Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.
SummaryFor kinase inhibitors, intracellular target selectivity is fundamental to pharmacological mechanism. Although a number of acellular techniques have been developed to measure kinase binding or enzymatic inhibition, such approaches can fail to accurately predict engagement in cells. Here we report the application of an energy transfer technique that enabled the first broad-spectrum, equilibrium-based approach to quantitatively profile target occupancy and compound affinity in live cells. Using this method, we performed a selectivity profiling for clinically relevant kinase inhibitors against 178 full-length kinases, and a mechanistic interrogation of the potency offsets observed between cellular and biochemical analysis. For the multikinase inhibitor crizotinib, our approach accurately predicted cellular potency and revealed improved target selectivity compared with biochemical measurements. Due to cellular ATP, a number of putative crizotinib targets are unexpectedly disengaged in live cells at a clinically relevant drug dose.
The therapeutic action of drugs is predicated on their physical engagement with cellular targets. Here we describe a broadly applicable method using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets within intact cells. Cell-permeable fluorescent tracers are used in a competitive binding format to quantify drug engagement with the target proteins fused to Nanoluc luciferase. The approach enabled us to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. Our analysis was directed particularly to the clinically approved prodrug FK228 (Istodax/Romidepsin) because of its unique and largely unexplained mechanism of sustained intracellular action. Analysis of the binding kinetics by BRET revealed remarkably long intracellular residence times for FK228 at HDAC1, explaining the protracted intracellular behaviour of this prodrug. Our results demonstrate a novel application of BRET for assessing target engagement within the complex milieu of the intracellular environment.
Mutations in Saccharomyces cerevisiae have been identified that derepress early meiotic genes functioning in separable pathways required for normal meiotic development. The phenotypes of these ume (unscheduled meiotic gene expression) mutations suggest that their wild-type alleles encode negative regulators acting downstream of both the celltype and nutritional controls of meiosis. These newly defined loci do not affect either general transcription or transcription of meiotic genes expressed later in meiosis and spore formation.Initiation of meiosis in Saccharomyces cerevisiae is under the control of two independent, convergent regulatory pathways, one responding to cell type and the other sensing nutritional status (1-3). The cell-type pathway operates through a transcriptional regulatory cascade in which the products of the MATa and MATa loci combine to form a negative regulator (4, 5) that inhibits the expression of RMEI (6, 7), which encodes a repressor of meiosis. RMEJ, in turn, negatively regulates IMEI, an inducer of meiosis (8), which positively regulates IME2 (9). Overexpression of either IMEI or IME2 allows meiotic functions to be expressed during mitosis (8, 9).The nutritional pathway senses glucose and nitrogen deprivation and involves a number of well-characterized genes, e.g., ARDI (10), BCYJ, CYR2, and CYR3 (11), and RAS2 (12,13). Evidence that the nutritional and cell-type pathways are initially independent is based on the observation that rmel mutants still require starvation conditions to enter meiosis (6) and, conversely, mutants that interfere with nutritional control, allowing meiosis in rich media, still require both MATa and MATa expression (14). IMEJ is regulated by both cell type and nutritional conditions and represents the first known point at which these pathways converge.The process of meiosis and gamete formation in yeast includes DNA replication, recombination, chromosome segregation at meioses I and II, and spore formation. A number of genes required for these events have been cloned and found to be developmentally regulated; i.e., they exhibit elevated message levels only during sporulation (15)(16)(17). Among these are SP013, a gene required for chromosome segregation at meiosis I (18), SPOIl, a gene involved in recombination (19), and SP016, a gene that affects the efficiency of early prophase events (R. T. Elder and R.E.E., unpublished results). The purpose of this study was to identify trans-acting regulators that directly control the expression of these genes. Our approach was to use a fusion reporter gene to recover regulatory mutations that derepress the mitotic expression of these meiosis-specific genes. Here we report the successful application of this method to meiotic control and the identification of five such trans-acting genes. MATERIALS AND METHODSStrains and Plasmids. Mutants were isolated in RSY10 (S. Frackman, University of Wisconsin-Milwaukee), an ade6 derivative of W303-1A (R. Rothstein, Columbia University College of Physicians and Surgeons): MATa ade...
The yeast Saccharomyces cerevisiae contains three heat-inducible hsp7O genes. We have characterized the promoter region of the hsp70 heat shock gene YGI00, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YGI00 contains multiple elements related to the Drosophia melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YGI00. The upstream activation site (UAS) of the yeast cytochrome c gene (CYCI) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YGI00 sequences that flank UASHs inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.The heat shock response of most cells is characterized by induced synthesis of a small set of proteins after the cells are subjected to temperature upshift. This response was first observed and extensively studied in Drosophila melanogaster and has provided a model system for eucaryotic inducible gene expression. Drosophila heat shock genes are coordinately induced in response to a number of stresses: a rapid shift to high temperature, recovery from anoxia, or exposure to a variety of metabolic inhibitors and chemicals (for reviews, see references 2, 3, 11, and 37). The major inducible gene, hsp70, encodes a 70,000-molecular-weight protein that has been conserved throughout the eucaryotic and procaryotic kingdoms (10). In addition to conservation of the protein-coding region, regulation of hsp70 synthesis in response to heat also appears to have been maintained during evolution (4,23,25).The yeast Saccharomyces cerevisiae contains eight genes (named YGIOO to YG107) related to Drosophila hsp70, three of which are heat inducible (12). The heat-inducible genes can be grouped into two classes based on their expression: (i) YG106 and YG107, which are not expressed significantly before heat shock; and (ii) YGIOO, which has a significant basal level of expression before heat shock.A current model for heat shock induction in eucaryotes is that an activated heat shock transcription factor binds discrete promoter sequences and participates with a factor already bound to the TATA box to form a promoter complex that stimulates RNA polymerase II initiation (39). The promoters of hsp70 and other Drosophila heat shock genes contain multiple copies of an imperfect palindromic sequence element, ...
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