Role and mechanism of chaperones calreticulin and ERP57 in restoring trafficking to mutant HERG‑A561V protein

Wu,Yujia; Huang,Xiaoyan; Zheng,Zequn; Yang,Xi; Ba,Yanna; Lian,Jiangfang

doi:10.3892/ijmm.2021.4992

Role and mechanism of chaperones calreticulin and ERP57 in restoring trafficking to mutant HERG‑A561V protein

Authors:
- Yujia Wu
- Xiaoyan Huang
- Zequn Zheng
- Xi Yang
- Yanna Ba
- Jiangfang Lian
View Affiliations

Affiliations: School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, P.R. China, Department of Cardiology, Lihuili Hospital, Ningbo University, Ningbo, Zhejiang 315100, P.R. China
Published online on: July 1, 2021 https://doi.org/10.3892/ijmm.2021.4992
Article Number: 159
Copyright: © Wu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Long QT syndrome type 2 is caused by a mutation in the human‑ether‑a‑go‑go‑related gene (HERG) gene encoding the rapidly activating delayed rectifier K‑current. HERG is a key cell membrane glycoprotein; however, whether the maturation process of HERG protein involves key molecules derived from the calnexin (CNX)/calreticulin (CRT) cycle and how these molecules work remains unknown. Using western blotting, the present study screened the key molecules CNX/CRT/endoplasmic reticulum protein 57 (ERP57) involved in this cycle, and it was revealed that the protein expression levels of CNX/CRT/ERP57 in wild‑type (WT)/A561V cells were increased compared with those in WT cells (n=3; P<0.05). Additionally, a co‑immunoprecipitation experiment was used to reveal that the ability of CNX/ERP57/CRT to interact with HERG was significantly increased in A561V and WT/A561V cells (n=3; P<0.05). A plasmid lacking the bb' domain of ERP57 was constructed and it was demonstrated that the key site of ERP57 binding to CRT and immature HERG protein is the bb' domain. The whole‑cell patch‑clamp technique detected that the tail current density increased by 46% following overexpression of CRT and by 53% following overexpression of ERP57 in WT/A561V cells. Overexpression of CRT and ERP57 could increased HERG protein levels on the membrane detected by confocal imaging. Furthermore, overexpression of ERP57 and CRT proteins could restore the HERG‑A561V mutant protein trafficking process and rescue the dominant‑negative suppression of WT. Overall, ERP57/CRT served a crucial role in the HERG‑A561V mutant protein trafficking deficiency and degradation process.

View Figures

View References

1	Wallace E, Howard L, Liu M, O'Brien T, Ward D, Shen S and Prendiville T: Long QT syndrome: Genetics and future perspective. Pediatr Cardiol. 40:1419–1430. 2019. View Article : Google Scholar : PubMed/NCBI
2	Kanner SA, Jain A and Colecraft HM: Development of a high-throughput flow cytometry assay to monitor defective trafficking and rescue of long QT2 mutant hERG channels. Front Physiol. 9:3972018. View Article : Google Scholar : PubMed/NCBI
3	Butler A, Helliwell MV, Zhang Y, Hancox JC and Dempsey CE: An update on the structure of hERG. Front Pharmacol. 10:15722020. View Article : Google Scholar : PubMed/NCBI
4	Perissinotti L, Guo J, Kudaibergenova M, Lees-Miller J, Ol'khovich M, Sharapova A, Perlovich GL, Muruve DA, Gerull B, Noskov SY and Duff HJ: The pore-lipid interface: Role of amino-acid determinants of lipophilic access by ivabradine to the hERG1 pore domain. Mol Pharmacol. 96:259–271. 2019. View Article : Google Scholar : PubMed/NCBI
5	Zhang KP, Yang BF and Li BX: Translational toxicology and rescue strategies of the hERG channel dysfunction: Biochemical and molecular mechanistic aspects. Acta Pharmacol Sin. 35:1473–1484. 2014. View Article : Google Scholar : PubMed/NCBI
6	Li G, Shi R, Wu J, Han W, Zhang A, Cheng G, Xue X and Sun C: Association of the hERG mutation with long-QT syndrome type 2, syncope and epilepsy. Mol Med Rep. 13:2467–2475. 2016. View Article : Google Scholar : PubMed/NCBI
7	Foo B, Williamson B, Young JC, Lukacs G and Shrier A: hERG quality control and the long QT syndrome. J Physiol. 594:2469–2481. 2016. View Article : Google Scholar : PubMed/NCBI
8	Araki K and Nagata K: Protein folding and quality control in the ER. Cold Spring Harb Perspect Biol. 3:a0075262011. View Article : Google Scholar : PubMed/NCBI
9	Marti L, Lia A, Reca IB, Roversi P, Santino A and Zitzmann N: In planta preliminary screening of ER glycoprotein folding quality control (ERQC) modulators. Int J Mol Sci. 19:21352018. View Article : Google Scholar :
10	Lamothe SM, Hulbert M, Guo J, Li W, Yang T and Zhang S: Glycosylation stabilizes hERG channels on the plasma membrane by decreasing proteolytic susceptibility. FASEB J. 32:1933–1943. 2018. View Article : Google Scholar : PubMed/NCBI
11	Patel C, Saad H, Shenkman M and Lederkremer GZ: Oxidoreductases in glycoprotein glycosylation, folding, and ERAD. Cells. 9:21382020. View Article : Google Scholar :
12	Tannous A, Pisoni GB, Hebert DN and Molinari M: N-linked sugar-regulated protein folding and quality control in the ER. Semin Cell Dev Biol. 41:79–89. 2015. View Article : Google Scholar :
13	Foo B, Barbier C, Guo K, Vasantharuban J, Lukacs GL and Shrier A: Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression. Sci Rep. 9:60662019. View Article : Google Scholar : PubMed/NCBI
14	Shi YQ, Yan M, Liu LR, Zhang X, Wang X, Geng HZ, Zhao X and Li BX: High glucose represses hERG K+ channel expression through trafficking inhibition. Cell Physiol Biochem. 37:284–296. 2015. View Article : Google Scholar : PubMed/NCBI
15	Hettinghouse A, Liu R and Liu CJ: Multifunctional molecule ERp57: From cancer to neurodegenerative diseases. Pharmacol Ther. 181:34–48. 2018. View Article : Google Scholar
16	Song D, Liu H, Wu J, Gao X, Hao J and Fan D: Insights into the role of ERp57 in cancer. J Cancer. 12:2456–2464. 2021. View Article : Google Scholar : PubMed/NCBI
17	Kozlov G and Gehring K: Calnexin cycle-structural features of the ER chaperone system. FEBS J. 287:4322–4340. 2020. View Article : Google Scholar : PubMed/NCBI
18	Pollock S, Kozlov G, Pelletier MF, Trempe JF, Jansen G, Sitnikov D, Bergeron JJ, Gehring K, Ekiel I and Thomas DY: Specific interaction of ERp57 and calnexin determined by NMR spectroscopy and an ER two-hybrid system. EMBO J. 23:1020–1029. 2004. View Article : Google Scholar : PubMed/NCBI
19	Wang Y, Shen T, Fang P, Zhou J, Lou K, Cen Z, Qian H, Zhou J, Liu N and Lian J: The role and mechanism of chaperones calnexin/calreticulin in which ALLN selectively rescues the trafficking defective of HERG-A561V mutation. Biosci Rep. Sep 7–2018.Epub ahead of print.
20	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar
21	Smith JL, Anderson CL, Burgess DE, Elayi CS, January CT and Delisle BP: Molecular pathogenesis of long QT syndrome type 2. J Arrhythm. 32:373–380. 2016. View Article : Google Scholar : PubMed/NCBI
22	Jenewein T, Kanner SA, Bauer D, Hertel B, Colecraft HM, Moroni A, Thiel G and Kauferstein S: The mutation L69P in the PAS domain of the hERG potassium channel results in LQTS by trafficking deficiency. Channels (Austin). 14:163–174. 2020. View Article : Google Scholar
23	Zhan G, Wang F, Ding YQ, Li XH, Li YX, Zhao ZR, Li JX, Liu Y, Zhao X, Yan CC and Li BX: Rutaecarpine targets hERG channels and participates in regulating electrophysiological properties leading to ventricular arrhythmia. J Cell Mol Med. 25:4938–4949. 2021. View Article : Google Scholar : PubMed/NCBI
24	Wang Y, Huang X, Zhou J, Yang X, Li D, Mao H, Sun HH, Liu N and Lian J: Trafficking-deficient G572R-hERG and E637K-hERG activate stress and clearance pathways in endoplasmic reticulum. PLoS One. 7:e298852012. View Article : Google Scholar : PubMed/NCBI
25	Ono M, Burgess DE, Schroder EA, Elayi CS, Anderson CL, January CT, Sun B, Immadisetty K, Kekenes-Huskey PM and Delisle BP: Long QT syndrome type 2: Emerging strategies for correcting class 2 KCNH2 (hERG) mutations and identifying new patients. Biomolecules. 10:11442020. View Article : Google Scholar :
26	Anderson CL, Delisle BP, Anson BD, Kilby JA, Will ML, Tester DJ, Gong Q, Zhou Z, Ackerman MJ and January CT: Most LQT2 mutations reduce Kv11.1 (hERG) current by a class 2 (trafficking-deficient) mechanism. Circulation. 113:365–373. 2006. View Article : Google Scholar : PubMed/NCBI
27	Du H, Zheng C, Aslam M, Xie X, Wang W, Yang Y and Liu X: Endoplasmic reticulum-mediated protein quality control and endoplasmic reticulum-associated degradation pathway explain the reduction of N-glycoprotein level under the lead stress. Front Plant Sci. 11:5985522021. View Article : Google Scholar : PubMed/NCBI
28	Ito Y, Takeda Y, Seko A, Izumi M and Kajihara Y: Functional analysis of endoplasmic reticulum glucosyltransferase (UGGT): Synthetic chemistry's initiative in glycobiology. Semin Cell Dev Biol. 41:90–98. 2015. View Article : Google Scholar
29	Tannous A, Patel N, Tamura T and Hebert DN: Reglucosylation by UDP-glucose: Glycoprotein glucosyltransferase 1 delays glycoprotein secretion but not degradation. Mol Biol Cell. 26:390–405. 2015. View Article : Google Scholar :
30	Ferris SP, Jaber NS, Molinari M, Arvan P and Kaufman RJ: UDP-glucose: Glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum. Mol Biol Cell. 24:2597–2608. 2013. View Article : Google Scholar : PubMed/NCBI
31	Guerriero CJ and Brodsky JL: The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology. Physiol Rev. 92:537–576. 2012. View Article : Google Scholar : PubMed/NCBI
32	Shenkman M, Ron E, Yehuda R, Benyair R, Khalaila I and Lederkremer GZ: Mannosidase activity of EDEM1 and EDEM2 depends on an unfolded state of their glycoprotein substrates. Commun Biol. 1:1722018. View Article : Google Scholar : PubMed/NCBI
33	Benyair R, Ogen-Shtern N, Mazkereth N, Shai B, Ehrlich M and Lederkremer GZ: Mammalian ER mannosidase I resides in quality control vesicles, where it encounters its glycoprotein substrates. Mol Biol Cell. 26:172–184. 2015. View Article : Google Scholar :
34	Ogen-Shtern N, Avezov E, Shenkman M, Benyair R and Lederkremer GZ: Mannosidase IA is in quality control vesicles and participates in glycoprotein targeting to ERAD. J Mol Biol. 428:3194–3205. 2016. View Article : Google Scholar : PubMed/NCBI
35	Chung H, Cho H, Perry C, Song J, Ylaya K, Lee H and Kim JH: Downregulation of ERp57 expression is associated with poor prognosis in early-stage cervical cancer. Biomarkers. 18:573–579. 2013. View Article : Google Scholar : PubMed/NCBI
36	Choe MH, Min JW, Jeon HB, Cho DH, Oh JS, Lee HG, Hwang SG, An S, Han YH and Kim JS: ERp57 modulates STAT3 activity in radioresistant laryngeal cancer cells and serves as a prognostic marker for laryngeal cancer. Oncotarget. 6:2654–2666. 2015. View Article : Google Scholar : PubMed/NCBI
37	Kozlov G, Pocanschi CL, Rosenauer A, Bastos-Aristizabal S, Gorelik A, Williams DB and Gehring K: Structural basis of carbohydrate recognition by calreticulin. J Biol Chem. 285:38612–38620. 2010. View Article : Google Scholar : PubMed/NCBI
38	Kiuchi T, Izumi M, Mukogawa Y, Shimada A, Okamoto R, Seko A, Sakono M, Takeda Y, Ito Y and Kajihara Y: Monitoring of glycoprotein quality control system with a series of chemically synthesized homogeneous native and misfolded glycoproteins. J Am Chem Soc. 140:17499–17507. 2018. View Article : Google Scholar : PubMed/NCBI
39	Ito Y, Kajihara Y and Takeda Y: Chemical-synthesis-based approach to glycoprotein functions in the endoplasmic reticulum. Chemistry. 26:15461–15470. 2020. View Article : Google Scholar : PubMed/NCBI
40	Ghemrawi R and Khair M: Endoplasmic reticulum stress and unfolded protein response in neurodegenerative diseases. Int J Mol Sci. 21:61272020. View Article : Google Scholar :
41	Wang SB, Shi Q, Xu Y, Xie WL, Zhang J, Tian C, Guo Y, Wang K, Zhang BY, Chen C, et al: Protein disulfide isomerase regulates endoplasmic reticulum stress and the apoptotic process during prion infection and PrP mutant-induced cytotoxicity. PLoS One. 7:e382212012. View Article : Google Scholar : PubMed/NCBI
42	Perri E, Parakh S and Atkin J: Protein disulphide isomerases: Emerging roles of PDI and ERp57 in the nervous system and as therapeutic targets for ALS. Expert Opin Ther Targets. 21:37–49. 2017. View Article : Google Scholar
43	Turano C, Gaucci E, Grillo C and Chichiarelli S: ERp57/GRP58: A protein with multiple functions. Cell Mol Biol Lett. 16:539–563. 2011. View Article : Google Scholar : PubMed/NCBI
44	Sepulveda M, Rozas P, Hetz C and Medinas DB: ERp57 as a novel cellular factor controlling prion protein biosynthesis: Therapeutic potential of protein disulfide isomerases. Prion. 10:50–56. 2016. View Article : Google Scholar : PubMed/NCBI
45	Hoffstrom BG, Kaplan A, Letso R, Schmid RS, Turmel GJ, Lo DC and Stockwell BR: Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins. Nat Chem Biol. 6:900–906. 2010. View Article : Google Scholar : PubMed/NCBI
46	Grek C and Townsend DM: Protein disulfide isomerase superfamily in disease and the regulation of apoptosis. Endoplasmic Reticulum Stress Dis. 1:4–17. 2014.PubMed/NCBI
47	Han A, Li C, Zahed T, Wong M, Smith I, Hoedel K, Green D and Boiko AD: Calreticulin is a critical cell survival factor in malignant neoplasms. PLoS Biol. 17:e30004022019. View Article : Google Scholar : PubMed/NCBI