Protein Activity In Living Cells Research Articles

Precisely Targeted Reporters Reveal ABCG2 Regulation in Live Lung Cancer Cells

Expression of the ABCG2 multidrug transporter is a marker of cancer stem cells and an indicator of poor disease outcome. Understanding the effect of pharmacotherapy on ABCG2 expression is critical in guiding therapy recommendations and may help rational drug development. Engineered reporter cell lines are useful in monitoring gene regulation and protein activity in live cells but rely on precise targeting to preserve native regulatory functions. Here, we generated a number of precision‐engineered, fluorescent human cell lines that interrogate ABCG2 regulation in live lung cancer cells. Using homology‐based CRISPR engineering, we targeted a promoterless eGFP to the translational start site of the ABCG2 gene, generating ABCG2 knockout and in situ tagged ABCG2 reporter cells. Using the engineered cell lines, we show that anti‐cancer medications, HDAC inhibitors and hypoxia‐mimicking agents induce ABCG2 expression. In addition, we provide evidence that the glucocorticoid receptor is a specific, positive regulator of the ABCG2 gene. To our knowledge, this is the first fluorescent reporter assay designed to follow the endogenous regulation of a human ABC transporter in live cells. The information gained may direct therapy recommendations and assist rational drug design.Support or Funding InformationThis research has been supported mainly by OTKA‐NKFIH grant NK 115375, but also by NKFIH K104903 and the NVKP_16‐1‐2016‐0005 program from the National Research, Development and Innovation Office. This work has also been supported by research grant OTKA‐NKFIH K128011, awarded to GV. A Borsy was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (HA: BO/00579/17/5), through the New National Excellence Program of the Ministry of Human Resources (HA: ÚNKP‐18‐4‐SE‐11).

Precision-engineered reporter cell lines reveal ABCG2 regulation in live lung cancer cells

Expression of the ABCG2 multidrug transporter is a marker of cancer stem cells and a predictor of recurrent malignant disease. Understanding how human ABCG2 expression is modulated by pharmacotherapy is crucial in guiding therapeutic recommendations and may aid rational drug development. Genome edited reporter cells are useful in investigating gene regulation and visualizing protein activity in live cells but require precise targeting to preserve native regulatory regions. Here, we describe a fluorescent reporter assay that allows the noninvasive assessment of ABCG2 regulation in human lung adenocarcinoma cells. Using CRISPR-Cas9 gene editing coupled with homology-directed repair, we targeted an EGFP coding sequence to the translational start site of ABCG2, generating ABCG2 knock-out and in situ tagged ABCG2 reporter cells. Using the engineered cell lines, we show that ABCG2 is upregulated by a number of anti-cancer medications, HDAC inhibitors, hypoxia-mimicking agents and glucocorticoids, supporting a model in which ABCG2 is under the control of a general stress response. To our knowledge, this is the first description of a fluorescent reporter assay system designed to follow the endogenous regulation of a human ABC transporter in live cells. The information gained may guide therapy recommendations and aid rational drug design.

Integrating Combinatorial Lipid Nanoparticle and Chemically Modified Protein for Intracellular Delivery and Genome Editing.

The use of protein to precisely manipulate cell signaling is an effective approach for controlling cell fate and developing precision medicine. More recently, programmable nucleases, such as CRISPR/Cas9, have shown extremely high potency for editing genetic flow of mammalian cells, and for treating genetic disorders. The therapeutic potential of proteins with an intracellular target, however, is mostly challenged by their low cell impermeability. Therefore, a developing delivery system to transport protein to the site of action in a spatiotemporal controlled manner is of great importance to expand the therapeutic index of the protein. In this Account, we first summarize our most recent advances in designing combinatorial lipid nanoparticles with diverse chemical structures for intracellular protein delivery. By designing parallel Michael addition or ring-opening reaction of aliphatic amines, we have generated a combinatorial library of cationic lipids, and identified several leading nanoparticle formulations for intracellular protein delivery both in vitro and in vivo. Moreover, we optimized the chemical structure of lipids to control lipid degradation and protein release inside cells for CRISPR/Cas9 genome-editing protein delivery. In the second part of this Account, we survey our recent endeavor in developing a chemical approach to modify protein, in particular, coupled with the nanoparticle delivery platform, to improve protein delivery for targeted diseases treatment and genome editing. Chemical modification of protein is a useful tool to modulate protein function and to improve the therapeutic index of protein drugs. Herein, we mostly summarize our recent advances on designing chemical approaches to modify protein with following unique findings: (1) chemically modified protein shows selective turn-on activity based on the specific intracellular microenvironment, with which we were able to protein-based targeted cancer therapy; (2) the conjugation of hyaluronic acid (HA) to protein allows cancer cell surface receptor-targeted delivery of protein; (3) the introduction of nonpeptidic boronic acid into protein enabled cell nucleus targeted delivery; this is the first report that a nonpeptidic signal can direct protein to subcellular compartment; and (4) the fusion of protein with negatively supercharged green fluorescent protein (GFP) facilitates the self-assembly of protein with lipid nanoparticle for genome-editing protein delivery. At the end of the Account, we give a perspective of expanding the chemistry that could be integrated to design biocompatible lipid nanocarriers for protein delivery and genome editing in vitro and in vivo, as well as the chemical approaches that we can harness to modulate protein activity in live cells for targeted diseases treatment.

Generation of Intramolecular FRET Probes via Noncanonical Amino Acid Mutagenesis.

Förster resonance energy transfer (FRET) probes are powerful tools to monitor protein-protein interactions and enzyme activities in a spatiotemporal manner in live cells. Using a combination of noncanonical amino acid (ncAA) mutagenesis and bioorthogonal labeling, we have developed intramolecular FRET probes consisting of a fluorescent protein and an organic dye within an individual protein. Herein we present a general approach to establish intramolecular FRET probes for imaging of protein activity in live cells.

Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors

Fluorescence resonance energy transfer (FRET)-based molecular biosensors serve as important tools for studying protein activity in live cells and have been widely used for this purpose over the past decade. However, FRET biosensors are rarely used in the context of the live organism because of the inherent high cellular complexity and imaging challenges associated with the three-dimensional environment. Here we provide a protocol for using single-chain intramolecular FRET-based biosensors in early development. We provide a general protocol for FRET ratio imaging in embryos, including the data-acquisition conditions and the algorithm for ratio image generation. We then use the pRaichu RacFRET biosensor to exemplify the adaptation and optimization of a particular biosensor for use in live zebrafish embryos. Once an optimized biosensor is available, the complete procedure, including introduction of the probes into embryos, imaging and data analysis, requires 2-3 d.

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Mutations that increase the dynamic range of a photoswitchable protein may be used to improve other such photoswitches and increase their potential for allosteric control of protein activity in live cells.