Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation
Jingyuan Sun, Pei Li, Honglian Gui, Laure Rittié, David B. Lombard, Katrin Rietscher, Thomas M. Magin, Qing Xie, Li Liu, M. Bishr Omary
Jingyuan Sun, Pei Li, Honglian Gui, Laure Rittié, David B. Lombard, Katrin Rietscher, Thomas M. Magin, Qing Xie, Li Liu, M. Bishr Omary
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Research Article Dermatology Hepatology

Deacetylation via SIRT2 prevents keratin-mutation-associated injury and keratin aggregation

Abstract

Keratin (K) and other intermediate filament (IF) protein mutations at conserved arginines disrupt keratin filaments into aggregates and cause human epidermolysis bullosa simplex (EBS; K14-R125C) or predispose to mouse liver injury (K18-R90C). The challenge for more than 70 IF-associated diseases is the lack of clinically utilized IF-targeted therapies. We used high-throughput drug screening to identify compounds that normalized mutation-triggered keratin filament disruption. Parthenolide, a plant sesquiterpene lactone, dramatically reversed keratin filament disruption and protected cells and mice expressing K18-R90C from apoptosis. K18-R90C became hyperacetylated compared with K18-WT and treatment with parthenolide normalized K18 acetylation. Parthenolide upregulated the NAD-dependent SIRT2, and increased SIRT2-keratin association. SIRT2 knockdown or pharmacologic inhibition blocked the parthenolide effect, while site-specific Lys-to-Arg mutation of keratin acetylation sites normalized K18-R90C filaments. Treatment of K18-R90C–expressing cells and mice with nicotinamide mononucleotide had a parthenolide-like protective effect. In 2 human K18 variants that associate with human fatal drug-induced liver injury, parthenolide protected K18-D89H– but not K8-K393R–induced filament disruption and cell death. Importantly, parthenolide normalized K14-R125C–mediated filament disruption in keratinocytes and inhibited dispase-triggered keratinocyte sheet fragmentation and Fas-mediated apoptosis. Therefore, keratin acetylation may provide a novel therapeutic target for some keratin-associated diseases.

Authors

Jingyuan Sun, Pei Li, Honglian Gui, Laure Rittié, David B. Lombard, Katrin Rietscher, Thomas M. Magin, Qing Xie, Li Liu, M. Bishr Omary

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Figure 6

PN normalizes K14-R125C–induced filament disruption and protects against Fas-induced apoptosis and cell response to mechanical stress.

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PN normalizes K14-R125C–induced filament disruption and protects against...
(A) Type I keratin–deficient keratinocytes were transduced with YFP-tagged K14-WT or K14-R125C. The transduced type I keratin pairs with endogenous type II keratins to form filaments (WT) or dots (R125C). Cells were then treated with vehicle (DMSO) or PN (5 μM, 48 hours). Scale bar: 50 μm. Quantification of the percentage cells with dots/group using pooled counts from 3 separate experiments is shown in the histogram on the right (*P = 0.013, ***P < 0.001). Imaging of the YFP-tagged K14 was done by converting the yellow YFP to a green pseudo color (i.e., from the yellow [570–590 nm] to the green [495–570 nm] wavelength). (B) Cells as in panel A were treated with dispase to test resistance of the epithelial sheet to mechanical stress. Fragments of the cell sheet were counted and quantified (***P < 0.001). (C) Representative images of TUNEL-stained keratinocytes transduced with K14-R125C and then treated with DMSO or PN (5 μM, 48 hours) followed by IFN-γ + Fas-L to induce apoptosis. Quantification of the percentage TUNEL+ cells using pooled counts from 3 separate experiments is shown on the right. ****P < 0.001 using unpaired, 2-tailed Student’s t test. Scale bar: 50 μm. (D) Lysates of cells, similar to those used in panel C, were blotted with antibodies against caspase-7 or cleaved caspase-7. For panels A and B, 1-way ANOVA was used for comparison between treatment groups, and Tukey’s post hoc test was used for 2-group comparisons.