RESUMEN
A common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.
Asunto(s)
Dimerización , Ingeniería Genética/métodos , Integrasas/genética , Recombinación Genética , Animales , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Compartimento Celular , Criptocromos/genética , Expresión Génica , Células HEK293 , Humanos , Integrasas/biosíntesis , Integrasas/metabolismo , Luz , RatonesRESUMEN
Synthetic regulation of gene expression provides a powerful approach to reprogram molecular and cellular processes and test the function of specific genes and gene products. In the last decade, optogenetic systems that allow light-dependent gene regulation have become valuable tools, providing tight spatiotemporal control of protein levels. Here we discuss and build on recent optogenetic approaches for regulating gene expression in mammalian cells using cryptochrome 2 (CRY2), a photoreceptor protein from Arabidopsis. We provide detailed protocols for using light to manipulate activity of a CRY2-based engineered photoactivatable Cre DNA recombinase, and to induce or disrupt transcription factor function. In addition, we provide instructions and software for building an inexpensive Rasberry-Pi-based programable LED device for optogenetic experiments, delivering pulsed light with customized control of illumination duration, frequency, and intensity.
Asunto(s)
Proteínas de Arabidopsis/genética , Criptocromos/genética , Regulación de la Expresión Génica/efectos de la radiación , Optogenética/métodos , Factores de Transcripción/metabolismo , Proteínas de Arabidopsis/metabolismo , Criptocromos/metabolismo , Genes Reporteros/genética , Células HEK293 , Humanos , Integrasas/metabolismo , Luz , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Optogenética/instrumentación , Programas InformáticosRESUMEN
Regulated secretion is critical for diverse biological processes ranging from immune and endocrine signaling to synaptic transmission. Botulinum and tetanus neurotoxins, which specifically proteolyze vesicle fusion proteins involved in regulated secretion, have been widely used as experimental tools to block these processes. Genetic expression of these toxins in the nervous system has been a powerful approach for disrupting neurotransmitter release within defined circuitry, but their current utility in the brain and elsewhere remains limited by lack of spatial and temporal control. Here we engineered botulinum neurotoxin B so that it can be activated with blue light. We demonstrate the utility of this approach for inducibly disrupting excitatory neurotransmission, providing a first-in-class optogenetic tool for persistent, light-triggered synaptic inhibition. In addition to blocking neurotransmitter release, this approach will have broad utility for conditionally disrupting regulated secretion of diverse bioactive molecules, including neuropeptides, neuromodulators, hormones, and immune molecules. VIDEO ABSTRACT.
Asunto(s)
Toxinas Botulínicas/farmacología , Optogenética/métodos , Transmisión Sináptica/efectos de los fármacos , Animales , Proteínas de Arabidopsis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Toxinas Botulínicas/genética , Toxinas Botulínicas/efectos de la radiación , Caenorhabditis elegans , Células Cultivadas , Criptocromos/genética , Femenino , Células HEK293 , Humanos , Luz , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/efectos de la radiación , Proteínas SNARE/metabolismo , Sinapsis/metabolismo , Sinapsis/fisiología , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN-SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.
Asunto(s)
Miocitos del Músculo Liso/fisiología , Fosfohidrolasa PTEN/metabolismo , Factor de Respuesta Sérica/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Músculo Liso Vascular/citología , Fosfohidrolasa PTEN/genética , Ratas , Ratas Sprague-Dawley , Factor de Respuesta Sérica/genéticaRESUMEN
Lysine residues are subject to a multitude of reversible post-translational modifications, including acetylation and SUMOylation. In the heart, enhancement of lysine acetylation or SUMOylation using histone deacetylase (HDAC) inhibitors or SUMO-1 gene transfer, respectively, has been shown to be cardioprotective. Here, we addressed whether there is crosstalk between lysine acetylation and SUMOylation in the heart. Treatment of cardiomyocytes and cardiac fibroblasts with pharmacological inhibitors of HDAC catalytic activity robustly increased conjugation of SUMO-1, but not SUMO-2/3, to several high molecular weight proteins in both cell types. The use of a battery of selective HDAC inhibitors and short hairpin RNAs demonstrated that HDAC2, which is a class I HDAC, is the primary HDAC isoform that controls cardiac protein SUMOylation. HDAC inhibitors stimulated protein SUMOylation in the absence of de novo gene transcription or protein synthesis, revealing a post-translational mechanism of HDAC inhibitor action. HDAC inhibition did not suppress the activity of de-SUMOylating enzymes, suggesting that increased protein SUMOylation in HDAC inhibitor-treated cells is due to stimulation of SUMO-1 conjugation rather than blockade of SUMO-1 cleavage. Consistent with this, multiple components of the SUMO conjugation machinery were capable of being acetylated in vitro. These findings reveal a novel role for reversible lysine acetylation in the control of SUMOylation in the heart, and suggest that cardioprotective actions of HDAC inhibitors are in part due to stimulation of protein SUMO-1-ylation in myocytes and fibroblasts.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Miocitos Cardíacos/metabolismo , Proteína SUMO-1/metabolismo , Sumoilación/efectos de los fármacos , Acetilación/efectos de los fármacos , Animales , Animales Recién Nacidos , Biocatálisis/efectos de los fármacos , Endopeptidasas/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HEK293 , Histona Desacetilasas/metabolismo , Humanos , Lisina/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Ratas Sprague-DawleyRESUMEN
The Ras-like GTPases RalA and RalB are important drivers of tumour growth and metastasis. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here we used protein structure analysis and virtual screening to identify drug-like molecules that bind to a site on the GDP-bound form of Ral. The compounds RBC6, RBC8 and RBC10 inhibited the binding of Ral to its effector RALBP1, as well as inhibiting Ral-mediated cell spreading of murine embryonic fibroblasts and anchorage-independent growth of human cancer cell lines. The binding of the RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasmon resonance and (1)H-(15)N transverse relaxation-optimized spectroscopy (TROSY) NMR spectroscopy. RBC8 and BQU57 show selectivity for Ral relative to the GTPases Ras and RhoA and inhibit tumour xenograft growth to a similar extent to the depletion of Ral using RNA interference. Our results show the utility of structure-based discovery for the development of therapeutics for Ral-dependent cancers.
Asunto(s)
Ensayos de Selección de Medicamentos Antitumorales , Terapia Molecular Dirigida , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas de Unión al GTP ral/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Simulación por Computador , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Ratones , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/metabolismo , Neoplasias/patología , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión al GTP ral/química , Proteínas de Unión al GTP ral/metabolismo , Proteínas ras/metabolismoRESUMEN
Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein-DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein-DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.
Asunto(s)
Cromatografía de Fase Inversa/métodos , ADN/aislamiento & purificación , ADN/química , Secuencias Invertidas RepetidasRESUMEN
Bacteria isolated from marine sponges, including the Silicibacter-Ruegeria (SR) subgroup of the Roseobacter clade, produce N-acylhomoserine lactone (AHL) quorum sensing signal molecules. This study is the first detailed analysis of AHL quorum sensing in sponge-associated bacteria, specifically Ruegeria sp. KLH11, from the sponge Mycale laxissima. Two pairs of luxR and luxI homologues and one solo luxI homologue were identified and designated ssaRI, ssbRI and sscI (sponge-associated symbiont locus A, B and C, luxR or luxI homologue). SsaI produced predominantly long-chain 3-oxo-AHLs and both SsbI and SscI specified 3-OH-AHLs. Addition of exogenous AHLs to KLH11 increased the expression of ssaI but not ssaR, ssbI or ssbR, and genetic analyses revealed a complex interconnected arrangement between SsaRI and SsbRI systems. Interestingly, flagellar motility was abolished in the ssaI and ssaR mutants, with the flagellar biosynthesis genes under strict SsaRI control, and active motility only at high culture density. Conversely, ssaI and ssaR mutants formed more robust biofilms than wild-type KLH11. AHLs and the ssaI transcript were detected in M. laxissima extracts, suggesting that AHL signalling contributes to the decision between motility and sessility and that it may also facilitate acclimation to different environments that include the sponge host.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Poríferos/microbiología , Percepción de Quorum/fisiología , Roseobacter/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Percepción de Quorum/genética , Roseobacter/genética , Roseobacter/crecimiento & desarrollo , Transducción de SeñalRESUMEN
Acyl-homoserine lactone (AHL) quorum sensing controls gene expression in hundreds of Proteobacteria including a number of plant and animal pathogens. Generally, the AHL receptors are members of a family of related transcription factors, and although they have been targets for development of antivirulence therapeutics there is very little structural information about this class of bacterial receptors. We have determined the structure of the transcription factor, QscR, bound to N-3-oxo-dodecanoyl-homoserine lactone from the opportunistic human pathogen Pseudomonas aeruginosa at a resolution of 2.55 Å. The ligand-bound QscR is a dimer with a unique symmetric "cross-subunit" arrangement containing multiple dimerization interfaces involving both domains of each subunit. The QscR dimer appears poised to bind DNA. Predictions about signal binding and dimerization contacts were supported by studies of mutant QscR proteins in vivo. The acyl chain of the AHL is in close proximity to the dimerization interfaces. Our data are consistent with an allosteric mechanism of signal transmission in the regulation of DNA binding and thus virulence gene expression.