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.

Figure 1. Cell-based (PC12) model of mutant huntingtin protein misfolding and cell toxicity

(a) Cells transfected with an inducible plasmid containing wild-type huntingtin (htt-Q25) show diffuse protein expression throughout the cytosol (24 hrs post-induction with the ecdysone analog tebufenozide, Teb). (b) Cells transfected with the same plasmid containing mutant, polyQ-expanded huntingtin (htt-Q103), show perinuclear inclusion bodies at 24 hrs post-induction (red arrowheads). (c) Cell viability of mutant-huntingtin-expressing cells is decreased to less than 20% of the wild-type expressing cells (measured by Alamar Blue fluorescence at 48 hrs post-induction). Cell death induced by htt-Q103 can be rescued by treatment with a general caspase inhibitor, Boc-D-FMK (FMK, 50 μM). (d) Primary screening results of 2,036 compounds showing effects on cell viability of induced Q25 and Q103 cells. Putative hit compounds that rescue Q103-induced cell death are shown in red, confirmed hit (thiomuscimol) is boxed, DMSO treated controls shown in green. Plasmid abbreviations: ecdysone responsive element (EcRE), wild-type huntingtin exon-1 (htt-Q25), mutant huntingtin exon-1 (htt-Q103), enhanced green fluorescent protein (EGFP), VP16-ecdysone receptor chimera (VBE), cytomegalovirus enhancer/beta-actin promoter (CMV-bA), neomycin resistance (PKG-neo^r).

Figure 2. Dose-response curves for hit compounds that suppress Q103-induced apoptosis

The viability of tebufenozide-induced htt-Q25 (blue) and htt-Q103 (red) cells was detected by Alamar Blue fluorescence and plotted as a percentage of uninduced cells at 48 hours post-induction. (a) 16F16, (b) arteannuin B, (c) BBC7M13, (d) BBC7E8, (e) thiomuscimol, (f) muscimol (inactive analog of thiomuscimol; single atom substitution shown in red).

Figure 3. Identification of small molecule target proteins using Huisgen cycloaddition chemistry

(a) Fluorescent tagging of small molecule target proteins. Step 1: alkyne derivatized-16F16 (16F16A) covalently binds to target proteins in PC12 lysate. Step 2: alkyne derivatized-16F16 (16F16A) is coupled to rhodamine-azide via Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. Step 3: fluorescently-tagged target proteins are affinity purified, analyzed by SDS-PAGE, and identified as by mass spectrometry (MS) as rat protein disulfide-isomerase precursors (PDIA1 and PDIA3). (b) Laser scanned gel of 16F16A-rhodamine tagged proteins (PDI = 53 kDa doublet, red arrowhead) in crude PC12 extract (16F16A-DC = inactive deschloro-16F16 propargyl analog, structure Fig. 4a). Pretreatment of PC12 extract with 16F16, arteannuin B (ART-B), BBC7M13 (M13), BBC7E8 (E8), thiomuscimol, or cystamine blocks 16F16A target binding. Inactive analogs muscimol and hypotaurine do not compete for 16F16A target binding. Purified bovine PDI (B-PDI) is covalently labeled by 16F16A-rhodamine (green arrowhead). (c) Anti-PDI Western blot of affinity purified 16F16A target proteins confirms the sequence data (full length 53 kDa and 38 kDa proteolytic PDI fragment; red arrowheads). Extract = crude PC12 extract, 16F16A = affinity-purified 16F16A, fluorescein-tagged target from PC12 extract, 16F16A comp = pre-treatment of PC12 extract with 20× 16F16, followed by addition of 16F16A, fluorescein-azide coupling, and affinity purification.

Figure 4. Compounds that bind to PDI and rescue htt-Q103-induced toxicity inhibit PDI reductase activity in vitro

(a) 16F16 and 16F16 propargyl analogs (structural modifications shown in blue), (b) thiomuscimol and its inactive analog muscimol, (c) cystamine and an inactive analog hypotaurine.

Figure 5. PDI accumulates at MAM contacts and induces MOMP

(a–c) Western blot and LI-COR quantification of PDIA1 in mitochondrial, cytosolic, and ER/microsomal cell fractions (nomalized to F₁-ATPase, actin, and calnexin respectively). Time-course analysis shows a 2.8-fold increase of Q103 mitochondrial PDI over Q25 mitochondrial PDI at 24 hrs post-induction. Induced Q103 cells rescued with 16F16 (7.8 μM) show a 5.3 fold increase of mitochondrial PDI over induced Q25 cells at 24 hrs. (d) Drawing of the MAM architecture. (e) Western blot analysis of purified mitochondrial and associated membrane (MAM) fractions. Crude (sucrose gradient purified) mitochondria isolated from Q103-expressing cells (Lane C) were further purified by Percoll gradient centrifugation to isolate the MAM fraction. The MAM fraction is shown to contain PDIA1 and PDIA3 protein. Abbreviations: crude mitochondria (C), microsomal/ER fraction (ER), purified mitochondrial fraction (MT), MAM fraction (MM). (f) LI-COR quantification of cytosolic cytochrome c in Q25 and Q103-expressing cells. (g) Mitochondrial MOMP assay using purified PC12 mitochondria. The mitochondria were then pelleted and the degree of MOMP calculated as a percentage of Smac released from the mitochondrial pellet (P) into the supernatant (S). PDI inhibitors 16F16, BBC7M13 (M13), BBC7E8 (E8), thiomuscimol (THIOM), and cystamine (CYS) suppress PDI-driven MOMP, whereas inactive analogs muscimol (MUSC) and hypotaurine (HYPT) do not suppress Smac release. Abbreviations: uninduced (U), induced (I). (h–i) Overexpressing PDIA1 or PDI-3 in PC12 cells induces apoptosis that is suppressed by PDI inhibitors (rescue over untreated cells calculated by ANOVA and Dunnett’s post hoc comparison test at the 0.01 confidence level). Supplementary Fig. 8 shows the characterization of securinine, which we identified as an additional PDI inhibitor.

Figure 6. Characterization of PDI-induced MOMP and validation in model systems

(a) Mitochondrial MOMP assay using mouse embryonic fibroblast (MEF) and Bax/Bak double knockout (BBKO) MEF cells. Western blot analysis for Smac and Bak protein demonstrates that PDI-induced MOMP takes place in the absence of cytosol and is mediated by oligomerization of mitochondrial Bak (lanes 1–6; Bak-M, D, and T correspond to Bak monomer, dimer, and trimer respectively). The Bak protein is oligomerized via oxidation of cystein residues (lanes 7–10). Oligomerization of Bak and mitochondrial release of Smac is suppressed by inhibiting PDI with thiomuscimol (THIOM; lanes 11 and 12). DSP= Dithiobis (succinimidyl) propionate; homobifunctional and membrane permeable crosslinker. (b) Schematic diagram of the rat corticostriatal brain slice assay (data in c–j). (c–e) Small molecule PDI inhibitors suppress htt-N90Q73-induced toxicity in brain slice MSNs (c) BBC7E8, (d) securinine, and (e) 16F16. (f) Knockdown of PDIA3 using a validated A3-3 shRNA targeting plasmid (Supplementary Fig. 6) suppresses htt-N90Q73 toxicity in brain slice MSNs. (g–i) PDI inhibitors and shRNA suppress APP/Aβ toxicity in brain slice pyramidal neurons (g) BBC7E8, (h) securinine, (i) 16F16, (j) PDIA3 shRNA. Controls and legend: YFP (transfection control), KW+CGS (chemical rescue of htt-N90Q73 by KW-6002 and CGS21680), NT (non-targeting shRNA), FMK (caspase inhibitor Boc-D-FMK, 100μM), PN (pyramidal neurons); yellow bars are inhibitor treated (PDI inhibitor or shRNA) htt-N90Q73 or APP/Aβ expressing cells; asterisks represent a statistically significant (ANOVA and Dunnett’s post hoc comparison test at the 0.05 confidence level) rescue of htt-Q73 or APP/Aβ toxicity.