UBR E3 Ligases and the PDIA3 Protease Control Degradation of Unfolded Antibody Heavy Chain by ERAD

Overview

Journal J Cell Biol

Specialty Cell Biology

Date 2020 Jun 20

PMID 32558906

Citations 6

Authors

Danming Tang

Wendy Sandoval

Cynthia Lam

Benjamin Haley

Peter Liu

Di Xue

Deepankar Roy

Tom Patapoff

Salina Louie

Brad Snedecor

Shahram Misaghi

Affiliations

Soon will be listed here.

Abstract

Accumulation of unfolded antibody chains in the ER triggers ER stress that may lead to reduced productivity in therapeutic antibody manufacturing processes. We identified UBR4 and UBR5 as ubiquitin E3 ligases involved in HC ER-associated degradation. Knockdown of UBR4 and UBR5 resulted in intracellular accumulation, enhanced secretion, and reduced ubiquitination of HC. In concert with these E3 ligases, PDIA3 was shown to cleave ubiquitinated HC molecules to accelerate HC dislocation. Interestingly, UBR5, and to a lesser degree UBR4, were down-regulated as cellular demand for antibody expression increased in CHO cells during the production phase, or in plasma B cells. Reducing UBR4/UBR5 expression before the production phase increased antibody productivity in CHO cells, possibly by redirecting antibody molecules from degradation to secretion. Altogether we have characterized a novel proteolysis/proteasome-dependent pathway involved in degradation of unfolded antibody HC. Proteins characterized in this pathway may be novel targets for CHO cell engineering.

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References

Adeli K, Macri J, Mohammadi A, Kito M, Urade R, Cavallo D . Apolipoprotein B is intracellularly associated with an ER-60 protease homologue in HepG2 cells. J Biol Chem. 1997; 272(36):22489-94. DOI: 10.1074/jbc.272.36.22489. View

Frakes A, Dillin A . The UPR: Sensor and Coordinator of Organismal Homeostasis. Mol Cell. 2017; 66(6):761-771. DOI: 10.1016/j.molcel.2017.05.031. View

Adams G, Weiner L . Monoclonal antibody therapy of cancer. Nat Biotechnol. 2005; 23(9):1147-57. DOI: 10.1038/nbt1137. View

Sriram S, Kim B, Kwon Y . The N-end rule pathway: emerging functions and molecular principles of substrate recognition. Nat Rev Mol Cell Biol. 2011; 12(11):735-47. DOI: 10.1038/nrm3217. View

Phu L, Izrael-Tomasevic A, Matsumoto M, Bustos D, Dynek J, Fedorova A . Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals. Mol Cell Proteomics. 2010; 10(5):M110.003756. PMC: 3098583. DOI: 10.1074/mcp.M110.003756. View

Loo T, Clarke D . Quality control by proteases in the endoplasmic reticulum. Removal of a protease-sensitive site enhances expression of human P-glycoprotein. J Biol Chem. 1998; 273(49):32373-6. DOI: 10.1074/jbc.273.49.32373. View

Elkabetz Y, Argon Y, Bar-Nun S . Cysteines in CH1 underlie retention of unassembled Ig heavy chains. J Biol Chem. 2005; 280(15):14402-12. DOI: 10.1074/jbc.M500161200. View

Lindquist J, Jensen O, Mann M, Hammerling G . ER-60, a chaperone with thiol-dependent reductase activity involved in MHC class I assembly. EMBO J. 1998; 17(8):2186-95. PMC: 1170563. DOI: 10.1093/emboj/17.8.2186. View

Henzel W, Rodriguez H, Watanabe C . Computer analysis of automated Edman degradation and amino acid analysis data. J Chromatogr. 1987; 404(1):41-52. DOI: 10.1016/s0021-9673(01)86835-7. View

10.

Lee R, Liu C, Harty C, McCracken A, Latterich M, Romisch K . Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein. EMBO J. 2004; 23(11):2206-15. PMC: 419910. DOI: 10.1038/sj.emboj.7600232. View

11.

Ma Y, Hendershot L . The stressful road to antibody secretion. Nat Immunol. 2003; 4(4):310-1. DOI: 10.1038/ni0403-310. View

12.

Kikuchi M, Doi E, Tsujimoto I, Horibe T, Tsujimoto Y . Functional analysis of human P5, a protein disulfide isomerase homologue. J Biochem. 2002; 132(3):451-5. DOI: 10.1093/oxfordjournals.jbchem.a003242. View

13.

Misaghi S, Ottosen S, Izrael-Tomasevic A, Arnott D, Lamkanfi M, Lee J . Association of C-terminal ubiquitin hydrolase BRCA1-associated protein 1 with cell cycle regulator host cell factor 1. Mol Cell Biol. 2009; 29(8):2181-92. PMC: 2663315. DOI: 10.1128/MCB.01517-08. View

14.

Omura F, Otsu M, Yoshimori T, Tashiro Y, Kikuchi M . Non-lysosomal degradation of misfolded human lysozymes with and without an asparagine-linked glycosylation site. Eur J Biochem. 1992; 210(2):591-9. DOI: 10.1111/j.1432-1033.1992.tb17459.x. View

15.

Chen C, Malchus N, Hehn B, Stelzer W, Avci D, Langosch D . Signal peptide peptidase functions in ERAD to cleave the unfolded protein response regulator XBP1u. EMBO J. 2014; 33(21):2492-506. PMC: 4283407. DOI: 10.15252/embj.201488208. View

16.

Sommeregger W, Mayrhofer P, Steinfellner W, Reinhart D, Henry M, Clynes M . Proteomic differences in recombinant CHO cells producing two similar antibody fragments. Biotechnol Bioeng. 2016; 113(9):1902-12. PMC: 4985663. DOI: 10.1002/bit.25957. View

17.

Gibbs D, Bacardit J, Bachmair A, Holdsworth M . The eukaryotic N-end rule pathway: conserved mechanisms and diverse functions. Trends Cell Biol. 2014; 24(10):603-11. DOI: 10.1016/j.tcb.2014.05.001. View

18.

Bhamidipati A, Denic V, Quan E, Weissman J . Exploration of the topological requirements of ERAD identifies Yos9p as a lectin sensor of misfolded glycoproteins in the ER lumen. Mol Cell. 2005; 19(6):741-51. DOI: 10.1016/j.molcel.2005.07.027. View

19.

Calame K, Lin K, Tunyaplin C . Regulatory mechanisms that determine the development and function of plasma cells. Annu Rev Immunol. 2003; 21:205-30. DOI: 10.1146/annurev.immunol.21.120601.141138. View

20.

Plemper R, Bohmler S, Bordallo J, Sommer T, Wolf D . Mutant analysis links the translocon and BiP to retrograde protein transport for ER degradation. Nature. 1997; 388(6645):891-5. DOI: 10.1038/42276. View