Molecular imaging of homodimeric protein-protein interactions in living subjects.
Homodimeric protein interactions are potent regulators of cellular functions, but are particularly challenging to study in vivo. We used a split synthetic renilla luciferase (hRLUC) complementation-based bioluminescence assay to study homodimerization of herpes simplex virus type 1 thymidine kinase (TK) in mammalian cells and in living mice. We quantified and imaged homodimerization of TK chimeras containing N-terminal (N-hRLUC) or C-terminal (C-hRLUC) fragments of hRLUC in the upstream and downstream positions, respectively (tail-to-head homodimer). This was monitored using luminometry (68-fold increase, and was significantly [P<0.01] above background light emission) and by CCD camera imaging of living mice implanted with ex vivo transfected 293T cells (2.7-fold increase, and is significantly [P<0.01] above background light emission). We also made a mutant-TK to generate N-hRLUC mutant TK and mutant TK-C-hRLUC by changing a single amino acid at position 318 from arginine to cysteine, a key site that has previously been reported to be essential for TK homo-dimerization, to support the specificity of the hRLUC complementation signal from TK homodimerization. Ex vivo substrate (8-3H Penciclovir) accumulation assays in 293T cells expressing the TK protein chimeras showed active TK enzyme. We also devised an experimental strategy by constructing variant TK chimeras (possessing extra N-hRLUC or C-hRLUC 'spacers') to monitor incremental lack of association of the tail-to-head TK homodimer. Application of this potentially generalizable assay to screen for molecules that promote or disrupt ubiquitous homodimeric protein-protein interactions could serve not only as an invaluable tool to understand biological networks but could also be applied to drug discovery and validation in living subjects. (+info)
Monoclonal antibodies assisting refolding of firefly luciferase.
The reactivation efficiency in the refolding of denatured luciferase in the presence and the absence of monoclonal antibodies (mAbs) has been studied. Luciferase could be partially reactivated when the protein was denatured in high concentrations of guanidium chloride (GdmCl; >4.5 M) and the refolding was carried out in very low protein concentrations. The refolding yield was, however, significantly lower when it was performed on luciferase that had been denatured with lower concentrations of GdmCl. The efficiency of refolding decreases when the formation of aggregates increases. Three of the five luciferase mAbs tested (4G3, N2E3, S2G10) dramatically increased the yield of reactivation and simultaneously eliminated the formation of aggregates. It is proposed that these mAbs assisted the refolding of luciferase by binding to the exposed hydrophobic surface of the refolding intermediate, thus preventing it from aggregating. The epitopes interacting with these refolding-assisting mAbs are all located in the A-subdomain of the N-terminal region of luciferase. These results have also shed light on the structural features of the intermediate and its interface involved in protein aggregate formation, contributing to the understanding of the protein folding mechanism. (+info)
Novel bidirectional vector strategy for amplification of therapeutic and reporter gene expression.
Molecular imaging methods have previously been employed to image tissue-specific reporter gene expression by a two-step transcriptional amplification (TSTA) strategy. We have now developed a new bidirectional vector system, based on the TSTA strategy, that can simultaneously amplify expression for both a target gene and a reporter gene, using a relatively weak promoter. We used the synthetic Renilla luciferase (hrl) and firefly luciferase (fl) reporter genes to validate the system in cell cultures and in living mice. When mammalian cells were transiently cotransfected with the GAL4-responsive bidirectional reporter vector and various doses of the activator plasmid encoding the GAL4-VP16 fusion protein, pSV40-GAL4-VP16, a high correlation (r(2) = 0.95) was observed between the expression levels of both reporter genes. Good correlations (r(2) = 0.82 and 0.66, respectively) were also observed in vivo when the transiently transfected cells were implanted subcutaneously in mice or when the two plasmids were delivered by hydrodynamic injection and imaged. This work establishes a novel bidirectional vector approach utilizing the TSTA strategy for both target and reporter gene amplification. This validated approach should prove useful for the development of novel gene therapy vectors, as well as for transgenic models, allowing noninvasive imaging for indirect monitoring and amplification of target gene expression. (+info)
Differential peroxisome proliferator-activated receptor-gamma isoform expression and agonist effects in normal and malignant prostate cells.
Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is being studied intensively for its role in carcinogenesis and in mediating the effects of prostate cancer treatment and prevention drugs. Prostate cancers express abundant and higher constitutive levels of PPAR-gamma than do normal prostate cells and are growth inhibited by ligand activation of PPAR-gamma. However, little is known about the role of PPARs in tumorigenesis or in normal prostate epithelial cells (EC). We examined the expression, phosphorylation patterns, and functions of the human PPAR (hPPAR)-gamma1 and hPPAR-gamma2 isoforms in normal prostate ECs to determine if activation of the receptor is sufficient for PPAR-gamma ligand activity in prostate cells. We found that ECs did not express either PPAR-gamma1 or PPAR-gamma2 protein and were not sensitive to growth inhibition by the PPAR-gamma ligand 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)). In contrast, prostate cancer cells (PC-3), which express PPAR-gamma1 receptor isoform, are growth inhibited by PPAR-gamma ligand. Forced expression of hPPAR-gamma1 or hPPAR-gamma2 made ECs sensitive to 15d-PGJ(2) and led to reduced cellular viability. The direct repeat-1 promoter containing PPAR response elements was transactivated in ECs expressing exogenous PPAR-gamma1 or PPAR-gamma2, indicating that either isoform can be active in these cells. 15-Lipoxygenase-2, expressed at high levels in ECs, was down-regulated by transfecting PPAR-gamma expression construct (either gamma1 or gamma2 isoform) into ECs. Addition of PPAR-gamma ligand 15-hydroxyeicosatetraenoic acid in the presence of PPAR-gamma expression caused further down-regulation of 15-lipoxygenase-2. Our data illustrate that a PPAR-gamma ligand (15d-PGJ(2)) activates PPAR-gamma1 and selectively induces cell death in human prostate cancer cells but not in normal prostate ECs. These findings have important implications for the development of PPAR-gamma-targeting agents that prevent or treat prostate cancer and spare normal prostate cells. (+info)
Inhibition of firefly luciferase by alkane analogues.
We reported that anesthetics increased the partial molal volume of firefly luciferase (FFL), while long-chain fatty acids (LCFA) decreased it. The present study measured the actions of dodecanol (neutral), dodecanoic acid (negatively charged), and dodecylamine (positively charged) hydrophobic molecules on FFL. The interaction modes are measured by (1) ATP-induced bioluminescence of FFL and (2) fluorescence of 2-(p-toluidino)naphthalene-6-sulfonate (TNS). TNS fluoresces brightly in hydrophobic media. It competes with the substrate luciferin on the FFL binding. From the Scatchard plot of TNS titration, the maximum binding number of TNS was 0.83, and its binding constant was 8.27 x 10(5) M(-1). Job's plot also showed that the binding number is 0.89. From the TNS titration of FFL, the binding constant was estimated to be 8.8 x 10(5) M(-1). Dodecanoic acid quenched the TNS fluorescence entirely. Dodecanol quenched about 25% of the fluorescence, whereas dodecylamine increased it. By comparing the fluorescence of TNS and bioluminescence of FFL, the binding modes and the inhibition mechanisms of these dodecane analogues are classified in three different modes: competitive (dodecanoic acid), noncompetitive (dodecylamine), and mixed (dodecanol). (+info)
Regulation of Hsp70 function by HspBP1: structural analysis reveals an alternate mechanism for Hsp70 nucleotide exchange.
HspBP1 belongs to a family of eukaryotic proteins recently identified as nucleotide exchange factors for Hsp70. We show that the S. cerevisiae ortholog of HspBP1, Fes1p, is required for efficient protein folding in the cytosol at 37 degrees C. The crystal structure of HspBP1, alone and complexed with part of the Hsp70 ATPase domain, reveals a mechanism for its function distinct from that of BAG-1 or GrpE, previously characterized nucleotide exchange factors of Hsp70. HspBP1 has a curved, all alpha-helical fold containing four armadillo-like repeats unlike the other nucleotide exchange factors. The concave face of HspBP1 embraces lobe II of the ATPase domain, and a steric conflict displaces lobe I, reducing the affinity for nucleotide. In contrast, BAG-1 and GrpE trigger a conserved conformational change in lobe II of the ATPase domain. Thus, nucleotide exchange on eukaryotic Hsp70 occurs through two distinct mechanisms. (+info)
Single copy shRNA configuration for ubiquitous gene knockdown in mice.
RNA interference through the expression of small hairpin RNA (shRNA) molecules has become a very promising tool in reverse mouse genetics as it may allow inexpensive and rapid gene function analysis in vivo. However, the prerequisites for ubiquitous and reproducible shRNA expression are not well defined. Here we show that a single copy shRNA-transgene can mediate body-wide gene silencing in mice when inserted in a defined locus of the genome. The most commonly used promoters for shRNA expression, H1 and U6, showed a comparably broad activity in this configuration. Taken together, the results define a novel approach for efficient interference with expression of defined genes in vivo. Moreover, we provide a rapid strategy for the production of gene knockdown mice combining recombinase mediated cassette exchange and tetraploid blastocyst complementation approaches. (+info)
Rapid bioassay for the determination of dioxins and dioxin-like PCDFs and PCBs in meat and animal feeds.
Over the past several years, the numerous contamination incidents have raised concerns over the presence of halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and related chemicals in foods and feeds. Here we applied a sensitive recombinant mouse hepatoma cell (H1L1.1c2) bioassay for the determination of dioxins and dioxin-like polychlorinated dibenzofurans (PCDFs) and biphenyls (PCBs) in meat and animal feeds. These cells responded to TCDD-like chemicals with dose-dependent induction of firefly luciferase activity, and the minimal detection limit of TCDD in the cell was 16 fg. Induction equivalency factors determined for pure TCDD-like polychlorinated dibenzo-p-dioxins (PCDDs), PCDFs, and PCBs in the bioassay were well-correlated with the World Health Organization's toxic equivalency factors. To determine the applicability of the bioassay system to detect those compounds presence in meat and feed samples, cell bioassays for 17 TCDD-like PCDDs and PCDFs congeners-spiked lipid extracted from beef or animal feed were performed. Mean recoveries of TCDD-like chlorinated PCDDs and PCDFs congeners from spiked beef or feed fat ranged from 61.2 to 122.3%. Within-laboratory coefficients of variation for analysis as index of precision were lower than 5.2%, and the calculated limits of detection and quantitation were 0.33 and 1 pg toxicity equivalency quantity (TEQ)/0.5 g fat, respectively. Correlation between bioassay- and high-resolution gas chromatography-mass spectrometry (HR-GC-MS)-determined TEQs for 10 meat samples was 0.85, with 1.2 times higher in bioassay than HR-GC-MS. The correlation between bioassay- and HR-GC-MS-determined TEQs in 10 animal feed products was 0.81, with 2.1 times higher in bioassay than HR-GC-MS. Overall, these results demonstrated that the recombinant cell bioassay can be used for the rapid detection and quantitation of PCDDs and dioxin-like PCDFs and PCBs in meats and animal feeds. (+info)