RESUMO
BACKGROUND: Molecular switch systems that activate gene expression by a small molecule are effective technologies that are widely used in applied biological research. Nuclear receptors are valuable candidates for these regulation systems due to their functional role as ligand activated transcription factors. Previously, our group engineered a variant of the retinoid x receptor to be responsive to the synthetic compound, LG335, but not responsive to its natural ligand, 9-cis-retinoic acid. RESULTS: This work focuses on characterizing a molecular switch system that quantitatively controls transgene expression. This system is composed of an orthogonal ligand/nuclear receptor pair, LG335 and GRQCIMFI, along with an artificial promoter controlling expression of a target transgene. GRQCIMFI is composed of the fusion of the DNA binding domain of the yeast transcription factor, Gal4, and a retinoid x receptor variant. The variant consists of the following mutations: Q275C, I310M, and F313I in the ligand binding domain. When introduced into mammalian cell culture, the switch shows luciferase activity at concentrations as low as 100 nM of LG335 with a 6.3 +/- 1.7-fold induction ratio. The developed one-component system activates transgene expression when introduced transiently or virally. CONCLUSIONS: We have successfully shown that this system can induce tightly controlled transgene expression and can be used for transient transfections or retroviral transductions in mammalian cell culture. Further characterization is needed for gene therapy applications.
Assuntos
Regulação da Expressão Gênica , Receptores X de Retinoides/genética , Transfecção/métodos , Transgenes , Alitretinoína , Animais , Humanos , Ligantes , Camundongos , Células NIH 3T3 , Plasmídeos , Regiões Promotoras Genéticas , Receptores X de Retinoides/metabolismo , Tretinoína/metabolismoRESUMO
ESPSearch is a computer program for rapidly identifying nucleic acid or amino acid sequences of any length within any source sequence from promoters to entire genomes to protein libraries. ESPSearch utilizes a user-constructed database to identify many sequences simultaneously, including target sequences with wildcards and mismatches and user-specified patterns of those recognized sequences. Here we use ESPSearch to identify a variety of possible binding sites for dimeric artificial transcription factors within several p53 recognition sites and the promoter of the BAX gene. Heterodimeric and homodimeric proteins are designed using human zinc fingers by identifying groups of zinc finger binding sites meeting particular pattern constraints. ESPSearch is also used to estimate the specificity of each artificial transcription factor by searching the entire genome. Next, the specificity of several possible small interfering RNA (siRNA) sequences is determined by searching both the whole genome and the library of known human mRNAs. Finally, ESPSearch identifies proteins containing different forms of the LXXLL motif used in nuclear receptor-coactivator interactions from the human proteome, making use of user-defined groups of amino acids. ESPSearch could also be applied to other tasks involving sequence and pattern recognition on small and large scales. ESPSearch is freely available at http://web.chemistry.gatech.edu/-doyle/espsearch/.
Assuntos
Sistemas de Gerenciamento de Base de Dados , Bases de Dados Genéticas , Armazenamento e Recuperação da Informação/métodos , Reconhecimento Automatizado de Padrão/métodos , Alinhamento de Sequência/métodos , Análise de Sequência/métodos , Software , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Homologia de SequênciaRESUMO
Entropy-enthalpy (SH) compensation occurs when a small change in DeltaG is caused by large, and nearly compensatory, changes in DeltaH and DeltaS. It is considered a ubiquitous property of reactions in water. Because water is intimately involved in protein stability, SH compensation among protein variants, if it exists, could lead to important knowledge about protein-water interactions. In light of recent theoretical work on SH compensation, we gathered thermodynamic data for >200 protein variants to seek evidence for the simplest quantitative model of SH compensation (i.e., The van't Hoff denaturation enthalpy divided by the van't Hoff denaturation entropy is a constant). We conclude that either the data are insufficient to support the idea that quantitative SH compensation is a general feature of variant proteins or that such compensation does not exist. This study reinforces the idea that DeltaH-versus-DeltaS plots should not be used to provide evidence for SH compensation.
Assuntos
Modelos Teóricos , Proteínas/química , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Entropia , Humanos , Nuclease do Micrococo/química , Nuclease do Micrococo/genética , Muramidase/química , Muramidase/genética , Mutação , Desnaturação Proteica , Ribonuclease T1/química , Ribonuclease T1/genética , TermodinâmicaRESUMO
Hydrogen peroxide (H2O2), produced in living cells by oxidases and by other biochemical reactions, plays an important role in cellular processes such as signaling and cell cycle progression. Nevertheless, H2O2 and other reactive oxygen species are capable of inducing damage to DNA and other cellular components, and oxidative stress caused by overproduction of cellular oxidants has been linked to pathologies such as inflammatory diseases and cancer. Therefore, new approaches for reducing the accumulation of cellular oxidants are of considerable interest from both a biotechnological and a therapeutic perspective. Recognizing that selenium is an essential component of the active sites of several antioxidant enzymes, we have developed a family of novel phenylaminoethyl selenide compounds that are readily taken up into cells and have low toxicity in vivo. We now report chemiluminescent imaging of hydrogen peroxide consumption by phenylaminoethyl selenides, via the use of peroxalate nanoparticle methodology. Further, we demonstrate the ability of phenylaminoethyl selenides to decrease lipopolysaccharide-induced oxidative stress in human embryonic kidney cells. We also report the successful encapsulation of a phenylaminoethyl selenide within poly(lactide-co-glycolide) nanoparticles, and we show that these selenide-loaded nanoparticles exhibit antioxidant activity in cells. Taken together, these results significantly enhance the attractiveness of phenylaminoethyl selenides as potential agents for supplementing cellular defenses against reactive oxygen species.
Assuntos
Antioxidantes/farmacologia , Compostos Organosselênicos/farmacologia , Antioxidantes/metabolismo , Biotecnologia , Etilaminas/farmacologia , Células HEK293 , Humanos , Peróxido de Hidrogênio/metabolismo , Lipopolissacarídeos/farmacologia , Luminescência , Nanopartículas/química , Nanotecnologia , Oxalatos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
We describe a set of novel histone deacetylase inhibitors (HDACi) equipped with either an antagonist or an agonist of the estrogen receptor (ER) to confer selective activity against breast cancers. These bifunctional compounds potently inhibit HDAC at nanomolar concentrations and either agonize or antagonize ERα and ERß. The ER antagonist activities of tamoxifen-HDACi conjugates (Tam-HDACi) are nearly identical to those of tamoxifen. Conversely, ethynyl-estradiol-HDACi conjugates (EED-HDACi) have attenuated ER agonist activities relative to the parent ethynyl-estradiol. In silico docking analysis provides structural basis for the trends of ER agonism/antagonism and ER subtype selectivity. Excitingly, lead Tam-HDACi conjugates show anticancer activity that is selectively more potent against MCF-7 (ERα positive breast cancer) compared to MDA-MB-231 (triple negative breast cancer), DU145 (prostate cancer), or Vero (noncancerous cell line). This dual-targeting approach illustrates the utility of designing small molecules with an emphasis on cell-type selectivity, not merely improved potency, working toward a higher therapeutic index at the earliest stages of drug development.
Assuntos
Moduladores de Receptor Estrogênico/síntese química , Inibidores de Histona Desacetilases/síntese química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Células Cultivadas , Descoberta de Drogas , Moduladores de Receptor Estrogênico/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Simulação de Acoplamento Molecular , Receptores de Estrogênio/agonistas , Receptores de Estrogênio/química , Relação Estrutura-AtividadeRESUMO
Genetic selection systems, such as the yeast two-hybrid system, are efficient methods to detect protein-protein and protein-ligand interactions. These systems have been further developed to assess negative interactions, such as inhibition, using the URA3 genetic selection marker. Previously, chemical complementation was used to assess positive selection in Saccharomyces cerevisiae. In this work, a new S. cerevisiae strain, called BAPJ69-4A, containing three selective markers ADE2, HIS3, and URA3 as well as the lacZ gene controlled by Gal4 response elements, was developed and characterized using the retinoid X receptor (RXR) and its ligand 9-cis retinoic acid (9cRA). Further characterization was performed using RXR variants and the synthetic ligand LG335. To assess the functionality of the strain, RXR was compared to the parent strain PJ69-4A in adenine, histidine, and uracil selective media. In positive selection, associating partners that lead to cell growth were observed in all media in the presence of ligand, whereas partners that did not associate due to the absence of ligand displayed no growth. Conversely, in negative selection, partners that did not associate in 5-FOA medium did not display cell death due to the lack of expression of the URA3 gene. The creation of the BAPJ69-4A yeast strain provides a high-throughput selection system, called negative chemical complementation, which can be used for both positive and negative selection, providing a fast, powerful tool for discovering novel ligand receptor pairs for applications in drug discovery and protein engineering.
Assuntos
Biologia Molecular/métodos , Saccharomyces cerevisiae/genética , Seleção Genética , Técnicas do Sistema de Duplo-Híbrido , Meios de Cultura/química , Humanos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily, involved in calcium and phosphate homeostasis; hence implicated in a number of diseases, such as Rickets and Osteoporosis. This receptor binds 1α,25-dihydroxyvitamin D(3) (also referred to as 1,25(OH)(2)D(3)) and other known ligands, such as lithocholic acid. Specific interactions between the receptor and ligand are crucial for the function and activation of this receptor, as implied by the single point mutation, H305Q, causing symptoms of Type II Rickets. In this work, further understanding of the significant and essential interactions between the ligand and the receptor was deciphered, through a combination of rational and random mutagenesis. A hVDR mutant, H305F, was engineered with increased sensitivity towards lithocholic acid, with an EC(50) value of 10 µM and 40±14 fold activation in mammalian cell assays, while maintaining wild-type activity with 1,25(OH)(2)D(3). Furthermore, via random mutagenesis, a hVDR mutant, H305F/H397Y, was discovered to bind a novel small molecule, cholecalciferol, a precursor in the 1α,25-dihydroxyvitamin D(3) biosynthetic pathway, which does not activate wild-type hVDR. This variant, H305F/H397Y, binds and activates in response to cholecalciferol concentrations as low as 100 nM, with an EC(50) value of 300 nM and 70±11 fold activation in mammalian cell assays. In silico docking analysis of the variant displays a dramatic conformational shift of cholecalciferol in the ligand binding pocket in comparison to the docked analysis of cholecalciferol with wild-type hVDR. This shift is hypothesized to be due to the introduction of two bulkier residues, suggesting that the addition of these bulkier residues introduces molecular interactions between the ligand and receptor, leading to activation with cholecalciferol.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Colecalciferol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Calcitriol/metabolismo , Linhagem Celular , Humanos , Mutagênese , Reação em Cadeia da Polimerase , Engenharia de Proteínas/métodos , Receptores de Calcitriol/química , Receptores de Calcitriol/genéticaRESUMO
Protein and metabolic engineering would greatly benefit from a general system linking the presence of a small molecule to the power of genetic selection. We use nuclear receptors to link the survival of Saccharomyces cerevisiae to the presence of small molecules through genetic selection, extending classical genetic complementation to a new "chemical complementation." In this system the Gal4 DNA-binding domain is fused to ligand-binding domains from two nuclear receptors, expressed in the strain PJ69-4A, and grown on plates containing known ligands for the receptors. Yeast survive on selective plates only in the presence of a nuclear receptor and the corresponding ligand. Mutagenesis can increase the sensitivity of chemical complementation. This system may be extended to engineer nuclear receptors for practically any small molecule through directed evolution coupled to genetic selection, and for performing metabolic engineering in yeast.
Assuntos
Teste de Complementação Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Seleção Genética , Fatores de Transcrição/metabolismo , Antineoplásicos/metabolismo , Antioxidantes/metabolismo , Proteínas de Ligação a DNA , Ligantes , Estrutura Molecular , Mutagênese Sítio-Dirigida , Paclitaxel/metabolismo , Receptor de Pregnano X , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptores X de Retinoides , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Tocoferóis/metabolismo , Fatores de Transcrição/genética , Tretinoína/metabolismoRESUMO
The nuclear receptor retinoid X receptor (RXR) is a ligand-activated transcription factor. To create receptors for a new ligand, a structure-based approach was used to generate a library of approximately 380,000 mutant RXR genes. To discover functional variants within the library, we used chemical complementation, a method of protein engineering that uses the power of genetic selection. Wild-type RXR has an EC50 of 500 nM for 9-cis retinoic acid (9cRA) and an EC50 of >10 microM for the synthetic retinoid-like compound LG335 in yeast. The library produced ligand-receptor pairs with LG335 that have a variety of EC50 values (40 nM to >2 microM) and activation levels (10-80% of wild-type RXR with 9cRA) in yeast. The variant I268V;A272V;I310L;F313M has an EC50 for LG335 of 40 nM and an EC50 for 9cRA of >10 microM in yeast. This variant has essentially the reverse ligand specificity of wild-type RXR and is transcriptionally active at a 10-fold-lower ligand concentration in yeast. This EC50 is 25-fold lower than the best receptor we have engineered through site-directed mutagenesis, Q275C;I310M;F313I. Furthermore, the variants' EC50 values and activation levels in yeast and mammalian cells correlate. This protein engineering method should be extendable to produce other functional ligand-receptor pairs, which can be selected and characterized from libraries within weeks. Coupling large library construction with chemical complementation could be used to engineer proteins that bind virtually any small molecule for conditional gene expression, applications in metabolic engineering, and biosensors and to engineer enzymes through genetic selection.
Assuntos
Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Substituição de Aminoácidos , Animais , Códon/genética , Biblioteca Gênica , Genes Reporter , Variação Genética , Cinética , Ligantes , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Retinoides/metabolismo , Transcrição Gênica , Tretinoína/metabolismoRESUMO
We assessed the cellular localization and relative concentration of the C-type natriuretic peptide (CNP) guanylate cyclase-B (GC-B) receptor in the adult rat heart ventricle by several techniques. In frozen sections of the ventricle, anti-receptor antibody stained the vasculature and cells interstitial to myocytes, but not the myocytes themselves. The same antibody detected GC-B in immunoblots of protein extracts of nonmyocytes, but not myocytes and recognized an equivalent protein in extracts of cultured cardiac fibroblasts, but not A7r5 rat smooth muscle cells. In functional assays, CNP-induced cGMP accumulation per milligram cell protein was an order of magnitude greater in cultured cardiac fibroblasts than in A7r5 smooth muscle cells and two orders of magnitude greater than in freshly isolated cardiac myocytes. Modulation of cGMP accumulation by phosphodiesterases (PDEs) was cell specific as determined by antagonist pharmacological profiles, PDE1 in fibroblasts, PDE2 in A7r5 cells, and PDE3 in myocytes, suggesting that significant but low-level cGMP response to CNP measured in heart myocytes is not due to nonmyocyte contamination. Fibroblasts of cardiac origin do not show an interactive relationship between receptor responsiveness to CNP, cGMP levels, and proliferation-related mitogen-activated signal transduction pathways. Whereas previous reports suggest CNP exerts significant effects in neonatal rat cardiomyocytes, our results suggest that fibroblasts are likely the most responsive cell type (cGMP production) in the adult rat heart.
Assuntos
Adenina/análogos & derivados , Guanilato Ciclase , Miocárdio/química , Receptores do Fator Natriurético Atrial/análise , 1-Metil-3-Isobutilxantina/farmacologia , Adenina/farmacologia , Animais , Células Cultivadas , Centrifugação com Gradiente de Concentração , GMP Cíclico/metabolismo , Fibroblastos/metabolismo , Imunofluorescência , Ventrículos do Coração/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso/metabolismo , Miocárdio/metabolismo , Peptídeo Natriurético Tipo C/farmacologia , Octoxinol/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Fosforilação , Ratos , Receptores do Fator Natriurético Atrial/metabolismo , Distribuição TecidualRESUMO
The presence of caveolin-3 was reinvestigated in smooth muscle cells in situ and in culture. Immunocytochemistry of aortic strips showed that both caveolins-1 and -3 are present, but that caveolin-3 is considerably less abundant and detected in only a fraction of the cells. Cultured smooth muscle cells of passage 4 or greater still expressed caveolin-1 but lacked caveolin-3, likely due to dedifferentiation. Cultures exposed to redifferentiation-inducing medium were found to re-express caveolin-3. The return of caveolin-3 protein in these cells was accompanied by a fourfold increase in cav-3 mRNA, as detected by quantitative PCR, suggesting that the loss of protein in untreated cells was due to transcriptional rather than translational inhibition. These results suggest that caveolin-3 has a specialized role associated with the differentiated state in certain types of smooth muscle cells.