A Suite of Engineered GFP Molecules for Oligomeric Scaffolding

Leibly, D.J.; Arbing, M.A.; Pashkov, I.; DeVore, Natasha M.; Waldo, Geoffrey S.; Terwilliger, Thomas C.; Yeates, Todd O.

doi:10.1016/j.str.2015.07.008

Cited by 32 publications

(26 citation statements)

References 45 publications

(67 reference statements)

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“…To determine the role of the multimericity of a soluble ligand in triggering CAR activation, we generated a green fluorescent protein (GFP)-binding CAR model system and compared two soluble ligands: monomeric superfolder GFP (sfGFP) versus homodimeric sfGFP linked by a disulfide bond introduced by a D117C substitution ( Fig. 1a ) 17 . The monomeric and dimeric states of the sfGFP ligands were verified by size-exclusion chromatography ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Rewiring T-cell responses to soluble factors with chimeric antigen receptors

et al. 2018

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Chimeric antigen receptor (CAR)-expressing T cells targeting surface-bound tumor antigens have yielded promising clinical outcomes, with two CD19 CAR-T cell therapies recently receiving FDA approval for the treatment of B-cell malignancies. The adoption of CARs for the recognition of soluble ligands, a distinct class of biomarkers in physiology and disease, could significantly broaden the utility of CARs in disease treatment. In this study, we demonstrate that CAR-T cells can be engineered to respond robustly to diverse soluble ligands, including CD19 ectodomain, GFP variants, and transforming growth factor beta (TGF-β). We additionally show that CAR signaling in response to soluble ligands relies on ligand-mediated CAR dimerization, and that CAR responsiveness to soluble ligands can be fine-tuned by adjusting the mechanical coupling between the CAR’s ligand-binding and signaling domains. Our results support a role for mechanotransduction in CAR signaling and demonstrate an approach to systematically engineer immune-cell responses to soluble, extracellular ligands.

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Section: Resultsmentioning

confidence: 99%

Rewiring T-cell responses to soluble factors with chimeric antigen receptors

et al. 2018

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“…Moreover, this system will enable the combinatory assembly of uncommon receptor combinations with desirable signaling potentials and capacities. The number of recruited synthetic receptors is only limited by the maximal number of ligands connected in one GFP:mCherry fusion protein or by alternative GFP/mCherry multimerization strategies 32 , 33 .…”

Section: Discussionmentioning

confidence: 99%

Synthetic cytokine receptors transmit biological signals using artificial ligands

et al. 2018

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Cytokine-induced signal transduction is executed by natural biological switches, which among many others control immune-related processes. Here, we show that synthetic cytokine receptors (SyCyRs) can induce cytokine signaling using non-physiological ligands. High-affinity GFP- and mCherry-nanobodies were fused to transmembrane and intracellular domains of the IL-6/IL-11 and IL-23 cytokine receptors gp130 and IL-12Rβ1/IL-23R, respectively. Homo- and heterodimeric GFP:mCherry fusion proteins as synthetic cytokine-like ligands were able to induce canonical signaling in vitro and in vivo. Using SyCyR ligands, we show that IL-23 receptor homodimerization results in its activation and IL-23-like signal transduction. Moreover, trimeric receptor assembly induces trans-phosphorylation among cytokine receptors with associated Janus kinases. The SyCyR technology allows biochemical analyses of transmembrane receptor signaling in vitro and in vivo, cell-specific activation through SyCyR ligands using transgenic animals and possible therapeutic regimes involving non-physiological targets during immunotherapy.

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“…Split green fluorescent proteins (sGFPs), where symmetric splits of the GFP β-barrel 1 or strategic removal of one or more of its 11 β-strands 2 – 6 are engineered to control the re-assembly of full-length GFPs (flGFPs), provide powerful approaches to study the β-strand structural stability of GFP as well as the photophysics and the photochemistry of its tripeptide chromophore (S65-Y66-G67) 5 – 9 . Such complementary sGFP fragments can additionally be employed as protein tags to assess the solubility of recombinantly expressed proteins 3 , study protein distributions in cells and animals by ensemble or single molecule fluorescence imaging 10 – 15 , target nanomaterials in cells 14 , 16 , 17 or design supramolecular protein nanostructures 18 , 19 . Amongst the various sGFPs available, those based on super-folder GFP 20 have been particularly useful for the aforementioned applications.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Split Fluorescent Protein Variants and Quantitative Analyses of Their Self-Assembly Process

Koker

Fernandez

Pinaud

2018

Sci Rep

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Many biotechniques use complementary split-fluorescent protein (sFPs) fragments to visualize protein-protein interactions, image cells by ensemble or single molecule fluorescence microscopy, or assemble nanomaterials and protein superstructures. Yet, the reassembly mechanisms of sFPs, including fragment binding rates, folding, chromophore maturation and overall photophysics remain poorly characterized. Here, we evolved asymmetric and self-complementing green, yellow and cyan sFPs together with their full-length equivalents (flFPs) and described their biochemical and photophysical properties in vitro and in cells. While re-assembled sFPs have spectral properties similar to flFPs, they display slightly reduced quantum yields and fluorescence lifetimes due to a less sturdy β-barrel structure. The complementation of recombinant sFPs expressed in vitro follows a conformational selection mechanism whereby the larger sFP fragments exist in a monomer-dimer equilibrium and only monomers are competent for fluorescence complementation. This bimolecular fragment interaction involves a slow and irreversible binding step, followed by chromophore maturation at a rate similar to that of flFPs. When expressed as fusion tags in cells, sFPs behave as monomers directly activated with synthetic complementary fragments. This study resulted in the development of sFP color variants having improved maturation kinetics, brightness, and photophysics for fluorescence microscopy imaging of cellular processes, including single molecule detection.

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A Suite of Engineered GFP Molecules for Oligomeric Scaffolding

Cited by 32 publications

References 45 publications

Rewiring T-cell responses to soluble factors with chimeric antigen receptors

Rewiring T-cell responses to soluble factors with chimeric antigen receptors

Synthetic cytokine receptors transmit biological signals using artificial ligands

Characterization of Split Fluorescent Protein Variants and Quantitative Analyses of Their Self-Assembly Process

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