Phenotypic screening of signaling motifs that efficiently induce cell proliferation.

Since cell proliferation is one of the fundamental cell fates, artificial control of cell proliferation based on a receptor-engineering approach is increasingly important in therapeutic and industrial applications. Since the signal transduction properties of cytokine receptors are greatly influenced by the amino acid sequence of tyrosine motifs, here we develop a phenotypic screening approach that can directly select cell proliferation-inducing tyrosine motifs from a synthetic library. In the tyrosine motif library, amino acid sequences around the tyrosine are randomized to attain diverse binding patterns of signaling molecules. Theoretically, engineered receptors with distinct tyrosine motifs would activate signaling molecules in diverse patterns. Thus, we investigated whether tyrosine motif sequences capable of inducing cell proliferation could be selected from the cellular library expressing the motif-engineered receptors. Consequently, the selected motifs induced similar levels of cell proliferation compared to the cytoplasmic signaling domain of a native receptor. The motif-screening system was applicable to cells that may differentiate or proliferate depending on cytokine signals. To our best knowledge, this is the first report demonstrating phenotypic screening of tyrosine motifs in living cells. Our approach would open up new possibilities in the field of artificial control of cell fate based on signal transduction engineering.

Canonical Wnt signaling activation by chimeric antigen receptors for efficient cardiac differentiation from mouse embryonic stem cells.

BACKGROUND: Canonical Wnt signaling is involved in a variety of biological processes including stem cell renewal and differentiation, embryonic development, and tissue regeneration. Previous studies reported the stage-specific roles of the Wnt signaling in heart development. Canonical Wnt signal activation by recombinant Wnt3a in the early phase of differentiation enhances the efficiency of myocardial cell production from pluripotent stem cells. However, the hydrophobicity of Wnt proteins results in high cost to produce the recombinant proteins and presents an obstacle to their preparation and application for therapeutics, cell therapy, or molecular analysis of Wnt signaling. METHODS: To solve this problem, we generated an inexpensive molecule-responsive differentiation-inducing chimeric antigen receptor (designated as diCAR) that can activate Wnt3a signaling. The extracellular domains of low-density-lipoprotein receptor-related protein 6 (LRP6) and frizzeled-8 (FZD8) were replaced with single-chain Fv of anti-fluorescein (FL) antibody, which can respond to FL-conjugated bovine serum albumin (BSA-FL) as a cognate ligand. We then analyzed the effect of this diCAR on Wnt signal activation and cardiomyocyte differentiation of mouse embryonic stem cells in response to BSA-FL treatment. RESULTS: Embryonic stem cell lines stably expressing this paired diCAR, named Wnt3a-diCAR, showed TCF/ß-catenin-dependent transactivation by BSA-FL in a dose-dependent manner. Treatment with either Wnt3a recombinant protein or BSA-FL in the early phase of differentiation revealed similar changes of global gene expressions and resulted in efficient myocardial cell differentiation. Furthermore, BSA-FL-mediated signal activation was not affected by a Wnt3a antagonist, Dkk1, suggesting that the signal transduction via Wnt3a-diCAR is independent of endogenous LRP6 or FZD8. CONCLUSION: We anticipate that Wnt3a-diCAR enables target-specific signal activation, and could be an economical and powerful tool for stem cell-based regeneration therapy.

Constitutively active GPR43 is crucial for proper leukocyte differentiation.

The G protein-coupled receptors, GPR43 (free fatty acid receptor 2, FFA2) and GPR41 (free fatty acid receptor 3, FFA3), are activated by short-chain fatty acids produced under various conditions, including microbial fermentation of carbohydrates. Previous studies have implicated this receptor energy homeostasis and immune responses as well as in cell growth arrest and apoptosis. Here, we observed the expression of both receptors in human blood cells and a remarkable enhancement in leukemia cell lines (HL-60, U937, and THP-1 cells) during differentiation. A reporter assay revealed that GPR43 is coupled with Gαi and Gα12/13 and is constitutively active without any stimuli. Specific blockers of GPR43, GLPG0974 and CATPB function as inverse agonists because treatment with these compounds significantly reduces constitutive activity. In HL-60 cells, enhanced expression of GPR43 led to growth arrest through Gα12/13 . In addition, the blockage of GPR43 activity in these cells significantly impaired their adherent properties due to the reduction of adhesion molecules. We further revealed that enhanced GPR43 activity induces F-actin formation. However, the activity of GPR43 did not contribute to butyrate-induced apoptosis in differentiated HL-60 cells because of the ineffectiveness of the inverse agonist on cell death. Collectively, these results suggest that GPR43, which possesses constitutive activity, is crucial for growth arrest, followed by the proper differentiation of leukocytes.

Intracellular Protein Photoactivation Using Sterically Bulky Caging.

Methods for intracellular protein photoactivation have been studied to elucidate the spatial and temporal roles of proteins of interest. In this study, an intracellular protein photoactivation method was developed using sterically bulky caging. The protein of interest was modified with biotin via a photocleavable linker, and then conjugated with streptavidin to sterically block the protein surface for inactivation. The caged protein was transduced into cells and reactivated by light-induced degradation of the conjugates. A cytotoxic protein, saporin, was caged and photoactivated both in vitro and in living cells with this method. This method achieved control of the cytotoxic activity in an off-on manner, introducing cell death selectively at the designed location using light. This simple and versatile photoactivation method is a promising tool for studying spatio-temporal cellular events that are related to intracellular proteins of interest.

Photoactivatable Materials for Versatile Single-Cell Patterning Based on the Photocaging of Cell-Anchoring Moieties through Lipid Self-Assembly.

Versatile methods for patterning multiple types of cells with single-cell resolution have become an increasingly important technology for cell analysis, cell-based device construction, and tissue engineering. Here, we present a photoactivatable material based on poly(ethylene glycol) (PEG)-lipids for patterning a variety of cells, regardless of their adhesion abilities. In this study, PEG-lipids bearing dual fatty acid chains were first shown to perfectly suppress cell anchoring on their coated substrate surfaces whereas those with single-chain lipids stably anchored cells through lipid-cell membrane interactions. From this finding, a PEG-lipid with one each of both normal and photocleavable fatty acid chains was synthesized as a material that could convert the chain number from two to one by exposure to light. On the photoconvertible PEG-lipid surface, cell anchoring was activated by light exposure. High-speed atomic force microscopy measurements revealed that this photocaging of the lipid-cell membrane interaction occurs because the hydrophobic dual chains self-assemble into nanoscale structures and cooperatively inhibit the anchoring. Light-induced dissociation of the lipid assembly achieved the light-guided fine patterning of multiple cells through local photoactivation of the anchoring interactions. Using this surface, human natural killer cells and leukemia cells could be positioned to interact one-by-one. The cytotoxic capacity of single immune cells was then monitored via microscopy, showing the proof-of-principle for applications in the high-throughput analysis of the heterogeneity in individual cell-cell communications. Thus, the substrate coated with our photoactivatable material can serve as a versatile platform for the accurate and rapid patterning of multiple-element cells for intercellular communication-based diagnostics.

Photo-Degradable Protein-Polymer Hybrid Shells for Caging Living Cells.

There is growing demand for the precise remote control of cellular functions in various fields. Herein, a method for caging mammalian cells by coating with photodegradable protein-polymer hybrid shells to photo-control their functions without genetic engineering is reported. A layer-by-layer assembly of photocleavable synthetic materials through biotin-streptavidin (SA) binding was employed for cell coating. The cell surfaces were first biotinylated with photocleavable biotinylated poly(ethylene glycol)(PEG)-lipid and then coated by repeatedly layering SA and micelles of the PEG-lipid and photocleavable biotinylated four-arm PEG. The cell extension and adhesion were suppressed with the shells and then triggered with the degradation of the shells by light exposure. Macrophage phagocytosis was also stopped by caging with the shells and restarted by light-guided uncaging. This study provides the first proof of principle that cellular functions can be remotely controlled by steric hinderance of cell surfaces with photodegradable materials.

Sterically Bulky Caging of Transferrin for Photoactivatable Intracellular Delivery.

Photoactivatable ligand proteins are potentially useful for light-induced intracellular delivery of therapeutic and diagnostic cargos through receptor-mediated cellular uptake. Here, we report the simple and effective caging of transferrin (Tf), a representative ligand protein with cellular uptake ability, which has been used in the delivery of various cargos. Tf was modified with several biotin molecules through a photocleavable linker, and then the biotinylated Tf (bTf) was conjugated with the biotin-binding protein, streptavidin (SA), to provide steric hindrance to block the interaction with the Tf receptor. Without exposure to light, the cellular uptake of the bTf-SA complex was effectively inhibited. In response to light exposure, the complex was degraded with the release of Tf, leading to cellular uptake of Tf. Similarly, the cellular uptake of Tf-doxorubicin (Dox) conjugates could be suppressed by caging with biotinylation and SA binding, and the intracellular delivery of Dox could be triggered in a light-dependent manner. The intracellularly accumulated Dox decreased the cell viability to 25% because of the cell growth inhibitory effect of Dox. These results provided proof of principle that the caged Tf can be employed as a photoactivatable molecular device for the intracellular delivery of cargos.

Photodegradable avidin-biotinylated polymer conjugate hydrogels for cell manipulation.

Protein-synthetic polymer hybrid hydrogels crosslinked via protein-ligand binding are promising materials for the three-dimensional culture of various cells, while photo-responsive hydrogels have been widely used for the spatio-temporal control of cell functions and patterning. Photo-responsive protein-polymer hybrid hydrogels are therefore attractive candidates for use in cell and artificial tissue fabrication; however, no examples combining these properties have been reported to date. Herein, a photodegradable hydrogel consisting of avidin and biotinylated polyethylene glycol (PEG) was developed as a multi-functional matrix for cell culture and sorting. A four-branched PEG with a biotinylated photocleavable group at the end of each chain was crosslinked with avidin to produce a photodegradable hydrogel. A cytokine-dependent immunocyte was successfully cultured in the hydrogel by supplying cytokine from a medium layered on the hydrogel. Additionally, the adhesion and survival of fibroblasts could be controlled by decorating the hydrogel with a biotinylated cell-adhesive peptide. Cells embedded in the hydrogels could be recovered without cell damage as a result of light-induced hydrogel degradation. Moreover, model target cells expressing red fluorescent protein were selectively liberated from a hydrogel containing cells of different colors by irradiating with a targeted light. Owing to both the selective biotin-binding ability of avidin and the photocleavable properties of the synthetic polymer, the hydrogels were easy to prepare and decorate with functional molecules; they provided an internal structure suitable for cell culture, and allowed light-guided cell manipulation. The hydrogels are therefore expected to contribute to various cell fabrication processes as useful cell engineering and sorting tools.

Reprogramming signal transduction through a designer receptor tyrosine kinase.

Controlling signal transduction with artificial designer receptors is a promising approach to realize future medicine for intractable diseases. Although several functional artificial receptors have been reported by domain engineering, more sophisticated engineering within domains has yet to be thoroughly investigated. Here we demonstrate motif-based engineering of a receptor tyrosine kinase for reprogramming signal transduction. We design a scaffold-less tyrosine kinase domain that does not recruit any signal transducers but retains its kinase function. The resultant scaffold-less tyrosine kinase domain is linked to a tyrosine motif that recruits a target signaling molecule upon its phosphorylation. The engineered tyrosine motif-kinase fusion protein is further connected to a small molecule- or light-dependent dimerizing domain that can switch on the kinase activity in response to an external stimulus. The resultant designer receptors attain specific chemical- or photo-activation of signaling molecules of interest in mammalian cells. Thus, our designer receptor tyrosine kinase proves the possibility of rationally reprogramming intracellular signal transduction on a motif basis. The motif-based receptor engineering may realize tailor-made functional receptors useful in the fields of biology and medicine.

A Novel Cell-Based Intracellular Protein-Protein Interaction Detection Platform (SOLIS) for Multimodality Screening.

Intervention in protein-protein interactions (PPIs) has tremendous effects in the molecular therapy of many diseases. To fulfill the requirements for targeting intracellular proteins, here we develop SOS-localization-based interaction screening (SOLIS), which elaborately mimics signaling via the Ras-mitogen-activated protein kinase pathway. SOLIS employs two chimeric proteins in which a membrane localization motif (CaaX) is fused at the C-terminus of a protein of interest and the catalytic domain of SOS is fused at the C-terminus of another protein of interest. Interaction between the two proteins of interest induces membrane localization of the SOS chimera and cell proliferation. Thus, the SOLIS system enables enrichment of superior binders based on cell proliferation in an intracellular PPI-dependent manner. This was verified by three major modalities against intracellular PPIs (small molecules, peptide aptamers, and intrabodies). The system worked over a broad range of affinities (KD = 0.32-140 nM). In a screening of a site-directed randomized library, novel intrabody clones were selected on the basis of the potency of cell proliferation. Three other PPI detection methods (NanoBiT, SPR, and pull-down assays) were employed to characterize the SOLIS system, and several intrabody clones were judged as false negatives in these assays. SOLIS signals would be less sensitive to the orientation/conformation of the chimeric proteins, and this feature emerges as the advantage of SOLIS as a mammalian cytosolic PPI detection system with few false negatives.

Photo-Cleavable Peptide-Poly(Ethylene Glycol) Conjugate Surfaces for Light-Guided Control of Cell Adhesion.

Photo-responsive cell attachment surfaces can simplify patterning and recovery of cells in microdevices for medicinal and pharmaceutical research. We developed a photo-responsive surface for controlling the attachment and release of adherent cells on a substrate under light-guidance. The surface comprises a poly(ethylene glycol) (PEG)-based photocleavable material that can conjugate with cell-adhesive peptides. Surface-bound peptides were released by photocleavage in the light-exposed region, where the cell attachment was subsequently suppressed by the exposed PEG. Simultaneously, cells selectively adhered to the peptide surface at the unexposed microscale region. After culture, the adhered and spread cells were released by exposure to a light with nontoxic dose level. Thus, the present surface can easily create both cell-adhesive and non-cell-adhesive regions on the substrate by single irradiation of the light pattern, and the adhered cells were selectively released from the light-exposed region on the cell micropattern without damage. This study shows that the photo-responsive surface can serve as a facile platform for the remote-control of patterning and recovery of adherent cells in microdevices.

Chimeric G-CSF Receptor-Mediated STAT3 Activation Contributes to Efficient Induction of Cardiomyocytes from Mouse Induced Pluripotent Stem Cells.

Producing a sufficient number of cardiomyocytes from pluripotent stem cells has been of great demand for cardiac regeneration therapy. However, it remains challenging to efficiently differentiate cardiomyocytes with low costs. Reportedly, granulocyte colony-stimulating factor (G-CSF) receptor (GCSFR) signaling activates signal transducers and activators of transcription (STAT) signaling and enhances cardiac differentiation from embryonic stem cells or induced pluripotent stem cells (iPSCs). To economically and efficiently produce cardiomyocytes from iPSCs through GCSFR/STAT axis activation, we constructed antibody/receptor chimeras that can respond to an inexpensive small molecule. Single-chain Fv of anti-fluorescein (FL) antibody was ligated to transmembrane/cytoplasmic domains of GCSFRs, enabling transduction of GCSFR signaling in response to FL-conjugated bovine serum albumin (BSA-FL) as an alternative ligand. Mouse iPSC lines constitutively expressing these chimeric receptors exhibited increased BSA-FL-induced STAT3 phosphorylation in a dose-dependent manner, which was abolished by an inhibitor of Janus tyrosine kinase (JAK). In addition, BSA-FL stimulation also increased the incidence of beating embryoid bodies and upregulated cardiac-specific gene expressions after differentiation in these iPSC lines. Therefore, the chimeric GCSFRs activated endogenous GCSFR signaling at least via the JAK/STAT3 pathway, thereby enhancing cardiac differentiation from iPSCs. This approach, as an economical strategy, could contribute to stem cell-based cardiac regeneration therapy.

Photo-responsive materials with strong cell trapping ability for light-guided manipulation of nonadherent cells.

We report a photo-cleavable material for tight trapping of nonadherent cells to substrate surfaces. Model immunocytes were selectively trapped in a non-irradiated area as single cells after the projection of a light pattern and withstood high-speed laminar flow, achieving light-guided cell release from the substrates.

An epitope-directed antibody affinity maturation system utilizing mammalian cell survival as readout.

Upon developing therapeutically potent antibodies, there are significant requirements, such as increasing their affinity, regulating their epitope, and using native target antigens. Many antibody selection systems, such as a phage display method, have been developed, but it is still difficult to fulfill these requirements at the same time. Here, we propose a novel epitope-directed antibody affinity maturation system utilizing mammalian cell survival as readout. This system is based on the competition of antibody binding, and can target membrane proteins expressed in a native form on a mammalian cell surface. Using this system, we successfully selected an affinity-matured anti-ErbB2 single-chain variable fragment variant, which had the same epitope as the original one. In addition, the affinity was increased mainly due to the decrease in the dissociation rate. This novel cell-based antibody affinity maturation system could contribute to directly obtaining therapeutically potent antibodies that are functional on the cell surface.

Selective intracellular delivery of perfluorocarbon nanodroplets for cytotoxicity threshold reduction on ultrasound-induced vaporization.

BACKGROUND: Phase-change nanodroplets (PCNDs), which are liquid perfluorocarbon nanoparticles, have garnered much attention as ultrasound-responsive nanomedicines. The vaporization phenomenon has been employed to treat tumors mechanically. However, the ultrasound pressure applied to induce vaporization must be low to avoid damage to nontarget tissues. AIMS: Here, we report that the pressure threshold for vaporization to induce cytotoxicity can be significantly reduced by selective intracellular delivery of PCNDs into targeted tumors. METHODS AND RESULTS: In vitro experiments revealed that selective intracellular delivery of PCNDs induced PCND aggregation specifically inside the targeted cells. This close-packed configuration decreased the pressure threshold for vaporization to induce cytotoxicity. Moreover, following ultrasound exposure, significant decrease was observed in the viability of cells that incorporated PCNDs (35%) but not in the viability of cells that did not incorporate PCNDs (88%). CONCLUSIONS: Intracellular delivery of PCNDs reduced ultrasound pressure applied for vaporization to induce cytotoxicity. Confocal laser scanning microscopy and flow cytometry revealed that prolonged PCND-cell incubation increased PCND uptake and aggregation. This aggregation effect might have contributed to the cytotoxicity threshold reduction effect.

A Suicide Switch Directly Eliminates Intracellular scFv Oligomers in the Cytoplasm of Mammalian Cells.

As intracellular antibodies (intrabodies) are highly promising tools for drug discovery, an innovative antibody screening platform in mammalian cells was previously developed by a single-chain Fv (scFv)-c-kit growth sensor, which successfully selected rabies nucleoprotein and phosphoprotein-specific intrabodies from a synthetic scFv library. Since the scFv-c-kit growth sensor releases a growth signal after forming oligomers due to binding to an oligomeric antigen, it is critical to use a library which does not contain self-oligomeric scFvs to avoid the off-target signal of the growth sensor. Here, a novel method to eliminate self-oligomeric scFvs directly in the cytoplasm of mammalian cells is presented. A suicide switch by fusing an scFv with a cell-death signaling domain to eliminate scFv oligomers is developed. It is found that among four cell-death signaling domains, a suicide switch by fusing scFv with Fas-associated death domain (FADD) can selectively reduce oligomeric scFvs. Furthermore, the library after eliminating scFv oligomers results in higher efficiency in the intrabody selection platform with a growth sensor. Collectively, the scFv-FADD suicide switch can be applied to eliminate oligomeric scFvs from a library, which can consequently improve the quality of intracellular scFv libraries and accelerate the discovery of intrabodies in the future.

Stepwise phosphorylation of leukotriene B4 receptor 1 defines cellular responses to leukotriene B4.

Leukotriene B4 (LTB4) receptor type 1 (BLT1) is abundant in phagocytic and immune cells and plays crucial roles in various inflammatory diseases. BLT1 is phosphorylated at several serine and threonine residues upon stimulation with the inflammatory lipid LTB4 Using Phos-tag gel electrophoresis to separate differentially phosphorylated forms of BLT1, we identified two distinct types of phosphorylation, basal and ligand-induced, in the carboxyl terminus of human BLT1. In the absence of LTB4, the basal phosphorylation sites were modified to various degrees, giving rise to many different phosphorylated forms of BLT1. Different concentrations of LTB4 induced distinct phosphorylation events, and these ligand-induced modifications facilitated additional phosphorylation events at the basal phosphorylation sites. Because neutrophils migrate toward inflammatory sites along a gradient of LTB4, the degree of BLT1 phosphorylation likely increases in parallel with the increase in LTB4 concentration as the cells migrate. At high concentrations of LTB4, deficiencies in these two types of phosphorylation events impaired chemotaxis and ß-hexosaminidase release, a proxy for degranulation, in Chinese hamster ovary (CHO-K1) and rat basophilic leukemia (RBL-2H3) cells, respectively. These results suggest that an LTB4 gradient around inflammatory sites enhances BLT1 phosphorylation in a stepwise manner to facilitate the precise migration of phagocytic and immune cells and the initiation of local responses, including degranulation.

Artificial Self-Sufficient Cytochrome P450 Containing Multiple Auxiliary Proteins Demonstrates Improved Monooxygenase Activity.

Most bacterial cytochrome P450 monooxygenases (P450s) do not work alone because their active species is generated by two electrons supplied through two separate auxiliary proteins. Artificial "self-sufficient" P450s, in which one molecule each of the two auxiliary proteins is arranged close to the P450s, have been developed but have not achieved the maximum catalytic turnover numbers of the P450s. In this study, the Pseudomonas putida P450 (P450cam) is assembled with multiple molecules of its auxiliary proteins, putidaredoxin (PdX) and putidaredoxin reductase (PdR), by fusion to a heterotrimeric protein. In the assembled P450cam containing one PdX and one PdR, kinetic analysis reveales that the catalytic cycle of P450cam is suspended twice awaiting the reduction of PdX by PdR. An increase in the number of PdR molecules stimulated the PdX reduction process. Assembly with two PdXs allows one PdX to be reduced during the binding of the other PdX to P450cam for the first electron transfer, eliminating one waiting step. Finally, P450cam assembled with two PdXs and three PdRs showes 92% of the maximum activity of free P450cam. Therefore, assembly with multiple molecules of auxiliary proteins will facilitate in vitro biotechnological applications of the P450s.

Designing Motif-Engineered Receptors To Elucidate Signaling Molecules Important for Proliferation of Hematopoietic Stem Cells.

The understanding of signaling events is critical for attaining long-term expansion of hematopoietic stem cells ex vivo. In this study, we aim to analyze the contribution of multiple signaling molecules in proliferation of hematopoietic stem cells. To this end, we design a bottom-up engineered receptor with multiple tyrosine motifs, which can recruit multiple signaling molecules of interest. This is followed by a top-down approach, where one of the multiple tyrosine motifs in the bottom-up engineered receptor is functionally knocked out by tyrosine-to-phenylalanine mutation. The combination of these two approaches demonstrates the importance of Shc in cooperation with STAT3 or STAT5 in the proliferation of hematopoietic stem cells. The platform developed herein may be applied for analyzing other cells and/or other cell fate regulation systems.

A Stable Artificial Multienzymatic Complex Using a Heterotrimeric Protein From Metallosphaera sedula.

Bacterial cytochrome P450 monooxygenases (P450s) are promising biocatalysts for chemical syntheses because they catalyze a variety of oxidations on non-activated hydrocarbons using O2 . However, the requirement of two auxiliary proteins, an electron transfer protein and a reductase, for the catalysis is a major bottleneck for in vitro applications of these monooxygenases. The authors previous study showed that artificial assembly of a bacterial P450 with its auxiliary proteins using a heterotrimeric proliferating cell nuclear antigen (PCNA) from Sulfolobus solfataricus yields a self-sufficient P450, but partial dissociation of P450 from the complex at catalytic concentrations reduces the apparent specific activity of this self-sufficient P450. In this study, a Metallosphaera sedula PCNA is used, which is currently the most stable heterotrimeric PCNA, to assemble a bacterial P450 with its auxiliary proteins at submicromolar protein concentrations. The apparent specific monooxygenase activity of the M. sedula PCNA-assembled P450 with auxiliary proteins is saturated at protein concentrations of 40 nM, and is 2.1-fold higher than that of the S. solfataricus PCNA-assembled P450. Therefore, M. sedula PCNA represents a versatile tool to facilitate multiple enzymatic reactions, including the P450 monooxygenase system.