RESUMO
Protein ubiquitination in eukaryotic cells is mediated by diverse E3 ligase enzymes that each target specific substrates. The cullin E3 ligase complexes are the most abundant class of E3 ligases; they contain various cullin components that serve as scaffolds for interaction with substrate-recruiting adaptor proteins. SPOP is a BTB-domain adaptor of the cullin-3 E3 ligase complexes; it selectively recruits substrates via its N-terminal MATH domain, whereas its BTB domain mediates dimerization and interactions with cullin-3. It has recently been recognized that the high-order oligomerization of SPOP enhances the ubiquitination of substrates. Here, a dimerization interface in the SPOP C-terminus is identified and it is shown that the dimerization interfaces of the BTB domain and of the C-terminus act independently and in tandem to generate high-order SPOP oligomers. The crystal structure of the dimeric SPOP C-terminal domain is reported at 1.5â Å resolution and it is shown that Tyr353 plays a critical role in high-order oligomerization. A model of the high-order SPOP oligomer is presented that depicts a helical organization that could enhance the efficiency of substrate ubiquitination.
Assuntos
Proteínas Culina/química , Proteínas Nucleares/química , Proteínas Repressoras/química , Ubiquitina-Proteína Ligases/química , Proteínas de Transporte/química , Cristalografia por Raios X , Proteínas Culina/genética , Proteínas Culina/ultraestrutura , Humanos , Microscopia de Força Atômica , Modelos Químicos , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestrutura , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética , Proteínas Repressoras/genética , Proteínas Repressoras/ultraestrutura , Especificidade por Substrato/genética , Ubiquitinação/genética , Regulação para Cima/genéticaRESUMO
Bach2 is a transcriptional repressor that is expressed during specific stages of B-cell development and in neuronal cells. It plays a critical role in modulating class-switch recombination during the differentiation of mature B cells to antibody-secreting plasma cells and it is also an important regulator of apoptotic responses to oxidative stress. Bach2 has been implicated both as an oncogene and as a tumour suppressor in human malignancy. The interaction of Bach2 with its target genes is mediated via its basic leucine-zipper region, whereas the N-terminal POZ domain recruits transcriptional co-repressors and class II histone deacetylases. Here, the crystal structure of the human Bach2 POZ domain is reported at 2.1 Å resolution. The Bach2 POZ-domain dimer resembles the POZ-domain dimers of the POZ zinc finger transcription factors and dimerization is independent of an N-terminal region that has previously been implicated in the dimerization of the POZ basic leucine-zipper protein Bach1. The Bach2 POZ domain crystallized in two forms which differed by the presence of an intersubunit disulfide bond. The intersubunit disulfide bond is present both in bacterially expressed Bach2 POZ domain in solution and in protein expressed in transfected eukaryotic cells. These crystal structures will be relevant for understanding the regulation of Bach2 in response to oxidative stress and for the design of therapeutics that target the Bach2 POZ domain in human malignancy.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/química , Dissulfetos/química , Multimerização Proteica , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células COS , Chlorocebus aethiops , Dissulfetos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Nac1 is a POZ-domain transcription factor that is involved in the self-renewal of embryonic stem cells. It is overexpressed in ovarian serous carcinoma and targeting the interactions of its POZ domain is a potential therapeutic strategy. Nac1 lacks a zinc-finger DNA-binding domain and thereby differs from most other POZ-domain transcription factors. Here, the crystal structure of the Nac1 POZ domain at 2.1 A resolution is reported. The Nac1 POZ domain crystallized as a dimer in which the interaction interfaces between subunits resemble those found in the POZ-zinc finger transcription factors. The organization of the Nac1 POZ-domain core resembles reported POZ-domain structures, whereas the C-terminus differs markedly. The C-terminal alpha-helix of the Nac1 POZ domain is shorter than that observed in most other POZ-domain transcription factors; variation in the organization of this region may be a general feature of POZ-domain structures.
Assuntos
Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Alinhamento de SequênciaRESUMO
The POZ/BTB domain is an evolutionarily conserved motif found in approximately 40 zinc-finger transcription factors (POZ-ZF factors). Several POZ-ZF factors are implicated in human cancer, and POZ domain interaction interfaces represent an attractive target for therapeutic intervention. Miz-1 (Myc-interacting zinc-finger protein) is a POZ-ZF factor that regulates DNA-damage-induced cell cycle arrest and plays an important role in human cancer by virtue of its interaction with the c-Myc and BCL6 oncogene products. The Miz-1 POZ domain mediates both self-association and the recruitment of non-POZ partners. POZ-ZF factors generally function as homodimers, although higher-order associations and heteromeric interactions are known to be physiologically important; crucially, the interaction interfaces in such large complexes have not been characterised. We report here the crystal structure of the Miz-1 POZ domain up to 2.1 A resolution. The tetrameric organisation of Miz-1 POZ reveals two types of interaction interface between subunits; an interface of alpha-helices resembles the dimerisation interface of reported POZ domain structures, whereas a novel beta-sheet interface directs the association of two POZ domain dimers. We show that the beta-sheet interface directs the tetramerisation of the Miz-1 POZ domain in solution and therefore represents a newly described candidate interface for the higher-order homo- and hetero-oligomerisation of POZ-ZF proteins in vivo.
Assuntos
Fatores de Transcrição Kruppel-Like/química , Dedos de Zinco , Sequência de Aminoácidos , Cristalografia por Raios X/métodos , Dimerização , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
BCL6 is a transcriptional repressor that is overexpressed in diffuse large B-cell lymphoma and follicular lymphoma. The N-terminal POZ domain of BCL6 interacts with transcriptional corepressors and targeting these associations is a promising therapeutic strategy. Previous structural studies of the BCL6 POZ domain have used a mutant form because of the low solubility of the wild-type recombinant protein. A method for the purification and crystallization of the wild-type BCL6 POZ domain is described and the crystal structure to 2.1 A resolution is reported. This will be relevant for the design of therapeutics that target BCL6 POZ-domain interaction interfaces.
Assuntos
Proteínas de Ligação a DNA/química , Estrutura Terciária de Proteína , Sítios de Ligação , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Nac1 (nucleus accumbens 1) is a POZ (poxvirus and zinc finger)-domain transcriptional repressor that is expressed at high levels in ovarian serous carcinoma. Here we identify Nac1 as a novel interacting partner of the POZ-domain transcriptional activator, Miz1 (Myc-interacting zinc-finger protein 1), and using chemical crosslinking we show that this association is mediated by a heterodimeric interaction of the Nac1 and Miz1 POZ domains. Nac1 is found in discrete bodies within the nucleus of mammalian cells, and we demonstrate the relocalization of Miz1 to these structures in transfected HeLa cells. We show that siRNA (small interfering RNA)-mediated knockdown of Nac1 in ovarian cancer cells results in increased levels of the Miz1 target gene product, p21Cip1. The interaction of Nac1 with Miz1 may thus be relevant to its mechanism of tumourigenesis in ovarian cancer.