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The serine/threonine protein kinase casein kinase 1α (CK1α) functions as a negative regulator of Wnt signaling, phosphorylating β-catenin at serine 45 (P-S45) to initiate its eventual ubiquitin-mediated degradation. We previously showed that the repurposed, FDA-approved anthelminthic drug pyrvinium potently inhibits Wnt signaling and Moreover, we proposed that pyrvinium's Wnt inhibitory activity was the result of its function as an activator of CK1α. An understanding of the mechanism by which pyrvinium activates CK1α is important because pyrvinium was given an orphan drug designation by the FDA to treat familial adenomatous polyposis, a precancerous condition driven by constitutive Wnt signaling. In the current study, we show that pyrvinium stimulates the phosphorylation of S45 β-catenin, a known CK1α substrate, in a cell-based assay, and does so in a dose- and time-dependent manner. Alternative splicing of CK1α results in four forms of the protein with distinct biological properties. We evaluated these splice products and identified the CK1α splice variant, CK1αS, as the form that exhibits the most robust response to pyrvinium in cells. Kinetic studies indicate that pyrvinium also stimulates the kinase activity of purified, recombinant CK1αS , increasing its catalytic efficiency (/) toward substrates. These studies provide strong and clear mechanistic evidence that pyrvinium enhances CK1α kinase activity.
The presynaptic dopamine transporter mediates rapid reuptake of synaptic dopamine. Although cell surface DAT trafficking recently emerged as an important component of DAT regulation, it has not been systematically investigated. Here, we apply our single quantum dot (Qdot) tracking approach to monitor DAT plasma membrane dynamics in several heterologous expression cell hosts with nanometer localization accuracy. We demonstrate that Qdot-tagged DAT proteins exhibited highly heterogeneous membrane diffusivity dependent on the local membrane topography. We also show that Qdot-tagged DATs were localized away from the flat membrane regions and were dynamically retained in the membrane protrusions and cell edges for the duration of imaging. Single quantum dot tracking of wildtype DAT and its conformation-defective coding variants (R60A and W63A) revealed a significantly accelerated rate of dysfunctional DAT membrane diffusion. We believe our results warrant an in-depth investigation as to whether compromised membrane dynamics is a common feature of brain disorder-derived DAT mutants.
Peptides and biologics provide unique opportunities to modulate intracellular targets not druggable by conventional small molecules. Most peptides and biologics are fused with cationic uptake moieties or formulated into nanoparticles to facilitate delivery, but these systems typically lack potency due to low uptake and/or entrapment and degradation in endolysosomal compartments. Because most delivery reagents comprise cationic lipids or polymers, there is a lack of reagents specifically optimized to deliver cationic cargo. Herein, we demonstrate the utility of the cytocompatible polymer poly(propylacrylic acid) (PPAA) to potentiate intracellular delivery of cationic biomacromolecules and nano-formulations. This approach demonstrates superior efficacy over all marketed peptide delivery reagents and enhances delivery of nucleic acids and gene editing ribonucleoproteins (RNPs) formulated with both commercially-available and our own custom-synthesized cationic polymer delivery reagents. These results demonstrate the broad potential of PPAA to serve as a platform reagent for the intracellular delivery of cationic cargo.
Cation-chloride cotransporters (CCCs) mediate the coupled, electroneutral symport of cations with chloride across the plasma membrane and are vital for cell volume regulation, salt reabsorption in the kidney, and γ-aminobutyric acid (GABA)-mediated modulation in neurons. Here we present cryo-electron microscopy (cryo-EM) structures of human potassium-chloride cotransporter KCC1 in potassium chloride or sodium chloride at 2.9- to 3.5-angstrom resolution. KCC1 exists as a dimer, with both extracellular and transmembrane domains involved in dimerization. The structural and functional analyses, along with computational studies, reveal one potassium site and two chloride sites in KCC1, which are all required for the ion transport activity. KCC1 adopts an inward-facing conformation, with the extracellular gate occluded. The KCC1 structures allow us to model a potential ion transport mechanism in KCCs and provide a blueprint for drug design.
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
BACKGROUND/AIMS - The prostaglandin E (PGE) EP3 receptor has a multifaceted role in metabolism. Drugs targeting EP3 have been proposed as therapeutics for diabetes; however, studies utilizing global EP3 knockout mice suggest that EP3 blockade increases obesity and insulin resistance. The present studies attempt to determine the effect of acute EP3 antagonist treatment on the diabetic phenotype.
METHODS - DG-041 was confirmed to be a high affinity antagonist at the mouse EP3 receptor by competition radioligand binding and by blockade of EP3-mediated responses. DG-041 pharmacokinetic studies were performed to determine the most efficacious route of administration. Male C57BL/6 × BALB/c (CB6F1) mice were fed diets containing 10%, 45%, or 60% calories from fat to induce obesity. Changes to the metabolic phenotype in these mice were evaluated after one week treatment with DG-041.
RESULTS - Subcutaneous injections of DG-041 at 20 mg/kg blocked the sulprostone-evoked rise in mean arterial pressure confirming the efficacy of this administration regime. Seven day treatment with DG-041 had minimal effect on body composition or glycemic control. DG-041 administration caused a reduction in skeletal muscle triglyceride content while showing a trend toward increased hepatic triglycerides.
CONCLUSION - Short term EP3 administration of DG-041 produced effective blockade of the EP3 receptor and decreased skeletal muscle triglyceride content but had no significant effects on the diabetic phenotype.
Published by Elsevier Inc.
BACKGROUND - Mutations in SLC6A1 have been associated mainly with myoclonic atonic epilepsy (MAE) and intellectual disability. We identified a novel missense mutation in a patient with Lennox-Gastaut syndrome (LGS) characterized by severe seizures and developmental delay.
METHODS - Exome Sequencing was performed in an epilepsy patient cohort. The impact of the mutation was evaluated by H γ-aminobutyric acid (GABA) uptake, structural modeling, live cell microscopy, cell surface biotinylation and a high-throughput assay flow cytometry in both neurons and non neuronal cells.
RESULTS - We discovered a heterozygous missense mutation (c700G to A [pG234S) in the SLC6A1 encoding GABA transporter 1 (GAT-1). Structural modeling suggests the mutation destabilizes the global protein conformation. With transient expression of enhanced yellow fluorescence protein (YFP) tagged rat GAT-1 cDNAs, we demonstrated that the mutant GAT-1(G234S) transporter had reduced total protein expression in both rat cortical neurons and HEK 293 T cells. With a high-throughput flow cytometry assay and live cell surface biotinylation, we demonstrated that the mutant GAT-1(G234S) had reduced cell surface expression. H radioactive labeling GABA uptake assay in HeLa cells indicated a reduced function of the mutant GAT-1(G234S).
CONCLUSIONS - This mutation caused instability of the mutant transporter protein, which resulted in reduced cell surface and total protein levels. The mutation also caused reduced GABA uptake in addition to reduced protein expression, leading to reduced GABA clearance, and altered GABAergic signaling in the brain. The impaired trafficking and reduced GABA uptake function may explain the epilepsy phenotype in the patient.
Copyright © 2019. Published by Elsevier Inc.
The unfolded protein response (UPR) detects and restores deficits in the endoplasmic reticulum (ER) protein folding capacity. Ceapins specifically inhibit the UPR sensor ATF6α, an ER-tethered transcription factor, by retaining it at the ER through an unknown mechanism. Our genome-wide CRISPR interference (CRISPRi) screen reveals that Ceapins function is completely dependent on the ABCD3 peroxisomal transporter. Proteomics studies establish that ABCD3 physically associates with ER-resident ATF6α in cells and in vitro in a Ceapin-dependent manner. Ceapins induce the neomorphic association of ER and peroxisomes by directly tethering the cytosolic domain of ATF6α to ABCD3's transmembrane regions without inhibiting or depending on ABCD3 transporter activity. Thus, our studies reveal that Ceapins function by chemical-induced misdirection which explains their remarkable specificity and opens up new mechanistic routes for drug development and synthetic biology.
© 2019, Torres et al.
Activation of the unfolded protein response (UPR)-associated transcription factor ATF6 has emerged as a promising strategy to reduce the secretion and subsequent toxic aggregation of destabilized, amyloidogenic proteins implicated in systemic amyloid diseases. However, the molecular mechanism by which ATF6 activation reduces the secretion of amyloidogenic proteins remains poorly defined. We employ a quantitative interactomics platform to define how ATF6 activation reduces secretion of a destabilized, amyloidogenic immunoglobulin light chain (LC) associated with light-chain amyloidosis (AL). Using this platform, we show that ATF6 activation increases the targeting of this destabilized LC to a subset of pro-folding ER proteostasis factors that retains the amyloidogenic LC within the ER, preventing its secretion. Our results define a molecular basis for the ATF6-dependent reduction in destabilized LC secretion and highlight the advantage for targeting this UPR-associated transcription factor to reduce secretion of destabilized, amyloidogenic proteins implicated in AL and related systemic amyloid diseases.
Copyright © 2019 Elsevier Ltd. All rights reserved.
The classic mode of G protein-coupled receptor (GPCR)-mediated transactivation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) transactivation occurs via matrix metalloprotease (MMP)-mediated cleavage of plasma membrane-anchored EGFR ligands. Herein, we show that the Gαs-activating GPCR ligands vasoactive intestinal peptide (VIP) and prostaglandin E (PGE ) transactivate EGFR through increased cell-surface delivery of the EGFR ligand transforming growth factor-α (TGFα) in polarizing madin-darby canine kidney (MDCK) and Caco-2 cells. This is achieved by PKA-mediated phosphorylation of naked cuticle homolog 2 (NKD2), previously shown to bind TGFα and direct delivery of TGFα-containing vesicles to the basolateral surface of polarized epithelial cells. VIP and PGE rapidly activate protein kinase A (PKA) that then phosphorylates NKD2 at Ser-223, a process that is facilitated by the molecular scaffold A-kinase anchoring protein 12 (AKAP12). This phosphorylation stabilized NKD2, ensuring efficient cell-surface delivery of TGFα and increased EGFR activation. Thus, GPCR-triggered, PKA/AKAP12/NKD2-regulated targeting of TGFα to the cell surface represents a new mode of EGFR transactivation that occurs proximal to ligand cleavage by MMPs.
© 2019 The Authors. Traffic published by John Wiley & Sons Ltd.
Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor (GPCR) subtypes and dozens of non-receptor signaling proteins. Here we describe the methods used to identify the interaction sites of arrestin-binding partners on arrestin-3 and the use of monofunctional individual arrestin-3 elements in cells. Our in vitro pull-down assay with purified proteins demonstrates that relatively few elements in arrestin engage each partner, whereas cell-based functional assays indicate that certain arrestin elements devoid of other functionalities can perform individual functions in living cells.