Green Fluorescent Protein Fusion Proteins Research Articles

Construction of cytomegalovirus promoter‐driven gene expression system in Laodelphax striatellus

AbstractThe small brown planthopper (SBPH, Laodelphax striatellus) is a significant rice pest, responsible for transmitting rice stripe virus (RSV) in a persistent and propagative manner. RSV is one of the most detrimental rice viruses, causing rice stripe disease, which results in considerable loss of rice grain yield. While RNA interference and gene knockout techniques have enabled gene downregulation in SBPH, no system currently exists for the overexpression of endogenous or exogenous genes. Consequently, the development of a protein expression system for SBPH is imperative to serve as a technical foundation for pest control and gene function investigations. This study aimed to construct an expression vector using the promoter of the constitutive‐expressed tubulin gene of SBPH, and promoter of human cytomegalovirus (CMV). Fluorescence experiments demonstrated that both tubulin and CMV promoter could drive green fluorescent protein (GFP) expression in SBPH, and could also facilitate the expression of a nucleocapsid protein (NP) ‐GFP fusion protein containing viral NP with comparable efficiency. Through expression vector optimization, we have identified that the 3 tandem CMV promoters display a significantly higher promoter activity compared with both the 2 tandem CMV promoters and the single CMV promoter. In addition, the incorporation of Star polycation nanoparticles significantly enhanced the expression efficiency in SBPH. These results provide a promising technical platform for investigating gene functions in SBPH.

Identification of short protein-destabilizing sequences in Arabidopsis cyclin-dependent kinase inhibitors, ICKs.

Plants have a family of cyclin-dependent kinase (CDK) inhibitors called interactors/inhibitors of CDK (ICKs) or Kip-related proteins (KRPs). ICK proteins have important functions in cell proliferation, endoreduplication, plant growth, and reproductive development, and their functions depend on the protein levels. However, understanding of how ICK protein levels are regulated is very limited. We fused Arabidopsis ICK sequences to green fluorescent protein (GFP) and determined their effects on the fusion proteins in plants, yeast, and Escherichia coli. The N-terminal regions of ICKs drastically reduced GFP fusion protein levels in Arabidopsis plants. A number of short sequences of 10-20 residues were found to decrease GFP fusion protein levels when fused at the N-terminus or C-terminus. Three of the four short sequences from ICK3 showed a similar function in yeast. Intriguingly, three short sequences from ICK1 and ICK3 caused the degradation of the fusion proteins in E. coli. In addition, computational analyses showed that ICK proteins were mostly disordered and unstructured except for the conserved C-terminal region, suggesting that ICKs are intrinsically disordered proteins. This study has identified a number of short protein-destabilizing sequences, and evidence suggests that some of them may cause protein degradation through structural disorder and instability.

Correction: Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands

[This corrects the article DOI: 10.1371/journal.pone.0230804.].

Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands.

Cytokine signaling is transmitted by cell surface receptors which act as natural biological switches to control cellular functions such as immune reactions. Recently, we have designed synthetic cytokine receptors (SyCyRs) consisting of green fluorescent protein (GFP)- and mCherry-nanobodies fused to the transmembrane and intracellular domains of cytokine receptors. Following stimulation with homo- and heterodimeric GFP-mCherry fusion proteins, the resulting receptors phenocopied signaling induced by physiologically occurring cytokines. GFP and mCherry fusion proteins were produced in E. coli or CHO-K1 cells, but the overall yield and stability was low. Therefore, we applied two alternative multimerization strategies and achieved immunoglobulin Fc-mediated dimeric and coiled-coil GCN4pII-mediated trimeric assemblies. GFP- and/or mCherry-Fc homodimers activated synthetic gp130 cytokine receptors, which naturally respond to Interleukin 6 family cytokines. Activation of these synthetic gp130 receptors resulted in STAT3 and ERK phosphorylation and subsequent proliferation of Ba/F3-gp130 cells. Half-maximal effective concentrations (EC50) of 8.1 ng/ml and 0.64 ng/ml were determined for dimeric GFP-Fc and mCherry-Fc, respectively. This is well within the expected EC50 range of the native cytokines. Moreover, we generated tetrameric and hexameric GFP-mCherry-Fc fusion proteins, which were also biologically active. This highlighted the importance of close juxtaposition of two cytokine receptors for efficient receptor activation. Finally, we used a trimeric GCN4pII motif to generate homo-trimeric GFP and mCherry complexes. These synthetic cytokines showed improved EC50 values (GFP3: 0.58 ng/ml; mCherrry3: 0.37 ng/ml), over dimeric Fc fused variants. In conclusion, we successfully generated highly effective and stable multimeric synthetic cytokine receptor ligands for activation of synthetic cytokine receptors.

Chemogenetic Control of Nanobodies.

We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows interrogation of their roles in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.

Identification of sequence-specific interactions of the CD44-intracellular domain with RUNX2 in the transcription of matrix metalloprotease-9 in human prostate cancer cells.

Aim: The Cluster of differentiation 44 (CD44) transmembrane protein is cleaved by γ-secretase, the inhibition of which blocks CD44 cleavage. This study aimed to determine the biological consequence of CD44 cleavage and its potential interaction with Runt-related transcription factor (RUNX2) in a sequence-specific manner in PC3 prostate cancer cells.Methods: Using full-length and C-terminal deletion constructs of CD44-ICD (D1-D5) expressed as stable green fluorescent protein-fusion proteins in PC3 cells, we located possible RUNX2-binding sequences.Results: Chromatin immunoprecipitation assays demonstrated that the C-terminal amino acid residues between amino acids 671 and 706 in D1 to D3 constructs were indispensable for sequence-specific binding of RUNX2. This binding was minimal for sequences in the D4 and D5 constructs. Correspondingly, an increase in matrix metalloprotease-9 (MMP-9) expression was observed at the mRNA and protein levels in PC3 cells stably expressing D1–D3 constructs.Conclusion: These results provide biochemical evidence for the possible sequence-specific CD44-ICD/RUNX2 interaction and its functional relationship to MMP-9 transcription in the promoter region.

High-Throughput Screening of T7 Promoter Mutants for Soluble Expression of Cephalosporin C Acylase in E. coli.

Cephalosporin C acylase (CCA) is the key enzyme in the production of 7-aminocephalosporanic acid (7-ACA) via a one-step enzymatic process. To improve the soluble expression level of CCA in recombinant Escherichia coli at elevated temperatures, a library of T7 promoter mutants was created by site-saturation mutagenesis, and a series of mutated promoters were subsequently screened. Green fluorescent protein (GFP) was fused to the C-terminus of CCA to facilitate library screening, and the expression of the CCA and GFP fusion proteins was investigated under the control of the T7 promoter. Twenty-four mutants were selected by detecting the fluorescence intensity of colonies on agar plates to form a library with different expression levels. The enzyme activities of the mutants were positively correlated with their fluorescence intensities. The highest enzyme activity among these mutant promoters was 1.3-fold higher than the enzyme activity resulting from the wild-type promoter when the cells were cultured at 32 °C for 16 h. In addition, the transcription and expression levels of several typical promoters were discussed, and the effects of GFP fusion on the enzyme activity of CCA were investigated.

Simvastatin Efficiently Reduces Levels of Alzheimer's Amyloid Beta in Yeast.

A large-scale epidemiology study on statins previously showed that simvastatin was unique among statins in reducing the incidence of dementia. Since amyloid beta (Aβ42) is the protein that is most associated with Alzheimer’s disease, this study has focused on how simvastatin influences the turnover of native Aβ42 and Aβ42 fused with green fluorescent protein (GFP), in the simplest eukaryotic model organism, Saccharomyces cerevisiae. Previous studies have established that yeast constitutively producing Aβ42 fused to GFP offer a convenient means of analyzing yeast cellular responses to Aβ42. Young cells clear the GFP fusion protein and do not have green fluorescence while the older population of cells retains the fusion protein and exhibits green fluorescence, offering a fast and convenient means of studying factors that affect Aβ42 turnover. In this study the proportion of cells having GFP fused to Aβ after exposure to simvastatin, atorvastatin and lovastatin was analyzed by flow cytometry. Simvastatin effectively reduced levels of the cellular Aβ42 protein in a dose-dependent manner. Simvastatin promoted the greatest reduction as compared to the other two statins. A comparison with fluconazole, which targets that same pathway of ergosterol synthesis, suggests that effects on ergosterol synthesis do not account for the reduced amounts of Aβ42 fused to GFP. The levels of native Aβ42 following treated with simvastatin were also examined using a more laborious approach, quantitative MALDI TOF mass spectrometry. Simvastatin efficiently reduced levels of native Aβ42 from the population. This work indicates a novel action of simvastatin in reducing levels of Aβ42 providing new insights into how simvastatin exerts its neuroprotective role. We hypothesize that this reduction may be due to protein clearance.

Pathogen-sourced enzyme redirected

Targeted Degradation Cells of all types have the means to degrade specific proteins. These pathways can be hijacked by some bacteria to diminish host immune responses. The enzymes involved can in turn be used in the lab to modulate the proteome of eukaryotic cells. Ludwicki et al. attached a bacterial ubiquitin ligase to a protein that binds green fluorescent protein (GFP), thus redirecting it to add ubiquitin to GFP and GFP fusion proteins, which causes their degradation. The authors used polyamines and mRNA-binding proteins to stabilize and deliver mRNAs encoding an engineered ubiquitin ligase to cells. The mRNA nanoplexes enabled proteome editing both in vitro and in mice. ACS Cent. Sci. 5 , 852 (2019).

Overexpression of BraLTP2, a Lipid Transfer Protein of Brassica napus, Results in Increased Trichome Density and Altered Concentration of Secondary Metabolites.

Plant non-specific lipid transfer proteins (nsLTPs) belong to a large multigene family that possesses complex physiological functions. Trichomes are present on the aerial surfaces of most plants and include both glandular secretory hairs and non-glandular hairs. In this study, BraLTP2 was isolated from Brassica rapa (B. rapa) and its function was characterized in the important oilseed crop Brassica napus (B. napus). B. rapa lipid transfer protein 2 (BraLTP2) belongs to the little-known Y class of nsLTPs and encodes a predicted secretory protein. In ProBraLTP2::GUS (β-glucuronidase) transgenic plants, strong GUS activity was observed in young leaves and roots, while low activity was observed in the anther. It is noteworthy that strong GUS activity was observed in trichomes of the first four leaves of 4-week-old and 8-week-old seedings, however, it disappeared in 12-week-old seedings. In transgenic plants expressing a BraLTP2::GFP (green fluorescent protein) fusion protein, GFP fluorescence localized in the extracellular space of epidermal cells and trichomes. Overexpression of BraLTP2 in B. napus caused an increase in trichome number and altered the accumulation of secondary metabolites in leaves, including 43 upregulated secondary metabolites. Moreover, transgenic plants showed significantly increased activities of antioxidant enzymes. These results suggest that BraLTP2, a new nsLTP gene, may play a role in trichome development and the accumulation of secondary metabolites.

Design, characterization and in vivo functioning of a light-dependent histidine protein kinase in the yeast Saccharomyces cerevisiae

Helical alignment of the α-helical linker of the LOV (light-oxygen-voltage) domain of YtvA from Bacillus subtilis with the α-helical linker of the histidine-protein kinase domain of the Sln1 kinase of the phospho-relay system for osmoregulation of Saccharomyces cerevisiae has been used to construct a light-modulatable histidine protein kinase. In vitro, illumination with blue light inhibits both the ATP-dependent phosphorylation of this hybrid kinase, as well as the phosphoryl transfer to Ypd1, the phosphoryl transfer domain of the Sln1 system. The helical alignment was carried out with conservation of the complete Jα helix of YtvA, as well as of the phosphorylatable histidine residue of the Sln1 kinase, with conservation of the hepta-helical motive of coiled-coil structures, recognizable in the helices of the two separate, constituent, proteins. Introduction of the gene encoding this hybrid histidine protein kinase into cells of S. cerevisiae in which the endogenous Sln1 kinase had been deleted, allowed us to modulate gene expression in the yeast cells with (blue) light. This was first demonstrated via the light-induced alteration of the expression level of the mannosyl-transferase OCH1, via a translational-fusion approach. As expected, illumination decreased the expression level of OCH1; the steady state decrease in saturating levels of blue light was about 40%. To visualize the in vivo functionality of this light-dependent regulation system, we fused the green fluorescent protein (GFP) to another regulatory protein, HOG1, which is also responsive to the Sln1 kinase. HOG1 is phosphorylated by the MAP-kinase-kinase Pbs2, which in turn is under control of the Sln1 kinase, via the phosphoryl transfer domain Ypd1. Fluorescence microscopy was used to show that illumination of cells that contained the combination of the hybrid kinase and the HOG1::GFP fusion protein, led to a persistent increase in the level of nuclear accumulation of HOG1, in contrast to salt stress, which—as expected—showed the well-characterized transient response. The system described in this study will be valuable in future studies on the role of cytoplasmic diffusion in signal transduction in eukaryotic cells.

The checkpoint kinase TOR (target of rapamycin) regulates expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) and modulates chloroplast ribosomal RNA synthesis in a unicellular red alga.

Chloroplasts are plant organelles that carry out oxygenic photosynthesis. Chloroplast biogenesis depends upon chloroplast ribosomes and their translational activity. However, regulation of chloroplast ribosome biogenesis remains an important unanswered question. In this study, we found that inhibition of target of rapamycin (TOR), a general eukaryotic checkpoint kinase, results in a decline in chloroplast ribosomal RNA (rRNA) transcription in the unicellular red alga, Cyanidioschyzon merolae. Upon TOR inhibition, transcriptomics and other analyses revealed increased expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) gene (CmRSH4b), which encodes a homolog of the guanosine 3'-diphosphate 5'-diphosphate (ppGpp) synthetases that modulate rRNA synthesis in bacteria. Using an Escherichia coli mutant lacking ppGpp, CmRSH4b was demonstrated to have ppGpp synthetase activity. Expression analysis of a green fluorescent protein-fused protein indicated that CmRSH4b localizes to the chloroplast, and overexpression of the CmRSH4b gene resulted in a decrease of chloroplast rRNA synthesis concomitant with growth inhibition and reduction of chloroplast size. Biochemical analyses using C. merolae cell lysates or purified recombinant proteins revealed that ppGpp inhibits bacteria-type RNA polymerase-dependent chloroplast rRNA synthesis as well as a chloroplast guanylate kinase. These results suggest that CmRSH4b-dependent ppGpp synthesis in chloroplasts is an important regulator of chloroplast rRNA transcription. Nuclear and mitochondrial rRNA transcription were both reduced by TOR inhibition, suggesting that the biogeneses of the three independent ribosome systems are interconnected by TOR in plant cells.

Overcoming Autofluorescence to Assess GFP Expression During Normal Physiology and Aging in Caenorhabditis elegans.

Green fluorescent protein (GFP) is widely used as a molecular tool to assess protein expression and localization. In C. elegans, the signal from weakly expressed GFP fusion proteins is masked by autofluorescence emitted from the intestinal lysosome-related gut granules. For instance, the GFP fluorescence from SKN-1 transcription factor fused to GFP is barely visible with common GFP (FITC) filter setups. Furthermore, this intestinal autofluorescence increases upon heat stress, oxidative stress (sodium azide), and during aging, thereby masking GFP expression even from proximal tissues. Here, we describe a triple band GFP filter setup that separates the GFP signal from autofluorescence, displaying GFP in green and autofluorescence in yellow. In addition, yellow fluorescent protein (YFP) remains distinguishable from both the yellowish autofluorescence and GFP with this triple band filter setup. Although some GFP intensity might be lost with the triple band GFP filter setup, the advantage is that no modification of currently used transgenic GFP lines is needed and these GFP filters are easy to install. Hence, by using this triple band GFP filter setup, the investigators can easily distinguish autofluorescence from GFP and YFP in their favorite transgenic C. elegans lines.

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.

Monomeric G-actin is uniformly distributed in pollen tubes and is rapidly redistributed via cytoplasmic streaming during pollen tube growth.

Dynamic assembly and disassembly of the actin cytoskeleton has been implicated in the regulation of pollen germination and subsequent tube growth. It is widely accepted that actin filaments are arrayed into distinct structures within different regions of the pollen tube. Maintenance of the equilibrium between monomeric globular actin (G-actin) and filamentous actin (F-actin) is crucial for actin assembly and array construction, and the local concentration of G-actin thus directly impacts actin assembly. The localization and dynamics of G-actin in the pollen tube, however, remain to be determined conclusively. To address this question, we created a series of fusion proteins between green fluorescent protein (GFP) and the Arabidopsis reproductive actin ACT11. Expression of a fusion protein with GFP inserted after methionine at position 49 within the DNase I-binding loop of ACT11 (GFPMet49 -ACT11) rescued the phenotypes in act11 mutants. Consistent with the notion that the majority of actin is in its monomeric form, GFPMet49 -ACT11 and GFP fusion proteins of four other reproductive actins generated with the same strategy do not obviously label filamentous structures. In further support of the functionality of these fusion proteins, we found that they can be incorporated into filamentous structures in jasplakinolide (Jasp)-treated pollen tubes. Careful observations showed that G-actin is distributed uniformly in the pollen tube and is rapidly redistributed via cytoplasmic streaming during pollen tube growth. Our study suggests that G-actin is readily available in the cytoplasm to support continuous actin polymerization during rapid pollen tube growth.

Detection of an interaction between prion protein and neuregulin I-β1 by fluorescence resonance energy transfer analysis.

Cellular prion protein (PrP) copurifies with neuregulin type I-β1 (NRG I-β1), but no interaction has been detected by a general immunoprecipitation study. We speculate that PrP interacts with NRG I-β1. Here, the interaction of PrP with NRG I-β1 was detected by measuring fluorescence resonance energy transfer (FRET) between enhanced blue (EBFP) and enhanced green (EGFP) fluorescent protein-fusion proteins. Full-length PrP interacted with EGFP in addition to NRG I-β1. From this result, we deduced that PrP interacts with EGFP through its unstructured N-terminal domain. We therefore detected FRET between PrP deleting the N-terminal domain and NRG I-β1. In contrast, the C-terminal domain of PrP interacted with NRG I-β1 and the proteins dissociated completely in the presence of sodium chloride. This interaction occurs at the nanomolar level, which is important for the reaction to be functional in organisms. We concluded that PrP interacted with NRG I-β1 through its C-terminal domain.

Development of a Novel FRET Assay to Characterize the Oligomerization State of Self-Associating Transmembrane Helices

Using genetic reporter assays, significant self-interaction has been discovered in over 50 transmembrane (TM) helices from single-pass membrane proteins. To understand the biological functions of these TM interactions, which include signal transfer across the membrane in cancer-related pathways, it is necessary to construct accurate structural models of the TM oligomer. However in most cases this is not possible because the oligomerization state (stoichiometry) is unknown. It is often assumed that the helices form a dimer, though biologically relevant TM oligomers include dimers, trimers, and pentamers, as exemplified by GpA, HLA II Ii, and phospholamban, respectively.To characterize the stoichiometry of interacting single-pass membrane proteins, we are establishing a novel method based on Forster resonance energy transfer between identical fluorophores (homo-FRET). In order to allow the rapid bacterial expression and the purification of a wide range of fluorophore-coupled TM helices, we have developed TM fusion proteins with green fluorescent protein (GFP) and affinity tags. The measured homoFRET (via steady-state anisotropy) is proportional to both the affinity of the helices and the oligomerization state. Using only a fluorescence microplate reader, we demonstrate significant homo-FRET-coupling for GFP fusion proteins. Theoretical studies have shown that gradual photobleaching should yield a pattern of polarization that depends on the oligomerization state. High-profile experimental studies have confirmed this in-vivo using advanced microscopy. We show for the first time that the “fingerprint”-like curves after laser photobleaching can be achieved in-vitro with relatively simple equipment, allowing us to differentiate between oligomerization states. This demonstrates the feasibility of a rapid homo-FRET assay to determine the stoichiometry of transmembrane proteins in liposomes.

RNA-ID, a Powerful Tool for Identifying and Characterizing Regulatory Sequences.

The identification and analysis of sequences that regulate gene expression is critical because regulated gene expression underlies biology. RNA-ID is an efficient and sensitive method to discover and investigate regulatory sequences in the yeast Saccharomyces cerevisiae, using fluorescence-based assays to detect green fluorescent protein (GFP) relative to a red fluorescent protein (RFP) control in individual cells. Putative regulatory sequences can be inserted either in-frame or upstream of a superfolder GFP fusion protein whose expression, like that of RFP, is driven by the bidirectional GAL1,10 promoter. In this chapter, we describe the methodology to identify and study cis-regulatory sequences in the RNA-ID system, explaining features and variations of the RNA-ID reporter, as well as some applications of this system. We describe in detail the methods to analyze a single regulatory sequence, from construction of a single GFP variant to assay of variants by flow cytometry, as well as modifications required to screen libraries of different strains simultaneously. We also describe subsequent analyses of regulatory sequences.

A histamine-independent itch pathway is required for allergic ocular itch

Itch is the cardinal symptom of allergic conjunctivitis and afflicts 15% to 20% of the population worldwide. Histamine produced by conjunctival mast cells has been implicated as the principal itch mediator that activates histamine receptors on primary sensory fibers to induce allergic ocular itch.1Ono S.J. Abelson M.B. Allergic conjunctivitis: update on pathophysiology and prospects for future treatment.J Allergy Clin Immunol. 2005; 115: 118-122Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar However, antihistamines cannot completely relieve ocular itch in many cases, suggesting the involvement of a histamine-independent itch pathway. Herein, we sought to identify the histamine-independent neural pathway involved in allergic conjunctivitis and to develop new therapeutic strategies for allergic ocular itch. Allergic ocular itch typically originates from the conjunctiva, a mucosal membrane that is anatomically distinct from the skin and covers the ocular surface over the sclera and the inner surface of the eyelid. However, our knowledge about the neural regulations of allergic ocular itch and its difference from skin itch is limited. Mast cells have been shown to secrete many bioactive compounds in addition to histamine.2Metcalfe D.D. Baram D. Mekori Y.A. Mast cells.Physiol Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1785) Google Scholar, 3Raap U. Stander S. Metz M. Pathophysiology of itch and new treatments.Curr Opin Allergy Clin Immunol. 2011; 11: 420-427Crossref PubMed Scopus (75) Google Scholar Yet, the contribution of histamine-independent mediators to allergic ocular itch in comparison to histamine, and the neural pathway mediating the histamine-independent components in ocular itch remain unclear. TRPA1 is a cation channel that is often colocalized with TRPV1 in a subpopulation of primary sensory neurons in the trigeminal ganglion (TG) and the dorsal root ganglion. TRPV1 is known to be the downstream transduction channel of histamine H1 receptor in sensory neurons.4Shim W.S. Tak M.H. Lee M.H. Kim M. Kim M. Koo J.Y. et al.TRPV1 mediates histamine-induced itching via the activation of phospholipase A2 and 12-lipoxygenase.J Neurosci. 2007; 27: 2331-2337Crossref PubMed Scopus (340) Google Scholar TRPA1 was recently found to be the downstream transduction channel of histamine-independent itch in the skin.5Wilson S.R. Gerhold K.A. Bifolck-Fisher A. Liu Q. Patel K.N. Dong X. et al.TRPA1 is required for histamine-independent, Mas-related G protein-coupled receptor-mediated itch.Nat Neurosci. 2011; 14: 595-602Crossref PubMed Scopus (433) Google Scholar However, it is yet unknown whether TRPA1 is required for mast cell–mediated allergic itch. In this study, we characterized the role of TRPA1 as a histamine-independent modulator in ocular itch associated with allergic conjunctivitis. To delineate the respective role of TRPA1 and TRPV1 in ocular itch, we first examined the acute behavioral responses of wild-type (WT), TRPA1 knockout (KO), and TRPV1 KO mice to pruritogens applied directly to their lower conjunctival sacs. We were able to differentiate ocular itch from pain using our behavioral models, in which itch-inducing compounds provoked mice to scratch the treated area using their hindpaw, whereas pain-inducing capsaicin elicited wiping behavior using the forelimb (Fig 1, A). We found that histamine challenge (46 μg in 2.5 μL PBS) induced 15 ± 1.8 scratching bouts in WT and 14.7 ± 3 scratching bouts in TRPA1 KO mice (Fig 1, B), but significantly less itch responses in TRPV1 KO mice (5 ± 1.3 bouts), suggesting that TRPV1—rather than TRPA1—is responsible for histamine-dependent ocular itch signaling. We then tested 2 histamine-independent pruritogens, chloroquine and serotonin, on evoking ocular itch. Chloroquine-induced (12.4 μg) ocular scratching was significantly decreased in TRPA1 KO mice (6.3 ± 1.1 bouts), compared with WT (16.6 ± 2 bouts) and TRPV1 KO mice (14.3 ± 2.3 bouts) (Fig 1, C), indicating that TRPA1 is required for histamine-independent itch induced by chloroquine. In contrast, there was no difference in ocular itch responses induced by serotonin (0.2 μg) among WT (11.1 ± 1.1 bouts), TRPA1 KO (12.3 ± 1.0 bouts) and TRPV1 KO mice (11.6 ± 1 bouts) (Fig 1, D). Vehicle control (PBS) induced only minimal scratching responses (Fig 1, E). These behavioral results indicate a pivotal role of TRPA1 in certain types of nonhistaminergic ocular itch. To further explore the underlying mechanisms, we examined whether TRPA1 is functionally required for the responsiveness of conjunctival sensory fibers to pruritogens. We found that the conjunctival mucosa is abundantly innervated by TRPA1+ and TRPV1+ neurons, but not cold-sensing TRPM8+ neurons, in TG (see Fig E1 in this article's Online Repository at www.jacionline.org). GCaMP3-assisted calcium imaging of conjunctival sensory fibers showed that on stimulation by pruritogens, subpopulations of conjunctival nerve fibers displayed moderate to high calcium mobilization (Fig 1, F-K). Interestingly, TRPA1 deficiency does not affect conjunctival nerve response to histamine stimulation, but significantly reduces the nerve response to chloroquine stimulation. In contrast, TRPV1 is required for nerve fiber response to histamine but not to chloroquine in the conjunctiva. Finally, deficiency in TRPA1 or TRPV1 did not affect serotonin-induced nerve activity (Fig 1, L). These data reveal a segregation of TRPA1-dependent and TRPV1-dependent pathways in conjunctiva-innervating sensory neurons. Involvement of TRPA1-mediated itch pathway in allergic ocular itch was next evaluated using an ocular allergy model. Mast cell–mediated allergic conjunctivitis was induced in mice using an ovalbumin (OVA) sensitization regime (see this article's Methods section in the Online Repository at www.jacionline.org). Topical OVA challenge (250 μg) into unilateral conjunctival sac provoked targeted scratching responses in WT mice (21.6 ± 2.1 bouts; Fig 2, A). This itch was caused by allergen (OVA)-specific immune reaction because OVA challenge before sensitization or vehicle treatment after sensitization did not elicit significant scratching responses (Fig 2, A). More importantly, our allergic conjunctivitis model recapitulates histologic changes associated with severe seasonal allergy or atopic keratoconjunctivitis in humans,6Hu Y. Matsumoto Y. Dogru M. Okada N. Igarashi A. Fukagawa K. et al.The differences of tear function and ocular surface findings in patients with atopic keratoconjunctivitis and vernal keratoconjunctivitis.Allergy. 2007; 62: 917-925Crossref PubMed Scopus (62) Google Scholar as evidenced by our serial OVA challenges leading to inflammatory cells infiltrations and loss of goblet cells in the conjunctiva (Fig 2, B). Interestingly, in this model, ocular itch was significantly attenuated in TRPA1 KO (10.8 ± 1.9 bouts) and TRPV1 KO (13.3 ± 2.9 bouts) mice, suggesting that both TRPA1 and TRPV1 are required for allergic ocular itch. The observed behavioral changes were not due to defects in mast cell activation of TRPA1 KO or TRPV1 KO mice because mast cell degranulation was comparable among WT, TRPA1 KO, and TRPV1 KO mice (see Fig E2 in this article's Online Repository at www.jacionline.org). To further explore the potential of TRPA1 as a therapeutic target for allergic conjunctivitis, we examined the effect of pharmacologically blocking TRPA1 in ocular allergy. Compared with the vehicle-treated group (23.4 ± 1.7 bouts), pretreatment with TRPA1 antagonist HC-030031 significantly alleviated allergic ocular itch in mice (10.7 ± 1.3 scratching bouts). More importantly, combined treatment of TRPA1 and TRPV1 antagonists abolished scratching responses to OVA challenge (Fig 2, C), indicating that the TRPA1 pathway complements the TRPV1 pathway in allergic ocular itch, and that combined pharmacological antagonism of TRPA1 and TRPV1 can be an effective and novel therapeutic strategy for allergy-induced ocular itch. Finally, we investigated potential endogenous histamine-independent itch mediators in ocular allergy. Platelet-activating factor (PAF) and prostaglandin E2 are key inflammatory factors released by mast cells during allergic reaction, and have been shown to cause ocular itch in guinea pigs.7Galli S.J. Tsai M. Piliponsky A.M. The development of allergic inflammation.Nature. 2008; 454: 445-454Crossref PubMed Scopus (1294) Google Scholar, 8Gill P. Jindal N.L. Jagdis A. Vadas P. Platelets in the immune response: revisiting platelet-activating factor in anaphylaxis.J Allergy Clin Immunol. 2015; 135: 1424-1432Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9Woodward D.F. Nieves A.L. Spada C.S. Williams L.S. Tuckett R.P. Characterization of a behavioral model for peripherally evoked itch suggests platelet-activating factor as a potent pruritogen.J Pharmacol Exp Ther. 1995; 272: 758-765PubMed Google Scholar Following this line of evidence, we found that topical PAF challenge evoked strong scratching responses (12 ± 1.2 bouts), but prostaglandin E2 induced only very mild itch in mice (4.4 ± 0.4 bouts) (Fig 2, D). Interestingly, PAF-induced ocular scratching was reduced in TRPA1 KO but not TRPV1 KO mice, suggesting that TRPA1 is required for PAF-induced itch (Fig 2, D). Indeed, GCaMP3-assisted calcium imaging demonstrated direct activation of a small population of TG neurons by PAF in the presence of TRPA1 (see Fig E3 in this article's Online Repository at www.jacionline.org). To determine whether PAF is involved in allergic ocular itch, mice were pretreated with Apafant, a specific PAF receptor antagonist. Apafant significantly alleviated itch in mice with allergic conjunctivitis (Fig 2, E), substantiating the involvement of PAF in ocular allergic itch. Taken together, our data suggest that PAF is one of the upstream itch mediators for histamine-independent TRPA1 pathway in allergic ocular itch. The essential role of TRPA1 in allergic ocular itch prompted us to ask whether TRPA1 plays a similar role in allergic skin itch. Allergic skin itch was only marginally reduced in TRPA1 KO mice (154.3 ± 16.1 bouts) compared with WT mice (206 ± 17.5 bouts). Surprisingly, allergic skin itch was not significantly reduced in TRPV1 KO mice (179.4 ± 28.0 bouts), arguing against a critical role of histamine-dependent pathway in this model of skin itch. Our data reveal the first disparity between skin and ocular itch. (For further information, please see Fig E4 in this article's Online Repository at www.jacionline.org.) Taken together, our results suggest that histamine-independent signaling is equally important as histamine-dependent signaling to mediate ocular itch in allergic conjunctivitis. Current therapeutic strategies for ocular itch management rely heavily on antihistamines and immunosuppressive drugs, and often have limited efficacy. Long-term use of these drugs may be associated with increased risks for ocular complications such as dry eye, glaucoma, and cataract.E1Ciprandi G. Buscaglia S. Cerqueti P.M. Canonica G.W. Drug treatment of allergic conjunctivitis: a review of the evidence.Drugs. 1992; 43: 154-176Crossref PubMed Scopus (57) Google Scholar, E2Friedlaender M.H. Ocular allergy.Curr Opin Allergy Clin Immunol. 2011; 11: 477-482Crossref PubMed Scopus (49) Google Scholar, E3Scheurer M.E. El-Zein R. Thompson P.A. Aldape K.D. Levin V.A. 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Allergic conjunctivitis: update on pathophysiology and prospects for future treatment.J Allergy Clin Immunol. 2005; 115: 118-122Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar TRPA1 can potentially serve as a novel therapeutic target for more effective management of allergic ocular itch, especially in cases refractory to conventional treatments. More importantly, our data also imply that targeting both TRPA1 and TRPV1 may achieve an even better and synergistic therapeutic outcome for ocular itch (Fig 2, F), while circumventing untoward effects of current antiocular itch therapies. We are grateful to Drs David C. Beebe, Hongzhen Hu, Brian S. Kim, Zhou-Feng Chen, and Todd P. Margolis for insightful discussions and comments on the manuscript. We thank Dr Xinzhong Dong for sharing PirtGCaMP3/+ mice. TRPV1 KO (Trpv1tm1Jul/J, RRID:MGI_MGI:3834761), TRPA1 KO (Trpa1tm1Kykw, RRID:MGI_MGI:3625358), and PirtGCaMP3/+ mice were backcrossed to C57BL/6 mice for at least 6 generations and maintained in the congenic background. TRPV1PLAP/+ mice (Trpv1tm2Bbm/J, RRID:IMSR_JAX:017623) were purchased from the Jackson Laboratory (Bar Harbor, Me). TRPM8EGFPf/+ mice were gifts from Dr Gina Story. Eight- to 12-week-old males were used for behavioral tests. All experimental procedures were performed in accordance with protocols approved by the Animal Studies Committee at the Washington University School of Medicine. Experimenters were masked to all mouse genotypes. For ocular itch model, 2.5 μL of testing compounds or vehicle was applied to an inferior conjunctival sac using a micropipette tip after acclimation. For skin itch model, 50 μL of histamine (322 μg), chloroquine (206 μg), or serotonin (10.6 μg) in PBS was injected intradermally around the nape of the back. Individual mouse was placed in a rodent behavior chamber. Scratching and wiping behaviors were video-recorded for 30 minutes and documented. A single scratching bout was defined as the animal lifting its hindpaw, scratching the treated area (typically multiple times within seconds), and putting the hindpaw to the floor or to its mouth. A wiping bout was defined as the animal lifting its forelimb, wiping the ocular area, and returning to its original start position. Histamine dihydrochloride, chloroquine diphosphate salt, serotonin hydrochloride, and allyl isothiocyanate were purchased from Sigma (St Louis, Mo). PAF and prostaglandin E2 were purchased from Cayman Chemical (Ann Arbor, Mich), dissolved in ethanol, and used at 100 μM. TRPV1 antagonist AMG-9810, TRPA1 antagonist HC-030031, and platelet-activating factor receptor antagonist Apafant were purchased from Tocris Bioscience (Bristol, United Kingdom). For behavioral pharmacology studies, 3 μL of 100 μM AMG-9810 (stock solution 50 mM in ethanol) dissolved in saline with 0.1% Tween-80, 200 μM HC-030031 (stock solution 100 mM in dimethyl sulfoxide) in saline, or both antagonists combined was topically applied to the lower conjunctival sac 10 minutes before OVA challenge. Apafant was used at 500 μM (stock solution 100 mM in ethanol). Control groups were treated with the corresponding vehicle. For whole-mount imaging of the conjunctiva, palpebral to fornical conjunctiva devoid of the hairy eyelid skin were isolated in cold, oxygenated (5% CO2, 95% O2) dissection buffer (composition in mM: sucrose 209, KCl 2, NaH2PO4 1.25, MgCl2 5, NaHCO3 26, glucose 10, and CaCl2 0.5) and recovered in recording buffer (composition in mM: NaCl 126, KCl 2.5, NaH2PO4 1.4, MgCl2 1.2, NaHCO3 25, dextrose 11, and CaCl2 2.4) at room temperature (∼25°C) for at least 30 minutes before test. Testing compounds were either perfused into the imaging chamber through a gravity-based delivery system (histamine, chloroquine, and KCl) or bath applied (serotonin and capsaicin). Calcium responses were acquired by an inverted Nikon fluorescence microscope with a CoolSnap HQ2 CCD camera (Photometrics, Tucson, Ariz). Data were quantified offline with the Nikon-NIS program. A minimal 10% increase in ΔF/F0 in GCaMP3 activity was used to threshold nerve terminal activity. Single nerve fiber activity was defined as an increase in the fluorescence intensity of a single identifiable fiber within 30 seconds of delivering chemicals. Any fluorescence changes before chemical application or after bath wash during image acquisition were omitted. Live sensory fibers or TG neurons were identified by eliciting depolarization with 100 mM KCl at the end of each experiment. OVA from chicken egg white from Sigma was diluted in sterile PBS to 0.02% (w/v) and emulsified with an equal volume of Imject Alum (Thermo Scientific, Rockford, Ill) immediately before use. Mice were injected with 200 μL of OVA/alum mixture intraperitoneally on the 1st and 10th days for sensitization. Sensitized mice were topically challenged with 250 μg OVA/PBS in the lower conjunctival sac, or intradermally injected with 50 μg OVA/PBS in the back skin, on the 17th day after acclimation. Scratching and wiping behaviors were video-recorded for 30 minutes immediately after OVA challenge and analyzed manually. In anesthetized mice, DiI solution (150 ng/μL dissolved in dimethyl sulfoxide and 1:5 diluted in sterile saline) was injected via a pulled glass pipette into the subconjunctival space (palpebral conjunctiva) of both lower eyelids. Five days after DiI injection, mice were transcardially perfused with PBS followed by 4% paraformaldehyde in PBS. Whole-mount conjunctiva staining for placental alkaline phosphatase was adapted from published protocols.E6Cavanaugh D.J. Chesler A.T. Jackson A.C. Sigal Y.M. Yamanaka H. Grant R. et al.Trpv1 reporter mice reveal highly restricted brain distribution and functional expression in arteriolar smooth muscle cells.J Neurosci. 2011; 31: 5067-5077Crossref PubMed Scopus (373) Google Scholar Immunofluorescence against green fluorescent protein (GFP) or TRPV1 was performed on sucrose-protected cryosections (15 μm). In brief, after blocking, sections were incubated at 4°C overnight in rabbit anti-GFP antibody (Life Technologies, Eugene, Ore; catalogue no. A11122, RRID:AB_10073917) or rabbit anti-TRPV1 antibody (Neuromics, Edina, Minn; catalogue no. RA14113-100, RRID:AB_2255202) at 1:1000 dilution in PBS containing 0.1% Triton-X100 (PBST). Sections were then washed and incubated at room temperature for 2 hours in Alexa 488–conjugated secondary antibodies (Life Technologies; catalogue no. A11008, RRID:AB_10563748). Of note, GCaMP3 is a fusion protein of GFP and calmodulin, and therefore can be recognized by the anti-GFP antibody. After final washes, slides were mounted with Fluoromount-G (Southern Biotech, Birmingham, Ala). In the mast cell–dependent allergy model, conjunctival and skin mast cells were identified by staining endogenous heparin with Alexa 488–conjugated avidin (Life Technologies; catalogue no. A21370). TG were dissected and rapidly frozen in optimal cutting temperature medium on a dry-ice/ethanol bath. Twenty-micrometer cryosections were collected and allowed to dry for at least 2 hours at room temperature. DiI fluorescence was imaged before in situ hybridization. Following imaging, all steps before hybridization were carried out under Rnase-free conditions. Sections were immersion-fixed in freshly made 4% PFA for 20 minutes at room temperature. Slides were then washed in fresh-Diethylpyrocarbonate (DEPC) PBS (1:1000 DEPC in PBS immediately before use), followed by wash in DEPC-pretreated PBS (1:1000 DEPC in PBS overnight, followed by autoclaving). Sections were immersed in freshly boiled antigen retrieval solution (10 mM citric acid, 0.05% Tween-20, pH = 6.0 with 1:1000 diluted DEPC) in a 95°C water bath for 20 minutes, and then allowed to cool at room temperature for 30 minutes. Sections were washed in DEPC-pretreated PBS, incubated in Proteinase K (25 μg/mL in DEPC-pretreated H2O) for 5 minutes, followed by washes in fresh-DEPC PBS and DEPC-pretreated PBS. Sections were then incubated in freshly made acetylation solution (0.1 M triethanolamine, 0.25% acetic anhydride in DEPC-pretreated H2O) for 10 minutes at room temperature. Next, slides were prehybridized in hybridization buffer (50% formamide, 5 × SSC, 0.3 mg/mL yeast tRNA, 100 μg/mL heparin, 1 × Denhardt's, 0.1% Tween-20, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 5 mM EDTA in Rnase-free H2O) at 62°C in a humidified chamber for 30 minutes. Following prehybridization, 1 to 2 ng/μL of Digoxigenin (DIG)-labeled riboprobe diluted in hybridization buffer was placed on the slide. Slides were incubated overnight under Parafilm coverslips at 62°C and then washed in 0.2 × SSC at 68°C. Following hybridization, slides were blocked in 20% lamb serum in PBST at room temperature for 1 hour. Sections were then incubated with AP-conjugated anti-DIG antibody (1:1000; Roche, catalogue no. 11093274910) in blocking buffer overnight at 4°C. Slides were washed in PBST and incubated overnight in darkness in alkaline phosphatase buffer: 100 mM Tris, pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20, 5 mM levamisole, 0.34 mg/mL 4-Nitro blue tetrazolium (Roche, Nutley, NJ; catalogue no. 11383213001) and 0.17 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate (Roche, catalogue no. 1138221001). Slides were then repeatedly rinsed in ddH2O, dried at 37°C for 1 hour, dehydrated in xylenes, and covered in Permount (Fisher Scientific, Pittsburgh, Pa; catalogue no. SP15). Data are shown as means ± SEM. One-way ANOVA was used for initial analyses among groups, followed by Turkey post hoc tests. Differences between groups were considered statistically significant when P < .05.Fig E2TRPA1 and TRPV1 are not required for allergen-induced mast cell degranulation. A, No difference in conjunctival mast cell degranulation was observed among 3 genotypes. White arrows, OVA-sensitized conjunctival mast cells. Red arrows, activated conjunctival mast cells by OVA. Scale bars represent 100 μm; 20 μm in insets. B, Quantitative analysis of mast cell degranulation among 3 genotypes. N ≥ 3 mice and >200 mast cells were analyzed per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3TRPA1 is required for PAF-induced Ca2+ mobilization in sensory neurons. A, PAF activates a subset of TRPA1+ neurons from the TG. Increased Ca2+ mobility on PAF (10 μM) and allyl isothiocyanate (AITC, TRPA1 agonist, 100 μM) treatment in PirtGCaMP3/+ TG neurons. Arrows indicate neurons that are sensitive to both PAF and AITC. B, Calcium traces of TG neurons that responded to PAF and AITC in A. C, PAF-induced Ca2+ mobility in TG neurons was significantly attenuated in TRPA1 KO mice. N ≥ 3 mice and >400 TG neurons were analyzed per group. Scale bar represents 100 μm. *P < .05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Behavioral responses of pruritogen-induced and allergy-induced skin itch. A, Scratching responses induced on the intradermal injection of OVA solution into the back skin in an allergic skin itch model. TRPA1 KO mice showed only marginally fewer scratching bouts than did the WT group, but did not differ from the TRPV1 KO mice (n = 9-14 per group). B, No difference in degranulation of skin mast cell was observed among 3 genotypes. N ≥ 3 mice and >500 mast cells were analyzed per group. C-E, Histamine-dependent itch was reduced in TRPV1 KO mice. Chloroquine-dependent itch was reduced in TRPA1 KO mice. No difference was shown among genotypes in serotonin-induced itch (n = 7-12 per group). Scale bars represent 50 μm; 10 μm in insets. ns, Not significant. ***P < .001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Mild and cost-effective green fluorescent protein purification employing small synthetic ligands

The green fluorescent protein (GFP) is a useful indicator in a broad range of applications including cell biology, gene expression and biosensing. However, its full potential is hampered by the lack of a selective, mild and low-cost purification scheme. In order to address this demand, a novel adsorbent was developed as a generic platform for the purification of GFP or GFP fusion proteins, giving GFP a dual function as reporter and purification tag. After screening a solid-phase combinatorial library of small synthetic ligands based on the Ugi-reaction, the lead ligand (A4C7) selectively recovered GFP with 94% yield and 94% purity under mild conditions and directly from Escherichia coli extracts. Adsorbents containing the ligand A4C7 maintained the selectivity to recover other proteins fused to GFP. The performance of A4C7 adsorbents was compared with two commercially available methods (immunoprecipitation and hydrophobic interaction chromatography), confirming the new adsorbent as a low-cost viable alternative for GFP purification.