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1.
Cell ; 153(5): 963-75, 2013 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-23706735

RESUMEN

The reprogramming factors that induce pluripotency have been identified primarily from embryonic stem cell (ESC)-enriched, pluripotency-associated factors. Here, we report that, during mouse somatic cell reprogramming, pluripotency can be induced with lineage specifiers that are pluripotency rivals to suppress ESC identity, most of which are not enriched in ESCs. We found that OCT4 and SOX2, the core regulators of pluripotency, can be replaced by lineage specifiers that are involved in mesendodermal (ME) specification and in ectodermal (ECT) specification, respectively. OCT4 and its substitutes attenuated the elevated expression of a group of ECT genes, whereas SOX2 and its substitutes curtailed a group of ME genes during reprogramming. Surprisingly, the two counteracting lineage specifiers can synergistically induce pluripotency in the absence of both OCT4 and SOX2. Our study suggests a "seesaw model" in which a balance that is established using pluripotency factors and/or counteracting lineage specifiers can facilitate reprogramming.


Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Madre Embrionarias/metabolismo , Fibroblastos/metabolismo , Factor de Transcripción GATA3/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Ratones , Modelos Biológicos , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Estómago/citología
2.
Proc Natl Acad Sci U S A ; 121(16): e2400077121, 2024 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-38598345

RESUMEN

Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to lower airway repair. Agents that promote the selective expansion of these cells might stimulate regeneration of the compromised alveolar epithelium, an etiology-defining event in several pulmonary diseases. From a high-content imaging screen of the drug repurposing library ReFRAME, we identified that dipeptidyl peptidase 4 (DPP4) inhibitors, widely used type 2 diabetes medications, selectively expand AEC2s and are broadly efficacious in several mouse models of lung damage. Mechanism of action studies revealed that the protease DPP4, in addition to processing incretin hormones, degrades IGF-1 and IL-6, essential regulators of AEC2 expansion whose levels are increased in the luminal compartment of the lung in response to drug treatment. To selectively target DPP4 in the lung with sufficient drug exposure, we developed NZ-97, a locally delivered, lung persistent DPP4 inhibitor that broadly promotes efficacy in mouse lung damage models with minimal peripheral exposure and good tolerability. This work reveals DPP4 as a central regulator of AEC2 expansion and affords a promising therapeutic approach to broadly stimulate regenerative repair in pulmonary disease.


Asunto(s)
Células Epiteliales Alveolares , Diabetes Mellitus Tipo 2 , Animales , Ratones , Células Epiteliales Alveolares/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Pulmón/metabolismo , Modelos Animales de Enfermedad
3.
Proc Natl Acad Sci U S A ; 120(28): e2305085120, 2023 07 11.
Artículo en Inglés | MEDLINE | ID: mdl-37399395

RESUMEN

Chronic cutaneous wounds remain a persistent unmet medical need that decreases life expectancy and quality of life. Here, we report that topical application of PY-60, a small-molecule activator of the transcriptional coactivator Yes-associated protein (YAP), promotes regenerative repair of cutaneous wounds in pig and human models. Pharmacological YAP activation enacts a reversible pro-proliferative transcriptional program in keratinocytes and dermal cells that results in accelerated re-epithelization and regranulation of the wound bed. These results demonstrate that transient topical administration of a YAP activating agent may represent a generalizable therapeutic approach to treating cutaneous wounds.


Asunto(s)
Calidad de Vida , Cicatrización de Heridas , Humanos , Animales , Porcinos , Cicatrización de Heridas/fisiología , Piel/lesiones , Queratinocitos/metabolismo , Administración Cutánea
5.
Nat Chem Biol ; 17(7): 767-775, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33723431

RESUMEN

The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anexina A2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/metabolismo , Administración Tópica , Células Madre Adultas/efectos de los fármacos , Células Madre Adultas/metabolismo , Animales , Anexina A2/metabolismo , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ratones , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Proteínas Señalizadoras YAP
6.
Proc Natl Acad Sci U S A ; 117(16): 8845-8849, 2020 04 21.
Artículo en Inglés | MEDLINE | ID: mdl-32253306

RESUMEN

The genetic incorporation of noncanonical amino acids (ncAAs) into proteins has been realized in bacteria, yeast, and mammalian cells, and recently, in multicellular organisms including plants and animals. However, the addition of new building blocks to the genetic code of tissues from human origin has not yet been achieved. To this end, we report a self-replicating Epstein-Barr virus-based episomal vector for the long-term encoding of ncAAs in human hematopoietic stem cells and reconstitution of this genetically engineered hematopoietic system in mice.


Asunto(s)
Aminoácidos/genética , Diferenciación Celular/genética , Vectores Genéticos/genética , Células Madre Hematopoyéticas/fisiología , Ingeniería de Proteínas/métodos , Animales , Sangre Fetal/citología , Técnicas de Transferencia de Gen , Código Genético , Células HEK293 , Trasplante de Células Madre Hematopoyéticas , Herpesvirus Humano 4/genética , Humanos , Ratones , Ratones Endogámicos NOD , Plásmidos/genética , Cultivo Primario de Células/métodos , Transfección/métodos , Quimera por Trasplante , Trasplante Heterólogo/métodos
7.
Int J Mol Sci ; 18(11)2017 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-29077054

RESUMEN

The treatment of patients with acute myeloid leukemia (AML) with targeted immunotherapy is challenged by the heterogeneity of the disease and a lack of tumor-exclusive antigens. Conventional immunotherapy targets for AML such as CD33 and CD123 have been proposed as targets for chimeric antigen receptor (CAR)-engineered T-cells (CAR-T-cells), a therapy that has been highly successful in the treatment of B-cell leukemia and lymphoma. However, CD33 and CD123 are present on hematopoietic stem cells, and targeting with CAR-T-cells has the potential to elicit long-term myelosuppression. C-type lectin-like molecule-1 (CLL1 or CLEC12A) is a myeloid lineage antigen that is expressed by malignant cells in more than 90% of AML patients. CLL1 is not expressed by healthy Hematopoietic Stem Cells (HSCs), and is therefore a promising target for CAR-T-cell therapy. Here, we describe the development and optimization of an anti-CLL1 CAR-T-cell with potent activity on both AML cell lines and primary patient-derived AML blasts in vitro while sparing healthy HSCs. Furthermore, in a disseminated mouse xenograft model using the CLL1-positive HL60 cell line, these CAR-T-cells completely eradicated tumor, thus supporting CLL1 as a promising target for CAR-T-cells to treat AML while limiting myelosuppressive toxicity.


Asunto(s)
Lectinas Tipo C/antagonistas & inhibidores , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Mitogénicos/antagonistas & inhibidores , Proteínas Recombinantes de Fusión , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoterapia Adoptiva/métodos , Lectinas Tipo C/inmunología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Ratones , Receptores de Antígenos de Linfocitos T/genética , Receptores Mitogénicos/inmunología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Angew Chem Int Ed Engl ; 56(18): 5096-5100, 2017 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-28371162

RESUMEN

The use of genetically encoded noncanonical amino acids (ncAAs) to construct crosslinks within or between proteins has emerged as a useful method to enhance protein stability, investigate protein-protein interactions, and improve the pharmacological properties of proteins. We report ncAAs with aryl carbamate side chains (PheK and FPheK) that can react with proximal nucleophilic residues to form intra- or intermolecular protein crosslinks. We evolved a pyrrolysyl-tRNA synthetase that incorporates site-specifically PheK and FPheK into proteins in both E. coli and mammalian cells. PheK and FPheK when incorporated into proteins showed good stability during protein expression and purification. FPheK reacted with adjacent Lys, Cys, and Tyr residues in thioredoxin in high yields. In addition, crosslinks could be formed between FPheK and Lys residue of two interacting proteins, including the heavy chain and light chain of an antibody Fab.


Asunto(s)
Aminoácidos/química , Carbamatos/química , Reactivos de Enlaces Cruzados/química , Proteínas/química , Aminoácidos/genética , Carbamatos/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Escherichia coli/química , Escherichia coli/genética , Células HEK293 , Humanos , Modelos Moleculares , Ingeniería de Proteínas/métodos , Proteínas/genética
9.
ACS Chem Biol ; 13(3): 578-581, 2018 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-29360343

RESUMEN

Here, we report the site-specific incorporation of a thioester containing noncanonical amino acid (ncAA) into recombinantly expressed proteins. Specifically, we genetically encoded a thioester-activated aspartic acid (ThioD) in bacteria in good yield and with high fidelity using an orthogonal nonsense suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. To demonstrate the utility of ThioD, we used native chemical ligation to label green fluorescent protein with a fluorophore in good yield.


Asunto(s)
Aminoácidos/química , Ésteres/química , Mutagénesis Sitio-Dirigida/métodos , Ingeniería de Proteínas/métodos , Compuestos de Azufre/química , Escherichia coli/genética , Proteínas Fluorescentes Verdes/química , Azufre
10.
ACS Chem Biol ; 10(7): 1599-603, 2015 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-25909834

RESUMEN

Here, we report the evolution of an orthogonal amber suppressor pyrrolysyl-tRNA synthetase (PylRS)/tRNACUA(Pyl) pair that genetically encodes the post-translationally modified amino acid, ε-N-2-hydroxyisobutyryl-lysine (HibK), in bacteria and mammalian cells. HibK is a new type of histone mark that is widely distributed in histone proteins. The ability to site-specifically incorporate HibK into proteins provides a useful tool to probe the biological function of this newly identified post-translational modification.


Asunto(s)
Histonas/genética , Lisina/análogos & derivados , Lisina/genética , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Células HEK293 , Histonas/metabolismo , Humanos , Modelos Moleculares , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
11.
Cell Res ; 25(2): 169-80, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25591928

RESUMEN

Members of the GATA protein family play important roles in lineage specification and transdifferentiation. Previous reports show that some members of the GATA protein family can also induce pluripotency in somatic cells by substituting for Oct4, a key pluripotency-associated factor. However, the mechanism linking lineage-specifying cues and the activation of pluripotency remains elusive. Here, we report that all GATA family members can substitute for Oct4 to induce pluripotency. We found that all members of the GATA family could inhibit the overrepresented ectodermal-lineage genes, which is consistent with previous reports indicating that a balance of different lineage-specifying forces is important for the restoration of pluripotency. A conserved zinc-finger DNA-binding domain in the C-terminus is critical for the GATA family to induce pluripotency. Using RNA-seq and ChIP-seq, we determined that the pluripotency-related gene Sall4 is a direct target of GATA family members during reprogramming and serves as a bridge linking the lineage-specifying GATA family to the pluripotency circuit. Thus, the GATA family is the first protein family of which all members can function as inducers of the reprogramming process and can substitute for Oct4. Our results suggest that the role of GATA family in reprogramming has been underestimated and that the GATA family may serve as an important mediator of cell fate conversion.


Asunto(s)
Reprogramación Celular , Factores de Transcripción GATA/metabolismo , Animales , Línea Celular , Linaje de la Célula , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factores de Transcripción GATA/química , Factores de Transcripción GATA/genética , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Proteína Homeótica Nanog , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Unión Proteica , Análisis de Secuencia de ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc
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