scholarly journals A foldable CFTRΔF508 biogenic intermediate accumulates upon inhibition of the Hsc70–CHIP E3 ubiquitin ligase

2004 ◽  
Vol 167 (6) ◽  
pp. 1075-1085 ◽  
Author(s):  
J. Michael Younger ◽  
Hong-Yu Ren ◽  
Liling Chen ◽  
Chun-Yang Fan ◽  
Andrea Fields ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Folding Defect ◽  
Cellular Components ◽  
Soluble State

CFTRΔF508 exhibits a correctable protein-folding defect that leads to its misfolding and premature degradation, which is the cause of cystic fibrosis (CF). Herein we report on the characterization of the CFTRΔF508 biogenic intermediate that is selected for proteasomal degradation and identification of cellular components that polyubiquitinate CFTRΔF508. Nonubiquitinated CFTRΔF508 accumulates in a kinetically trapped, but folding competent conformation, that is maintained in a soluble state by cytosolic Hsc70. Ubiquitination of Hsc70-bound CFTRΔF508 requires CHIP, a U box containing cytosolic cochaperone. CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a. Inactivation of the Hsc70–CHIP E3 leads CFTRΔF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface. Inhibition of CFTRΔF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.

scholarly journals Protein kinase C regulates organic anion transporter 1 through phosphorylating ubiquitin ligase Nedd4–2

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhou Yu ◽  
Chenchang Liu ◽  
Jinghui Zhang ◽  
Zhengxuan Liang ◽  
Guofeng You
Keyword(s):  
Protein Kinase C ◽  
Cell Surface ◽  
Ubiquitin Ligase ◽  
Organic Anion ◽  
Surface Expression ◽  
Organic Anion Transporter ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Kinase C ◽  
Anion Transporter

Abstract Background Organic anion transporter 1 (OAT1) is a drug transporter expressed on the basolateral membrane of the proximal tubule cells in kidneys. It plays an essential role in the disposition of numerous clinical therapeutics, impacting their pharmacological and toxicological properties. The activation of protein kinase C (PKC) is shown to facilitate OAT1 internalization from cell surface to intracellular compartments and thereby reducing cell surface expression and transport activity of the transporter. The PKC-regulated OAT1 internalization occurs through ubiquitination, a process catalyzed by a E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated 4–2 (Nedd4–2). Nedd4–2 directly interacts with OAT1 and affects ubiquitination, expression and stability of the transporter. However, whether Nedd4–2 is a direct substrate for PKC-induced phosphorylation is unknown. Results In this study, we investigated the role of Nedd4–2 phosphorylation in the PKC regulation of OAT1. The results showed that PKC activation enhanced the phosphorylation of Nedd4–2 and increased the OAT1 ubiquitination, which was accompanied by a decreased OAT1 cell surface expression and transport function. And the effects of PKC could be reversed by PKC-specific inhibitor staurosporine. We further discovered that the quadruple mutant (T197A/S221A/S354A/S420A) of Nedd4–2 partially blocked the effects of PKC on Nedd4–2 phosphorylation and on OAT1 transport activity. Conclusions Our investigation demonstrates that PKC regulates OAT1 likely through direct phosphorylation of Nedd4–2. And four phosphorylation sites (T197, S221, S354, and S420) of Nedd4–2 in combination play an important role in this regulatory process.

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.

Characterization of the biosynthesis and cell surface expression of avian metapneumovirus attachment glycoprotein

2010 ◽  
Vol 147 (2) ◽  
pp. 189-194 ◽  
Author(s):  
Lizhong Luo ◽  
Krista Nishi ◽  
Li Liu ◽  
Marta I. Sabara ◽  
Yan Li
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Avian Metapneumovirus

scholarly journals Characterization of thrombospondin synthesis, secretion and cell surface expression by human tumor cells

1989 ◽  
Vol 7 (3) ◽  
pp. 265-276 ◽  
Author(s):  
James Varani ◽  
Bruce L. Riser ◽  
Lisa A. Hughes ◽  
Thomas E. Carey ◽  
Suzanne E. G. Fligiel ◽  
...  
Keyword(s):  
Cell Surface ◽  
Tumor Cells ◽  
Human Tumor ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Tumor Cells

scholarly journals Calreticulin Negatively Regulates the Cell Surface Expression of Cystic Fibrosis Transmembrane Conductance Regulator

2006 ◽  
Vol 281 (18) ◽  
pp. 12841-12848 ◽  
Author(s):  
Kazutsune Harada ◽  
Tsukasa Okiyoneda ◽  
Yasuaki Hashimoto ◽  
Keiko Ueno ◽  
Kimitoshi Nakamura ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

Characterization of porcine UL16-binding protein 1 endothelial cell surface expression

2008 ◽  
Vol 15 (2) ◽  
pp. 136-144 ◽  
Author(s):  
Benjamin G. Lilienfeld ◽  
Anita Schildknecht ◽  
Lukas L. Imbach ◽  
Nicolas J. Mueller ◽  
Mårten K. J. Schneider ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Surface ◽  
Binding Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Endothelial Cell Surface
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