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. 2015 Nov 6;117(11):956-66.
doi: 10.1161/CIRCRESAHA.115.306783. Epub 2015 Sep 17.

COP9 signalosome controls the degradation of cytosolic misfolded proteins and protects against cardiac proteotoxicity

Huabo Su  1 , Jie Li  1 , Hanming Zhang  1 , Wenxia Ma  1 , Ning Wei  1 , Jinbao Liu  2 , Xuejun Wang  2
Affiliations

COP9 signalosome controls the degradation of cytosolic misfolded proteins and protects against cardiac proteotoxicity

Huabo Su et al. Circ Res. .

Abstract

Rationale: Impaired degradation of misfolded proteins is associated with a large subset of heart diseases. Misfolded proteins are degraded primarily by the ubiquitin-proteasome system, but the ubiquitin ligases responsible for the degradation remain largely unidentified. The cullin deneddylation activity of the COP9 signalosome (CSN) requires all 8 CSN subunits (CSN1 through CSN8) and regulates cullin-RING ligases, thereby controlling ubiquitination of a large number of proteins; however, neither CSN nor cullin-RING ligases is known to regulate the degradation of cytosolic misfolded proteins.

Objective: We sought to investigate the role of CSN8/CSN in misfolded protein degradation and cardiac proteinopathy.

Methods and results: Cardiac CSN8 knockout causes mouse premature death; hence, CSN8 hypomorphism (CSN8(hypo)) mice were used. Myocardial neddylated forms of cullins were markedly increased, and myocardial capacity of degrading a surrogate misfolded protein was significantly reduced by CSN8 hypomorphism. When introduced into proteinopathic mice in which a bona fide misfolded protein R120G missense mutation of αβ-crystallin (CryAB(R120G)) is overexpressed in the heart, CSN8 hypomorphism aggravated CryAB(R120G)-induced restrictive cardiomyopathy and shortened the lifespan of CryAB(R120G) mice, which was associated with augmented accumulation of protein aggregates, increased neddylated proteins, and reduced levels of total ubiquitinated proteins and LC3-II in the heart. In cultured cardiomyocytes, both CSN8 knockdown and cullin-RING ligase inactivation suppressed the ubiquitination and degradation of CryAB(R120G) but not native CryAB, resulting in accumulation of protein aggregates and exacerbation of CryAB(R120G) cytotoxicity.

Conclusions: (1) CSN8/CSN promotes the ubiquitination and degradation of misfolded proteins and protects against cardiac proteotoxicity, and (2) cullin-RING ligases participate in degradation of cytosolic misfolded proteins.

Keywords: COP9 signalosome; Cops8; autophagy; desmin-related cardiomyopathy; proteasome; ubiquitin.

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Conflict of interest statement

DISCLOSURE

The authors declare there is no conflict of interest to disclose.

Figures

Figure 1
Figure 1. Decreased deneddylation activity in CSN8neoflox/− (CSN8hypo) mouse hearts
Age-matched littermate CSN8neoflox/+ mice were used as controls (CTL). (A) Csn8-targeted alleles described in this study. The numbered light grey rectangles denote the exons. In the neoflox allele (CSN8neoflox), a neomycin phosphotransferase II gene (NEO) flanked by Frt sites (empty triangles) are inserted between exon 3 and 4; and exons 4 through 6 are flanked by LoxP sites (solid triangles). In CSN8 knockout allele (CSN8−), the NEO cassette and exons 4 through 6 are deleted by FLP- and Cre- mediated recombination. (B, C) Western blot analyses of indicated proteins. Representative images (B) and pooled densitometry data (C) are shown. GAPDH serves as a loading control. (D, E) Representative images (D) and pooled densitometry data (E) from western blot analyses of indicated cullin proteins in mouse hearts. Arrows mark the neddylated form of cullins. (F, G) Representative images (F) and pooled densitometry data (G) from western blot analyses of neddylated proteins in mouse hearts. Total neddylated proteins (revealed by long-time exposure) and neddylated cullin (revealed by short-time exposure) are increased in CSN8hypo hearts. N=3 mice/group; *p<0.05, #p<0.01 vs. CTL; t-test.
Figure 2
Figure 2. Effect of CSN8hypo on myocardial UPS function
Mice were created via cross-breeding between CSN8neoflox/neoflox and CSN8+/−::GFPdgn mice. The resultant 8-week-old CTL::GFPdgn mice and CSN8hypo::GFPdgn mice were used. (A, B) Images (A) and pooled densitometry data (B) from western blot analyses for myocardial GFPdgn. Alpha-actinin was probed as a loading control. n= 3 or 5 mice per group, #p < 0.01 vs. CTL. (C) GFPdgn mRNA levels are assessed by real time RT-PCR using total RNA extracted from mouse ventricular myocardium. (D) Proteasomal peptidase activity assays of ventricular myocardium. n=4~6 per group, t-test.
Figure 3
Figure 3. DRC exacerbation by CSN8hypo
(A, B) Western blot analysis for the indicated CSN subunits in mouse hearts with tg overexpression of wild type CryAB (WT) or CryABR120G (R120G). Representative images (A) and pooled densitometry data (B) are shown. n=4 mice/group; *p<0.05 vs. Ntg or WT; ANOVA followed by the Scheffé test. (C) Kaplan-Meier survival analysis of a cohort of mixed-sex littermate CTL, CSN8hypo, CTL::CryABR120G, and CSN8hypo::CryABR120G mice. Log-rank test. (D, E) Representative image (D) and pooled densitometry data (E) of western blot analysis for myocardial NEDD8 conjugates (conj.) of mice of indicated genotypes at 8 weeks (n=4 mice/group); t-test.
Figure 4
Figure 4. CSN8hypo aggravates CryABR120G-induced aberrant protein aggregation and decreases the ubiquitination in mouse hearts
Littermate mice with the indicated genotypes were used at the age of 8 weeks. (A) Representative confocal fluorescent images of myocardial sections immunostained for CryAB (white). Scale bar = 20 µm. (B) Quantitative analysis of the relative abundance of the CryAB-positive protein aggregates in the heart. n=3 mice/group. *p<0.05 vs. CTL::CryABR120G, t-test. (C) Filter trapping assays for CryAB and ubiquitin (Ub) in ventricular myocardium of mice with indicated genotypes. The proteins retained on the filter were immunostained for CryAB or Ub. The relative density of each dot is shown below the blots. (D) Western blot analyses of myocardial total ubiquitinated proteins. β-tubulin was probed as a loading control.
Figure 5
Figure 5. CSN8 knockdown stabilizes CryABR120G in cardiomyocytes
NRVMs were infected with adenoviruses expressing HA-CryABR120G (Ad-HA- CryABR120G) or β-Gal as indicated. The cells were also transfected with siRNAs against either luciferase (siLuci) or CSN8 (siCSN8). At 72 hours after the siRNA transfection, the cells were harvested for the analyses (A, B) or treated with cycloheximide (CHX, 100 µmol/L) for the indicated times (C). (A) Representative western blot images of indicated proteins in the Triton X-100 soluble and insoluble fraction of cell lysate. GAPDH and α-actinin were probed as loading controls. (B) Immunofluorescent images showing increased protein aggregates in CSN8 knockdown cells. HA-tag (green) and Sec61α(red) were stained for CryABR120G and aggresomes, respectively. Scale bar=50 µm. (C, D) Cycloheximide (CHX) chase assay for HA-CryABR120G. HA-CryABR120G protein levels at the indicated time points were measured using western blot analyses for HA-tag. A representative image (C) and a summary of the relative levels of HA-CryABR120G (D) are shown; *p<0.05 vs. the siLuci+CryABR120G group, n=3 repeats; t-test.
Figure 6
Figure 6. Inhibition of CRLs impairs CryABR120G ubiquitination and degradation in cardiomyocytes
(A) CSN8 knockdown impairs CryABR120G ubiquitination. NVRMs were treated as described in Figure 5. Seventy-two hours after siRNA transfection, MG132 (5 µmol/L) treatment was initiated and lasted for 6 hours before the cells were harvested. Representative images of western blot analyses (IB) of the indicated proteins in immunoprecipitated (IP) HA-CryABR120G are shown. (B, C) MLN4024 stabilizes CryABR120G in cultured NRVMs. MLN4924 (1µmol/L) or vehicle control (DMSO) treatment was initiated at 48 hours after Ad-HA-CryABR120G infection in cultured NRVMs. Cycloheximide (CHX, 50µmol/L) was added to the culture media at 30min after initiating MLN4924 treatment. Cells were harvested at the indicated time points for extraction of total proteins. Representative images (B) and pooled densitometry data (C) of western blot analyses of HA-CryABR120G are shown. *p<0.05 vs. the DMSO group, n=3 repeats; t-test.
Figure 7
Figure 7. CSN8 deficiency mitigates autophagic responses in mouse hearts and augments proteotoxicity in cardiomyocytes
(A, B) Western blot analyses for myocardial LC3 and p62. CSN8 hypomorphism was cross-bred into CryABR120G (R120G) mice. Ventricular myocardial total protein extracts from 8-week-old mice were used. Representative images (A) and pooled densitometry data (B) are shown. n=4~6 mice/group. #p<0.01 vs. CTL; ‡p<0.05 vs. CTL::R120G. (C, D) CSN8 knock-down augments CryABR120G-induced cardiomyocyte injury. NRVMs were treated as described in Figure 5. The assays were performed 72 hours after siRNA transfection. LDH activities in the cultured medium (C) were measured to assess cell injury. Cell viability was assessed by the MTT assay (D). #p<0.01 vs. siLuci; ‡p<0.01 vs. siLuci+R120G; n=4~6. ANOVA followed by the Scheffé test.
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