Kathryn E. Luker

Imaging 26S proteasome activity and inhibition in living mice

The ubiquitin-proteasome pathway is the central mediator of regulated proteolysis in cells, and defects in this pathway are associated with cancer and neurodegenerative diseases. To assess 26S proteasome function in living animals, we developed a ubiquitin-luciferase reporter for bioluminescence imaging. The reporter was degraded rapidly under steady-state conditions and stabilized in a dose- and time-dependent manner in response to proteasome inhibitors. Using bioluminescence imaging after one dose of the chemo-therapeutic proteasome inhibitor bortezomib (PS-341), proteasome function in tumor xenografts was blocked within 30 min and returned to nearly baseline by 46 h. After a 2-week regimen of bortezomib, however, imaging of target tumors showed significantly enhanced proteasome inhibition that no longer returned to baseline. The ubiquitin-luciferase reporter enables repetitive tissue-specific analysis of 26S proteasome activity in vivo and should facilitate development and validation of proteasome inhibitors in mouse models, as well as investigations of the ubiquitin-proteasome pathway in disease pathogenesis.

Kinetic analysis of plasmepsins I and II aspartic proteases of the Plasmodium falciparum digestive vacuole.

Plasmepsins I and II are Plasmodium falciparum aspartic proteases implicated in hemoglobin degradation. Using a synthetic fluorogenic peptide substrate based on the initial hemoglobin cleavage site, we have analyzed kinetic parameters of the two enzymes in native and recombinant forms. Both native plasmepsins cleave the model substrate well. Recombinant plasmepsin II behaves similarly to native enzyme, substantiating its usefulness for inhibition and structural studies. In contrast, recombinant plasmepsin I does not resemble its native homolog kinetically. A hybrid molecule, in which the polyproline loop of plasmepsin I has been replaced by the homologous sequence from plasmepsin II, still maintains the specificity/kinetics of plasmepsin II. This suggests that the polyproline loop, important for substrate recognition in the mammalian aspartic protease renin, does not play a similar role in the plasmepsins.

Optimizing luciferase protein fragment complementation for bioluminescent imaging of protein-protein interactions in live cells and animals

Publisher Summary This chapter discusses the optimization of luciferase protein fragment complementation for bioluminescent imaging of protein–protein interactions in live cells and animals. Protein–protein interactions regulate a variety of cellular functions, including cell cycle progression, signal transduction, and metabolic pathways. The strategies for detecting protein-binding partners using conventional cell biology assays with the intent of identifying properties that might be exploited for imaging applications are presented. Two-hybrid systems exploit the modular nature of transcription factors, many of which can be separated into discrete DNA-binding and activation domains. Protein–fragment complementation assays depend on the division of a monomeric reporter enzyme into two separate inactive components that can reconstitute function upon association. Luciferase complementation imaging of protein interactions in cells and small animal models has been developed to permit the rapid and repetitive measurement of protein pairs of interest. It is found that optimized luciferase complementation, quantified by imaging with a charge coupled device camera, equipped with appropriate software, can provide an accurate measure of relative protein association in animals and quantitative measurement of protein association in live cells.

Tracheal cytotoxin structural requirements for respiratory epithelial damage in pertussis

The respiratory epithelial pathology of pertussis (whooping cough) can be reproduced by tracheal cyto‐toxin (TCT), a disaccharide‐tetrapeptide released by Bordetella pertussis. TCT is a muramyl peptide, a class of peptidoglycan‐derived compounds which have many biological activities including adjuvanticity, somnogenicity, pyrogenicity, and cytotoxicity. The structural requirements for muramyl peptides to produce some of these biological effects have been partially characterized. Using in vitro assays with respiratory epithelial cells and tissue, we have previously determined that the disaccharide moiety of TCT is not involved in toxicity and that the side‐chain functional groups of diaminopimelic acid (A2pm) are crucial for toxicity. In this study, we determine the importance of every amino acid, functional group and chiral centre in the peptide portion of TCT. Although lactyl tetrapeptides are the most toxic of the TCT fragments, producing dose‐response curves identical to TCT, the smallest analogues of TCT which are active in our assay are of the form X‐γ‐(d)‐Glu‐meso‐A2pm, where X may be an amino acid or a blocking group. Within this active substructure, main‐chain chirality and all functional groups are essential for toxicity. This definition of the core region of TCT indicates that the TCT interaction site is unlike almost all other muramyl peptide interaction sites for which structure‐activity data are available.

In vitro and in vivo characterization of a dual-function green fluorescent protein-HSV1-thymidine kinase reporter gene driven by the human elongation factor 1α promoter

Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1a a (EF-1a-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[ 3 H]ganciclovir (8-[ 3 H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[ 3 H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([ 18 F]FHBG) � 8-[ 3 H]penciclovir (8-[ 3 H]PCV) < 2 0 -fluoro2 0 -deoxy-5-iodouracil-beta-D-arabinofuranoside (2-[ 14 C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[ 3 H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques. Mol Imaging (2002) 1, 65 – 73.

Fluorescence Lifetime Imaging of Apoptosis.

Genetically encoded fluorescence resonance energy transfer (FRET) reporters are powerful tools for analyzing cell signaling and function at single-cell resolution in standard 2D cell cultures, but these reporters rarely have been applied to 3D environments. FRET interactions between donor and acceptor molecules typically are determined by changes in relative fluorescence intensities, but wavelength-dependent differences in light absorption complicate this analysis method in 3D settings. Herein we report fluorescence lifetime imaging microscopy (FLIM) with phasor analysis, a method that displays fluorescence lifetimes on a pixel-wise basis in real time to quantify apoptosis in breast cancer cells stably expressing a genetically encoded FRET reporter. This microscopic imaging technology allowed us to identify treatment-induced apoptosis in single breast cancer cells in environments ranging from 2D cell culture, spheroids with cancer and bone marrow stromal cells, and living mice with orthotopic human breast cancer xenografts. Using this imaging strategy, we showed that combined metabolic therapy targeting glycolysis and glutamine pathways significantly reduced overall breast cancer metabolism and induced apoptosis. We also determined that distinct subpopulations of bone marrow stromal cells control the resistance of breast cancer cells to chemotherapy, suggesting heterogeneity of treatment responses of malignant cells in different bone marrow niches. Overall, this study establishes FLIM with phasor analysis as an imaging tool for apoptosis in cell-based assays and living mice, enabling real-time, cellular-level assessment of treatment efficacy and heterogeneity.