RESUMO
The ligand-sensing transcription factor retinoid X receptor (RXR) is the universal heterodimer partner of nuclear receptors and involved in multiple physiological processes. Its pharmacological modulation holds therapeutic potential in cancer and neurodegeneration but many available RXR ligands lack specificity. The sesquiterpenoid valerenic acid has been identified as RXR agonist with unprecedented subtype and homodimer preference. Here, we identified simplified mimetics of the complex natural product by rational design and virtual screening that exhibited similar activity profiles on RXR and informed about structural elements contributing to the favorable activity.
Assuntos
Indenos , Sesquiterpenos , Receptores X de Retinoides , Receptores do Ácido Retinoico/química , Sesquiterpenos/farmacologiaRESUMO
IMPORTANCE: A common hypothesis holds that bats (order Chiroptera) are outstanding reservoirs for zoonotic viruses because of a special antiviral interferon (IFN) system. However, functional studies about key components of the bat IFN system are rare. RIG-I is a cellular sensor for viral RNA signatures that activates the antiviral signaling chain to induce IFN. We cloned and functionally characterized RIG-I genes from two species of the suborders Yangochiroptera and Yinpterochiroptera. The bat RIG-Is were conserved in their sequence and domain organization, and similar to human RIG-I in (i) mediating virus- and IFN-activated gene expression, (ii) antiviral signaling, (iii) temperature dependence, and (iv) recognition of RNA ligands. Moreover, RIG-I of Rousettus aegyptiacus (suborder Yinpterochiroptera) and of humans were found to recognize SARS-CoV-2 infection. Thus, members of both bat suborders encode RIG-Is that are comparable to their human counterpart. The ability of bats to harbor zoonotic viruses therefore seems due to other features.
Assuntos
Quirópteros , Receptores do Ácido Retinoico , SARS-CoV-2 , Animais , Humanos , Quirópteros/metabolismo , COVID-19 , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , SARS-CoV-2/fisiologia , Vírus , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismoRESUMO
We found previously that nuclear receptors (NRs) compete for heterodimerization with their common partner, retinoid X receptor (RXR), in a ligand-dependent manner. To investigate potential competition in their DNA binding, we monitored the mobility of retinoic acid receptor (RAR) and vitamin D receptor (VDR) in live cells by fluorescence correlation spectroscopy. First, specific agonist treatment and RXR coexpression additively increased RAR DNA binding, while both agonist and RXR were required for increased VDR DNA binding, indicating weaker DNA binding of the VDR/RXR dimer. Second, coexpression of RAR, VDR, and RXR resulted in competition for DNA binding. Without ligand, VDR reduced the DNA-bound fraction of RAR and vice versa, i.e., a fraction of RXR molecules was occupied by the competing partner. The DNA-bound fraction of either RAR or VDR was enhanced by its own and diminished by the competing NR's agonist. When treated with both ligands, the DNA-bound fraction of RAR increased as much as due to its own agonist, whereas that of VDR increased less. RXR agonist also increased DNA binding of RAR at the expense of VDR. In summary, competition between RAR and VDR for RXR is also manifested in their DNA binding in an agonist-dependent manner: RAR dominates over VDR in the absence of agonist or with both agonists present. Thus, side effects of NR-ligand-based (retinoids, thiazolidinediones) therapies may be ameliorated by other NR ligands and be at least partly explained by reduced DNA binding due to competition. Our results also complement the model of NR action by involving competition both for RXR and for DNA sites.
Assuntos
Receptores de Calcitriol , Receptores do Ácido Retinoico , Receptores X de Retinoides , DNA/metabolismo , Ligantes , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Receptores Citoplasmáticos e Nucleares , Receptores X de Retinoides/química , Receptores X de Retinoides/metabolismo , Tretinoína/farmacologia , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismoRESUMO
BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.
Assuntos
Receptores de Glucocorticoides , Receptores do Ácido Retinoico , Animais , DNA , Dimerização , Humanos , Cetosteroides , Ligantes , Filogenia , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Glucocorticoides/genética , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/química , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismoRESUMO
The screening of compounds with endocrine disrupting effects has been attracting increasing attention due to the continuous release of emerging chemicals into the environment. Testing the (ant)agonistic activities of these chemicals on the retinoic acid receptor α (RARα), a vital nuclear receptor, is necessary to explain their perturbation in the endocrine system in vivo. In the present study, MCF-7 breast carcinoma cells were transiently transfected with a RARα expression vector (pEF1α-RARα-RFP) and a reporter vector containing a retinoic acid reaction element (pRARE-TA-Luc). Under optimized conditions, the performance of the newly constructed system was evaluated for its feasibility in screening the (ant)agonistic effects of emerging phenolic compounds on RARα. The results showed that this transient transfection cell model responded well to stimulation with (ant)agonists of RARα, and the EC50 and IC50 values were 0.87 nM and 2.67 µM for AM580 and Ro41-5253, respectively. Its application in testing several emerging phenolic compounds revealed that triclosan (TCS) and tetrabromobisphenol A (TBBPA) exerted notable RARα antagonistic activities. This newly developed bioassay based on MCF-7 is promising in identifying the agonistic or antagonistic activities of xenobiotics on RARα and has good potential for studying RARα signaling-involved toxicological effects of emerging chemicals of concern.
Assuntos
Formigas , Neoplasias da Mama , Animais , Bioensaio , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Detecção Precoce de Câncer , Feminino , Humanos , Células MCF-7 , Fenóis/toxicidade , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , TransfecçãoRESUMO
The use of light-responsive proteins to control both living or synthetic cells, is at the core of the expanding fields of optogenetics and synthetic biology. It is thus apparent that a richer reaction toolbox for the preparation of such systems is of fundamental importance. Here, we provide a proof-of-principle demonstration that Morita-Baylis-Hillman adducts can be employed to perform a facile site-specific, irreversible and diastereoselective click-functionalization of a lysine residue buried into a lipophilic binding pocket and yielding an unnatural chromophore with an extended π-system. In doing so we effectively open the path to the inâ vitro preparation of a library of synthetic proteins structurally reminiscent of xanthopsin eubacterial photoreceptors. We argue that such a library, made of variable unnatural chromophores inserted in an easy-to-mutate and crystallize retinoic acid transporter, significantly expand the scope of the recently introduced rhodopsin mimics as both optogenetic and "lab-on-a-molecule" tools.
Assuntos
Receptores do Ácido Retinoico/metabolismo , Rodopsina/metabolismo , Química Click , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Receptores do Ácido Retinoico/química , Rodopsina/química , EstereoisomerismoRESUMO
Cytochrome P450 27C1 (P450 27C1) is a retinoid desaturase expressed in the skin that catalyzes the formation of 3,4-dehydroretinoids from all-trans retinoids. Within the skin, retinoids are important regulators of proliferation and differentiation. In vivo, retinoids are bound to cellular retinol-binding proteins (CRBPs) and cellular retinoic acid-binding proteins (CRABPs). Interaction with these binding proteins is a defining characteristic of physiologically relevant enzymes in retinoid metabolism. Previous studies that characterized the catalytic activity of human P450 27C1 utilized a reconstituted in vitro system with free retinoids. However, it was unknown whether P450 27C1 could directly interact with holo-retinoid-binding proteins to receive all-trans retinoid substrates. To assess this, steady-state kinetic assays were conducted with free all-trans retinoids and holo-CRBP-1, holo-CRABP-1, and holo-CRABP-2. For holo-CRBP-1 and holo-CRABP-2, the kcat/Km values either decreased 5-fold or were equal to the respective free retinoid values. The kcat/Km value for holo-CRABP-1, however, decreased â¼65-fold in comparison with reactions with free all-trans retinoic acid. These results suggest that P450 27C1 directly accepts all-trans retinol and retinaldehyde from CRBP-1 and all-trans retinoic acid from CRABP-2, but not from CRABP-1. A difference in substrate channeling between CRABP-1 and CRABP-2 was also supported by isotope dilution experiments. Analysis of retinoid transfer from holo-CRABPs to P450 27C1 suggests that the decrease in kcat observed in steady-state kinetic assays is due to retinoid transfer becoming rate-limiting in the P450 27C1 catalytic cycle. Overall, these results illustrate that, like the CYP26 enzymes involved in retinoic acid metabolism, P450 27C1 interacts with cellular retinoid-binding proteins.
Assuntos
Família 27 do Citocromo P450/química , Receptores do Ácido Retinoico/química , Retinoides/química , Proteínas Celulares de Ligação ao Retinol/química , Família 27 do Citocromo P450/metabolismo , Humanos , Receptores do Ácido Retinoico/metabolismo , Retinoides/metabolismo , Proteínas Celulares de Ligação ao Retinol/metabolismoRESUMO
Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) form heterodimers that activate target gene transcription by recruiting co-activator complexes in response to ligand binding. The nuclear receptor (NR) co-activator TIF2 mediates this recruitment by interacting with the ligand-binding domain (LBD) of NRs trough the nuclear receptor interaction domain (TIF2NRID) containing three highly conserved α-helical LxxLL motifs (NR-boxes). The precise binding mode of this domain to RXR/RAR is not clear due to the disordered nature of TIF2. Here we present the structural characterization of TIF2NRID by integrating several experimental (NMR, SAXS, Far-UV CD, SEC-MALS) and computational data. Collectively, the data are in agreement with a largely disordered protein with partially structured regions, including the NR-boxes and their flanking regions, which are evolutionary conserved. NMR and X-ray crystallographic data on TIF2NRID in complex with RXR/RAR reveal a multisite binding of the three NR-boxes as well as an active role of their flanking regions in the interaction.
Assuntos
Coativador 2 de Receptor Nuclear/química , Coativador 2 de Receptor Nuclear/metabolismo , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/química , Receptores X de Retinoides/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Polarização de Fluorescência , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Ligantes , Camundongos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
A detailed understanding of the interactions between small-molecule ligands and their proposed binding targets is of the utmost importance for modern drug-development programs. Cellular retinoic acid-binding proteins I and II (CRABPI and CRABPII) facilitate a number of vital retinoid signalling pathways in mammalian cells and offer a gateway to manipulation of signalling that could potentially reduce phenotypes in serious diseases, including cancer and neurodegeneration. Although structurally very similar, the two proteins possess distinctly different biological functions, with their signalling influence being exerted through both genomic and nongenomic pathways. In this article, crystal structures are presented of the L29C mutant of Homo sapiens CRABPI in complex with naturally occurring fatty acids (1.64â Å resolution) and with the synthetic retinoid DC645 (2.41â Å resolution), and of CRABPII in complex with the ligands DC479 (1.80â Å resolution) and DC645 (1.71â Å resolution). DC645 and DC479 are two potential drug compounds identified in a recent synthetic retinoid development program. In particular, DC645 has recently been shown to have disease-modifying capabilities in neurodegenerative disease models by activating both genomic and nongenomic signalling pathways. These co-crystal structures demonstrate a canonical binding behaviour akin to that exhibited with all-trans-retinoic acid and help to explain how the compounds are able to exert an influence on part of the retinoid signalling cascade.
Assuntos
Receptores do Ácido Retinoico , Retinoides/metabolismo , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Doenças Neurodegenerativas/metabolismo , Ligação Proteica , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Relação Estrutura-AtividadeRESUMO
Activated by retinoids, metabolites of vitamin A, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) play important roles in a wide variety of biological processes, including embryo development, homeostasis, cell proliferation, differentiation and death. In this review, we summarized the functional roles of nuclear receptor RAR/RXR heterodimers in liver physiology. Specifically, RAR/RXR modulate the synthesis and metabolism of lipids and bile acids in hepatocytes, regulate cholesterol transport in macrophages, and repress fibrogenesis in hepatic stellate cells. We have also listed the specific genes that carry these functions and how RAR/RXR regulate their expression in liver cells, providing a mechanistic view of their roles in liver physiology. Meanwhile, we pointed out many questions regarding the detailed signaling of RAR/RXR in regulating the expression of liver genes, and hope future studies will address these issues.
Assuntos
Regulação da Expressão Gênica , Fígado/fisiologia , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/metabolismo , Animais , Humanos , Fígado/citologia , Fígado/metabolismo , Multimerização Proteica , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides/química , Receptores X de Retinoides/genética , Transdução de SinaisAssuntos
Antineoplásicos/farmacologia , Família 26 do Citocromo P450/genética , Resistencia a Medicamentos Antineoplásicos/genética , Retinoides/farmacologia , Tretinoína/farmacologia , Animais , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)mobility by fluorescence (cross-)correlation spectroscopy (F(C)CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation and facilitates comapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed, and an extension of the molecular switch model of the nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR-RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. The RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.
Assuntos
DNA/química , Receptores do Ácido Retinoico/química , Receptores X de Retinoides/química , Sítios de Ligação , Dimerização , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Microscopia de Fluorescência , Receptores do Ácido Retinoico/agonistas , Células Tumorais CultivadasRESUMO
Uveal coloboma is a potentially blinding congenital ocular malformation caused by the failure of optic fissure closure during the fifth week of human gestation. We performed custom capture high-throughput screening of 38 known coloboma-associated genes in 66 families. Suspected causative novel variants were identified in TFAP2A and CHD7, as well as two previously reported variants of uncertain significance in RARB and BMP7. The variant in RARB, unlike previously reported disease mutations in the ligand-binding domain, was a missense change in the highly conserved DNA-binding domain predicted to affect the protein's DNA-binding ability. In vitro studies revealed lower steady-state protein levels, reduced transcriptional activity, and incomplete nuclear localization of the mutant RARB protein compared with wild-type. Zebrafish studies showed that human RARB messenger RNA partially reduced the ocular phenotype caused by morpholino knockdown of rarga gene, a zebrafish homolog of human RARB. Our study indicates that sequence alterations in known coloboma genes account for a small percentage of coloboma cases and that mutations in the RARB DNA-binding domain could result in human disease.
Assuntos
Coloboma/diagnóstico , Coloboma/genética , Proteínas de Ligação a DNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Domínios e Motivos de Interação entre Proteínas , Receptores do Ácido Retinoico/metabolismo , Adulto , Animais , Criança , Análise Mutacional de DNA , Proteínas de Ligação a DNA/química , Feminino , Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Células HEK293 , Humanos , Lactente , Masculino , Modelos Moleculares , Linhagem , Fenótipo , Receptores do Ácido Retinoico/química , Relação Estrutura-Atividade , Peixe-ZebraRESUMO
Cellular retinoic acid-binding protein 2 (CRABP2) delivers all-trans retinoic acid (atRA) to retinoic acid receptors (RARs), allowing for the activation of specific gene transcription. The structural similarities between free and atRA-bound CRABP2 raise the questions of how atRA binding occurs and how the atRA:CRABP2 complex is recognized by downstream binding partners. Thus, to gain insights into these questions, we conducted a detailed atRA-CRABP2 interaction study using nuclear magnetic resonance spectroscopy. The data showed that free CRABP2 displays widespread intermediate-time scale dynamics that is effectively suppressed upon atRA binding. This effect is mirrored by the fast-time scale dynamics of CRABP2. Unexpectedly, CRABP2 rigidification in response to atRA binding leads to the stabilization of a homodimerization interface, which encompasses residues located on helix α2 and the ßC-ßD loop as well as residues on strands ßI-ßA and the ßH-ßI loop. Critically, this rigidification also affects CRABP2's nuclear localization signal and RAR-binding motif, suggesting that the loss of conformational entropy upon atRA binding may be the key for the diverse cellular functions of CRABP2.
Assuntos
Multimerização Proteica , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Tretinoína/química , Tretinoína/metabolismo , Núcleo Celular/metabolismo , Cristalização , Entropia , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Ligação Proteica , Estrutura Secundária de Proteína , Receptores do Ácido Retinoico/genéticaRESUMO
The rapidly accelerated fibrosarcoma (Raf) kinase is canonically activated by growth factors that regulate multiple cellular processes. In this kinase cascade Raf activation ultimately results in extracellular regulated kinase 1/2 (Erk1/2) activation, which requires Ras binding to the Ras binding domain (RBD) of Raf. We recently reported that all-trans retinoic acid (atRA) rapidly (within minutes) activates Erk1/2 to modulate cell cycle progression in stem cells, which is mediated by cellular retinoic acid binding protein 1 (Crabp1). But how atRA-bound Crabp1 regulated Erk1/2 activity remained unclear. We now report Raf kinase as the direct target of atRA-Crabp1. Molecularly, Crabp1 acts as a novel atRA-inducible scaffold protein for Raf/Mek/Erk in cells without growth factor stimulation. However, Crabp1 can also compete with Ras for direct interaction with the RBD of Raf, thereby negatively modulating growth factor-stimulated Raf activation, which can be enhanced by atRA binding to Crabp1. NMR heteronuclear single quantum coherence (HSQC) analyses reveal the 6-strand ß-sheet face of Crabp1 as its Raf-interaction surface. We identify a new atRA-mimicking and Crabp1-selective compound, C3, that can also elicit such an activity. This study uncovers a new signal crosstalk between endocrine (atRA-Crabp1) and growth factor (Ras-Raf) pathways, providing evidence for atRA-Crabp1 as a novel modulator of cell growth. The study also suggests a new therapeutic strategy by employing Crabp1-selective compounds to dampen growth factor stimulation while circumventing RAR-mediated retinoid toxicity.
Assuntos
Receptores do Ácido Retinoico/metabolismo , Transdução de Sinais , Quinases raf/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Camundongos , Ligação Proteica , Conformação Proteica em Folha beta , Receptores do Ácido Retinoico/química , Tretinoína/análogos & derivados , Tretinoína/metabolismo , Quinases raf/químicaRESUMO
Retinoic acid (RA), an active derivative of vitamin A, is critical for the neural system development. During the neural development, the RA/RA receptor (RAR) pathway suppresses BMP signaling-mediated proliferation and differentiation of neural progenitor cells. However, how the stability of RAR is regulated during neural system development and how BMP pathway genes expression in neural tissue from human fetuses affected with neural tube defects (NTDs) remain elusive. Here, we report that FBXO30 acts as an E3 ubiquitin ligase and targets RARγ for ubiquitination and proteasomal degradation. In this way, FBXO30 positively regulates BMP signaling in mammalian cells. Moreover, RA treatment leads to suppression of BMP signaling by reducing the level of FBXO30 in mammalian cells and in mouse embryos with NTDs. In samples from human NTDs with high levels of retinol, downregulation of BMP target genes was observed, along with aberrant FBXO30 levels. Collectively, our results demonstrate that RARγ levels are controlled by FBXO30-mediated ubiquitination and that FBXO30 is a key regulator of BMP signaling. Furthermore, we suggest a novel mechanism by which high-retinol levels affect the level of FBXO30, which antagonizes BMP signaling during early stage development.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteínas F-Box/metabolismo , Defeitos do Tubo Neural/metabolismo , Receptores do Ácido Retinoico/metabolismo , Animais , Proteína Morfogenética Óssea 2/antagonistas & inibidores , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/farmacologia , Modelos Animais de Doenças , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Proteínas F-Box/genética , Feminino , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Células HEK293 , Humanos , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/genética , Ligação Proteica , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Transdução de Sinais/genética , Tretinoína/farmacologia , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/genética , Receptor gama de Ácido RetinoicoRESUMO
Retinoid acid receptor (RAR)-dependent signalling pathways are essential for the regulation and maintenance of essential biological functions and are recognized targets of disruptive anthropogenic compounds. Recent studies put forward the inability of mollusc RARs to bind and respond to the canonical vertebrate ligand, retinoic acid: a feature that seems to have been lost during evolution. Yet, these studies were carried out in a limited number of molluscs. Therefore, using an in vitro transactivation assay, the present work aimed to characterize phylogenetically relevant mollusc RARs, as monomers or as functional units with RXR, not only in the presence of vertebrate bone fine ligands but also known endocrine disruptors, described to modulate retinoid-dependent pathways. In general, none of the tested mollusc RARs were able to activate reporter gene transcription when exposed to retinoic acid isomers, suggesting that the ability to respond to retinoic acid was lost across molluscs. Similarly, the analysed mollusc RAR were unresponsive towards organochloride pesticides. In contrast, transcriptional repressions were observed with the RAR/RXR unit upon exposure to retinoids or RXR-specific ligands. Loss-of-function and gain-of-function mutations further corroborate the obtained results and suggest that the repressive behaviour, observed with mollusc and human RAR/RXR heterodimers, is possibly mediated by ligand biding to RXR.
Assuntos
Disruptores Endócrinos/toxicidade , Evolução Molecular , Moluscos/genética , Moluscos/metabolismo , Receptores do Ácido Retinoico/metabolismo , Sequência de Aminoácidos , Animais , Genes Reporter , Humanos , Luciferases/metabolismo , Mutação/genética , Filogenia , Multimerização Proteica , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/química , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/genética , Tretinoína/farmacologia , Poluentes Químicos da Água/toxicidadeRESUMO
For decades, retinoids and their synthetic derivatives have been well established anticancer treatments due to their ability to regulate cell growth and induce cell differentiation and apoptosis. Many studies have reported the promising role of retinoids in attaining better outcomes for adult or pediatric patients suffering from several types of cancer, especially acute myeloid leukemia and neuroblastoma. However, even this promising differentiation therapy has some limitations: retinoid toxicity and intrinsic or acquired resistance have been observed in many patients. Therefore, the identification of molecular markers that predict the therapeutic response to retinoid treatment is undoubtedly important for retinoid use in clinical practice. The purpose of this review is to summarize the current knowledge on candidate markers, including both genetic alterations and protein markers, for retinoid resistance and sensitivity in human malignancies.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias/tratamento farmacológico , Retinoides/farmacologia , Retinoides/uso terapêutico , Animais , Antineoplásicos Fitogênicos/química , Biomarcadores , Diferenciação Celular/efeitos dos fármacos , Ensaios Clínicos como Assunto , Gerenciamento Clínico , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/metabolismo , Prognóstico , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/metabolismo , Retinoides/química , Resultado do TratamentoRESUMO
The folding of predominantly ß-sheet proteins is complicated by the presence of a large number of non-local interactions in their native states, which increase the ruggedness of their folding energy landscapes. However, forming non-local contacts early in folding or even in the unfolded state can smooth the energy landscape and facilitate productive folding. We report that several sequence regions of a ß-barrel protein, cellular retinoic acid-binding protein 1 (CRABP1), populate native-like secondary structure to a significant extent in the denatured state in 8 M urea. In addition, we provide evidence for both local and non-local interactions in the denatured state of CRABP1. NMR chemical shift perturbations (CSPs) under denaturing conditions upon substitution of single residues by mutation support the presence of several non-local interactions in topologically key sites, arguing that the denatured state is conformationally restricted and contains topological information for the native fold. Among the most striking non-local interactions are those between the N- and C-terminal regions, which are involved in closure of the native ß-barrel. In addition, CSPs support the presence of two features in the denatured state: a major hydrophobic cluster involving residues from various parts of the sequence and a native-like interaction similar to one identified in previous studies as forming early in folding (Budyak et al., Structure 21, 476 [2013]). Taken together, our data support a model in which transient structures involving nonlocal interactions prime early folding interactions in CRABP1, determine its barrel topology, and may protect this predominantly ß-sheet protein against aggregation.
Assuntos
Receptores do Ácido Retinoico/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Agregados Proteicos , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
Allostery tweaks innumerable biological processes and plays a fundamental role in human disease and drug discovery. Exploration of allostery has thus been regarded as a crucial requirement for research on biological mechanisms and the development of novel therapeutics. Here, based on our previously developed allosteric data and methods, we present an interactive platform called AlloFinder that identifies potential endogenous or exogenous allosteric modulators and their involvement in human allosterome. AlloFinder automatically amalgamates allosteric site identification, allosteric screening and allosteric scoring evaluation of modulator-protein complexes to identify allosteric modulators, followed by allosterome mapping analyses of predicted allosteric sites and modulators in human proteome. This web server exhibits prominent performance in the reemergence of allosteric metabolites and exogenous allosteric modulators in known allosteric proteins. Specifically, AlloFinder enables identification of allosteric metabolites for metabolic enzymes and screening of potential allosteric compounds for disease-related targets. Significantly, the feasibility of AlloFinder to discover allosteric modulators was tested in a real case of signal transduction and activation of transcription 3 (STAT3) and validated by mutagenesis and functional experiments. Collectively, AlloFinder is expected to contribute to exploration of the mechanisms of allosteric regulation between metabolites and metabolic enzymes, and to accelerate allosteric drug discovery. The AlloFinder web server is freely available to all users at http://mdl.shsmu.edu.cn/ALF/.