RESUMEN
Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.
Asunto(s)
Transferasas Intramoleculares/metabolismo , Biosíntesis de Proteínas , Seudouridina/metabolismo , ARN de Transferencia/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Diferenciación Celular , Factores Eucarióticos de Iniciación/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Transferasas Intramoleculares/antagonistas & inhibidores , Transferasas Intramoleculares/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Síndromes Mielodisplásicos/patología , Conformación de Ácido Nucleico , Fosfoproteínas/metabolismo , Proteína I de Unión a Poli(A)/antagonistas & inhibidores , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Nicho de Células MadreRESUMEN
Hematopoietic stem cells (HSCs) reside and self-renew in the bone marrow (BM) niche. Overall, the signaling that regulates stem cell dormancy in the HSC niche remains controversial. Here, we demonstrate that TGF-ß type II receptor-deficient HSCs show low-level Smad activation and impaired long-term repopulating activity, underlining the critical role of TGF-ß/Smad signaling in HSC maintenance. TGF-ß is produced as a latent form by a variety of cells, so we searched for those that express activator molecules for latent TGF-ß. Nonmyelinating Schwann cells in BM proved responsible for activation. These glial cells ensheathed autonomic nerves, expressed HSC niche factor genes, and were in contact with a substantial proportion of HSCs. Autonomic nerve denervation reduced the number of these active TGF-ß-producing cells and led to rapid loss of HSCs from BM. We propose that glial cells are components of a BM niche and maintain HSC hibernation by regulating activation of latent TGF-ß.
Asunto(s)
Médula Ósea/fisiología , Células Madre Hematopoyéticas/citología , Células de Schwann/citología , Factor de Crecimiento Transformador beta3/metabolismo , Animales , Antígenos CD34/metabolismo , Células Madre Hematopoyéticas/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Neuroglía/metabolismo , Células de Schwann/fisiología , SimpatectomíaRESUMEN
Stem cell transplantation is a cornerstone in the treatment of blood malignancies. The most common method to harvest stem cells for transplantation is by leukapheresis, requiring mobilization of CD34+ hematopoietic stem and progenitor cells (HSPCs) from the bone marrow into the blood. Identifying the genetic factors that control blood CD34+ cell levels could reveal new drug targets for HSPC mobilization. Here we report the first large-scale, genome-wide association study on blood CD34+ cell levels. Across 13 167 individuals, we identify 9 significant and 2 suggestive associations, accounted for by 8 loci (PPM1H, CXCR4, ENO1-RERE, ITGA9, ARHGAP45, CEBPA, TERT, and MYC). Notably, 4 of the identified associations map to CXCR4, showing that bona fide regulators of blood CD34+ cell levels can be identified through genetic variation. Further, the most significant association maps to PPM1H, encoding a serine/threonine phosphatase never previously implicated in HSPC biology. PPM1H is expressed in HSPCs, and the allele that confers higher blood CD34+ cell levels downregulates PPM1H. Through functional fine-mapping, we find that this downregulation is caused by the variant rs772557-A, which abrogates an MYB transcription factor-binding site in PPM1H intron 1 that is active in specific HSPC subpopulations, including hematopoietic stem cells, and interacts with the promoter by chromatin looping. Furthermore, PPM1H knockdown increases the proportion of CD34+ and CD34+90+ cells in cord blood assays. Our results provide the first large-scale analysis of the genetic architecture of blood CD34+ cell levels and warrant further investigation of PPM1H as a potential inhibition target for stem cell mobilization.
Asunto(s)
Estudio de Asociación del Genoma Completo , Células Madre Hematopoyéticas , Antígenos CD34/metabolismo , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/metabolismo , HumanosRESUMEN
Even though hematopoietic stem cells (HSC) are characterized by their ability to self-renew and differentiate, they primarily reside in quiescence. Despite the immense importance of this quiescent state, its maintenance and regulation is still incompletely understood. Schlafen2 (Slfn2) is a cytoplasmic protein known to be involved in cell proliferation, differentiation, quiescence, interferon response, and regulation of the immune system. Interestingly, Slfn2 is highly expressed in primitive hematopoietic cells. In order to investigate the role of Slfn2 in the regulation of HSC we have studied HSC function in the elektra mouse model, where the elektra allele of the Slfn2 gene contains a point mutation causing loss of function of the Slfn2 protein. We found that homozygosity for the elektra allele caused a decrease of primitive hematopoietic compartments in murine bone marrow. We further found that transplantation of elektra bone marrow and purified HSC resulted in a significantly reduced regenerative capacity of HSC in competitive transplantation settings. Importantly, we found that a significantly higher fraction of elektra HSC (as compared to wild-type HSC) were actively cycling, suggesting that the mutation in Slfn2 increases HSC proliferation. This additionally caused an increased amount of apoptotic stem and progenitor cells. Taken together, our findings demonstrate that dysregulation of Slfn2 results in a functional deficiency of primitive hematopoietic cells, which is particularly reflected by a drastically impaired ability to reconstitute the hematopoietic system following transplantation and an increase in HSC proliferation. This study thus identifies Slfn2 as a novel and critical regulator of adult HSC and HSC quiescence.
Asunto(s)
Proteínas de Ciclo Celular , Hematopoyesis , Células Madre Hematopoyéticas , Animales , Ratones , Médula Ósea , Diferenciación Celular/genética , Proliferación Celular , Células Madre Hematopoyéticas/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMEN
The ability to capture alterations in the genome or transcriptome by next-generation sequencing has provided critical insight into molecular changes and programs underlying cancer biology. With the rapid technological development in single-cell sequencing, it has become possible to study individual cells at the transcriptional, genetic, epigenetic, and protein level. Using single-cell analysis, an increased resolution of fundamental processes underlying cancer development is obtained, providing comprehensive insights otherwise lost by sequencing of entire (bulk) samples, in which molecular signatures of individual cells are averaged across the entire cell population. Here, we provide a concise overview on the application of single-cell analysis of different modalities within cancer research by highlighting key articles of their respective fields. We furthermore examine the potential of existing technologies to meet clinical diagnostic needs and discuss current challenges associated with this translation.
Asunto(s)
Pruebas Genéticas/métodos , Neoplasias/genética , RNA-Seq/métodos , Análisis de la Célula Individual/métodos , Investigación Biomédica Traslacional/métodos , Animales , Pruebas Genéticas/normas , Humanos , Neoplasias/diagnóstico , RNA-Seq/normas , Análisis de la Célula Individual/normas , Investigación Biomédica Traslacional/normasRESUMEN
Airway basal cells are crucial for regeneration of the human lung airway epithelium and are believed to be important contributors to chronic obstructive pulmonary disease (COPD) and other lung disorders. To reveal how basal cells contribute to disease and to discover novel therapeutic targets, these basal cells need to be further characterized. In this study, we optimized a flow cytometry-based cell sorting protocol for primary human airway basal cells dependent on cell size and NGFR (nerve-growth factor receptor) expression. The basal cell population was found to be molecularly and functionally heterogeneous, in contrast to cultured basal cells. In addition, significant differences were found, such as KRT14 expression exclusively existing in cultured cells. Also, colony-forming capacity was significantly increased in cultured cells showing a clonal enrichment in vitro. Next, by single-cell RNA sequencing on primary basal cells from healthy donors and patients with Global Initiative for Chronic Obstructive Lung Disease stage IV COPD, the gene expression revealed a continuum ranging from healthy basal cell signatures to diseased basal cell phenotypes. We identified several upregulated genes that may indicate COPD, such as stress response-related genes GADD45B and AHSA1, together with with genes involved in the response to hypoxia, such as CITED2 and SOD1. Taken together, the presence of healthy basal cells in stage IV COPD demonstrates the potential for regeneration through the discovery of novel therapeutic targets. In addition, we show the importance of studying primary basal cells when investigating disease mechanisms as well as for developing future cell-based therapies in the human lung.
Asunto(s)
Células Epiteliales/metabolismo , Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Mucosa Respiratoria/metabolismo , Antígenos de Diferenciación/metabolismo , Células Cultivadas , Células Epiteliales/patología , Humanos , Queratina-14/metabolismo , Pulmón/patología , Chaperonas Moleculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Receptores de Factor de Crecimiento Nervioso/metabolismo , Mucosa Respiratoria/patologíaRESUMEN
Understanding leukemia heterogeneity is critical for the development of curative treatments as the failure to eliminate therapy-persistent leukemic stem cells (LSCs) may result in disease relapse. Here we have combined high-throughput immunophenotypic screens with large-scale single-cell gene expression analysis to define the heterogeneity within the LSC population in chronic phase chronic myeloid leukemia (CML) patients at diagnosis and following conventional tyrosine kinase inhibitor (TKI) treatment. Our results reveal substantial heterogeneity within the putative LSC population in CML at diagnosis and demonstrate differences in response to subsequent TKI treatment between distinct subpopulations. Importantly, LSC subpopulations with myeloid and proliferative molecular signatures are proportionally reduced at a higher extent in response to TKI therapy compared with subfractions displaying primitive and quiescent signatures. Additionally, cell surface expression of the CML stem cell markers CD25, CD26, and IL1RAP is high in all subpopulations at diagnosis but downregulated and unevenly distributed across subpopulations in response to TKI treatment. The most TKI-insensitive cells of the LSC compartment can be captured within the CD45RA- fraction and further defined as positive for CD26 in combination with an aberrant lack of cKIT expression. Together, our results expose a considerable heterogeneity of the CML stem cell population and propose a Lin-CD34+CD38-/lowCD45RA-cKIT-CD26+ population as a potential therapeutic target for improved therapy response.
Asunto(s)
Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Análisis de la Célula Individual/métodos , ADP-Ribosil Ciclasa 1/deficiencia , ADP-Ribosil Ciclasa 1/genética , ADP-Ribosil Ciclasa 1/inmunología , Antígenos CD34/genética , Antígenos CD34/inmunología , Biomarcadores de Tumor/inmunología , Estudios de Casos y Controles , Linaje de la Célula/inmunología , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/inmunología , Expresión Génica , Heterogeneidad Genética , Humanos , Inmunofenotipificación , Proteína Accesoria del Receptor de Interleucina-1/genética , Proteína Accesoria del Receptor de Interleucina-1/inmunología , Subunidad alfa del Receptor de Interleucina-2/genética , Subunidad alfa del Receptor de Interleucina-2/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Antígenos Comunes de Leucocito/deficiencia , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/patología , Proteínas Proto-Oncogénicas c-kit/deficiencia , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/inmunología , Resultado del TratamientoRESUMEN
BACKGROUND: Metabolomics represents a powerful tool for exploring modulation of the human metabolome in response to food intake. However, the choice of multivariate statistical approach is not always evident, especially for complex experimental designs with repeated measurements per individual. Here we have investigated the serum metabolic responses to two breakfast meals: an egg and ham based breakfast and a cereal based breakfast using three different multivariate approaches based on the Projections to Latent Structures framework. METHODS: In a cross over design, 24 healthy volunteers ate the egg and ham breakfast and cereal breakfast on four occasions each. Postprandial serum samples were subjected to metabolite profiling using 1H nuclear magnetic resonance spectroscopy and metabolites were identified using 2D nuclear magnetic resonance spectroscopy. Metabolic profiles were analyzed using Orthogonal Projections to Latent Structures with Discriminant Analysis and Effect Projections and ANOVA-decomposed Projections to Latent Structures. RESULTS: The Orthogonal Projections to Latent Structures with Discriminant Analysis model correctly classified 92 and 90% of the samples from the cereal breakfast and egg and ham breakfast, respectively, but confounded dietary effects with inter-personal variability. Orthogonal Projections to Latent Structures with Effect Projections removed inter-personal variability and performed perfect classification between breakfasts, however at the expense of comparing means of respective breakfasts instead of all samples. ANOVA-decomposed Projections to Latent Structures managed to remove inter-personal variability and predicted 99% of all individual samples correctly. Proline, tyrosine, and N-acetylated amino acids were found in higher concentration after consumption of the cereal breakfast while creatine, methanol, and isoleucine were found in higher concentration after the egg and ham breakfast. CONCLUSIONS: Our results demonstrate that the choice of statistical method will influence the results and adequate methods need to be employed to manage sample dependency and repeated measurements in cross-over studies. In addition, 1H nuclear magnetic resonance serum metabolomics could reproducibly characterize postprandial metabolic profiles and identify discriminatory metabolites largely reflecting dietary composition. TRIAL REGISTRATION: Registered with ClinicalTrials.gov, identifier: NCT02039596 . Date of registration: January 17, 2014.
Asunto(s)
Aminoácidos/sangre , Desayuno/fisiología , Ingestión de Alimentos/fisiología , Metabolómica/métodos , Espectroscopía de Protones por Resonancia Magnética , Adulto , Estudios Cruzados , Grano Comestible , Huevos , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Carne de Cerdo , Periodo PosprandialRESUMEN
A high-affinity polypeptide conjugate 4-C25L22-DQ, has been developed for the molecular recognition of the human C-reactive protein, CRP, a well-known inflammation biomarker. CRP is one of the most frequently quantified targets in diagnostic applications and a target in drug development. With the exception of antibodies, most molecular constructs take advantage of the known affinity for CRP of phosphocholine that depends on Ca2+ for its ability to bind. 4-C25L22-DQ which is unrelated to phosphocholine binds in the absence of Ca2+ with a dissociation constant of 760 nM, an order of magnitude lower than that of phosphocholine, the KD of which is 5 µM. The small organic molecule 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) was designed based on the structural similarities between three hits from a set of compounds selected from a building block collection and evaluated with regards to affinity for CRP by NMR spectroscopy. 4-C25L22-DQ was shown in a competition experiment to bind CRP three orders of magnitude more strongly than DQ itself, and in a pull-down experiment 4-C25L22-DQ was shown to extract CRP from human serum. The development of a robust and phosphocholine-independent recognition element provides unprecedented opportunities in bioanalytical applications in vivo and in vitro under conditions where the concentration of Ca2+ ions is low, or where Ca2+ binding agents such as EDTA or heparin are needed to prevent blood coagulation. The identification from a compound library of a small organic molecule and its conjugation to a small set of polypeptides, none of which were previously known to bind CRP, illustrates a convenient and general route to selective high-affinity binders for proteins with dissociation constants in the µM to nM range for which no small molecule ligands are known.
Asunto(s)
Proteína C-Reactiva/metabolismo , Fosforilcolina/metabolismo , Secuencia de Aminoácidos , Proteína C-Reactiva/química , Diseño de Fármacos , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
The class IId bacteriocin lactococcin A and the pediocin-like bacteriocins induce membrane leakage and cell death by specifically binding the mannose phophotransferase system (man-PTS) on their target cells. The bacteriocins' cognate immunity proteins that protect the producer cell from its own bacteriocin recognize and bind to the bacteriocin-man-PTS complex and thereby block membrane leakage. In this study, we have determined the three-dimensional structure of the lactococcin A immunity protein (LciA) by the use of nuclear magnetic resonance spectroscopy. LciA forms a four-helix bundle structure with a flexible C-terminal tail. Despite the low degree of sequence similarity between LciA and the pediocin-like immunity proteins, they share the same fold. However, there are certain differences between the structures. The C-terminal helix in LciA is considerably shorter than that observed in the pediocin-like immunity proteins, and the surface potentials of the immunity proteins differ. Truncated variants of LciA in which 6 or 10 of the C-terminal residues were removed yielded a reduced degree of protection, indicating that the unstructured C-terminal tail is important for the functionality of the immunity proteins.
Asunto(s)
Bacteriocinas/genética , Análisis Mutacional de ADN/métodos , Lactococcus lactis/genética , Espectroscopía de Resonancia Magnética/métodos , Bacteriocinas/química , Bacteriocinas/metabolismo , Lactococcus lactis/metabolismo , Modelos Moleculares , Mutación , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross-links, or caps the filament ends have been identified and the actin cross-linker α-actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α-actinin is believed to be required for infection. To better understand the role of α-actinin in the infectious process we have determined the solution structure of the C-terminal calmodulin-like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium-binding EF-hand motifs, connected with a mobile linker.
Asunto(s)
Actinina/química , Calcio/química , Calmodulina/química , Entamoeba histolytica/química , Proteínas Protozoarias/química , Factores de Virulencia/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinina/genética , Actinina/metabolismo , Secuencia de Aminoácidos , Calcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Clonación Molecular , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
BACKGROUND: Single cell gene expression assays have become a powerful tool with which to dissect heterogeneous populations. While methods and software exist to interrogate such data, what has been lacking is a unified solution combining analysis and visualisation which is also accessible and intuitive for use by non-bioinformaticians, as well as bioinformaticians. RESULTS: We present the Single cell expression visualiser (SCExV), a webtool developed to expedite the analysis of single cell qRT-PCR data. SCExV is able to take any data matrix of Ct values as an input, but can handle files exported by the Fluidigm Biomark platform directly. In addition, SCExV also accepts and automatically integrates cell surface marker intensity values which are measured during index sorting. This allows the user to directly visualise relationships between a single cell gene expression profile and the immunophenotype of the interrogated cell. CONCLUSIONS: SCExV is a freely available webtool created to import, filter, analyse, and visualise single cell gene expression data whilst being able to simultaneously consider cellular immunophenotype. SCExV is designed to be intuitive to use whilst maintaining advanced functionality and flexibility in how analyses are performed.
Asunto(s)
ADN/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Interfaz Usuario-Computador , Animales , Internet , Ratones , Células Madre/citología , Células Madre/metabolismoRESUMEN
In an attempt to discover novel growth factors for hematopoietic stem and progenitor cells (HSPCs), we have assessed cytokine responses of cord blood (CB)-derived CD34(+) cells in a high-content growth factor screen. We identify the immunoregulatory chemokine (C-C motif) ligand 28 (CCL28) as a novel growth factor that directly stimulates proliferation of primitive hematopoietic cells from different ontogenetic origins. CCL28 enhances the functional progenitor cell content of cultured cells by stimulating cell cycling and induces gene expression changes associated with survival. Importantly, addition of CCL28 to cultures of purified putative hematopoietic stem cells (HSCs) significantly increases the ability of the cells to long-term repopulate immunodeficient mice compared with equivalent input numbers of fresh cells. Together, our findings identify CCL28 as a potent growth-promoting factor with the ability to support the in vitro and in vivo functional properties of cultured human hematopoietic cells.
Asunto(s)
Proliferación Celular , Quimiocinas CC/fisiología , Células Madre Hematopoyéticas/fisiología , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Quimiocinas CC/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Recién Nacido , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Células Madre/fisiologíaRESUMEN
Hematopoietic stem cells (HSCs) constitute a rare population of tissue-specific cells that can self-renew and differentiate into all lineages of the blood cell system. These properties are critical for tissue regeneration and clinical applications of HSCs. Cord blood is an easily accessible source of HSCs. However, the number of HSCs from one unit is too low to effectively transplant most adult patients, and expansion of HSCs in vitro has met with limited success because of incomplete knowledge regarding mechanisms regulating self-renewal. Members of the TGF-ß superfamily have been shown to regulate HSCs through the Smad signaling pathway; however, its role in human HSCs has remained relatively uncharted in vivo. Therefore, we asked whether enforced expression of the common-Smad, Smad4, could reveal a role for TGF-ß in human hematopoietic stem/progenitor cells (HSPCs) from cord blood. Using a lentiviral overexpression approach, we demonstrate that Smad4 overexpression sensitizes HSPCs to TGF-ß, resulting in growth arrest and apoptosis in vitro. This phenotype translates in vivo into reduced HSPC reconstitution capacity yet intact lineage distribution. This suggests that the Smad pathway regulates self-renewal independently of differentiation. These findings demonstrate that the Smad signaling circuitry negatively regulates the regeneration capacity of human HSPCs in vivo.
Asunto(s)
Proliferación Celular , Células Madre Hematopoyéticas/metabolismo , Proteína Smad4/genética , Animales , Antígenos CD34/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Lentivirus/genética , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Modelos Biológicos , Proteína Smad4/metabolismo , TransfecciónRESUMEN
The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease.
Asunto(s)
Proteínas de Unión al ADN/genética , Células Madre Hematopoyéticas/metabolismo , Trastornos Mieloproliferativos/genética , Proteínas Proto-Oncogénicas/genética , Animales , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Eritropoyesis/genética , Expresión Génica , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Linfopoyesis/genética , Ratones , Mielopoyesis/genética , Trastornos Mieloproliferativos/metabolismo , Fenotipo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Activación Transcripcional , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Mucosa-associated lymphoid tissue lymphoma-translocation protein 1 (MALT1) is an attractive target for the development of modulatory compounds in the treatment of lymphoma and other cancers. While the three-dimensional structure of MALT1 has been previously determined through X-ray analysis, its dynamic behaviour in solution has remained unexplored. We present here dynamic analyses of the apo MALT1 form along with the E549A mutation. This investigation used NMR 15N relaxation and NOE measurements between side-chain methyl groups. Our findings confirm that MALT1 exists as a monomer in solution, and demonstrate that the domains display semi-independent movements in relation to each other. Our dynamic study, covering multiple time scales, along with the assessment of conformational populations by Molecular Dynamic simulations, Alpha Fold modelling and PCA analysis, put the side chain of residue W580 in an inward position, shedding light at potential mechanisms underlying the allosteric regulation of this enzyme.
Asunto(s)
Simulación de Dinámica Molecular , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Regulación Alostérica , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/química , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Humanos , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , MutaciónRESUMEN
The MUC2 mucin protects the colonic epithelium by a two-layered mucus with an inner attached bacteria-free layer and an outer layer harboring commensal bacteria. CysD domains are 100 amino-acid-long sequences containing 10 cysteines that separate highly O-glycosylated proline, threonine, serine (PTS) regions in mucins. The structure of the second CysD, CysD2, of MUC2 is now solved by nuclear magnetic resonance. CysD2 shows a stable stalk region predicted to be partly covered by adjacent O-glycans attached to neighboring PTS sequences, whereas the CysD2 tip with three flexible loops is suggested to be well exposed. It shows transient dimer interactions at acidic pH, weakened at physiological pH. This transient interaction can be stabilized in vitro and in vivo by transglutaminase 3-catalyzed isopeptide bonds, preferring a specific glutamine residue on one flexible loop. This covalent dimer is modeled suggesting that CysD domains act as connecting hubs for covalent stabilization of mucins to form a protective mucus.
Asunto(s)
Mucina 2 , Dominios Proteicos , Transglutaminasas , Mucina 2/metabolismo , Mucina 2/química , Humanos , Transglutaminasas/metabolismo , Transglutaminasas/química , Modelos Moleculares , Cisteína/metabolismo , Cisteína/química , Secuencia de Aminoácidos , Multimerización de Proteína , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismoRESUMEN
Immunological control of residual leukemia cells is thought to occur in patients with chronic myeloid leukemia (CML) that maintain treatment-free remission (TFR) following tyrosine kinase inhibitor (TKI) discontinuation. To study this, we analyzed 55 single-cell RNA and T cell receptor (TCR) sequenced samples (scRNA+TCRαß-seq) from patients with CML (n = 13, N = 25), other cancers (n = 28), and healthy (n = 7). The high number and active phenotype of natural killer (NK) cells in CML separated them from healthy and other cancers. Most NK cells in CML belonged to the active CD56dim cluster with high expression of GZMA/B, PRF1, CCL3/4, and IFNG, with interactions with leukemic cells via inhibitory LGALS9-TIM3 and PVR-TIGIT interactions. Accordingly, upregulation of LGALS9 was observed in CML target cells and TIM3 in NK cells when co-cultured together. Additionally, we created a classifier to identify TCRs targeting leukemia-associated antigen PR1 and quantified anti-PR1 T cells in 90 CML and 786 healthy TCRß-sequenced samples. Anti-PR1 T cells were more prevalent in CML, enriched in bone marrow samples, and enriched in the mature, cytotoxic CD8 + TEMRA cluster, especially in a patient maintaining TFR. Our results highlight the role of NK cells and anti-PR1 T cells in anti-leukemic immune responses in CML.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Humanos , Receptor 2 Celular del Virus de la Hepatitis A , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Análisis de la Célula IndividualRESUMEN
Members of the transforming growth factor beta (TGF-beta) superfamily of growth factors have been shown to regulate the in vitro proliferation and maintenance of hematopoietic stem cells (HSCs). Working at a common level of convergence for all TGF-beta superfamily signals, Smad4 is key in orchestrating these effects. The role of Smad4 in HSC function has remained elusive because of the early embryonic lethality of the conventional knockout. We clarify its role by using an inducible model of Smad4 deletion coupled with transplantation experiments. Remarkably, systemic induction of Smad4 deletion through activation of MxCre was incompatible with survival 4 wk after induction because of anemia and histopathological changes in the colonic mucosa. Isolation of Smad4 deletion to the hematopoietic system via several transplantation approaches demonstrated a role for Smad4 in the maintenance of HSC self-renewal and reconstituting capacity, leaving homing potential, viability, and differentiation intact. Furthermore, the observed down-regulation of notch1 and c-myc in Smad4(-/-) primitive cells places Smad4 within a network of genes involved in the regulation HSC renewal.