ER-associated degradation is required for vasopressin prohormone processing and systemic water homeostasis
Guojun Shi, … , Martin Spiess, Ling Qi
Guojun Shi, … , Martin Spiess, Ling Qi
Published September 18, 2017
Citation Information: J Clin Invest. 2017;127(10):3897-3912. https://doi.org/10.1172/JCI94771.
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Research Article Cell biology Endocrinology Article has an altmetric score of 38

ER-associated degradation is required for vasopressin prohormone processing and systemic water homeostasis

Abstract

Peptide hormones are crucial regulators of many aspects of human physiology. Mutations that alter these signaling peptides are associated with physiological imbalances that underlie diseases. However, the conformational maturation of peptide hormone precursors (prohormones) in the ER remains largely unexplored. Here, we report that conformational maturation of proAVP, the precursor for the antidiuretic hormone arginine-vasopressin, within the ER requires the ER-associated degradation (ERAD) activity of the Sel1L-Hrd1 protein complex. Serum hyperosmolality induces expression of both ERAD components and proAVP in AVP-producing neurons. Mice with global or AVP neuron–specific ablation of Se1L-Hrd1 ERAD progressively developed polyuria and polydipsia, characteristics of diabetes insipidus. Mechanistically, we found that ERAD deficiency causes marked ER retention and aggregation of a large proportion of all proAVP protein. Further, we show that proAVP is an endogenous substrate of Sel1L-Hrd1 ERAD. The inability to clear misfolded proAVP with highly reactive cysteine thiols in the absence of Sel1L-Hrd1 ERAD causes proAVP to accumulate and participate in inappropriate intermolecular disulfide–bonded aggregates, promoted by the enzymatic activity of protein disulfide isomerase (PDI). This study highlights a pathway linking ERAD to prohormone conformational maturation in neuroendocrine cells, expanding the role of ERAD in providing a conducive ER environment for nascent proteins to reach proper conformation.

Authors

Guojun Shi, Diane R.M. Somlo, Geun Hyang Kim, Cristina Prescianotto-Baschong, Shengyi Sun, Nicole Beuret, Qiaoming Long, Jonas Rutishauser, Peter Arvan, Martin Spiess, Ling Qi

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Figure 6

The majority of proAVP proteins form aggregates in the absence of SEL1L-HRD1 ERAD.

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The majority of proAVP proteins form aggregates in the absence of SEL1L-...
(A and B) Representative immunofluorescence staining of WT and G57S-mutant proAVP and endogenous BiP in transfected WT (A) and Sel1L-deficient N2a cells (B). (C and D) Immunogold (proAVP, anti-NPII) coupled with TEM analysis of WT (C) or Sel1L-deficient (D) N2a cells expressing WT or ΔE47 human disease mutant proAVP. Higher-magnification images are shown below. Arrows indicate proAVP in ER sheets; asterisks mark fibrillar proAVP aggregates. (E and F) Immunogold labeling and TEM analysis of endogenous proAVP in the PVN of 10-week-old mice, with higher-magnification images shown on the right. Arrows indicate proAVP-containing dense secretory granules, and asterisks mark aggregates. n = 2 male mice each. (G and H) Sucrose fractionation followed by Western blot analyses of the proAVP protein complex in (G) WT HEK293T cells transfected with WT, G57S proAVP, or both at a ratio of 1:1, or (H) HRD1-deficient HEK293T cells transfected with WT proAVP. Fractions were collected from top (no. 1) to bottom (no. 6) and the redissolved pellet as fraction no. 7, followed by Western blot analysis under nonreducing conditions. HSP90 and calnexin represent cytosolic and ER protein controls, respectively. HMW, high-molecular-weight complexes. Data shown are representative of at least 2 independent experiments.