RESUMEN
The pathogenesis of idiopathic pulmonary fibrosis (IPF), an intractable interstitial lung disease, is unclear. Recessive mutations in some genes implicated in Hermansky-Pudlak syndrome (HPS) cause HPS-associated interstitial pneumonia (HPSIP), a clinical entity that is similar to IPF. We previously reported that HPS1-/- embryonic stem cell-derived 3D lung organoids showed fibrotic changes. Here, we show that the introduction of all HPS mutations associated with HPSIP promotes fibrotic changes in lung organoids, while the deletion of HPS8, which is not associated with HPSIP, does not. Genome-wide expression analysis revealed the upregulation of interleukin-11 (IL-11) in epithelial cells from HPS mutant fibrotic organoids. IL-11 was detected predominantly in type 2 alveolar epithelial cells in end-stage IPF, but was expressed more broadly in HPSIP. Finally, IL-11 induced fibrosis in WT organoids, while its deletion prevented fibrosis in HPS4-/- organoids, suggesting IL-11 as a therapeutic target. hPSC-derived 3D lung organoids are, therefore, a valuable resource to model fibrotic lung disease.
Asunto(s)
Síndrome de Hermanski-Pudlak/patología , Interleucina-11/metabolismo , Modelos Biológicos , Organoides/patología , Células Madre Pluripotentes/patología , Fibrosis Pulmonar/patología , Adulto , Anciano , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Síndrome de Hermanski-Pudlak/epidemiología , Síndrome de Hermanski-Pudlak/genética , Síndrome de Hermanski-Pudlak/metabolismo , Humanos , Interleucina-11/genética , Masculino , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Organoides/metabolismo , Células Madre Pluripotentes/metabolismo , Fibrosis Pulmonar/epidemiología , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismoRESUMEN
The specific cellular physiology of hematopoietic stem cells (HSCs) is underexplored, and their maintenance in vitro remains challenging. We discovered that culture of HSCs in low calcium increased their maintenance as determined by phenotype, function, and single-cell expression signature. HSCs are endowed with low intracellular calcium conveyed by elevated activity of glycolysis-fueled plasma membrane calcium efflux pumps and a low-bone-marrow interstitial fluid calcium concentration. Low-calcium conditions inhibited calpain proteases, which target ten-eleven translocated (TET) enzymes, of which TET2 was required for the effect of low calcium conditions on HSC maintenance in vitro. These observations reveal a physiological feature of HSCs that can be harnessed to improve their maintenance in vitro.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Hematopoyéticas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Animales , Calpaína/metabolismo , Autorrenovación de las Células , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Dioxigenasas , Glucólisis , Hematopoyesis , Homeostasis , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de la Célula Individual , TranscriptomaRESUMEN
PURPOSE: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. EXPERIMENTAL DESIGN: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. RESULTS: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. CONCLUSIONS: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji/genética , Leucemia de Células T/genética , Receptor Notch1/genética , Peptidasa Específica de Ubiquitina 7/genética , Animales , Carcinogénesis/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Terapia Genética , Humanos , Células Jurkat , Leucemia de Células T/patología , Leucemia de Células T/terapia , Ratones , Transducción de Señal/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although strategies for directed differentiation of human pluripotent stem cells (hPSCs) into lung and airway have been established, terminal maturation of the cells remains a vexing problem. We show here that in collagen I 3D cultures in the absence of glycogen synthase kinase 3 (GSK3) inhibition, hPSC-derived lung progenitors (LPs) undergo multilineage maturation into proximal cells, type I alveolar epithelial cells and morphologically mature type II cells. Enhanced cell cycling, one of the signaling outputs of GSK3 inhibition, plays a role in the maturation-inhibiting effect of GSK3 inhibition. Using this model, we show NOTCH signaling induced a distal cell fate at the expense of a proximal and ciliated cell fate, whereas WNT signaling promoted a proximal club cell fate, thus implicating both signaling pathways in proximodistal specification in human lung development. These findings establish an approach to achieve multilineage maturation of lung and airway cells from hPSCs, demonstrate a pivotal role of GSK3 in the maturation of lung progenitors and provide novel insight into proximodistal specification during human lung development.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Linaje de la Célula , Glucógeno Sintasa Quinasa 3/metabolismo , Células Madre Pluripotentes Inducidas/citología , Pulmón/citología , Piridinas/farmacología , Animales , Tipificación del Cuerpo/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Colágeno Tipo I/metabolismo , Genoma Humano , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/ultraestructura , Ratones , Receptores Notch/metabolismo , Reproducibilidad de los Resultados , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
PRDM16 is a transcriptional coregulator involved in translocations in acute myeloblastic leukemia (AML), myelodysplastic syndromes, and T acute lymphoblastic leukemia that is highly expressed in and required for the maintenance of hematopoietic stem cells (HSCs), and can be aberrantly expressed in AML. Prdm16 is expressed as full-length (fPrdm16) and short (sPrdm16) isoforms, the latter lacking the N-terminal PR domain. The role of both isoforms in normal and malignant hematopoiesis is unclear. We show here that fPrdm16 was critical for HSC maintenance, induced multiple genes involved in GTPase signaling, and repressed inflammation, while sPrdm16 supported B cell development biased toward marginal zone B cells and induced an inflammatory signature. In a mouse model of human MLL-AF9 leukemia, fPrdm16 extended latency, while sPrdm16 shortened latency and induced a strong inflammatory signature, including several cytokines and chemokines that are associated with myelodysplasia and with a worse prognosis in human AML. Finally, in human NPM1-mutant and in MLL-translocated AML, high expression of PRDM16, which negatively impacts outcome, was associated with inflammatory gene expression, thus corroborating the mouse data. Our observations demonstrate distinct roles for Prdm16 isoforms in normal HSCs and AML, and identify sPrdm16 as one of the drivers of prognostically adverse inflammation in leukemia.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Leucémica de la Expresión Génica , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Células Madre Hematopoyéticas/patología , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/patología , Nucleofosmina , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Factores de Transcripción/genéticaRESUMEN
Maintenance of stem cell plasticity is determined by the ability to balance opposing forces that control gene expression. Regulation of transcriptional networks, signaling cues and chromatin-modifying mechanisms constitute crucial determinants of tissue equilibrium. Histone modifications can affect chromatin compaction, therefore co-transcriptional events that influence their deposition determine the propensities toward quiescence, self-renewal, or cell specification. The Paf1 complex (Paf1C) is a critical regulator of RNA PolII elongation that controls gene expression and deposition of histone modifications, however few studies have focused on its role affecting stem cell fate decisions. Here we delineate the functions of Paf1C in pluripotency and characterize its impact in deposition of H2B ubiquitylation (H2BK120-ub) and H3K79 methylation (H3K79me), 2 fundamental histone marks that shape transcriptional regulation. We identify that H2BK120-ub is increased in the absence of Paf1C on its embryonic stem cell targets, in sharp contrast to H3K79me, suggesting opposite functions in the maintenance of self-renewal. Furthermore, we found that core pluripotency genes are characterized by a dual gain of H2BK120-ub and loss of H3K79me on their gene bodies. Our findings elucidate molecular mechanisms of cellular adaptation and reveal novel functions of Paf1C in the regulation of the self-renewal network.
Asunto(s)
Proteínas Portadoras/metabolismo , Histonas/metabolismo , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Autorrenovación de las Células , Cromatina/metabolismo , Proteínas de Unión al ADN , Metilación , Ratones , Células Madre Embrionarias de Ratones , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN , Transactivadores , UbiquitinaciónRESUMEN
Pluripotent embryonic stem cells (ESCs) self-renew or differentiate into all tissues of the developing embryo and cell-specification factors are necessary to balance gene expression. Here we delineate the function of the PHD-finger protein 5a (Phf5a) in ESC self-renewal and ascribe its role in regulating pluripotency, cellular reprogramming and myoblast specification. We demonstrate that Phf5a is essential for maintaining pluripotency, since depleted ESCs exhibit hallmarks of differentiation. Mechanistically, we attribute Phf5a function to the stabilization of the Paf1 transcriptional complex and control of RNA polymerase II elongation on pluripotency loci. Apart from an ESC-specific factor, we demonstrate that Phf5a controls differentiation of adult myoblasts. Our findings suggest a potent mode of regulation by Phf5a in stem cells, which directs their transcriptional programme, ultimately regulating maintenance of pluripotency and cellular reprogramming.
Asunto(s)
Proteínas Portadoras/genética , Diferenciación Celular/genética , Reprogramación Celular/genética , Células Madre Embrionarias de Ratones/citología , Mioblastos/citología , Células Madre Pluripotentes/citología , Transcripción Genética , Envejecimiento , Animales , Línea Celular , Proliferación Celular/genética , Proteínas de Unión al ADN , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al ARN , TransactivadoresRESUMEN
Little is known on post-transcriptional regulation of adult and embryonic stem cell maintenance and differentiation. Here we characterize the role of Ddb1, a component of the CUL4-DDB1 ubiquitin ligase complex. Ddb1 is highly expressed in multipotent hematopoietic progenitors and its deletion leads to abrogation of both adult and fetal hematopoiesis, targeting specifically transiently amplifying progenitor subsets. However, Ddb1 deletion in non-dividing lymphocytes has no discernible phenotypes. Ddb1 silencing activates Trp53 pathway and leads to significant effects on cell cycle progression and rapid apoptosis. The abrogation of hematopoietic progenitor cells can be partially rescued by simultaneous deletion of Trp53. Conversely, depletion of DDB1 in embryonic stem cell (ESC) leads to differentiation albeit negative effects on cell cycle and apoptosis. Mass spectrometry reveals differing protein interactions between DDB1 and distinct DCAFs, the substrate recognizing components of the E3 complex, between cell types. Our studies identify CUL4-DDB1 complex as a novel post-translational regulator of stem and progenitor maintenance and differentiation.
Asunto(s)
Diferenciación Celular , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/fisiología , Animales , Proteínas de Unión al ADN/genética , Eliminación de Gen , Regulación de la Expresión Génica , Silenciador del Gen , Homeostasis , Ratones , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Protein degradation plays key roles in diverse pathways in cell division, growth and differentiation. Aberrant stabilization of crucial proteins participating in oncogenic pathways is often observed in cancer. The importance of proper protein turnover is exemplified by the SCF(Fbxw7) ubiquitin ligase, which is frequently mutated in human cancer, including T cell acute lymphoblastic leukemia. Recent studies have revealed novel substrates of Fbxw7 and shed light on its role on differentiation of stem cells and expansion of stem-cell-like cells driving tumorigenesis. Detailed understanding of the contribution of the Fbxw7-regulated network of proteins in initiation and progression of cancer will facilitate the identification of candidate intervention targets in human cancer.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Leucemia/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Diferenciación Celular , Reprogramación Celular , Proteína 7 que Contiene Repeticiones F-Box-WD , Humanos , Células Madre/enzimología , Ubiquitina/metabolismoRESUMEN
T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy with a dismal overall prognosis, including a relapse rate of up to 25%, mainly because of the lack of non-cytotoxic targeted therapy options. Drugs that target the function of key epigenetic factors have been approved in the context of haematopoietic disorders, and mutations that affect chromatin modulators in a variety of leukaemias have recently been identified; however, 'epigenetic' drugs are not currently used for T-ALL treatment. Recently, we described that the polycomb repressive complex 2 (PRC2) has a tumour-suppressor role in T-ALL. Here we delineated the role of the histone 3 lysine 27 (H3K27) demethylases JMJD3 and UTX in T-ALL. We show that JMJD3 is essential for the initiation and maintenance of T-ALL, as it controls important oncogenic gene targets by modulating H3K27 methylation. By contrast, we found that UTX functions as a tumour suppressor and is frequently genetically inactivated in T-ALL. Moreover, we demonstrated that the small molecule inhibitor GSKJ4 (ref. 5) affects T-ALL growth, by targeting JMJD3 activity. These findings show that two proteins with a similar enzymatic function can have opposing roles in the context of the same disease, paving the way for treating haematopoietic malignancies with a new category of epigenetic inhibitors.
Asunto(s)
Histona Demetilasas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Animales , Benzazepinas/farmacología , Epigénesis Genética/efectos de los fármacos , Histona Demetilasas/genética , Histonas/química , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Lisina/metabolismo , Metilación/efectos de los fármacos , Ratones , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Pirimidinas/farmacología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Tissue homeostasis depends largely on the ability to replenish impaired or aged cells. Thus, tissue-resident stem cells need to provide functional progeny throughout the lifetime of an organism. Significant work in the past years has characterized how stem cells integrate signals from their environment to shape regulatory transcriptional networks and chromatin-regulating factors that control stem cell differentiation or maintenance. There is increasing interest in how post-translational modifications, and specifically ubiquitylation, control these crucial decisions. Ubiquitylation modulates the stability and function of important factors that regulate key processes in stem cell behavior. In this review, we analyze the role of ubiquitylation in embryonic stem cells and different adult multipotent stem cell systems and discuss the underlying mechanisms that control the balance between quiescence, self-renewal, and differentiation. We also discuss deregulated processes of ubiquitin-mediated protein degradation that lead to the development of tumor-initiating cells.
Asunto(s)
Células Madre/fisiología , Ubiquitinación , Animales , Diferenciación Celular , División Celular , Cromatina/fisiología , Epigénesis Genética , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Células-Madre Neurales , Proteolisis , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
Protein-protein interactions are typically identified by either biochemical purification coupled to mass spectrometry or genetic approaches exemplified by the yeast two-hybrid assay; however, neither assay works well for the identification of cofactors for poorly soluble proteins. Solubility of a poorly soluble protein is thought to increase upon cofactor binding, possibly by masking otherwise exposed hydrophobic domains. We have exploited this notion to develop a high-throughput genetic screen to identify interacting partners of an insoluble protein fused to chloramphenicol acetyltransferase by monitoring the survival of bacteria in the presence of a drug. In addition to presenting proof-of-principle experiments, we apply this screen to activation-induced cytidine deaminase (AID), a poorly soluble protein that is essential for antibody diversification. We identify a unique cofactor, RING finger protein 126 (RNF126), verify its interaction by traditional techniques, and show that it has functional consequences as RNF126 is able to ubiquitylate AID. Our results underpin the value of this screening technique and suggest a unique form of AID regulation involving RNF126 and ubiquitylation.
Asunto(s)
Citidina Desaminasa/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos B/metabolismo , Secuencia Conservada , Citidina Desaminasa/química , Citidina Desaminasa/deficiencia , Citidina Desaminasa/genética , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Dominios RING Finger , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Solubilidad , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
Although transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for posttranslational modifications in these processes. A key mechanism of posttranslational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here, using shotgun proteomics, we map the ubiquitinated protein landscape during embryonic stem cell (ESC) differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify additional regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7, and characterize their importance in ESC pluripotency and cellular reprogramming. This global characterization of the UPS as a key regulator of stem cell pluripotency opens the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates.
Asunto(s)
Reprogramación Celular/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Proliferación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas F-Box/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Semivida , Ratones , Estabilidad Proteica , Proteolisis , Proteoma/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/metabolismo , Transactivadores/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
D-type cyclins form complexes with cyclin-dependent kinases (CDK4/6) and promote cell cycle progression. Although cyclin D functions appear largely tissue specific, we demonstrate that cyclin D3 has unique functions in lymphocyte development and cannot be replaced by cyclin D2, which is also expressed during blood differentiation. We show that only combined deletion of p27(Kip1) and retinoblastoma tumor suppressor (Rb) is sufficient to rescue the development of Ccnd3(-/-) thymocytes. Furthermore, we show that a small molecule targeting the kinase function of cyclin D3:CDK4/6 inhibits both cell cycle entry in human T cell acute lymphoblastic leukemia (T-ALL) and disease progression in animal models of T-ALL. These studies identify unique functions for cyclin D3:CDK4/6 complexes and suggest potential therapeutic protocols for this devastating blood tumor.
Asunto(s)
Ciclina D3/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Animales , Ciclina D2/fisiología , Ciclina D3/fisiología , Quinasa 4 Dependiente de la Ciclina/fisiología , Quinasa 6 Dependiente de la Ciclina/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/fisiología , Humanos , Linfocitos/fisiología , Ratones , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiología , Receptor Notch1/fisiología , Proteína de Retinoblastoma/fisiologíaRESUMEN
Editing of adenosine (A) to inosine (I) at the first anticodon position in tRNA is catalyzed by adenosine deaminases acting on tRNA (ADATs). This essential reaction in bacteria and eukarya permits a single tRNA to decode multiple codons. Bacterial ADATa is a homodimer with two bound essential Zn(2+). The ADATa crystal structure revealed residues important for substrate binding and catalysis; however, such high resolution structural information is not available for eukaryotic tRNA deaminases. Despite significant sequence similarity among deaminases, we continue to uncover unexpected functional differences between Trypanosoma brucei ADAT2/3 (TbADAT2/3) and its bacterial counterpart. Previously, we demonstrated that TbADAT2/3 is unique in catalyzing two different deamination reactions. Here we show by kinetic analyses and inductively coupled plasma emission spectrometry that wild type TbADAT2/3 coordinates two Zn(2+) per heterodimer, but unlike any other tRNA deaminase, mutation of one of the key Zn(2+)-coordinating cysteines in TbADAT2 yields a functional enzyme with a single-bound zinc. These data suggest that, at least, TbADAT3 may play a role in catalysis via direct coordination of the catalytic Zn(2+). These observations raise the possibility of an unusual Zn(2+) coordination interface with important implications for the function and evolution of editing deaminases.