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Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins

Abstract

A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction and oxidation of disulfides. Through a small molecule screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated ER membranes and trigger apoptotic cell death via mitochondrial outer-membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon 1 and Aβ peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI represents a new mechanism linking protein misfolding and apoptotic cell death.

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Figure 1: Cell-based (PC12) model of mutant huntingtin protein misfolding and cell toxicity.
Figure 2: Dose-response curves for hit compounds that suppress Q103-induced apoptosis.
Figure 3: Identification of small-molecule target proteins using Huisgen cycloaddition chemistry. (a) Fluorescent tagging of small-molecule target proteins.
Figure 4: Compounds that bind to PDI and rescue HTT-Q103–induced toxicity inhibit PDI reductase activity in vitro.
Figure 5: PDI accumulates at MAM contacts and induces MOMP.
Figure 6: Characterization of PDI-induced MOMP and validation in model systems.

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Acknowledgements

We thank E. Schweitzer (University of California–Los Angeles) for the transfected PC12 cell lines; C. Ross (Johns Hopkins University) for HTT DNAs upon which the constructs used here were based; I. Smukste for assistance with organic synthesis; A. Bauer for compound library assembly library; D. Dunn for additional brain-slice experiments; C. Thompson (University of Pennsylvania) for the Bax/Bak double-knockout MEF cells; L. Moore (Gage laboratory, Salk Institute) for tebufenozide; A. Speers, J. Alexander and B. Cravatt for reagents and advice on the cycloaddition reactions; E. Miller, E. Signer, A. Tobin, N. Wexler, C. Johnson, R. Pacifici and M. Finn for useful discussions; and E. Miller and S. Hoffschmidt for editorial comments on the manuscript. This research was supported by grants from the US National Institutes of Health (NIGMS 1RO1GM085081 to B.R.S. and NINDS R21NS048181 to D.C.L.), the Hereditary Disease Foundation (D.C.L., B.R.S.), the High Q Foundation (B.R.S.), CHDI Foundation (D.C.L., B.R.S.), the Arnold and Mabel Beckman Foundation (B.R.S.), the Training Program in Molecular Biophysics (T32GM008281 to A.K.) and a Burroughs Wellcome Fund Career Award at the Scientific Interface (B.R.S.). B.G.H. was supported in part by a postdoctoral fellowship from the High Q Foundation. B.R.S. is an Early Career Scientist of the Howard Hughes Medical Institute.

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B.G.H. and B.R.S designed the experiments. B.G.H. performed the experiments and analyzed the data with B.R.S. A.K. assisted with the library screening, characterization of hit compounds, analysis of synthesized analogs, validation studies and structural identification of securinine. R.L. performed the PDI and Bcl-2 overexpression experiments. R.S.S., G.J.T. and D.C.L. provided the cortical-striatal brain-slice data. B.G.H. and B.R.S. wrote the manuscript.

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Correspondence to Brent R Stockwell.

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Hoffstrom, B., Kaplan, A., Letso, R. et al. Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins. Nat Chem Biol 6, 900–906 (2010). https://doi.org/10.1038/nchembio.467

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