Tailoring Chimeric Ligands for Studying and Biasing ErbB Receptor Family Interactions

Krueger, Andrew T.; Kroll, Carsten; Sánchez, Edgar N.; Griffith, Linda G.; Imperiali, Barbara

doi:10.1002/anie.201307869

Cited by 24 publications

(31 citation statements)

References 43 publications

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“…However, the affibody polyproteam showed good stability over time and to heating. Polyproteams should be a powerful platform to dissect the spatial requirements for cellular signaling, such as in immunity and differentiation (51)(52)(53). Other applications of this simple route to new biological architectures may include vaccination (2), biomaterials (34,(54)(55)(56), multienzyme organization (9), and enhancing capture of circulating tumor cells (57).…”

Section: Discussionmentioning

confidence: 99%

Programmable polyproteams built using twin peptide superglues

Veggiani

Nakamura

Brenner

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

282

360

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Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Here we achieved sequenceprogrammed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. SpyTag peptide is engineered to spontaneously form an isopeptide bond with SpyCatcher protein. By engineering the adhesin RrgA from Streptococcus pneumoniae, we developed the peptide SnoopTag, which formed a spontaneous isopeptide bond to its protein partner SnoopCatcher with >99% yield and no crossreaction to SpyTag/SpyCatcher. Solid-phase attachment followed by sequential SpyTag or SnoopTag reaction between building-blocks enabled iterative extension. Linear, branched, and combinatorial polyproteins were synthesized, identifying optimal combinations of ligands against death receptors and growth factor receptors for cancer cell death signal activation. This simple and modular route to programmable "polyproteams" should enable exploration of a new area of biological space.synthetic biology | protein engineering | nanobiotechnology | split protein | antibody

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Section: Discussionmentioning

confidence: 99%

Programmable polyproteams built using twin peptide superglues

Veggiani

Nakamura

Brenner

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

282

360

View full text Add to dashboard Cite

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“…SrtA catalyzes a peptide exchange process of the general form: (R)- LPXTG + G GG -(R’) = (R)- LPXT G GG -(R’) + G . This transpeptidase reaction is now an established protein engineering tool, used to ligate large protein subdomains together or to link proteins with synthetic polymers (24, 28, 29). The reversibility of SrtA-mediated reactions (28, 29), which is a shortcoming in most protein engineering applications, led us to investigate whether SrtA mutants could be used to disassemble synthetic ECM crosslinked with defined peptides while preserving crucial extracellular signaling proteins.…”

Section: Introductionmentioning

confidence: 99%

On-demand dissolution of modular, synthetic extracellular matrix reveals local epithelial-stromal communication networks

et al. 2017

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Methods to parse paracrine epithelial-stromal communication networks are a vital need in drug development, as disruption of these networks underlies diseases ranging from cancer to endometriosis. Here, we describe a modular, synthetic, and dissolvable extracellular matrix (MSD-ECM) hydrogel that fosters functional 3D epithelial-stromal co-culture, and that can be dissolved on-demand to recover cells and paracrine signaling proteins intact for subsequent analysis. Specifically, synthetic polymer hydrogels, modified with cell-interacting adhesion motifs and crosslinked with peptides that include a substrate for cell-mediated proteolytic remodeling, can be rapidly dissolved by an engineered version of the microbial transpeptidase Sortase A (SrtA) if the crosslinking peptide includes a SrtA substrate motif and a soluble second substrate. SrtA-mediated dissolution affected only 1 of 31 cytokines and growth factors assayed, whereas standard protease degradation methods destroyed about half of these same molecules. Using co-encapsulated endometrial epithelial and stromal cells as one model system, we show that the dynamic cytokine and growth factor response of co-cultures to an inflammatory cue is richer and more nuanced when measured from SrtA-dissolved gel microenvironments than from the culture supernate. This system employs accessible, reproducible reagents and facile protocols; hence, has potential as a tool in identifying and validating therapeutic targets in complex diseases.

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“…We have previously shown maleimide functionalized pNIPAAm to yield homodimers up to 21%, 2 4 . but the polymers used for those conjugations were much longer (15.5 kDa).…”

Section: Resultsmentioning

confidence: 99%

“…38 The tetrazine- trans -cyclooctene ligation proceeds very rapidly ( k 2 up to 10 6 M −1 s −1 ) without a catalyst and at almost 100% conversion at micromolar concentrations at room temperature. 39 This ligation has been employed to prepare dimeric PEG-based conjugate species for studying receptor interactions through the use of a tetrazine-norbornene ligation, 4 but has not been explored with CRP-based polymers. Due to the discriminating nature of this ligation and rate of the tetrazine- trans -cyclooctene ligation, we hypothesized that homotelechelic tetrazine-polymers will enhance protein dimerization yields at significantly decreased times compared to existing dimerization methods.…”

Section: Introductionmentioning

confidence: 99%

Homodimeric Protein–Polymer Conjugates via the Tetrazine–trans-Cyclooctene Ligation

et al. 2015

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Tetrazine end-functionalized telechelic polymers were synthesized by controlled radical polymerization (CRP) and employed to generate T4 Lysozyme homodimers. Mutant T4 Lysozyme (V131C), containing a single surface-exposed cysteine, was modified with a protein-reactive trans-cyclooctene (T4L-TCO). Reversible addition-fragmentation chain transfer (RAFT) polymerization yielded poly(N-isopropylacrylamide) (pNIPAAm) with a number average molecular weight (Mn by 1H-NMR) of 2.0 kDa and a dispersity (Đ by GPC) of 1.05. pNIPAAm was then modified at both ends by post-polymerization with 6-methyl tetrazine. For comparison, 2.0 kDa bis-tetrazine poly(ethylene glycol) (PEG) and 2.0 kDa bis-maleimide pNIPAAm were synthesized. Ligation of T4L-TCO to bis-tetrazine pNIPAAm or bis-tetrazine PEG resulted in protein homodimer in 38% yield and 37% yield, respectively, after only 1 hour, whereas bis-maleimide pNIPAAm resulted in only 5% yield of dimer after 24 h. This work illustrates the advantage of employing tetrazine ligation over maleimide thiol-ene chemistry for the synthesis of protein homodimer conjugates.

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Tailoring Chimeric Ligands for Studying and Biasing ErbB Receptor Family Interactions

Cited by 24 publications

References 43 publications

Programmable polyproteams built using twin peptide superglues

Programmable polyproteams built using twin peptide superglues

On-demand dissolution of modular, synthetic extracellular matrix reveals local epithelial-stromal communication networks

Homodimeric Protein–Polymer Conjugates via the Tetrazine–trans-Cyclooctene Ligation

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