A Stabilizing Influence: CAL PDZ Inhibition Extends the Half‐Life of ΔF508‐CFTR

Cushing, Patrick R.; Vouillème, Lars; Pellegrini, Maria; Boisguérin, Prisca; Madden, Dean R.

doi:10.1002/anie.201005585

Cited by 63 publications

(78 citation statements)

References 31 publications

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“…This structure allows this protein to interact with both cargo and trafficking machinery. Interestingly, GOPC promotes the lysosomal targeting of CFTR and Muc3 but promotes cell surface targeting of receptors such as Frizzled (Cheng et al, 2010a; Cheng et al, 2002; Cheng et al, 2004; Cushing et al, 2010; Pelaseyed and Hansson, 2011; Yao et al, 2001). The enhanced lysosomal targeting of CFTR and Muc3 may relate to the fact that GOPC interacts with the autophagy machinery, and is required for development of autophagic vacuoles after activation of the cell surface antigen CD46 (Meiffren et al, 2010; Richetta et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Scaffolding protein GOPC regulates tight junction structure

Stewart

Wilson

2015

Cell Tissue Res

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GOPC (FIG/PIST/CAL) is a PDZ-domain scaffolding protein that regulates the trafficking of a wide array of proteins, including small GTPases, receptors, and cell surface molecules such as cadherin 23 and CFTR. In Madin-Darby Canine Kidney (MDCK) cells, we find that GOPC localizes to the Trans-Golgi Network (TGN), but not the cis or trans Golgi cisternae. There is also colocalization with the early endosome Rab GTPase Rab5 and a TGN/endosome marker Rab14, but not with Rab11, a marker of recycling endosomes. There is no localization of GOPC to the lateral membranes or tight junctions. We find that knockdown of GOPC in MDCK cells results in decreased transepithelial resistance and increased paracellular flux. This may be due to compromised trafficking of tight junction components from the TGN, as GOPC knockdown cells have decreased lateral labeling of the tight junction protein claudin-1 and decreased protein levels of claudin-2. GOPC may mediate trafficking of newly synthesized tight junction proteins from the TGN to the cell surface or recycling of these proteins from specialized endosomal compartments.

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Section: Introductionmentioning

confidence: 99%

Scaffolding protein GOPC regulates tight junction structure

Stewart

Wilson

2015

Cell Tissue Res

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“…The CFTR-associated ligand (CAL) controls the apical membrane half-life of CFTR and is a validated therapeutic target for the disease cystic fibrosis [5], [9], [10]. We have previously described a peptide array-based approach that enabled us to design a selective inhibitor for the CAL PDZ (CALP) domain.…”

Section: Introductionmentioning

confidence: 99%

Chemically Modified Peptide Scaffolds Target the CFTR-Associated Ligand PDZ Domain

et al. 2014

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PDZ domains are protein-protein interaction modules that coordinate multiple signaling and trafficking pathways in the cell and that include active therapeutic targets for diseases such as cancer, cystic fibrosis, and addiction. Our previous work characterized a PDZ interaction that restricts the apical membrane half-life of the cystic fibrosis transmembrane conductance regulator (CFTR). Using iterative cycles of peptide-array and solution-binding analysis, we targeted the PDZ domain of the CFTR-Associated Ligand (CAL), and showed that an engineered peptide inhibitor rescues cell-surface expression of the most common CFTR disease mutation ΔF508. Here, we present a series of scaffolds containing chemically modifiable side chains at all non-motif positions along the CAL PDZ domain binding cleft. Concordant equilibrium dissociation constants were determined in parallel by fluorescence polarization, isothermal titration calorimetry, and surface plasmon resonance techniques, confirming robust affinity for each scaffold and revealing an enthalpically driven mode of inhibitor binding. Structural studies demonstrate a conserved binding mode for each peptide, opening the possibility of combinatorial modification. Finally, we diversified one of our peptide scaffolds with halogenated substituents that yielded modest increases in binding affinity. Overall, this work validates our approach and provides a stereochemical foundation for further CAL inhibitor design and screening.

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“…Peptide affinities for the two domains were determined by Fluorescence Polarization (FP) using well characterized assays [42, 49, 57] (see Materials and Methods). The affinity of each domain for a fluorescently labeled reporter peptide was measured first, enabling competition assays to determine apparent inhibition constants, K i , of designed and native peptides (see Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

“…Fluorescence Polarization (FP) assays were performed to measure domain-peptide dissociation and inhibition constants ( K d and K i , respectively) generally as described elsewhere in the literature [42], with some minor changes. Purified domain proteins were dialyzed into FP buffer A (25 mM Tris, pH 7.5, 150 mM NaCl, 0,1 mM TCEP).…”

Section: Methodsmentioning

confidence: 99%

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Computational Design of Selective Peptides to Discriminate between Similar PDZ Domains in an Oncogenic Pathway

Zheng

Jewell

Fitzpatrick

et al. 2015

Journal of Molecular Biology

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Reagents that target protein-protein interactions to rewire signaling are of great relevance in biological research. Computational protein design may offer a means of creating such reagents on demand, but methods for encoding targeting selectivity are sorely needed. This is especially challenging when targeting interactions with ubiquitous recognition modules—e.g., PDZ domains, which bind C-terminal sequences of partner proteins. Here we consider the problem of designing selective PDZ inhibitor peptides in the context of an oncogenic signaling pathway, in which two PDZ domains (NHERF-2 PDZ2—N2P2 and MAGI-3 PDZ6—M3P6) compete for a receptor C-terminus to differentially modulate oncogenic activities. Because N2P2 increases tumorigenicity and M3P6 decreases it, we sought to design peptides that inhibit N2P2 without affecting M3P6. We developed a structure-based computational design framework that models peptide flexibility in binding, yet is efficient enough to rapidly analyze tradeoffs between affinity and selectivity. Designed peptides showed low-micromolar inhibition constants for N2P2 and no detectable M3P6 binding. Peptides designed for reverse discrimination bound M3P6 tighter than N2P2, further testing our technology. Experimental and computational analysis of selectivity determinants revealed significant indirect energetic coupling in the binding site. Successful discrimination between N2P2 and M3P6, despite their overlapping binding preferences, is highly encouraging for computational approaches to selective PDZ targeting, especially because design relied on a homology model of M3P6. Still, we demonstrate specific deficiencies of structural modeling that must be addressed to enable truly robust design. The presented framework is general and can be applied in many scenarios to engineer selective targeting.

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A Stabilizing Influence: CAL PDZ Inhibition Extends the Half‐Life of ΔF508‐CFTR

Cited by 63 publications

References 31 publications

Scaffolding protein GOPC regulates tight junction structure

Scaffolding protein GOPC regulates tight junction structure

Chemically Modified Peptide Scaffolds Target the CFTR-Associated Ligand PDZ Domain

Computational Design of Selective Peptides to Discriminate between Similar PDZ Domains in an Oncogenic Pathway

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