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S-Nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration

Nature volume 441, pages 513–517 (2006)Cite this article

Abstract

Stress proteins located in the cytosol or endoplasmic reticulum (ER) maintain cell homeostasis and afford tolerance to severe insults1,2,3. In neurodegenerative diseases, several chaperones ameliorate the accumulation of misfolded proteins triggered by oxidative or nitrosative stress, or of mutated gene products4,5. Although severe ER stress can induce apoptosis2,6, the ER withstands relatively mild insults through the expression of stress proteins or chaperones such as glucose-regulated protein (GRP) and protein-disulphide isomerase (PDI), which assist in the maturation and transport of unfolded secretory proteins. PDI catalyses thiol–disulphide exchange, thus facilitating disulphide bond formation and rearrangement reactions7,8,9,10. PDI has two domains that function as independent active sites with homology to the small, redox-active protein thioredoxin7,8. During neurodegenerative disorders and cerebral ischaemia, the accumulation of immature and denatured proteins results in ER dysfunction11, but the upregulation of PDI represents an adaptive response to protect neuronal cells12,13,14. Here we show, in brains manifesting sporadic Parkinson's or Alzheimer's disease, that PDI is S-nitrosylated, a reaction transferring a nitric oxide (NO) group to a critical cysteine thiol to affect protein function15,16,17,18. NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leads to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. S-Nitrosylation also abrogates PDI-mediated attenuation of neuronal cell death triggered by ER stress, misfolded proteins or proteasome inhibition. Thus, PDI prevents neurotoxicity associated with ER stress and protein misfolding, but NO blocks this protective effect in neurodegenerative disorders through the S-nitrosylation of PDI.

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Figure 1: S -Nitrosylation of PDI in vitro and in vivo.
Figure 2: S -Nitrosylation of PDI regulates its enzymatic activity.
Figure 3: NMDA stimulates the accumulation of polyubiquitinated proteins and UPR pathway.
Figure 4: Neuroprotection by PDI against ER stress, proteasome inhibition, or Pael receptor expression.

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Acknowledgements

We thank X. Fang for preparation of cerebrocortical cultures, T. William for technical assistance with the analysis of mass spectra, and R. Takahashi for the Pael receptor construct. T.U. was supported in part by the Mitsubishi Pharma Research Foundation and a Grant-in-Aid from the Ministry of Education, Culture, Sports and Technology of Japan. S.A.L. was supported in part by grants from the NIH, the American Parkinson's Disease Association, San Diego Chapter, and an Ellison Senior Scholars Award in Aging. Author Contributions T.U. and T.N. performed most of the experiments, contributing equally to the work, and helped to write the manuscript. D.Y., Z.Q.S. and Z.G. provided the biochemical data, and also contributed equally to the work. Y.M. analyzed the mass spectrometry data. E.M. provided the human subjects, and Y.N. provided constructs and advice. S.A.L., the senior author, designed the project, helped to analyse the data, wrote the manuscript and provided the financial support.

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Authors and Affiliations

  1. Center for Neuroscience and Aging,

    Takashi Uehara, Tomohiro Nakamura, Dongdong Yao, Zhong-Qing Shi, Zezong Gu & Stuart A. Lipton

  2. Proteomic Facility, Burnham Institute for Medical Research, La Jolla, 10901 North Torrey Pines Road, California, 92037, USA

    Yuliang Ma

  3. Department of Neurosciences, University of California at San Diego, La Jolla, 9500 Gilman Drive, California, 92039, USA

    Eliezer Masliah & Stuart A. Lipton

  4. Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, 060-0812, Japan

    Takashi Uehara & Yasuyuki Nomura

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Correspondence to Stuart A. Lipton.

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Supplementary information

Supplementary Figure 1

Supplementary Figure 1 a and b detail S-Nitrosylation of PDI in cells. (PDF 513 kb)

Supplementary Figure 2

LC-MS/MS analysis of PDI showing modified cysteine thiol groups in the C-terminal CGHC motif. (PDF 106 kb)

Supplementary Figure 3

S-Nitrosylation of PDI in human brains. (PDF 173 kb)

Supplementary Figure 4

Effect of oxidative and nitrosative stress on PDI activity. (PDF 127 kb)

Supplementary Figure 5

Colocalization of overexpressed PDI protein with the ER marker Calreticulin in SH-SY5Y cells. (PDF 384 kb)

Supplementary Figure 6

Effect of endogenous NO and transduced PDI on cell survival. (PDF 213 kb)

Supplementary Figure 7

Overexpression of PDI by adenoviral infection. (PDF 669 kb)

Supplementary Table 1

List of human brain subjects used in this study. (PDF 175 kb)

Supplementary Notes

This file contains Supplementary Methods and additional references. (DOC 68 kb)

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Uehara, T., Nakamura, T., Yao, D. et al. S-Nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration. Nature 441, 513–517 (2006). https://doi.org/10.1038/nature04782

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