RESUMO
The inhibitors of DNA binding Id1-4 are helix-loop-helix (HLH) proteins that exert their biological function by interacting with members of the basic-HLH (bHLH) transcription-factor family. The HLH domains of the Id and bHLH proteins allow both self- and hetero-association. Due to their abnormal expression in cancer cells, the Id proteins are potential protein targets for cancer treatment. Suitable Id-protein inactivators should promote self-association and/or prevent hetero-association. In this work we evaluated the ability of the Id-protein HLH domain to recognize itself in form of short sequences extracted from the helical and loop regions. We performed a peptide scan of the Id1 HLH domain 64-106 based on three-residue overlapping octapeptides. Interaction of each octapeptide with the natively folded Id1 HLH domain was investigated by CD and fluorescence spectroscopy. The results from both techniques showed that the helix-based but not the loop-based octapeptides interacted with the Id1 HLH domain in the low-micromolar range. In contrast, a nitrotyrosine-containing analog of the Id1 HLH region, which was unable to reproduce the native-like conformation, quenched only the 2-amino-benzoyl-(Abz)-labeled loop-based octapeptides. This opposite self-recognition pattern suggests that the short helix-based and loop-based sequences should be able to distinguish different folding states of the Id1 HLH domain. This feature may be biologically relevant, as the Id proteins are predicted to behave as intrinsically disordered proteins, being in equilibrium between rapidly exchanging monomeric conformations and structurally better-defined homo-/heterodimers displaying the parallel four-helix bundle.
Assuntos
Sequências Hélice-Alça-Hélice , Proteína 1 Inibidora de Diferenciação/antagonistas & inibidores , Oligopeptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Ensaios de Triagem em Larga Escala , Humanos , Proteína 1 Inibidora de Diferenciação/química , Cinética , Ligantes , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Ligação Proteica , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Coloração e Rotulagem , Tirosina/análogos & derivados , Tirosina/química , ortoaminobenzoatos/químicaRESUMO
Inhibitor of differentiation or DNA binding-1 (ID1) is an important helix-loop-helix (HLH) transcription factor involved in diverse biological functions including cell differentiation, proliferation, apoptosis, and senescence. Recently, it was reported that ID1 can activate the NF-κB signaling pathway in a variety of cancer cells and a T cell line, but the mechanisms involved in ID1-mediated transactivation of NF-κB are not clear. In this study, we demonstrate by both in vitro pull-down assays and a cell-based in vivo two-hybrid system that ID1-mediated NF-κB activation is due to its physical interaction with p65. We have identified that the transcriptional activation domain (TAD) in p65 and the HLH domain in ID1 are vital for their interaction. Interestingly, a single site mutation (Leu76) in the HLH domain of ID1 protein drastically decreased its ability to bind with p65. Using a dual-luciferase assay, we demonstrated that the interaction between ID1 and p65 modulates activation of the NF-κB signaling pathway in vivo. In addition, we demonstrated that, by affecting the nuclear translocation of p65, ID1 is essential in regulating TNF-α-induced p65 recruitment to its downstream target, the cellular inhibitor of apoptosis protein 2 (cIAP2) promoter.
Assuntos
Proteína 1 Inibidora de Diferenciação/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Sequência de Aminoácidos , Apoptose/fisiologia , Proteína 3 com Repetições IAP de Baculovírus , Linhagem Celular Tumoral , Regulação da Expressão Gênica/fisiologia , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Proteína 1 Inibidora de Diferenciação/química , Proteína 1 Inibidora de Diferenciação/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas/fisiologia , Fator de Transcrição RelA/química , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína LigasesRESUMO
Synthetic peptides reproducing the helix-loop-helix (HLH) domains of the Id proteins fold into highly stable helix bundles upon self-association. Recently, we have shown that the replacement of the dipeptide Val-Ser at the loop-helix-2 junction with the corresponding O-acyl iso-dipeptide leads to a completely unfolded state that only refolds after intramolecular O --> N acyl migration. Herein, we report on an Id HLH analog based on the substitution of the Pro-Ser motif at the helix-1-loop junction with the corresponding O-acyl iso-dipeptide. This analog has been successfully synthesized by solid-phase Fmoc chemistry upon suppression of DKP formation. No secondary structure could be detected for the O-acyl iso-peptide before its conversion into the native form by O --> N acyl shift. These results show that the loop-helix junctions are determinant for the folded/unfolded state of the Id HLH domain. Further, despite the high risk of DKP formation, peptides containing O-acyl iso-Pro-Ser/Thr units are synthetically accessible by Fmoc chemistry.
Assuntos
Sequências Hélice-Alça-Hélice , Proteína 1 Inibidora de Diferenciação/química , Proteína 1 Inibidora de Diferenciação/genética , Peptídeos/química , Peptídeos/síntese química , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos/genética , Dobramento de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
MyoD is a DNA-binding protein capable of specific interactions that involve the helix-loop-helix (HLH) domain. The HLH motif of MyoD can form oligomers with the HLH motif of Id1 (the inhibitor of DNA-binding proteins) that folds into a highly stable helical conformation stabilized by the self-association. The Id family consists of four related proteins that contain a highly conserved dimerization motif known as the HLH domain. In signaling pathways, Id proteins act as dominant negative antagonists of the basic helix-loop-helix (bHLH) family of transcription factors which play important roles in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize bHLH proteins by binding as dominant negative HLH proteins to form high-affinity heterodimers with other bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of differentiation-associated genes. The goal of this study is to design and synthesize peptide fragments of MyoD with high affinity for Id1 to interrupt the interactions among Id1, MyoD, and other bHLH DNA-binding proteins and to inhibit the proliferation of cancer cells. Affinity of each peptide for Id1 was determined by surface plasmon resonance (SPR) technology. The secondary structure of each peptide was studied by circular dichroism (CD) spectroscopy. Biological effects of each peptide in several cancer cells such as breast and colon cancer cells were analyzed. Results demonstrated that the peptide 3C (H-Tyr-Ile-Glu-Gly-Leu-Gln-Ala-Leu-Leu-Arg-Asp-Gln-NH(2)) not only showed high affinity for Id1 but also exhibited antiproliferative effects in HT-29 and MCF-7 cancer cells; the IC(50) value of 3C was determined as 25 microM in both cells. The percentage of sub-G1 in the cell cycle of the cancer cells treated with 5 microM of 3C was increased, indicating the induced apoptosis of cancer cells by 3C. Taken together, the peptide 3C is a promising lead compound for the development of antiproliferative agents.
Assuntos
Proteína 1 Inibidora de Diferenciação/química , Proteína MyoD/química , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Citometria de Fluxo , Células HT29 , Humanos , Fragmentos de Peptídeos/químicaRESUMO
Id proteins play important roles in cellular differentiation and proliferation by negatively regulating basic helix-loop-helix transcription factors. Although their intracellular localization may change depending on the biological situation, little is known about the molecular determinants underlying such changes. Here we report the identification of a nuclear export signal (NES) in Id1. The identified NES was different from that of Id2, but had the ability to confine heterologous green fluorescent protein to the cytoplasm. Thus, our results indicate that the intracellular localization of Id1 is regulated differently from that of Id2.
Assuntos
Proteína 1 Inibidora de Diferenciação/química , Sinais de Exportação Nuclear , Sequência de Aminoácidos , Animais , Citoplasma/química , Proteínas de Fluorescência Verde , Proteína 2 Inibidora de Diferenciação , Camundongos , Células NIH 3T3 , TransfecçãoRESUMO
The family of inhibitor of differentiation (Id) proteins is a group of evolutionarily conserved molecules, which play important regulatory roles in organisms ranging from Drosophila to humans. Id proteins are small polypeptides harboring a helix-loop-helix (HLH) motif, which are best known to mediate dimerization with other basic HLH proteins, primarily E proteins. Because Id proteins do not possess the basic amino acids adjacent to the HLH motif necessary for DNA binding, Id proteins inhibit the function of E protein homodimers, as well as heterodimers between E proteins and tissue-specific bHLH proteins. However, Id proteins have also been shown to have E protein-independent functions. The Id genes are broadly but differentially expressed in a variety of cell types. Transcription of the Id genes is controlled by transcription factors such as C/EBPß and Egr as well as by signaling pathways triggered by different stimuli, which include bone morphogenic proteins, cytokines, and ligands of T cell receptors. In general, Id proteins are capable of inhibiting the differentiation of progenitors of different cell types, promoting cell-cycle progression, delaying cellular senescence, and facilitating cell migration. These properties of Id proteins enable them to play significant roles in stem cell maintenance, vasculogenesis, tumorigenesis and metastasis, the development of the immune system, and energy metabolism. In this review, we intend to highlight the current understanding of the function of Id proteins and discuss gaps in our knowledge about the mechanisms whereby Id proteins exert their diverse effects in multiple cellular processes.
Assuntos
Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Adipogenia/fisiologia , Sequência de Aminoácidos , Animais , Regulação da Expressão Gênica , Humanos , Sistema Imunitário/metabolismo , Proteína 1 Inibidora de Diferenciação/química , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Proteínas Inibidoras de Diferenciação/química , Dados de Sequência Molecular , Neoplasias/metabolismo , Células-Tronco/fisiologiaRESUMO
Among the many abnormally expressed proteins in ovarian cancer, the prominent cancer in women, ID1 (inhibitors of DNA binding protein 1) is a potential one among other several targets. Interaction of ID1 with ETS-1 (transcriptional activator of p16(INK4a)) suppresses the transcription of p16(INK4a) and causes abnormal cell proliferation. A peptide aptamer (ID1/3-PA7) has been designed to prevent this interaction and thereby leading to the transcription of p16(INK4a). However, the structural basis behind the molecular interaction of ID1 with ETS-1 (agonist) and ID1/3-PA7 (antagonist) is poorly understood. In order to understand this structural recognition and their interaction mechanism, in silico methods were used. From this interaction analysis, the residues of ETS-1 involved in interaction with the p16(INK4a) promoter were found to be targeted by ID1. Subsequently, ETS-1 binding residues of ID1 were found to be targeted by its aptamer- ID1/3-PA7. These results suggest that both ETS-1 and ID1/3-PA7 binds at the same region harbored by the residues-H97, D100, R103, D104, L107, A144, C145, D149, D150 and C154 of ID1. All these observations correlate with the experimental reports, suggesting that the identified residues might play a crucial role in promulgating the oncogenic effects of ID1. In silico alanine scanning mutagenesis also confirms the role of identified hot spot residues in p16(INK4a) regulation. Finally, the molecular dynamic simulation studies reveal the prolonged stability of the aforementioned interacting complexes. The obtained results throw light on the structure and residues of ID1 involved in transcriptional regulation of p16(INK4a).
Assuntos
Aptâmeros de Peptídeos/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteína 1 Inibidora de Diferenciação/antagonistas & inibidores , Proteína 1 Inibidora de Diferenciação/química , Proteína Proto-Oncogênica c-ets-1/metabolismo , Aptâmeros de Peptídeos/farmacologia , Sítios de Ligação , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Desenho de Fármacos , Feminino , Humanos , Proteína 1 Inibidora de Diferenciação/agonistas , Proteína 1 Inibidora de Diferenciação/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neoplasias Ovarianas/tratamento farmacológico , Ligação Proteica , Estrutura Terciária de Proteína , Proteína Proto-Oncogênica c-ets-1/química , Transcrição GênicaRESUMO
The inhibitors of DNA binding and cell differentiation Id1-4 are helix-loop-helix (HLH) proteins that negatively regulate DNA transcription by forming inactive dimers with ubiquitous and tissue-specific bHLH proteins, including E47 and MyoD, respectively. Their highly conserved HLH domains are essential for heterodimerization, but can also self-associate to highly stable, alpha-helix-rich structures at low micromolar peptide concentrations. Here, we show that the introduction of an O-acyl isodipeptide unit involving the putative N-cap serine residue of the C-terminal helix completely abrogates the propensity of the Id HLH analogue for any secondary and tertiary structure, resulting in a random coil, as shown by CD measurements in nonbuffered aqueous solutions. However, the HLH fold reappears as soon as an O-->N intramolecular acyl migration, which occurs spontaneously under physiological conditions, restores the native N-cap serine residue. These results show that changes addressing the N-terminus of the C-terminal helix can dramatically influence the HLH structure, and suggest that local interactions at the junction between the loop and the C-terminal helix might be crucial during the HLH folding process. Furthermore, the present study contributes to the evaluation of the O-acyl isodipeptide unit as a powerful tool to introduce a conformational switch into peptides.
Assuntos
Peptídeos/química , Sequência de Aminoácidos , Química/métodos , Dimerização , Sequências Hélice-Alça-Hélice , Humanos , Concentração de Íons de Hidrogênio , Proteína 1 Inibidora de Diferenciação/química , Modelos Químicos , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Solubilidade , Ativação TranscricionalRESUMO
Id-1 is a member of the HLH protein family that regulates a wide range of cellular processes such as cell proliferation, apoptosis, senescence and overexpression of Id-1 was recently suggested to play roles in the development and progression of different cancers. Previously, Id-1 was shown to physically interact with the viral protein E1A. Meanwhile, Id-1 expression was found to be regulated by several of the virus-encoded proteins, suggesting that Id-1 may be a common cellular target of the viral proteins. Here, we report that Id-1 interacts with the Hepatitis-B virus (HBV)-encoded protein HBX and regulates its stability in hepatocellular carcinoma (HCC) cells. We found that in HCC cells, ectopic Id-1 expression significantly decreased the half-life of the HBX protein, indicating that HBX is destabilized by Id-1. Meanwhile, the Id-1-induced HBX degradation was found to be inhibited by treatment with proteasome inhibitor, suggesting that this process is mediated through the proteasome pathway. Interestingly, while Id-1 did not induce HBX-ubiquitination, we found that removal of all the lysine residues of the HBX protein protects it from the effect of Id-1, indicating that ubiquitination is still required for the Id-1-mediated HBX degradation. Meanwhile, we found that Id-1 binds to the proteasome subunit C8 and facilitates its interaction with the HBX protein and disruption of this interaction completely abolishes the negative effect of Id-1 on HBX protein stability. Taken together, our results demonstrated a novel function of Id-1 in regulating HBX protein stability through interaction with the proteasome.
Assuntos
Proteína 1 Inibidora de Diferenciação/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Virais Reguladoras e Acessórias/metabolismo , Linhagem Celular Tumoral , Humanos , Proteína 1 Inibidora de Diferenciação/química , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/metabolismo , Termodinâmica , Transativadores , Ubiquitinação , Proteínas Virais Reguladoras e Acessórias/químicaRESUMO
The basic helix-loop-helix transcription factor, Id1, was shown to induce tetraploidy in telomerase-immortalized nasopharyngeal epithelial cells in this study. Using both transient and stable Id1-expressing cell models, multiple mitotic aberrations were detected, including centrosome amplification, binucleation, spindle defects, and microtubule perturbation. Many of these abnormal phenotypes have previously been reported in cells overexpressing Aurora A. Further experiments showed that Id1 could stabilize Aurora A, whereas knocking down Aurora A expression in Id1-expressing cells could rescue some of the mitotic defects. The mechanisms by which Aurora A could be modulated by Id1 were explored. DNA amplification of the Aurora A locus was not involved. Id1 could only weakly activate the transcriptional activity of the Aurora A promoter. We found that Id1 overexpression could affect Aurora A degradation, leading to its stabilization. Aurora A is normally degraded from mitosis exit by the APC/C(Cdh1)-mediated proteasomal proteolysis pathway. Our results revealed that Id1 and Cdh1 are binding partners. The association of Id1 and Cdh1 was found to be dependent on the canonical destruction box motif of Id1, the increased binding of which may compete with the interaction between Cdh1 and Aurora A, leading to stabilization of Aurora A in Id1-overexpressing cells.
Assuntos
Proteína 1 Inibidora de Diferenciação/metabolismo , Mitose , Poliploidia , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Aurora Quinases , Linhagem Celular , Polaridade Celular , Centríolos/enzimologia , Citocinese , Regulação para Baixo , Estabilidade Enzimática , Amplificação de Genes , Humanos , Proteína 1 Inibidora de Diferenciação/química , Proteína 1 Inibidora de Diferenciação/deficiência , Interfase , Microtúbulos/enzimologia , Mutação/genética , Neoplasias/enzimologia , Neoplasias/patologia , Fenótipo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático/enzimologia , Especificidade por Substrato , Ativação Transcricional , Complexos Ubiquitina-Proteína Ligase/metabolismo , Regulação para CimaRESUMO
Growth factors and peptides playing important roles during early development of the central nervous system have also been shown to maintain their regulation of cell genesis in the adult brain. We have previously described that endogenous opioids, expressed in the developing hippocampus, regulate proliferation and differentiation in the adult rat hippocampus. The aim of this study was to investigate the effects of the opioid beta-endorphin on gene expression and glial differentiation in cultures of adult rat hippocampal progenitors (AHPs). Changes in gene expression after stimulation of AHPs with beta-endorphin for 48 h were investigated using cDNA arrays. Confirmation experiments verified that stimulation with beta-endorphin increased the mRNA levels of myelin basic protein, glutathione S-transferase pi, c-junD and rab16 (P < 0.05), genes that are associated with oligodendrogenesis. Furthermore, beta-endorphin increased the levels of Id1, but not Id3, mRNA on the arrays. Incubation of AHPs with beta-endorphin resulted in a threefold increase in oligodendrogenesis (P < 0.01) but no significant change in astrogliogenesis. No effect on oligodendrogenesis was observed in the presence of the opioid antagonist naloxone. Coincubation of beta-endorphin with Id1 antisense oligonucleotides for 10 days also entirely blocked the induced oligodendrogenesis in our AHP cultures. Moreover, a subpopulation of AHPs (25%) showed nuclear expression of the proneural transcriptional activator Mash1 that was reduced to approximately 5% of the cells when exposed to beta-endorphin. We suggest a requirement for Id1 in opioid-induced oligodendrogenesis in cultured AHPs possibly acting on opioid-responsive AHPs expressing the proneural transcriptional activator Mash1.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Hipocampo/citologia , Proteína 1 Inibidora de Diferenciação/fisiologia , Oligodendroglia , Células-Tronco/efeitos dos fármacos , beta-Endorfina/farmacologia , Animais , Western Blotting/métodos , Contagem de Células/métodos , Células Cultivadas , Interações Medicamentosas , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica/efeitos dos fármacos , Glutationa S-Transferase pi/metabolismo , Imuno-Histoquímica/métodos , Proteína 1 Inibidora de Diferenciação/química , Proteína Básica da Mielina/metabolismo , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligorribonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
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.