scholarly journals IRE1α kinase–mediated unconventional protein secretion rescues misfolded CFTR and pendrin

2020 ◽  
Vol 6 (8) ◽  
pp. eaax9914
Author(s):  
Hak Park ◽  
Dong Hoon Shin ◽  
Ju-Ri Sim ◽  
Sowon Aum ◽  
Min Goo Lee
Keyword(s):  
Cell Surface ◽  
Protein Secretion ◽  
Protein Misfolding ◽  
Mammalian Cells ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Downstream Effector ◽  
Mouse Colon ◽  
Δf508 Cftr ◽  
Kinase Pathway

The most prevalent pathogenic mutations in the CFTR (ΔF508) and SLC26A4/pendrin (p.H723R), which cause cystic fibrosis and congenital hearing loss, respectively, evoke protein misfolding and subsequent defects in their cell surface trafficking. Here, we report that activation of the IRE1α kinase pathway can rescue the cell surface expression of ΔF508-CFTR and p.H723R-pendrin through a Golgi-independent unconventional protein secretion (UPS) route. In mammalian cells, inhibition of IRE1α kinase, but not inhibition of IRE1α endonuclease and the downstream effector XBP1, inhibited CFTR UPS. Treatment with the IRE1α kinase activator, (E)-2-(2-chlorostyryl)-3,5,6-trimethyl-pyrazine (CSTMP), rescued cell surface expression and functional activity of ΔF508-CFTR and p.H723R-pendrin. Treatment with a nontoxic dose of CSTMP to ΔF508-CFTR mice restored CFTR surface expression and CFTR-mediated anion transport in the mouse colon. These findings suggest that UPS activation via IRE1α kinase is a strategy to treat diseases caused by defective cell surface trafficking of membrane proteins, including ΔF508-CFTR and p.H723R-pendrin.

scholarly journals ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter

2018 ◽  
Vol 23 (8) ◽  
pp. 823-831
Author(s):  
Puay-Wah Phuan ◽  
Guido Veit ◽  
Joseph-Anthony Tan ◽  
Ariel Roldan ◽  
Walter E. Finkbeiner ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Nucleotide Binding ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Nucleotide Binding Domain ◽  
Δf508 Cftr ◽  
Nucleotide Binding Domain 1 ◽  
Cftr Modulators

The most common cystic fibrosis–causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (∆F508). The ∆F508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify ∆F508-CFTR modulators that act on NBD1. A biochemical screen for ΔF508-NBD1 cell surface expression was done in Madin–Darby canine kidney cells expressing a chimeric reporter consisting of ΔF508-NBD1, the CD4 transmembrane domain, and an extracellular horseradish peroxidase (HRP) reporter. Using a luminescence readout of HRP activity, the screen was robust with a Z′ factor of 0.7. The screening of ~20,000 synthetic small molecules allowed the identification of compounds from four chemical classes that increased ∆F508-NBD1 cell surface expression by up to 4-fold; for comparison, a 12-fold increased cell surface expression was found for a wild-type NBD1 chimera. While the compounds were inactive as correctors of full-length ΔF508-CFTR, several carboxamide-benzothiophenes had potentiator activity with low micromolar EC50. Interestingly, the potentiators did not activate G551D or wild-type CFTR. Our results provide a proof of concept for a cell-based NBD1 domain screen to identify ∆F508-CFTR modulators that target the NBD1 domain.

scholarly journals The CFTR-Associated Ligand Arrests the Trafficking of the Mutant ΔF508 CFTR Channel in the ER Contributing to Cystic Fibrosis

2018 ◽  
Vol 45 (2) ◽  
pp. 639-655 ◽  
Author(s):  
Emily Bergbower ◽  
Clement Boinot ◽  
Inna Sabirzhanova ◽  
William Guggino ◽  
Liudmila Cebotaru
Keyword(s):  
Cell Surface ◽  
Cell Biology ◽  
Pdz Domain ◽  
Coiled Coil ◽  
Surface Expression ◽  
26S Proteasome ◽  
Scaffolding Protein ◽  
Cell Surface Expression ◽  
Membrane Expression ◽  
Δf508 Cftr

Background/Aims: The CFTR-Associated Ligand (CAL), a PDZ domain containing protein with two coiled-coil domains, reduces cell surface WT CFTR through degradation in the lysosome by a well-characterized mechanism. However, CAL’s regulatory effect on ΔF508 CFTR has remained almost entirely uninvestigated. Methods: In this study, we describe a previously unknown pathway for CAL by which it regulates the membrane expression of ΔF508 CFTR through arrest of ΔF508 CFTR trafficking in the endoplasmic reticulum (ER) using a combination of cell biology, biochemistry and electrophysiology. Results: We demonstrate that CAL is an ER localized protein that binds to ΔF508 CFTR and is degraded in the 26S proteasome. When CAL is inhibited, ΔF508 CFTR retention in the ER decreases and cell surface expression of mature functional ΔF508 CFTR is observed alongside of enhanced expression of plasma membrane scaffolding protein NHERF1. Chaperone proteins regulate this novel process, and ΔF508 CFTR binding to HSP40, HSP90, HSP70, VCP, and Aha1 changes to improve ΔF508 CFTR cell surface trafficking. Conclusion: Our results reveal a pathway in which CAL regulates the cell surface availability and intracellular retention of ΔF508 CFTR.

scholarly journals Targeting CAL as a Negative Regulator of ΔF508-CFTR Cell-Surface Expression

2006 ◽  
Vol 282 (11) ◽  
pp. 8099-8109 ◽  
Author(s):  
Michael Wolde ◽  
Abigail Fellows ◽  
Jie Cheng ◽  
Aleksandr Kivenson ◽  
Bonita Coutermarsh ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Negative Regulator ◽  
Cell Surface Expression ◽  
Δf508 Cftr

Lidocaine Promotes the Trafficking and Functional Expression of Nav1.8 Sodium Channels in Mammalian Cells

2007 ◽  
Vol 98 (1) ◽  
pp. 467-477 ◽  
Author(s):  
Juan Zhao ◽  
Rahima Ziane ◽  
Aurélien Chatelier ◽  
Michael E. O'Leary ◽  
Mohamed Chahine
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Biophysical Properties ◽  
Nociceptive Neurons ◽  
Mammalian Cell Lines ◽  
Na Currents

Nociceptive neurons of the dorsal root ganglion (DRG) express a combination of rapidly gating TTX-sensitive and slowly gating TTX-resistant Na currents, and the channels that produce these currents have been cloned. The Nav1.7 and Nav1.8 channels encode for the rapidly inactivating TTX-sensitive and slowly inactivating TTX-resistant Na currents, respectively. Although the Nav1.7 channel expresses well in cultured mammalian cell lines, attempts to express the Nav1.8 channel using similar approaches has been met with limited success. The inability to heterologously express Nav1.8 has hampered detailed characterization of the biophysical properties and pharmacology of these channels. In this study, we investigated the determinants of Nav1.8 expression in tsA201 cells, a transformed variant of HEK293 cells, using a combination of biochemistry, immunochemistry, and electrophysiology. Our data indicate that the unusually low expression levels of Nav1.8 in tsA201 cells results from a trafficking defect that traps the channel protein in the endoplasmic reticulum. Incubating the cultured cells with the local anesthetic lidocaine dramatically enhanced the cell surface expression of functional Nav1.8 channels. The biophysical properties of the heterologously expressed Nav1.8 channel are similar but not identical to those of the TTX-resistant Na current of native DRG neurons, recorded under similar conditions. Our data indicate that the lidocaine acts as a molecular chaperone that promotes efficient trafficking and increased cell surface expression of Nav1.8 channels.

scholarly journals Secretory carrier-associated membrane protein 2 (SCAMP2) regulates cell surface expression of T-type calcium channels

2022 ◽  
Vol 15 (1) ◽  
Author(s):  
Leos Cmarko ◽  
Robin N. Stringer ◽  
Bohumila Jurkovicova-Tarabova ◽  
Tomas Vacik ◽  
Lubica Lacinova ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Neuronal Excitability ◽  
Low Voltage ◽  
Activity Patterns ◽  
Surface Expression ◽  
Single Amino Acid ◽  
Cell Surface Expression ◽  
Charge Movement

AbstractLow-voltage-activated T-type Ca2+ channels are key regulators of neuronal excitability both in the central and peripheral nervous systems. Therefore, their recruitment at the plasma membrane is critical in determining firing activity patterns of nerve cells. In this study, we report the importance of secretory carrier-associated membrane proteins (SCAMPs) in the trafficking regulation of T-type channels. We identified SCAMP2 as a novel Cav3.2-interacting protein. In addition, we show that co-expression of SCAMP2 in mammalian cells expressing recombinant Cav3.2 channels caused an almost complete drop of the whole cell T-type current, an effect partly reversed by single amino acid mutations within the conserved cytoplasmic E peptide of SCAMP2. SCAMP2-induced downregulation of T-type currents was also observed in cells expressing Cav3.1 and Cav3.3 channel isoforms. Finally, we show that SCAMP2-mediated knockdown of the T-type conductance is caused by the lack of Cav3.2 expression at the cell surface as evidenced by the concomitant loss of intramembrane charge movement without decrease of total Cav3.2 protein level. Taken together, our results indicate that SCAMP2 plays an important role in the trafficking of Cav3.2 channels at the plasma membrane.

scholarly journals MEK1/2 activity modulates TREM2 cell surface recruitment

2020 ◽  
pp. jbc.RA120.014352
Author(s):  
Jason Schapansky ◽  
Yelena Y Grinberg ◽  
David M Osiecki ◽  
Emily A Freeman ◽  
Stephen G Walker ◽  
...  
Keyword(s):  
Cell Surface ◽  
Myeloid Cells ◽  
Surface Expression ◽  
Mek Inhibitor ◽  
Therapeutic Benefit ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Loss Of Function ◽  
Downstream Effector ◽  
Surface Receptor

Rare sequence variants in the microglial cell surface receptor TREM2 have been shown to increase the risk for Alzheimer’s disease (AD). Disease-linked TREM2 mutations seem to confer a partial loss of function, and increasing TREM2 cell surface expression and thereby its function(s) might have therapeutic benefit in AD. However, druggable targets that could modulate microglial TREM2 surface expression are not known. To identify such targets, we conducted a screen of small molecule compounds with known pharmacology using human myeloid cells, searching for those that enhance TREM2 protein at the cell surface Inhibitors of the kinases MEK1/2 displayed the strongest and most consistent increases in cell surface TREM2 protein, identifying a previously unreported pathway for TREM2 regulation. Unexpectedly, inhibitors of the downstream effector ERK kinases did not have the same effect, suggesting that non-canonical MEK signaling regulates TREM2 trafficking. In addition, siRNA knockdown experiments confirmed that decreased MEK1 and MEK2 was required for this recruitment. In iPSC-derived microglia, MEK inhibition increased cell surface TREM2 only modestly, so various cytokines were used to alter iPSC microglia phenotype, making cells more sensitive to MEK inhibitor-induced TREM2 recruitment. Of those tested, only IFN-gamma priming prior to MEK inhibitor treatment resulted in greater TREM2 recruitment. These data identify the first known mechanisms for increasing surface TREM2 protein and TREM2-regulated function in human myeloid cells, and are the first to show a role for MEK1/MEK2 signaling in TREM2 activity.

Increased functional cell surface expression of CFTR and ΔF508-CFTR by the anthracycline doxorubicin

2001 ◽  
Vol 280 (5) ◽  
pp. C1031-C1037 ◽  
Author(s):  
Rangan Maitra ◽  
Collin M. Shaw ◽  
Bruce A. Stanton ◽  
Joshua W. Hamilton
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Protein Expression ◽  
Surface Expression ◽  
Chloride Secretion ◽  
Cell Surface Expression ◽  
Common Mutation ◽  
Transmembrane Conductance Regulator ◽  
Δf508 Cftr ◽  
Cftr Protein

Cystic fibrosis (CF) is a disease that is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, ΔF508, accounts for 70% of all CF alleles and results in a protein that is defective in folding and trafficking to the cell surface. However, ΔF508-CFTR is functional when properly localized. We report that a single, noncytotoxic dose of the anthracycline doxorubicin (Dox, 0.25 μM) significantly increased total cellular CFTR protein expression, cell surface CFTR protein expression, and CFTR-associated chloride secretion in cultured T84 epithelial cells. Dox treatment also increased ΔF508-CFTR cell surface expression and ΔF508-CFTR-associated chloride secretion in stably transfected Madin-Darby canine kidney cells. These results suggest that anthracycline analogs may be useful for the clinical treatment of CF.

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