RESUMO
The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral transcription is the first step in the control of latency and depends upon the hijacking of the host cell RNA polymerase II (Pol II) machinery by the 5' HIV LTR. Consequently, "block and lock" or "shock and kill" strategies for an HIV cure depend upon a full understanding of HIV transcriptional control. The HIV trans-activating protein, Tat, controls HIV latency as part of a positive feed-forward loop that strongly activates HIV transcription. The recognition of the TATA box and adjacent sequences of HIV essential for Tat trans-activation (TASHET) of the core promoter by host cell pre-initiation complexes of HIV (PICH) has been shown to be necessary for Tat trans-activation, yet the protein composition of PICH has remained obscure. Here, DNA-affinity chromatography was employed to identify the mitotic deacetylase complex (MiDAC) as selectively recognizing TASHET. Using biophysical techniques, we show that the MiDAC subunit DNTTIP1 binds directly to TASHET, in part via its CTGC DNA motifs. Using co-immunoprecipitation assays, we show that DNTTIP1 interacts with MiDAC subunits MIDEAS and HDAC1/2. The Tat-interacting protein, NAT10, is also present in HIV-bound MiDAC. Gene silencing revealed a functional role for DNTTIP1, MIDEAS, and NAT10 in HIV expression in cellulo. Furthermore, point mutations in TASHET that prevent DNTTIP1 binding block the reactivation of HIV by latency reversing agents (LRA) that act via the P-TEFb/7SK axis. Our data reveal a key role for MiDAC subunits DNTTIP1, MIDEAS, as well as NAT10, in Tat-activated HIV transcription and latency. DNTTIP1, MIDEAS and NAT10 emerge as cell cycle-regulated host cell transcription factors that can control activated HIV gene expression, and as new drug targets for HIV cure strategies.
Assuntos
Regulação Viral da Expressão Gênica , Infecções por HIV , HIV-1 , Regiões Promotoras Genéticas , Latência Viral , Humanos , HIV-1/genética , HIV-1/fisiologia , Infecções por HIV/virologia , Infecções por HIV/metabolismo , Infecções por HIV/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Transcrição ViralRESUMO
Cyclin D2 (CCND2) stabilization underpins a range of macrocephaly-associated disorders through mutation of CCND2 or activating mutations in upstream genes encoding PI3K-AKT pathway components. Here, we describe three individuals with overlapping macrocephaly-associated phenotypes who carry the same recurrent de novo c.179G>A (p.Arg60Gln) variant in Myc-associated factor X (MAX). The mutation, located in the b-HLH-LZ domain, causes increased intracellular CCND2 through increased transcription but it does not cause stabilization of CCND2. We show that the purified b-HLH-LZ domain of MAXArg60Gln (Max∗Arg60Gln) binds its target E-box sequence with a lower apparent affinity. This leads to a more efficient heterodimerization with c-Myc resulting in an increase in transcriptional activity of c-Myc in individuals carrying this mutation. The recent development of Omomyc-CPP, a cell-penetrating b-HLH-LZ-domain c-Myc inhibitor, provides a possible therapeutic option for MAXArg60Gln individuals, and others carrying similar germline mutations resulting in dysregulated transcriptional c-Myc activity.
Assuntos
Megalencefalia , Proteínas Proto-Oncogênicas c-myc , Humanos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Dimerização , Megalencefalia/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Compounds beyond the rule-of-five are generating interest as they expand the molecular toolbox for modulating targets previously considered "undruggable". Macrocyclic peptides are an efficient class of molecules for modulating protein-protein interactions. However, predicting their permeability is difficult as they differ from small molecules. Although constrained by macrocyclization, they generally retain some conformational flexibility associated with an enhanced ability to cross biological membranes. In this study, we investigated the relationship between the structure of semi-peptidic macrocycles and their membrane permeability through structural modifications. Based on a scaffold of four amino acids and a linker, we synthesized 56 macrocycles incorporating modifications in either stereochemistry, N-methylation, or lipophilicity and assessed their passive permeability using the parallel artificial membrane permeability assay (PAMPA). Our results show that some semi-peptidic macrocycles have adequate passive permeability even with properties outside the Lipinski rule of five. We found that N-methylation in position 2 and the addition of lipophilic groups to the side chain of tyrosine led to an improvement in permeability with a decrease in tPSA and 3D-PSA. This enhancement could be attributed to the shielding effect of the lipophilic group on some regions of the macrocycle, which in turn, facilitates a favorable macrocycle conformation for permeability, suggesting some degree of chameleonic behavior.
Assuntos
Aminoácidos , Peptídeos , Peptídeos/química , Conformação Molecular , Permeabilidade , TirosinaRESUMO
Medicinal chemistry is constantly searching for new approaches to develop more effective and targeted therapeutic molecules. The design of peptidomimetics is a promising emerging strategy that is aimed at developing peptides that mimic or modulate the biological activity of proteins. Among these, stapled peptides stand out for their unique ability to stabilize highly frequent helical motifs, but they have failed to be systematically reported. Here, we exploit chemically diverse helix-inducing i, i + 4 constraints-lactam, hydrocarbon, triazole, double triazole and thioether-on two distinct short sequences derived from the N-terminal peptidase domain of hACE2 upon structural characterization and in silico alanine scan. Our overall objective was to provide a sequence-independent comparison of α-helix-inducing staples using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. We identified a 9-mer lactam stapled peptide derived from the hACE2 sequence (His34-Gln42) capable of reaching its maximal helicity of 55% with antiviral activity in bioreporter- and pseudovirus-based inhibition assays. To the best of our knowledge, this study is the first comprehensive investigation comparing several cyclization methods with the goal of generating stapled peptides and correlating their secondary structures with PPI inhibitions using a highly topical model system (i.e., the interaction of SARS-CoV-2 Spike RBD with hACE2).
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Ciclização , Lactamas , Peptídeos/farmacologia , TriazóisRESUMO
Miz-1 (ZBTB17) is a poly-zinc finger BTB/POZ transcription factor with 12 consecutive C2H2 zinc fingers (ZFs) that binds transcriptional start sites (TSSs) to regulate the expression of genes involved in cell development and proliferation. As of now, it is not known which of the 12 consecutive ZFs are responsible for the recognition of the 24 base pair consensus sequence found at these TSSs. Evidence suggests ZFs 7-12 plays this role. We provide validation for this and describe the structural and dynamical characterization of unprecedented conformational exchange in the linker between ZFs 10 and 11. This conformational exchange uncouples ZFs 7-10 from 11 and 12 and promotes a scanning-recognition mechanism through which the two segments cooperate to bind two sub-sites at both ends of the consensus. We further show that this can result in the coiling of TSSs as part of Miz-1's mechanism of transcriptional transactivation.
Assuntos
Fatores de Transcrição , Dedos de Zinco , Sequência de Aminoácidos , Sítios de Ligação , DNA/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like , Fatores de Transcrição/metabolismoRESUMO
The b-HLH-LZ domain of c-Myc is a key target for the development of cancer therapies by blunting its binding to DNA with cell penetrant b-HLH-LZs and/or by stabilizing it into a state that cannot recognize Max to activate and amplify transcription of oncogenic genes. Although recent milestones have been reached with DNA binding blunting of c-Myc with the cell penetrant b-HLH-LZ Omomyc, the targeting of its b-HLH-LZ with small molecules, peptides, or proteins is lagging. As reviewed recently, the main problem relies in the intrinsically disordered nature of the b-HLH-LZ of c-Myc. This greatly complicates the classical approach of targeting a docking site with inhibitors. The solution state methods such as NMR are progressing towards the characterization of the ensembles of structures or states the b-HLH-LZ can adopt. However, the delicate balance that dictates the population of these dynamically interchanging states relies on its primary structure and the weak polar, electrostatic and hydrophobic interactions allowed. In this context, it is of the utmost importance to study the b-HLH-LZ of c-Myc in its WT background and avoid the use of tags such as His-tags. These tags could disrupt the balance of forces which could alter the conformational and physical transitions and states it can undergo and adopt. Here, we describe a robust protocol to express the WT b-HLH-LZ in E. coli and purify it, without the need of tags, to obtain the required quantities for solution state biophysical characterization such as NMR.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/isolamento & purificação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/isolamento & purificação , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , DNA/química , DNA/genética , Dimerização , Escherichia coli/genética , Expressão Gênica , Genes myc , Sequências Hélice-Alça-Hélice , Humanos , Zíper de Leucina/genética , Zíper de Leucina/fisiologia , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Multimerização Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Rab4a is a small GTPase associated with endocytic compartments and a key regulator of early endosomes recycling. Gathering evidence indicates that its expression and activation are required for the development of metastases. Rab4a-intrinsic GTPase properties that control its activity, i.e. nucleotide exchange and hydrolysis rates, have not yet been thoroughly studied. The determination of these properties is of the utmost importance to understand its functions and contributions to tumorigenesis. Here, we used the constitutively active (Rab4aQ67L) and dominant negative (Rab4aS22N) mutants to characterize the thermodynamical and structural determinants of the interaction between Rab4a and GTP (GTPγS) as well as GDP. We report the first 1H, 13C, 15N backbone NMR assignments of a Rab GTPase family member with Rab4a in complex with GDP and GTPγS. We also provide a qualitative description of the extent of structural and dynamical changes caused by the Q67L and S22N mutations. Using a real-time NMR approach and the two aforementioned mutants as controls, we evaluated Rab4a intrinsic nucleotide exchange and hydrolysis rates. Compared to most small GTPases such as Ras, a rapid GTP exchange rate along with slow hydrolysis rate were observed. This suggests that, in a cellular context, Rab4a can self-activate and persist in an activated state in absence of regulatory mechanisms. This peculiar profile is uncommon among the Ras superfamily members, making Rab4a an atypical fast-cycling GTPase and may explain, at least in part, how it contributes to metastases.
Assuntos
GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Nucleotídeos/química , Nucleotídeos/metabolismo , Linhagem Celular Tumoral , Células HeLa , Humanos , Hidrólise , Cinética , Espectroscopia de Ressonância Magnética/métodosRESUMO
Pseudogenes are mutated copies of protein-coding genes that cannot be translated into proteins, but a small subset of pseudogenes has been detected at the protein level. Although ubiquitin pseudogenes represent one of the most abundant pseudogene families in many organisms, little is known about their expression and signaling potential. By re-analyzing public RNA-sequencing and proteomics datasets, we here provide evidence for the expression of several ubiquitin pseudogenes including UBB pseudogene 4 (UBBP4), which encodes UbKEKS (Q2K, K33E, Q49K, N60S). The functional consequences of UbKEKS conjugation appear to differ from canonical ubiquitylation. Quantitative proteomics shows that UbKEKS modifies specific proteins including lamins. Knockout of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. Our work suggests that a subset of proteins reported as ubiquitin targets may instead be modified by ubiquitin variants that are the products of wrongly annotated pseudogenes and induce different functional effects.
Assuntos
Lamina Tipo A/metabolismo , Pseudogenes/genética , Ubiquitina/genética , Sistemas CRISPR-Cas/genética , Divisão Celular , Núcleo Celular/metabolismo , Clonagem Molecular , Conjuntos de Dados como Assunto , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Proteômica , RNA-Seq , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
The signaling mechanisms of the angiotensin II type 2 receptor (AT2R), a heptahelical receptor, have not yet been clearly and completely defined. In the present contribution, we set out to identify the molecular determinants involved in AT2R activation. Although AT2R has not been shown to engage Gq/11, G12, Gi2, and ß-arrestin (ßarr) pathways as does the AT1R upon angiotensin II (AngII) stimulation, the atypical positioning of helix VIII in the recently published AT2R structure may play a role in the receptor's capacity to couple to downstream effectors. In the AT2R structure, helix VIII points inwards and towards intracellular loop 3 (ICL3) to form tertiary interactions with transmembrane domain 6 (TM6), possibly impeding access to signaling effectors. On the other hand, in most class A GPCRs, helix VIII is found to be engaged in tertiary interactions with ICL1 and away from the effector binding site. Upon closer examination of the AT2R structure, we found that the residues contained within intracellular loop 1 (ICL1) may be involved in driving this unusual conformation of helix VIII. To explore this hypothesis, we designed a series of AT1R/AT2R receptor chimeras to validate the roles of ICL1 and helix VIII in AT2R signaling. Substituting the AT1R ICL1 into AT2R led to a mutant receptor that coupled to Gi2. The substitution of the helix VIII and C-terminal domains of AT2R into the AT1R backbone led to a mutant receptor that retained AT1R-like signaling properties. These results suggest that the C-terminal portion of AT2R is compatible with canonical GPCR signaling and that ICL1 of AT2R is involved in repositioning helix VIII, which impedes engagement of classical GPCR effectors such as G proteins or ßarrs.
Assuntos
Sequências Hélice-Alça-Hélice/fisiologia , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Receptor Tipo 2 de Angiotensina/química , Receptor Tipo 2 de Angiotensina/metabolismo , Angiotensina II/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Relação Dose-Resposta a Droga , Células HEK293 , Sequências Hélice-Alça-Hélice/efeitos dos fármacos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor Tipo 2 de Angiotensina/agonistasRESUMO
Inhibiting MYC has long been considered unfeasible, although its key role in human cancers makes it a desirable target for therapeutic intervention. One reason for its perceived undruggability was the fear of catastrophic side effects in normal tissues. However, we previously designed a dominant-negative form of MYC called Omomyc and used its conditional transgenic expression to inhibit MYC function both in vitro and in vivo. MYC inhibition by Omomyc exerted a potent therapeutic impact in various mouse models of cancer, causing only mild, well-tolerated, and reversible side effects. Nevertheless, Omomyc has been so far considered only a proof of principle. In contrast with that preconceived notion, here, we show that the purified Omomyc mini-protein itself spontaneously penetrates into cancer cells and effectively interferes with MYC transcriptional activity therein. Efficacy of the Omomyc mini-protein in various experimental models of non-small cell lung cancer harboring different oncogenic mutation profiles establishes its therapeutic potential after both direct tissue delivery and systemic administration, providing evidence that the Omomyc mini-protein is an effective MYC inhibitor worthy of clinical development.
Assuntos
Peptídeos Penetradores de Células/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/patologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/farmacocinética , Peptídeos Penetradores de Células/uso terapêutico , DNA/metabolismo , Modelos Animais de Doenças , Elementos E-Box/genética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacocinética , Fragmentos de Peptídeos/uso terapêutico , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/farmacocinética , Proteínas Proto-Oncogênicas c-myc/farmacologia , Proteínas Proto-Oncogênicas c-myc/uso terapêuticoRESUMO
The use of direct calorimetric methods such as isothermal titration calorimetry for measuring the affinity and specificity of protein-ligand interactions requires large amounts of proteins and ligands. When material is scarce and/or in the absence of calorimeters, thermal Shift Assays (TSA) using Circular Dichroism (CD) or other spectroscopic methods offers an alternative and quantitative method for the determination of apparent or indirect thermodynamical parameters describing the affinity of ligands for proteins. Indeed, the binding constants of ligands (Kb) and other parameters such as the enthalpy and Gibbs free energy of binding may be estimated from the changes in the stability curves ΔGu(T) of a protein in the presence of a ligand. Here we describe the application of two different procedures proposed by Layton and Hellinga et al. (Biochemistry 49:10831-10841, 2010) to evaluate the apparent Kb of testosterone to the START (StAR-related lipid transfer domain) domains.
Assuntos
Dicroísmo Circular , Ligantes , Proteínas de Membrana Transportadoras/metabolismo , Clonagem Molecular , Expressão Gênica , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Ligação Proteica , Proteínas Recombinantes , Análise Espectral , TermodinâmicaRESUMO
G protein coupled receptors (GPCRs) produce pleiotropic effects by their capacity to engage numerous signaling pathways once activated. Functional selectivity (also called biased signaling), where specific compounds can bring GPCRs to adopt conformations that enable selective receptor coupling to distinct signaling pathways, continues to be significantly investigated. However, an important but often overlooked aspect of functional selectivity is the capability of ligands such as angiotensin II (AngII) to adopt specific conformations that may preferentially bind to selective GPCRs structures. Understanding both receptor and ligand conformation is of the utmost importance for the design of new drugs targeting GPCRs. In this study, we examined the properties of AngII cyclic analogs to impart biased agonism on the angiotensin type 1 receptor (AT1R). Positions 3 and 5 of AngII were substituted for cysteine and homocysteine residues ([Sar1Hcy3,5]AngII, [Sar1Cys3Hcy5]AngII and [Sar1Cys3,5]AngII) and the resulting analogs were evaluated for their capacity to activate the Gq/11, G12, Gi2, Gi3, Gz, ERK and ß-arrestin (ßarr) signaling pathways via AT1R. Interestingly, [Sar1Hcy3,5]AngII exhibited potency and full efficacy on all pathways tested with the exception of the Gq pathway. Molecular dynamic simulations showed that the energy barrier associated with the insertion of residue Phe8 of AngII within the hydrophobic core of AT1R, associated with Gq/11 activation, is increased with [Sar1Hcy3,5]AngII. These results suggest that constraining the movements of molecular determinants within a given ligand by introducing cyclic structures may lead to the generation of novel ligands providing more efficient biased agonism.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Angiotensina II/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Angiotensina II/química , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/química , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Receptor Tipo 1 de Angiotensina/química , Transdução de Sinais/fisiologiaRESUMO
The Gαs subunit is classically involved in the signal transduction of G protein-coupled receptors (GPCRs) at the plasma membrane. Recent evidence has revealed noncanonical roles for Gαs in endosomal sorting of receptors to lysosomes. However, the mechanism of action of Gαs in this sorting step is still poorly characterized. Here, we report that Gαs interacts with ubiquitin to regulate the endosomal sorting of receptors for lysosomal degradation. We reveal that the N-terminal extremity of Gαs contains a ubiquitin-interacting motif (UIM), a sorting element usually found in the endosomal sorting complex required for transport (ESCRT) machinery responsible for sorting ubiquitinated receptors into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). Mutation of the UIM in Gαs confirmed the importance of ubiquitin interaction for the sorting of epidermal growth factor receptor (EGFR) into ILVs for lysosomal degradation. These findings demonstrate a role for Gαs as an integral component of the ubiquitin-dependent endosomal sorting machinery and highlight the dual role of Gαs in receptor trafficking and signaling for the fine-tuning of the cellular response.
Assuntos
Endossomos/metabolismo , Receptores ErbB/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Sinais Direcionadores de Proteínas , Transporte ProteicoRESUMO
Caspases are cysteinyl peptidases involved in inflammation and apoptosis during which hundreds of proteins are cleaved by executioner caspase-3 and -7. Despite the fact that caspase-3 has a higher catalytic activity, caspase-7 is more proficient at cleaving poly(ADP ribose) polymerase 1 (PARP1) because it uses an exosite within its N-terminal domain (NTD). Here, we demonstrate that molecular determinants also located in the NTD enhance the recognition and proteolysis of the Hsp90 co-chaperone p23. Structure-activity relationship analyses using mutagenesis of the caspase-7 NTD and kinetics show that residues 36-45 of caspase-7, which overlap with residues necessary for efficacious PARP1 cleavage, participate in p23 recognition. We also demonstrate using chimeric and truncated proteins that the caspase-7 NTD binds close to the cleavage site in the C-terminal tail of p23. Moreover, because p23 is cleaved at a site bearing a P4 Pro residue (PEVD142↓G), which is far from the optimal sequence, we tested all residues at that position and found notable differences in the preference of caspase-7 and magnitude of differences between residues compared to the results of studies that have used small peptidic substrate libraries. Finally, bioinformatics shows that the regions we identified in caspase-7 and p23 are intrinsically disordered regions that contain molecular recognition features that permit a transient interaction between these two proteins. In summary, we characterized the binding mode for a caspase that is tailored to the specific recognition and cleavage of a substrate, highlighting the importance of studying the peptidase-substrate pair to understand the modalities of substrate recognition by caspases.
Assuntos
Caspase 7/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Caspase 7/genética , Dicroísmo Circular , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Cinética , Chaperonas Moleculares/genética , Mutação , Fosfoproteínas/genética , Prostaglandina-E Sintases , Domínios Proteicos , Relação Estrutura-AtividadeRESUMO
It is classically recognized that the physiological and oncogenic functions of Myc proteins depend on specific DNA binding enabled by the dimerization of its C-terminal basic-region-Helix-Loop-Helix-Leucine Zipper (b-HLH-LZ) domain with that of Max. However, a new paradigm is emerging, where the binding of the c-Myc/Max heterodimer to non-specific sequences in enhancers and promoters drives the transcription of genes involved in diverse oncogenic programs. Importantly, Max can form a stable homodimer even in the presence of c-Myc and bind DNA (specific and non-specific) with comparable affinity to the c-Myc/Max heterodimer. Intriguingly, alterations in the Max gene by germline and somatic mutations or changes in the gene product by alternative splicing (e.g. ΔMax) were recently associated with pheochromocytoma and glioblastoma, respectively. This has led to the proposition that Max is, by itself, a tumor suppressor. However, the actual mechanism through which it exerts such an activity remains to be elucidated. Here, we show that contrary to the WT motif, the b-HLH-LZ of ΔMax does not homodimerize in the absence of DNA. In addition, although ΔMax can still bind the E-box sequence as a homodimer, it cannot bind non-specific DNA in that form, while it can heterodimerize with c-Myc and bind E-box and non-specific DNA as a heterodimer with high affinity. Taken together, our results suggest that the WT Max homodimer is important for attenuating the binding of c-Myc to specific and non-specific DNA, whereas ΔMax is unable to do so. Conversely, the splicing of Max into ΔMax could provoke an increase in overall chromatin bound c-Myc. According to the new emerging paradigm, the splicing event and the stark reduction in homodimer stability and DNA binding should promote tumorigenesis impairing the tumor suppressor activity of the WT homodimer of Max.
Assuntos
Processamento Alternativo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , DNA/metabolismo , Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , DNA/química , Elementos E-Box , Humanos , Modelos Moleculares , Neoplasias/metabolismo , Multimerização Proteica , Proteínas Repressoras/química , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
c-Myc is a basic helix-loop-helix leucine zipper (b-HLH-LZ) transcription factor deregulated in the majority of human cancers. As a heterodimer with Max, another b-HLH-LZ transcription factor, deregulated and persistent c-Myc accumulates at transcriptionally active promoters and enhancers and amplifies transcription. This leads to the so-called transcriptional addiction of tumor cells. Recent studies have showed that c-Myc transcriptional activities can be reversed by its association with Miz-1, a POZ transcription factor containing 13 classical zinc fingers. Although evidences have led to suggest that c-Myc interacts with both Miz-1 and Max to form a ternary repressive complex, earlier evidences also suggest that Miz-1 and Max may compete to engage c-Myc. In such a scenario, the Miz-1/c-Myc complex would be the entity responsible for the inhibition of c-Myc transcriptional amplification. Considering the implications of the Miz-1/c-Myc interaction, it is highly important to solve this duality. While two potential c-Myc interacting domains (hereafter termed MID) have been identified in Miz-1 by yeast two-hybrid, with the b-HLH-LZ as a bait, the biophysical characterization of these interactions has not been reported so far. Here, we report that the MID located between the 12th and 13th zinc finger of Miz-1 and the b-HLH-LZ of Max compete to form a complex with the b-HLH-LZ of c-Myc. Our results support the notion that the repressive action of Miz-1 on c-Myc does not rely on the formation of a ternary complex. The implications of these observations for the mechanism of inhibition of c-Myc transcriptional activity by Miz-1 are discussed. Proteins 2017; 85:199-206. © 2016 Wiley Periodicals, Inc.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Fatores de Transcrição Kruppel-Like/química , Proteínas Proto-Oncogênicas c-myc/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação , Dicroísmo Circular , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
c-Myc-interacting zinc finger protein-1 (Miz-1) is a poly-Cys2His2 zinc finger (ZF) transcriptional regulator of many cell cycle genes. A Miz-1 DNA sequence consensus has recently been identified and has also unveiled Miz-1 functions in other cellular processes, underscoring its importance in the cell. Miz-1 contains 13 ZFs, but it is unknown why Miz-1 has so many ZFs and whether they recognize and bind DNA sequences in a typical fashion. Here, we used NMR to deduce the role of Miz-1 ZFs 1-4 in detecting the Miz-1 consensus sequence and preventing nonspecific DNA binding. In the construct containing the first 4 ZFs, we observed that ZFs 3 and 4 form an unusual compact and stable structure that restricts their motions. Disruption of this compact structure by an electrostatically mismatched A86K mutation profoundly affected the DNA binding properties of the WT construct. On the one hand, Miz1-4WT was found to bind the Miz-1 DNA consensus sequence weakly and through ZFs 1-3 only. On the other hand, the four ZFs in the structurally destabilized Miz1-4A86K mutant bound to the DNA consensus with a 30-fold increase in affinity (100 nm). The formation of such a thermodynamically stable but nonspecific complex is expected to slow down the rate of DNA scanning by Miz-1 during the search for its consensus sequence. Interestingly, we found that the motif stabilizing the compact structure between ZFs 3 and 4 is conserved and enriched in other long poly-ZF proteins. As discussed in detail, our findings support a general role of compact inter-ZF structures in minimizing the formation of off-target DNA complexes.
Assuntos
DNA/metabolismo , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/metabolismo , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Alinhamento de Sequência , Dedos de ZincoRESUMO
Objective: Review the impact of StAR (STARD1) mutations on steroidogenesis and fertility in LCAH patients. Examine the endocrine mechanisms underlying the pathology of the disorder and the appropriate therapy for promoting fertility and pregnancies. Design: Published data in the literature and a detailed 38-year follow-up of two sibling LCAH patients. Molecular structure and modeling of the STARD1 L275P mutation. Setting: University hospital. Patients: Patient A (46,XY female phenotype) and patient B (46,XX female) with LCAH bearing the L275P mutation in STARD1. Interventions: Since early-age diagnosis, both patients underwent corticoid replacement therapy. Patient A received estrogen therapy at pubertal age. Clomiphene therapy was given to Patient B to induce ovulation. Pregnancies were protected with progesterone administration. Main Outcome Measures: Clinical and molecular assessment of adrenal and gonadal functions. Results: Both patients have classic manifestations of corticosteroid deficiency observed in LCAH. Time of onset and severity were different. Patient A developed into a female phenotype due to early and severe damage of Leydig cells. Patient B started a progressive pubertal development, menarche and regular non-ovulatory cycle. She was able to have successful pregnancies. Conclusions: Understanding the molecular structure and function of STARD1 in all steroidogenic tissues is the key for comprehending the heterogeneous clinical manifestations of LCAH, and the development of an appropriate strategy for the induction of ovulation and protecting pregnancies in this disease.
RESUMO
A combination of the CXCR4 inverse agonist T140 with N-terminal CXCL12 oligopeptides has produced the first nanomolar synthetic CXCR4 agonists. In these agonists, the inverse agonistic portion provides affinity whereas the N-terminal CXCL12 sequence induces receptor activation. Several CXCR4 crystal structures exist with either CVX15, an inverse agonist closely related to T140 and IT1t, a small molecule; we therefore attempted to produce another CXCL12 oligopeptide combination with IT1t. For this purpose, a primary amino group was introduced by total synthesis into one of the methyl groups of IT1t, serving as an anchoring point for the oligopeptide graft. The introduction of the oligopeptides on this analog however yielded antagonists, one compound displaying high affinity. On the other hand, the amino-substituted analogue itself proved to be an inverse agonist with a binding affinity of 2.6 nM compared to 11.5 nM for IT1t. This IT1t-like analog is hitherto one of the most potent non-peptidic CXCR4 inverse agonists reported.
Assuntos
Desenho de Fármacos , Receptores CXCR4/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/metabolismo , Técnicas de Química Sintética , Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Células HEK293 , Humanos , Ligantes , Simulação de Dinâmica Molecular , Conformação Proteica , Receptores CXCR4/agonistas , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
The CXCR4 receptor binds with meaningful affinities only CXCL12 and synthetic antagonists/inverse agonists. We recently described high affinity synthetic agonists for this chemokine receptor, obtained by grafting the CXCL12 N-terminus onto the inverse agonist T140. While those chimeric molecules behave as agonists for CXCR4, their binding and activation mode are unknown. The present SAR of those CXCL12-oligopeptide grafts reveals the key determinants involved in CXCR4 activation. Position 3 (Val) controls affinity, whereas position 7 (Tyr) acts as an efficacy switch. Chimeric molecules bearing aromatic residues in position 3 possess high binding affinities for CXCR4 and are Gαi full agonists with robust chemotactic properties. Fine-tuning of electron-poor aromatic rings in position 7 enhances receptor activation. To rationalize these results, a homology model of a receptor-ligand complex was built using the published crystal structures of CXCR4. Molecular dynamics simulations reveal further details accounting for the observed SAR for this series.