RESUMEN
The inhibitor of DNA binding and cell differentiation 2 (Id2) is a helix-loop-helix (HLH) protein that acts as negative dominant regulator of basic-HLH transcription factors during development and in cancer. The structural properties of Id2 have been investigated so far by using synthetic or recombinant fragments reproducing single domains (N-terminus, HLH, C-terminus): the HLH domain tends to dimerize into a four-helix bundle, whereas the flanking regions are flexible. In this work, the intact protein was expressed in E. coli, solubilized from inclusion bodies with urea, purified and dissolved in water at pH~4. Under these conditions, Id2 was obtained with both cysteine residues disulfide-bonded to ß-mercaptoethanol that was present during the solubilization process. Moreover, it existed in a self-assembled state, in which the N-terminus remained highly flexible, while the HLH domain and, surprisingly, part of the C-terminus, which corresponds to the nuclear export signal (NES), both were involved in slowly tumbling, rigid structures. The protein oligomers also formed twisted fibrils that were several micrometers long and up to 80 nm thick. These results show that self-assembly decreases the backbone flexibility of those two protein regions (HLH and NES) that are important for interaction with basic-HLH transcription factors or for nucleocytoplasmic shuttling.
Asunto(s)
Escherichia coli/crecimiento & desarrollo , Proteína 2 Inhibidora de la Diferenciación/química , Proteína 2 Inhibidora de la Diferenciación/genética , Transporte Activo de Núcleo Celular , Dicroismo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Secuencias Hélice-Asa-Hélice , Humanos , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Modelos Moleculares , Señales de Exportación Nuclear , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Inhibitor of DNA binding proteins (Id1-Id4) function to inhibit differentiation and promote proliferation of many different cell types. Among the Id family members, Id2 has been most extensively studied in the central nervous system (CNS). Id2 contributes to cultured neural precursor cell (NPC) proliferation as well as to the proliferation of CNS tumors such as glioblastoma that are likely to arise from NPC-like cells. We identified three phosphorylation sites near the N-terminus of Id2 in NPCs. To interrogate the importance of Id2 phosphorylation, Id2(-/-) NPCs were modified to express wild type (WT) Id2 or an Id2 mutant protein that could not be phosphorylated at the identified sites. We observed that NPCs expressing this mutant lacking phosphorylation near the N-terminus had higher steady-state levels of Id2 when compared to NPCs expressing WT Id2. This elevated level was the result of a longer half-life and reduced proteasome-mediated degradation. Moreover, NPCs expressing constitutively de-phosphorylated Id2 proliferated more rapidly than NPCs expressing WT Id2, a finding consistent with the well-characterized function of Id2 in driving proliferation. Observing that phosphorylation of Id2 modulates the degradation of this important cell-cycle regulator, we sought to identify a phosphatase that would stabilize Id2 enhancing its activity in NPCs and extended our analysis to include human glioblastoma-derived stem cells (GSCs). We found that expression of the phosphatase PP2A altered Id2 levels. Our findings suggest that inhibition of PP2A may be a novel strategy to regulate the proliferation of normal NPCs and malignant GSCs by decreasing Id2 levels. Stem Cells 2016;34:1321-1331.
Asunto(s)
Proteína 2 Inhibidora de la Diferenciación/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Proteolisis , Secuencia de Aminoácidos , Animales , Ciclo Celular , Proliferación Celular , Glioblastoma/patología , Proteína 2 Inhibidora de la Diferenciación/química , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Fosfatasa 2/metabolismoRESUMEN
FANCD2 and FANCI function together in the Fanconi anemia network of deoxyribonucleic acid (DNA) crosslink repair. These proteins form the dimeric ID2 complex that binds DNA and becomes monoubiquitinated upon exposure of cells to DNA crosslinking agents. The monoubiquitinated ID2 complex is thought to facilitate DNA repair via recruitment of specific nucleases, translesion DNA polymerases and the homologous recombination machinery. Using the ubiquitin conjugating enzyme (E2) UBE2T and ubiquitin ligase (E3) FANCL, monoubiquitination of human FANCD2 and FANCI was examined. The ID2 complex is a poor substrate for monoubiquitination, consistent with the published crystal structure showing the solvent inaccessibility of the target lysines. Importantly, FANCD2 monoubiquitination within the ID2 complex is strongly stimulated by duplex or branched DNA, but unstructured single-stranded DNA or chromatinized DNA is ineffective. Interaction of FANCL with the ID2 complex is indispensable for its E3 ligase efficacy. Interestingly, mutations in FANCI that impair its DNA binding activity compromise DNA-stimulated FANCD2 monoubiquitination. Moreover, we demonstrate that in the absence of FANCD2, DNA also stimulates FANCI monoubiquitination, but in a FANCL-independent manner. These results implicate the role of a proper DNA ligand in FANCD2 and FANCI monoubiquitination, and reveal regulatory mechanisms that are dependent on protein-protein and protein-DNA interactions.
Asunto(s)
ADN Viral/química , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Ubiquitinación , Sustitución de Aminoácidos , Animales , ADN Circular/química , Proteína del Grupo de Complementación L de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Humanos , Proteína 2 Inhibidora de la Diferenciación/química , Nucleosomas/química , Plásmidos/química , Unión Proteica , Células Sf9 , Spodoptera , Especificidad por SustratoRESUMEN
The ID proteins are named for their role as inhibitors of DNA binding and differentiation. They contain a helix-loop-helix (HLH) domain but lack a basic DNA-binding domain. In complex with basic HLH (bHLH) transcription factors, gene expression is regulated by DNA-binding inactivation. Although the HLH domain is highly conserved and shares a similar topology, the IDs preferentially bind class I bHLH-group members such as E47 (TCF3) but not the class III bHLH member Myc. A structure of an ID protein could potentially shed light on its mechanism. Owing to their short half-lives in vivo and reported in vitro instability, this paper describes the strategies that went into expressing sufficient soluble and stable ID2 to finally obtain diffraction-quality crystals. A 2.1â Å resolution data set was collected from a crystal belonging to space group P3(1)21 with unit-cell parameters a=b=51.622, c=111.474â Å, α=ß=90, γ=120° that was obtained by hanging-drop vapour diffusion in a precipitant solution consisting of 0.1â M MES pH 6.5, 2.0â M potassium acetate. The solvent content was consistent with the presence of one or two molecules in the asymmetric unit.
Asunto(s)
Proteína 2 Inhibidora de la Diferenciación/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Unión Competitiva , Cromatografía de Afinidad , Clonación Molecular , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , ADN/química , Humanos , Proteína 2 Inhibidora de la Diferenciación/aislamiento & purificación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de ProteínaRESUMEN
Inhibitors of DNA binding and differentiation (ID) proteins, a dominant-negative group of helix-loop-helix (HLH) transcription regulators, are well-characterized key players in cellular fate determination during development in mammals as well as Drosophila. Although not oncogenes themselves, their upregulation by various oncogenic proteins (such as Ras, Myc) and their inhibitory effects on cell cycle proteins (such as pRb) hint at their possible roles in tumorigenesis. Furthermore, their potency as inhibitors of cellular differentiation, through their heterodimerization with subsequent inactivation of the ubiquitous E proteins, suggest possible novel roles in engineering induced pluripotent stem cells (iPSCs). We present the high-resolution 2.1Å crystal structure of ID2 (HLH domain), coupled with novel biochemical insights in the presence of a divalent ion, possibly calcium (Ca2+), in the loop of ID proteins, which appear to be crucial for the structure and activity of ID proteins. These new insights will pave the way for new rational drug designs, in addition to current synthetic peptide options, against this potent player in tumorigenesis as well as more efficient ways for stem cells reprogramming.
Asunto(s)
Cationes Bivalentes/química , Proteína 2 Inhibidora de la Diferenciación/química , Proteínas Inhibidoras de la Diferenciación/química , Proteínas de Neoplasias/química , Multimerización de Proteína , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Sitios de Unión/genética , Calcio/química , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Cristalografía por Rayos X , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/genética , Humanos , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Proteína MioD/química , Proteína MioD/genética , Proteína MioD/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Factor de Transcripción 3/química , Factor de Transcripción 3/genética , Factor de Transcripción 3/metabolismoRESUMEN
Inhibitor of differentiation protein-2 (Id2) is a dominant negative helix-loop-helix (HLH) protein, and a positive regulator of proliferation, in various cells. The N-terminal region of Id2 contains a consensus cdk2 phosphorylation sequence SPVR, which may be involved with the induction of apoptosis, at least in myeloid 32d.3 cells. However, the role of Id2 phosphorylation at serine 5 in skeletal muscle cells is unknown. The objective of this study was to determine if the phosphorylation of Id2 at serine 5 alters its cellular localization and its role in apoptosis in C2C12 myoblasts. Overexpression of wild type Id2 decreased MyoD protein expression, which corresponded to the increased binding of Id2 to basic HLH proteins E47 and E12. Bromodeoxyuridine incorporation was significantly decreased by the overexpression of phospho-ablated Id2 (S5A); conversely, overexpression of wild type Id2 increased cellular proliferation. The subcellular localization of Id2 and phospho-mimicking Id2 (S5D) were predominantly nuclear compared to S5A. The decreased nuclear localization of S5A corresponded to a decrease in cellular proliferation, and an increase in apoptosis. These data suggest that unphosphorylated Id2 is primarily localized in the cytosol, where it is growth suppressive and potentially pro-apoptotic. These results imply that reducing unphosphorylated Id2 may improve the pool of myoblasts available for differentiation by increasing proliferation and inhibiting apoptosis.
Asunto(s)
Apoptosis/fisiología , División Celular/fisiología , Proliferación Celular , Proteína 2 Inhibidora de la Diferenciación/fisiología , Mioblastos/citología , Animales , Secuencia de Bases , Bromodesoxiuridina/metabolismo , Cartilla de ADN , Electroporación , Citometría de Flujo , Inmunohistoquímica , Inmunoprecipitación , Proteína 2 Inhibidora de la Diferenciación/química , Proteína 2 Inhibidora de la Diferenciación/genética , Ratones , Mutagénesis Sitio-Dirigida , FosforilaciónRESUMEN
The Id proteins are negative regulators of several basic-helix-loop-helix (HLH) transcription factors, including the ubiquitous E factors and the tissue-specific myogenin-regulating factors. Id1 through Id4 contain highly identical HLH domains but different N- and C-terminal extensions. Beside the heterodimerization with the parent HLH factors, Id2 was shown to additionally interact with the retinoblastoma protein and to be overexpressed in neuroblastoma. Thus, Id2 represents an interesting target for cancer therapy based on the inhibition of protein-protein interactions. Here we present the synthesis and circular dichroism (CD) analysis of peptides derived from point mutations and N-/C-terminal truncations of Id2. The helix character of the HLH domain (residues 36-76) was reduced upon substitution of Met39/-62 and Cys42 with Nle and Ser, respectively, suggesting a structural role of these side chains. The largest sequence that could be obtained by stepwise solid-phase peptide synthesis (SPPS) with Fmoc strategy spanned the entire HLH motif (with Cys42 replaced by Ser) and part of the C-terminus (residues 77-110). This 75-residue long fragment was less helical than the isolated HLH domain and had propensity to aggregate, which was correlated with the presence of the flanking residues C-terminal to helix-2. By CD analysis of an equimolar mixture of the sequence 36-110 with the N-terminus 1-35, noncovalent interactions between the two peptides were detected, which, however, changed upon aging. In contrast, the mixture of the HLH sequence 36-76 with the N-terminus was characterized by a stabilized helix structure that was maintained also upon aging. Presumably, the N-terminal region interacted with the folded HLH motif in a specific manner, whereas only unspecific, weak contacts occurred with the partly unfolded HLH domain and/or the immediate flanking residues 77-110.
Asunto(s)
Secuencias Hélice-Asa-Hélice , Proteína 2 Inhibidora de la Diferenciación/síntesis química , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Cromatografía Líquida de Alta Presión/métodos , Dicroismo Circular/métodos , Proteína 2 Inhibidora de la Diferenciación/química , Proteína 2 Inhibidora de la Diferenciación/genética , Mutación Puntual/genética , Conformación ProteicaRESUMEN
Id2 is a natural inhibitor of the basic helix-loop-helix transcription factors and the retinoblastoma tumor suppressor protein. Active Id2 prevents differentiation and promotes cell-cycle progression and tumorigenesis in the nervous system. A key event that regulates Id2 activity during differentiation is translocation from the nucleus to the cytoplasm. Here we show that the actin-associated protein enigma homolog (ENH) is a cytoplasmic retention factor for Id2. ENH contains three LIM domains, which bind to the helix-loop-helix domain of Id proteins in vitro and in vivo. ENH is up-regulated during neural differentiation, and its ectopic expression in neuroblastoma cells leads to translocation of Id2 from the nucleus to the cytoplasm, with consequent inactivation of transcriptional and cell-cycle-promoting functions of Id2. Conversely, silencing of ENH by RNA interference prevents cytoplasmic relocation of Id2 in neuroblastoma cells differentiated with retinoic acid. Finally, the differentiated neural crest-derived tumor ganglioneuroblastoma coexpresses Id2 and ENH in the cytoplasm of ganglionic cells. These data indicate that ENH contributes to differentiation of the nervous system through cytoplasmic sequestration of Id2. They also suggest that ENH is a restraining factor of the oncogenic activity of Id proteins in neural tumors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Citoplasma/metabolismo , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Proteínas de Microfilamentos/metabolismo , Sistema Nervioso/citología , Sistema Nervioso/metabolismo , Neuroblastoma/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Chlorocebus aethiops , Secuencias Hélice-Asa-Hélice , Humanos , Proteína 2 Inhibidora de la Diferenciación/química , Proteína 2 Inhibidora de la Diferenciación/genética , Proteínas con Dominio LIM , Ratones , Proteínas de Microfilamentos/genética , Sistema Nervioso/química , Sistema Nervioso/patología , Neuroblastoma/genética , Neuroblastoma/patología , Unión Proteica , Transporte de Proteínas , ARN Interferente PequeñoRESUMEN
Id proteins are dominant negative regulators of the helix-loop-helix (HLH) transcription factors and are important during development, especially by preventing cell differentiation while inducing cell proliferation. In contrast, they are poorly expressed in healthy adults but are found in several tumor types. The Id HLH motif is responsible for the inhibitory activity, whereas not much is known about the role of the N- and C-termini. In the presented work, synthetic peptides reproducing the HLH, the N-terminal region, and the C-terminal region of the Id proteins were characterized by CD. The four HLH sequences built highly stable helical conformations, whereas the N- and C-termini were unstructured, with the exception of an alanine-rich fragment preceding the Id4 HLH motif. Deletion of the loop connecting the two helices led to helix destabilization for all four Id HLH peptides. In addition, modifications of the amino acid composition within the hydrophobic face of the helices of the Id1 HLH peptide induced conformational changes, mostly associated with loss of helix content. Moreover, a fragment containing the helix-2 and the C-terminus of the Id1 protein did not show any helical character. Therefore, both the helix propensity and stability of the HLH domain were shown to be strongly dependent on favorable interhelical contacts. In contrast, it is suggested that the regions beyond this domain could rather play a destabilizing role, for example, by increasing the flexibility of the folded protein.