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 2

PN decreases K18 acetylation via SIRT2 in cultured cells and mouse liver.

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PN decreases K18 acetylation via SIRT2 in cultured cells and mouse liver...
(A) GFP-K18-WT and GFP-K18-R90C lentivirus–transduced A549 cells were treated with DMSO or PN (5 μM, 48 hours). Acetylated proteins were immunoprecipitated using an anti-AcK antibody followed by blotting with an anti-K18 antibody. (B) GFP-K18-R90C lentivirus–transduced A549 cells were used for immunoprecipitation with an anti-GFP antibody then blotted with an anti-AcK antibody. An input blot is also shown. (C) Lysates from GFP-K18-WT/GFP-K18-R90C–transduced cells were blotted with antibodies against SIRT2 or acetylated α-tubulin (actin blot is included as loading control). The average relative intensity of the indicated bands from 3 separate experiments is included beneath each blot. *P < 0.05 using unpaired, 2-tailed Student’s t test (comparing R90C DMSO with PN). (D) The percentage activation of SIRT2 in the presence of PN, NMN (activator control), and NAM (inhibitor control). *P < 0.05 using the least significant difference test, compared with PN (= 0). (E) Immunofluorescent double staining of K18 and SIRT2 in A549 cells expressing K18-R90C treated with DMSO/PN. Scale bar: 50 μm. (F) Anti-SIRT2 (and control nonimmune IgG) immunoprecipitates were prepared from cells used in panel E, and then blotted with anti-SIRT2/anti-K18 antibodies. (G) Liver lysates from DMSO/PN-treated mice expressing K18-R90C were used for immunoprecipitation with an anti-AcK antibody (separate livers/lane) followed by immunoblotting with an anti-K18 antibody (input lysate was similarly blotted). n = 3 (DMSO group), n = 2 (PN group). (H) SIRT2 immunoprecipitates were prepared from liver extracts isolated from K18-R90C–expressing mice pretreated with DMSO/PN. K18 was analyzed by immunoblotting. Input lysates were also blotted with anti-SIRT2 (separate livers/lane are shown). n = 3 (DMSO group), n = 3 (PN). For panels G and H, the mean relative band intensity for each of the 2 groups is included beneath each blot. *P < 0.05, ***P < 0.001 using unpaired, 2-tailed Student’s t test (comparing DMSO with PN).