HELZ directly interacts with CCR4-NOT and causes decay of bound mRNAs.

Eukaryotic superfamily (SF) 1 helicases have been implicated in various aspects of RNA metabolism, including transcription, processing, translation, and degradation. Nevertheless, until now, most human SF1 helicases remain poorly understood. Here, we have functionally and biochemically characterized the role of a putative SF1 helicase termed "helicase with zinc-finger," or HELZ. We discovered that HELZ associates with various mRNA decay factors, including components of the carbon catabolite repressor 4-negative on TATA box (CCR4-NOT) deadenylase complex in human and Drosophila melanogaster cells. The interaction between HELZ and the CCR4-NOT complex is direct and mediated by extended low-complexity regions in the C-terminal part of the protein. We further reveal that HELZ requires the deadenylase complex to mediate translational repression and decapping-dependent mRNA decay. Finally, transcriptome-wide analysis of Helz-null cells suggests that HELZ has a role in the regulation of the expression of genes associated with the development of the nervous system.

A conserved CAF40-binding motif in metazoan NOT4 mediates association with the CCR4-NOT complex.

The multisubunit CCR4-NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression. The NOT4 E3 ubiquitin ligase is a stable component of the CCR4-NOT complex in yeast but does not copurify with the human or Drosophila melanogaster complex. Here we show that the C-terminal regions of human and D. melanogaster NOT4 contain a conserved sequence motif that directly binds the CAF40 subunit of the CCR4-NOT complex (CAF40-binding motif [CBM]). In addition, nonconserved sequences flanking the CBM also contact other subunits of the complex. Crystal structures of the CBM-CAF40 complex reveal a mutually exclusive binding surface for NOT4 and Roquin or Bag of marbles mRNA regulatory proteins. Furthermore, CAF40 depletion or structure-guided mutagenesis to disrupt the NOT4-CAF40 interaction impairs the ability of NOT4 to elicit decay of tethered reporter mRNAs in cells. Together with additional sequence analyses, our results reveal the molecular basis for the association of metazoan NOT4 with the CCR4-NOT complex and show that it deviates substantially from yeast. They mark the NOT4 ubiquitin ligase as an ancient but nonconstitutive cofactor of the CCR4-NOT deadenylase with potential recruitment and/or effector functions.

The Ccr4-Not Complex: Architecture and Structural Insights.

The Ccr4-Not complex is an essential multi-subunit protein complex that plays a fundamental role in eukaryotic mRNA metabolism and has a multitude of different roles that impact eukaryotic gene expression . It has a conserved core of three Not proteins, the Ccr4 protein, and two Ccr4 associated factors, Caf1 and Caf40. A fourth Not protein, Not4, is conserved, but is only a stable subunit of the complex in yeast. Certain subunits have been duplicated during evolution, with functional divergence, such as Not3 in yeast, and Ccr4 or Caf1 in human. However the complex includes only one homolog for each protein. In addition, species-specific subunits are part of the complex, such as Caf130 in yeast or Not10 and Not11 in human. Two conserved catalytic functions are associated with the complex, deadenylation and ubiquitination . The complex adopts an L-shaped structure, in which different modules are bound to a large Not1 scaffold protein. In this chapter we will summarize our current knowledge of the architecture of the complex and of the structure of its constituents.

Structure of a Human 4E-T/DDX6/CNOT1 Complex Reveals the Different Interplay of DDX6-Binding Proteins with the CCR4-NOT Complex.

The DEAD-box protein DDX6 is a central component of translational repression mechanisms in maternal mRNA storage in oocytes and microRNA-mediated silencing in somatic cells. DDX6 interacts with the CCR4-NOT complex and functions in concert with several post-transcriptional regulators, including Edc3, Pat1, and 4E-T. We show that the conserved CUP-homology domain (CHD) of human 4E-T interacts directly with DDX6 in both the presence and absence of the central MIF4G domain of CNOT1. The 2.1-Å resolution structure of the corresponding ternary complex reveals how 4E-T CHD wraps around the RecA2 domain of DDX6 and contacts CNOT1. Although 4E-T CHD lacks recognizable sequence similarity with Edc3 or Pat1, it shares the same DDX6-binding surface. In contrast to 4E-T, however, the Edc3 and Pat1 FDF motifs dissociate from DDX6 upon CNOT1 MIF4G binding in vitro. The results underscore the presence of a complex network of simultaneous and/or mutually exclusive interactions in DDX6-mediated repression.

Insights into the binding modes of CC chemokine receptor 4 (CCR4) inhibitors: a combined approach involving homology modelling, docking, and molecular dynamics simulation studies.

CC chemokine receptor 4 (CCR4), a G protein-coupled receptor (GPCR), plays a vital role in the progression of asthma, T-cell lymphoma, inflammation, and Alzheimer's disease. To date, the structure of CCR4 has not been determined. Therefore, the nature of the interactions between inhibitors and CCR4 is not well known. In this study, we used CCR5 as a template to model the structure of CCR4. Docking studies were performed for four naphthalene-sulphonamide derivatives and crucial ligand-protein interactions were analysed. Molecular dynamics (MD) simulations of these complexes (100 ns each) were carried out to gain insights into the interactions between ligands and CCR4. MD simulations revealed that the residues identified by the docking were displaced and new residues were inserted near the ligands. Results of a principal component analysis (PCA) suggested that CCR4 unfolds at the extracellular site surrounding the ligands. Our simulations identified crucial residues involved in CCR4 antagonism, which were supported by previous mutational studies. Additionally, we identified Ser3.29, Leu3.33, Ser5.39, Phe6.47, Ile7.35, Thr7.38, Thr7.40, and Ala7.42 as residues that play crucial roles in CCR4 antagonism. Mutational studies will help elucidate the significance of these residues in CCR4 antagonism. An understanding of ligand-CCR4 interactions might aid in the design of novel CCR4 inhibitors.

Gain-of-function CCR4 mutations in adult T cell leukemia/lymphoma.

Adult T cell leukemia/lymphoma (ATLL) is an aggressive malignancy caused by human T cell lymphotropic virus type-I (HTLV-I) without curative treatment at present. To illuminate the pathogenesis of ATLL we performed whole transcriptome sequencing of purified ATLL patient samples and discovered recurrent somatic mutations in CCR4, encoding CC chemokine receptor 4. CCR4 mutations were detected in 14/53 ATLL samples (26%) and consisted exclusively of nonsense or frameshift mutations that truncated the coding region at C329, Q330, or Y331 in the carboxy terminus. Functionally, the CCR4-Q330 nonsense isoform was gain-of-function because it increased cell migration toward the CCR4 ligands CCL17 and CCL22, in part by impairing receptor internalization. This mutant enhanced PI(3) kinase/AKT activation after receptor engagement by CCL22 in ATLL cells and conferred a growth advantage in long-term in vitro cultures. These findings implicate somatic gain-of-function CCR4 mutations in the pathogenesis of ATLL and suggest that inhibition of CCR4 signaling might have therapeutic potential in this refractory malignancy.

Structural basis for the Nanos-mediated recruitment of the CCR4-NOT complex and translational repression.

The RNA-binding proteins of the Nanos family play an essential role in germ cell development and survival in a wide range of metazoan species. They function by suppressing the expression of target mRNAs through the recruitment of effector complexes, which include the CCR4-NOT deadenylase complex. Here, we show that the three human Nanos paralogs (Nanos1-3) interact with the CNOT1 C-terminal domain and determine the structural basis for the specific molecular recognition. Nanos1-3 bind CNOT1 through a short CNOT1-interacting motif (NIM) that is conserved in all vertebrates and some invertebrate species. The crystal structure of the human Nanos1 NIM peptide bound to CNOT1 reveals that the peptide opens a conserved hydrophobic pocket on the CNOT1 surface by inserting conserved aromatic residues. The substitutions of these aromatic residues in the Nanos1-3 NIMs abolish binding to CNOT1 and abrogate the ability of the proteins to repress translation. Our findings provide the structural basis for the recruitment of the CCR4-NOT complex by vertebrate Nanos, indicate that the NIMs are the major determinants of the translational repression mediated by Nanos, and identify the CCR4-NOT complex as the main effector complex for Nanos function.

Distinct conformations of the chemokine receptor CCR4 with implications for its targeting in allergy.

CC chemokine receptor 4 (CCR4) is expressed by Th2 and regulatory T cells and directs their migration along gradients of the chemokines CCL17 and CCL22. Both chemokines and receptor are upregulated in allergic disease, making CCR4 a therapeutic target for the treatment of allergy. We set out to assess the mechanisms underlying a previous report that CCL22 is a dominant ligand of CCR4, which may have implications for its therapeutic targeting. Human T cells expressing endogenous CCR4 and transfectants engineered to express CCR4 were assessed for receptor function, using assays of calcium release, chemotaxis, receptor endocytosis, and ligand binding. Despite the two ligands having equal potency in calcium flux and chemotaxis assays, CCL22 showed dominance in both receptor endocytosis assays and heterologous competitive binding assays. Using two different CCR4-specific Abs, we showed that CCR4 exists in at least two distinct conformations, which are differentially activated by ligand. A major population is activated by both CCL17 and CCL22, whereas a minor population is activated only by CCL22. Mutation of a single C-terminal residue K310 within a putative CCR4 antagonist binding site ablated activation of CCR4 by CCL17, but not by CCL22, despite having no effect on the binding of either ligand. We conclude that CCL17 and CCL22 are conformationally selective ligands of CCR4 and interact with the receptor by substantially different mechanisms. This finding suggests that the selective blockade of CCR4 in allergy may be feasible when one CCR4 ligand dominates, allowing the inhibition of Th2 signaling via one ligand while sparing regulatory T cell recruitment via another.

Structural basis for the recruitment of the human CCR4-NOT deadenylase complex by tristetraprolin.

Tristetraprolin (TTP) is an RNA-binding protein that controls the inflammatory response by limiting the expression of several proinflammatory cytokines. TTP post-transcriptionally represses gene expression by interacting with AU-rich elements (AREs) in 3' untranslated regions of target mRNAs and subsequently engenders their deadenylation and decay. TTP accomplishes these tasks, at least in part, by recruiting the multisubunit CCR4-NOT deadenylase complex to the mRNA. Here we identify an evolutionarily conserved C-terminal motif in human TTP that directly binds a central domain of CNOT1, a core subunit of the CCR4-NOT complex. A high-resolution crystal structure of the TTP-CNOT1 complex was determined, providing the first structural insight, to our knowledge, into an ARE-binding protein bound to the CCR4-NOT complex. Mutations at the CNOT1-TTP interface impair TTP-mediated deadenylation, demonstrating the significance of this interaction in TTP-mediated gene silencing.

The Not3/5 subunit of the Ccr4-Not complex: a central regulator of gene expression that integrates signals between the cytoplasm and the nucleus in eukaryotic cells.

The Ccr4-Not complex is a conserved multi-subunit complex in eukaryotes that carries 2 enzymatic activities: ubiquitination mediated by the Not4 RING E3 ligase and deadenylation mediated by the Ccr4 and Caf1 orthologs. This complex has been implicated in all aspects of the mRNA life cycle, from synthesis of mRNAs in the nucleus to their degradation in the cytoplasm. More recently the complex has also been implicated in many aspects of the life cycle of proteins, from quality control during synthesis of peptides, to assembly of protein complexes and protein degradation. Consistently, the Ccr4-Not complex is found both in the nucleus, where it is connected to transcribing ORFs, and in the cytoplasm, where it was revealed to be both associated with translating ribosomes and in RNA processing bodies. This functional and physical presence of the Ccr4-Not complex at all stages of gene expression raises the question of its fundamental role. This review will summarize recent evidence designing the Not3/5 module of the Ccr4-Not complex as a functional module involved in coordination of the regulation of gene expression between the nucleus and the cytoplasm.

The Ccr4--not complex.

The Ccr4-Not complex is a unique, essential and conserved multi-subunit complex that acts at the level of many different cellular functions to regulate gene expression. Two enzymatic activities, namely ubiquitination and deadenylation, are provided by different subunits of the complex. However, studies over the last decade have demonstrated a tantalizing multi-functionality of this complex that extends well beyond its identified enzymatic activities. Most of our initial knowledge about the Ccr4-Not complex stemmed from studies in yeast, but an increasing number of reports on this complex in other species are emerging. In this review we will discuss the structure and composition of the complex, and describe the different cellular functions with which the Ccr4-Not complex has been connected in different organisms. Finally, based upon our current state of knowledge, we will propose a model to explain how one complex can provide such multi-functionality. This model suggests that the Ccr4-Not complex might function as a "chaperone platform".

The Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the human Ccr4-Not complex contribute to the prevention of cell death and senescence.

A key step in cytoplasmic mRNA degradation is the shortening of the poly(A) tail, which involves several deadenylase enzymes. Relatively little is known about the importance of these enzymes for the cellular physiology. Here we focused on the role of the highly similar Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the Ccr4-Not complex. In addition to a role in cell proliferation, Ccr4a and Ccr4b play a role in cell survival, in contrast to the Caf1a (CNOT7) and Caf1b (CNOT8) deadenylase subunits or the CNOT1 and CNOT3 noncatalytic subunits of the Ccr4-Not complex. Underscoring the differential contributions of the deadenylase subunits, we found that knockdown of Caf1a/Caf1b or Ccr4a/Ccr4b differentially affects the formation of cytoplasmic foci by processing-body components. Furthermore, we demonstrated that the amino-terminal leucine-rich repeat (LRR) domain of Ccr4b influenced its subcellular localization but was not required for the deadenylase activity of Ccr4b. Moreover, overexpression of Ccr4b lacking the LRR domain interfered with cell cycle progression but not with cell viability. Finally, gene expression profiling indicated that distinct gene sets are regulated by Caf1a/Caf1b and Ccr4a/Ccr4b and identified Ccr4a/Ccr4b as a key regulator of insulin-like growth factor-binding protein 5, which mediates cell cycle arrest and senescence via a p53-dependent pathway.

Screening of thiourea derivatives and carbonyl-2-aminothiazole derivatives for potential CCR4 antagonists using capillary zone electrophoresis.

CC chemokine receptor 4 (CCR4) is a kind of G-protein-coupled receptors with a characteristic seven-transmembrane structure and selectively expressed on Th2-type CD4+ T-cells. CCR4 has been identified as a potentially important drug target for the treatment of T cell-mediated allergic inflammatory diseases. In this study, a novel series of CCR4 antagonists were screened by investigating the interactions between the compounds and the human CCR4 N-terminal peptide ML40 using capillary zone electrophoresis (CZE) for the first time. Both qualitative and quantitative characterizations of the compound-peptide binding were determined. The results showed that, compared with positive control, ten of the compounds were interacted with ML40, which were A3C223, A3C231, A4C238, A3C241, A4C241, A4C239, ZXF0337, ZXF0432, ZXF0519 and ZXF0637A, and their binding constants were calculated from the Scatchard plot by regression. The binding constants of the compounds to ML40 were calculated and the binding constant of ZXF0432 was the largest among them [(7.6334±0.1907)×10(4)M(-1)]. Here, a sensitive and selective high-performance analytical method based on CZE was developed for screening of thiourea derivatives and C-arbonyl-2-aminothiazole derivatives for potential CCR4 antagonists for the first time. The methodology presented should be generally applicable to study compounds-ML40 interactions as a powerful, sensitive and fast screening method for CCR4 antagonist discovery.

Potent CCR4 antagonists: synthesis, evaluation, and docking study of 2,4-diaminoquinazolines.

A series of CC chemokine receptor-4 (CCR4) antagonists were examined in a previous report in an attempt to improve metabolic stability in human liver microsomes. In this study, the cycloheptylamine moiety of N-cycloheptyl-6,7-dimethoxy-2-(4-pyrrolidin-1-ylpiperidin-1-yl)quinazolin-4-amine 1 was replaced with the p-chloroaniline moiety, and the resulting compound, N-(4-chlorophenyl)-6,7-dimethoxy-2-(4-pyrrolidin-1-ylpiperidin-1-yl)quinazolin-4-amine (8c), retained its potency ([(35)S]GTPgammaS-binding inhibition and CCL22-induced chemotaxis in humans/mice). Based on the structure-activity relationships (SAR), a homology model was constructed for CCR4 to explain the binding mode of 8c. Overall, there was good agreement between the docking pose of the CCR4 homology model and the human [(35)S]GTPgammaS assay results. Administration of 8c in a murine model of acute dermatitis showed anti-inflammatory activity (oxazolone-induced contact hypersensitivity test).

In silico identified CCR4 antagonists target regulatory T cells and exert adjuvant activity in vaccination.

Adjuvants are substances that enhance immune responses and thus improve the efficacy of vaccination. Few adjuvants are available for use in humans, and the one that is most commonly used (alum) often induces suboptimal immunity for protection against many pathogens. There is thus an obvious need to develop new and improved adjuvants. We have therefore taken an approach to adjuvant discovery that uses in silico modeling and structure-based drug-design. As proof-of-principle we chose to target the interaction of the chemokines CCL22 and CCL17 with their receptor CCR4. CCR4 was posited as an adjuvant target based on its expression on CD4(+)CD25(+) regulatory T cells (Tregs), which negatively regulate immune responses induced by dendritic cells (DC), whereas CCL17 and CCL22 are chemotactic agents produced by DC, which are crucial in promoting contact between DC and CCR4(+) T cells. Molecules identified by virtual screening and molecular docking as CCR4 antagonists were able to block CCL22- and CCL17-mediated recruitment of human Tregs and Th2 cells. Furthermore, CCR4 antagonists enhanced DC-mediated human CD4(+) T cell proliferation in an in vitro immune response model and amplified cellular and humoral immune responses in vivo in experimental models when injected in combination with either Modified Vaccinia Ankara expressing Ag85A from Mycobacterium tuberculosis (MVA85A) or recombinant hepatitis B virus surface antigen (rHBsAg) vaccines. The significant adjuvant activity observed provides good evidence supporting our hypothesis that CCR4 is a viable target for rational adjuvant design.

An intracellular allosteric site for a specific class of antagonists of the CC chemokine G protein-coupled receptors CCR4 and CCR5.

A novel mechanism for antagonism of the human chemokine receptors CCR4 and CCR5 has been discovered with a series of small-molecule compounds that seems to interact with an allosteric, intracellular site on the receptor. The existence of this site is supported by a series of observations: 1) intracellular access of these antagonists is required for their activity; 2) specific, saturable binding of a radiolabeled antagonist requires the presence of CCR4; and 3) through engineering receptor chimeras by reciprocal transfer of C-terminal domains between CCR4 and CCR5, compound binding and the selective structure-activity relationships for antagonism of these receptors seem to be associated with the integrity of that intracellular region. Published antagonists from other chemical series do not seem to bind to the novel site, and their interaction with either CCR4 or CCR5 is not affected by alteration of the C-terminal domain. The precise location of the proposed binding site remains to be determined, but the known close association of the C-terminal domain, including helix 8, as a proposed intracellular region that interacts with transduction proteins (e.g., G proteins and beta-arrestin) suggests that this could be a generic allosteric site for chemokine receptors and perhaps more broadly for class A G protein-coupled receptors. The existence of such a site that can be targeted for drug discovery has implications for screening assays for receptor antagonists, which would need, therefore, to consider compound properties for access to this intracellular site.

Defucosylated anti CC chemokine receptor 4 monoclonal antibody combined with immunomodulatory cytokines: a novel immunotherapy for aggressive/refractory Mycosis fungoides and Sezary syndrome.

PURPOSE: Sézary syndrome (SS) and Mycosis fungoides (MF) in the advanced stage have dismal prognoses. Because CC chemokine receptor 4 (CCR4) has an important role in the skin-homing capacity of MF/SS cells, we postulated that anti-CCR4 monoclonal antibody (mAb) could represent a novel therapeutic agent against aggressive/refractory MF/SS. EXPERIMENTAL DESIGN: The defucosylated next-generation therapeutic mAb KM2760 induces enhanced antibody-dependent cellular cytotoxicity (ADCC). Here, we assessed the therapeutic potential of this antibody against aggressive MF/SS tumor cells in vitro and in animal models in vivo. RESULTS: KM2760 induced robust ADCC by peripheral blood mononuclear cell (PBMC) from healthy controls against a MF/SS cell line as well as against primary tumor cells from patients with aggressive MF/SS. KM2760 also showed significant antitumor activity in disseminated and nondisseminated MF/SS mouse models. In addition, approximately 30% of autologous MF/SS tumor cells were killed in in vitro assays of KM2760-induced ADCC mediated by patients' PBMC after only 4 h, despite the low numbers of natural killer cells present in these PBMCs. It is also shown that ADCC induced by defucosylated therapeutic mAb can be greatly augmented by the immunomodulatory cytokines interleukin-12, IFN-alpha-2b, and IFN-gamma. CONCLUSIONS: The present study has encouraged us in the conducting of a phase I clinical trial of a completely defucosylated anti-CCR4 mAb in patients with CCR4-positive T-cell lymphomas, including aggressive MF/SS (ClinicalTrials.gov identifier: NCT00355472). In the near future, the efficacy not only of defucosylated anti-CCR4 mAb single-agent treatment but also of combination therapy with immunomodulatory cytokines will be clinically established to target aggressive/refractory MF/SS.

Studies on the interactions between lactam analogs and the N-terminal extracellular tail of CC chemokine receptor 4 by CZE.

CC chemokine receptor 4 (CCR4) is a kind of G-protein-coupled receptors with a characteristic seven-transmembrane structure and selectively expressed on Th2-type CD4+ T-cells, which play a pivotal role in allergic inflammation. In this study, the interactions between 2-(2-(2,4-dichloro-phenyl)-4-{[(2-methyl-3-chloro-phenyl)-1-ylmethyl]-carbamoyl}-methyl)-5-oxo-pyrrole-1-yl)-N-(3-piperidinyl-propyl)-acetamide (compound A), a known CCR4 antagonist, and ML40 were studied by CZE for the first time. Both qualitative and quantitative characterizations of the drug-peptide binding were determined. The binding constant of the interaction between the trans-diastereomer of compound A and ML40, calculated from the Scatchard plot by regression, was (1.06 +/- 0.11)x10(5)/M. Also, it was confirmed that the trans-diastereomer was more potent affinity with CCR4 than its cis-counterpart. The experimental results show that this reported method by CZE for the determination of the compound A and ML40 interactions is powerful, sensitive, and fast, requires less amounts of reagents, and further, it can be employed as one of the reliable screening methods to a series of lactam analogs in the drug discovery for allergic inflammation diseases.