fate in the thymus is determined by mutually exclusive expression of the transcription factors ThPOK and Runx3, with ThPOK imposing the CD4(+) lineage fate and Runx3 promoting the CD8(+) lineage fate. While it is known that cytokine signals induce thymocytes to express Runx3, it is not known how ThPOK prevents thymocytes from expressing Runx3 and adopting the CD8(+) lineage fate, nor is it understood why ThPOK itself imposes the CD4(+) lineage fate on thymocytes. We now report that genes encoding members of the SOCS (suppressor of cytokine signaling) family are critical targets of ThPOK and that their induction by ThPOK represses Runx3 expression and promotes the CD4(+) lineage fate. Thus, induction of SOCS-encoding genes is the main mechanism by which ThPOK imposes the CD4(+) lineage fate in the thymus.
Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Linaje de la Célula , Subunidad alfa 3 del Factor de Unión al Sitio Principal/fisiología , Proteínas Supresoras de la Señalización de Citocinas/genética , Factores de Transcripción/fisiología , Animales , Linfocitos T CD8-positivos/fisiología , Ratones , Ratones Endogámicos C57BLRESUMEN
Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.
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Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas , División del Núcleo Celular , Quinasa Tipo Polo 1RESUMEN
With the increasing number of sequencing projects involving families, quality control tools optimized for family genome sequencing are needed. However, accurately quantifying contamination in a DNA mixture is particularly difficult when genetically related family members are the sources. We developed TrioMix, a maximum likelihood estimation (MLE) framework based on Mendel's law of inheritance, to quantify DNA mixture between family members in genome sequencing data of parent-offspring trios. TrioMix can accurately deconvolute any intrafamilial DNA contamination, including parent-offspring, sibling-sibling, parent-parent, and even multiple familial sources. In addition, TrioMix can be applied to detect genomic abnormalities that deviate from Mendelian inheritance patterns, such as uniparental disomy (UPD) and chimerism. A genome-wide depth and variant allele frequency plot generated by TrioMix facilitates tracing the origin of Mendelian inheritance deviations. We showed that TrioMix could accurately deconvolute genomes in both simulated and real data sets.
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Contaminación de ADN , Genoma , Humanos , Mapeo Cromosómico , Disomía Uniparental , Bases de Datos GenéticasRESUMEN
The maintenance of naive CD8(+) T cells is necessary for lifelong immunocompetence but for unknown reasons requires signaling via both interleukin 7 (IL-7) and the T cell antigen receptor (TCR). We now report that naive CD8(+) T cells required IL-7 signaling to be intermittent, not continuous, because prolonged IL-7 signaling induced naive CD8(+) T cells to proliferate, produce interferon-γ (IFN-γ) and undergo IFN-γ-triggered cell death. Homeostatic engagement of the TCR interrupted IL-7 signaling and thereby supported the survival and quiescence of CD8(+) T cells. However, CD8(+) T cells with insufficient TCR affinity for self ligands received prolonged IL-7 signaling and died during homeostasis. In this study we identified regulation of the duration of IL-7 signaling by homeostatic engagement of the TCR as the basis for in vivo CD8(+) T cell homeostasis.
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Linfocitos T CD8-positivos/inmunología , Homeostasis/inmunología , Interleucina-7/genética , Receptores de Antígenos de Linfocitos T/genética , Transducción de Señal/inmunología , Animales , Linfocitos T CD8-positivos/citología , Muerte Celular/inmunología , Proliferación Celular , Supervivencia Celular/inmunología , Regulación de la Expresión Génica , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-7/inmunología , Activación de Linfocitos , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/inmunología , Factores de TiempoRESUMEN
Drug addiction therapies commonly fail because continued drug use promotes the release of excessive and pleasurable dopamine levels. Because the connection between pleasure and drug use becomes hard-wired in the nucleus accumbens (NAc), which interfaces motivation, effective therapies need to modulate this mesolimbic reward system. Here, we report that mice with knockdown of the cation channel TRPA1 (transient receptor potential ankyrin 1) were resistant to the drug-seeking behavior and reward effects of cocaine compared to their wildtype litter mates. In our study, we demonstrate that TRPA1 inhibition in the NAc reduces cocaine activity and dopamine release, and conversely, that TRPA1 is critical for cocaine-induced synaptic strength in dopamine receptor 1-expressing medium spiny neurons. Taken together, our data support that cocaine-induced reward-related behavior and synaptic release of dopamine in the NAc are controlled by TRPA1 and suggest that TRPA1 has therapeutic potential as a target for drug misuse therapies.
RESUMEN
CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from Scytonema hofmanni (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB's tetrameric architecture is stabilized by a different set of protein-protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5' nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor-DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue "hook" that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cianobacterias , Elementos Transponibles de ADN , Transposasas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón , Cianobacterias/enzimología , Cianobacterias/genética , Proteínas de Unión al ADN/metabolismo , Transposasas/genética , Transposasas/metabolismoRESUMEN
The selenium-binding protein 1 (SELENBP1) has been reported to be up-regulated in the prefrontal cortex (PFC) of schizophrenia patients in postmortem reports. However, no causative link between SELENBP1 and schizophrenia has yet been established. Here, we provide evidence linking the upregulation of SELENBP1 in the PFC of mice with the negative symptoms of schizophrenia. We verified the levels of SELENBP1 transcripts in postmortem PFC brain tissues from patients with schizophrenia and matched healthy controls. We also generated transgenic mice expressing human SELENBP1 (hSELENBP1 Tg) and examined their neuropathological features, intrinsic firing properties of PFC 2/3-layer pyramidal neurons, and frontal cortex (FC) electroencephalographic (EEG) responses to auditory stimuli. Schizophrenia-like behaviors in hSELENBP1 Tg mice and mice expressing Selenbp1 in the FC were assessed. SELENBP1 transcript levels were higher in the brains of patients with schizophrenia than in those of matched healthy controls. The hSELENBP1 Tg mice displayed negative endophenotype behaviors, including heterotopias- and ectopias-like anatomical deformities in upper-layer cortical neurons and social withdrawal, deficits in nesting, and anhedonia-like behavior. Additionally, hSELENBP1 Tg mice exhibited reduced excitabilities of PFC 2/3-layer pyramidal neurons and abnormalities in EEG biomarkers observed in schizophrenia. Furthermore, mice overexpressing Selenbp1 in FC showed deficits in sociability. These results suggest that upregulation of SELENBP1 in the PFC causes asociality, a negative symptom of schizophrenia.