Ube2v1 Positively Regulates Protein Aggregation by Modulating Ubiquitin Proteasome System Performance Partially Through K63 Ubiquitination - PubMed (original) (raw)

Ube2v1 Positively Regulates Protein Aggregation by Modulating Ubiquitin Proteasome System Performance Partially Through K63 Ubiquitination

Na Xu et al. Circ Res. 2020.

Abstract

Rationale: Compromised protein quality control can result in proteotoxic intracellular protein aggregates in the heart, leading to cardiac disease and heart failure. Defining the participants and understanding the underlying mechanisms of cardiac protein aggregation is critical for seeking therapeutic targets. We identified Ube2v1 (ubiquitin-conjugating enzyme E2 variant 1) in a genome-wide screen designed to identify novel effectors of the aggregation process. However, its role in the cardiomyocyte is undefined.

Objective: To assess whether Ube2v1 regulates the protein aggregation caused by cardiomyocyte expression of a mutant αB crystallin (CryABR120G) and identify how Ube2v1 exerts its effect.

Methods and results: Neonatal rat ventricular cardiomyocytes were infected with adenoviruses expressing either wild-type CryAB (CryABWT) or CryABR120G. Subsequently, loss- and gain-of-function experiments were performed. Ube2v1 knockdown decreased aggregate accumulation caused by CryABR120G expression. Overexpressing Ube2v1 promoted aggregate formation in CryABWT and CryABR120G-expressing neonatal rat ventricular cardiomyocytes. Ubiquitin proteasome system performance was analyzed using a ubiquitin proteasome system reporter protein. Ube2v1 knockdown improved ubiquitin proteasome system performance and promoted the degradation of insoluble ubiquitinated proteins in CryABR120G cardiomyocytes but did not alter autophagic flux. Lys (K) 63-linked ubiquitination modulated by Ube2v1 expression enhanced protein aggregation and contributed to Ube2v1's function in regulating protein aggregate formation. Knocking out Ube2v1 exclusively in cardiomyocytes by using AAV9 (adeno-associated virus 9) to deliver multiplexed single guide RNAs against Ube2v1 in cardiac-specific Cas9 mice alleviated CryABR120G-induced protein aggregation, improved cardiac function, and prolonged lifespan in vivo.

Conclusions: Ube2v1 plays an important role in protein aggregate formation, partially by enhancing K63 ubiquitination during a proteotoxic stimulus. Inhibition of Ube2v1 decreases CryABR120G-induced aggregate formation through enhanced ubiquitin proteasome system performance rather than autophagy and may provide a novel therapeutic target to treat cardiac proteinopathies.

Keywords: cardiomyocytes; heart failure; protein aggregates; signal transduction.

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Figures

Figure 1.

Figure 1.. Ube2v1 knockdown inhibits CryABR120G-induced protein aggregation.

A, Western blot analysis showed effective siRNA knockdown of Ube2v1 in NRVMs. GAPDH was used as a loading control. B, Data shown in panel A were quantitated. C, NRVMs were infected with AdGFP-CryABR120G and then transfected with either a scrambled siRNA (Scramble) or siRNAs to Ube2v1 as indicated. At 5 days post-infection, NRVMs were fixed and immunostained with troponin I (TnI; red) to identify the cardiomyocytes and DAPI (nuclear staining; blue). Scale bar=100 μm. D, Aggregates in NRVMs were quantitated using NIS-elements software. At least 100 cells per group were quantitated. E and F, siUbe2v1 #1 was transfected into NRVMs and Western blots used to quantitate CryAB protein in the soluble and insoluble fractions as indicated. GAPDH and α-actinin were used as loading controls as indicated. One-way ANOVA with Tukey’s post-hoc multiple comparisons for B and D. Data are shown as mean±SEM. ns; non-significant (_P_>0.05). Scr; scrambled siRNA control, siv1; siUbe2v1. R120G; AdGFP-CryABR120G. MOCK; mock transfected cells.

Figure 2.

Figure 2.. The expression profile of Ube2v1 in CryABR120G hearts.

A, Western blot showing Ube2v1 expression levels in 5-month old CryABR120G hearts (n=3, two males and one female per group). GAPDH was used as a loading control. B, Density analyses of the complete Western blot data. C, A 5-month old heart section was immunostained with Ube2v1 (green) and the cardiomyocyte marker troponin I (TnI; red). Nuclei were counterstained with DAPI (blue). Scale bar=100 μm. Unpaired, two-tailed Student’s t-test for B. Data are shown as mean±SEM. Ntg; non-transgenic, R120G; CryABR120G transgenic.

Figure 3.

Figure 3.. Ube2v1 overexpression promotes protein aggregate formation.

A, Schematic representation of the recombinant adenovirus encoding Ube2v1-c-myc fusion protein. B, NRVMs were infected with AdUbe2v1-myc at different multiplicities of infection. The Western blot shows the protein expression levels of the c-Myc tag and Ube2v1. GAPDH was used as a loading control. C, NRVMs were coinfected with either AdGFP-CryABWT or AdGFP-CryABR120G and AdUbe2v1-myc. At 5 days post-infection, NRVMs were fixed and immunostained with troponin I (TnI; red) and DAPI (nuclear staining; blue). Scale bar=100 μm. D, Aggregates in NRVMs were quantitated using NIS-elements software. At least 100 cells per group were quantitated. E, Soluble CryAB expression was detected by Western blot. GAPDH was used as a loading control. F, Insoluble CryAB expression was detected by western blot after protein solubilization. α-actinin was used as a loading control. One-way ANOVA with Tukey’s post-hoc multiple comparisons for D. Data are shown as mean±SEM. ns; non-significant (_P_>0.05). Ctr; non-infected cells, CMV; cytomegalovirus, WT; AdGFP-CryABWT, R120G; AdGFP-CryABR120G.

Figure 4.

Figure 4.. Ube2v1 impacts cardiomyocyte protein ubiquitination.

A, NRVMs were transfected as indicated and subjected to immunohistochemical analyses using anti-ubiquitin (red) and anti-myosin binding protein C (MyBP-C) to detect the cardiomyocytes (purple). Scale bar=100 μm. B, Quantification of ubiquitinated protein levels in NRVMs using NIS-elements software. At least 100 cells per group were analyzed. C, Western blot showing insoluble ubiquitinated proteins. α-actinin was used as a loading control. Scr; scrambled siRNA control, siv1; siUbe2v1. D, Western blot showing insoluble ubiquitinated protein levels in NRVMs coinfected with AdUbe2v1-myc and AdGFP-CryABR120G. α-actinin was used as a loading control. One-way ANOVA with Tukey’s post-hoc multiple comparisons for B. Data are shown as mean±SEM. ns; non-significant (_P_>0.05). Ctr; non-transfected cells, MOCK; mock transfected NRVMs, R120G; AdGFP-CryABR120G infected NRVMs.

Figure 5.

Figure 5.. Proteasomal activity is negatively affected by Ube2v1 expression.

A, Chymotrypsin-like proteasomal activity in NRVMs infected with Ad-Ube2v1. Epx; epoxomicin, AU; arbitrary unit. B, NRVMs were coinfected with an adenovirus vector expressing an inverse reporter of the UPS; AdGFPu, and adenovirus containing a Flag-tagged CryABR120G (AdFlag-R120G). NRVMs were subsequently transfected with scrambled (Scr) or Ube2v1 (siv1) siRNA and cultured for 4 days. Cells were fixed and immunostained with troponin (TnI) to identify the cardiomyocytes and the nuclei stained with DAPI. Scale bar=100 μm. C, Quantitation of GFP levels in NRVMs using NIS-elements software. At least 100 cells per group were quantitated. D, NRVMs were coinfected with AdGFPu and AdUbe2v1-myc. Five days later, cells were fixed and immunostained with TnI and DAPI. Scale bar=100 μm. E, Quantitation of GFP levels in NRVMs using NIS-elements software. At least 100 cells per group were quantitated. F and H, Western blots of GFP expression levels. GAPDH was used as a loading control. G and I, The Western blots were quantitated. One-way ANOVA with Tukey’s post-hoc multiple comparisons for A, E and I. Two-way ANOVA with Tukey’s post-hoc multiple comparisons for C and G. Data are shown as mean±SEM. ns; non-significant (_P_>0.05). Ctr; untreated cells.

Figure 6.

Figure 6.. NRVM autophagic flux is not affected by Ube2v1 knockdown.

Cells were infected with AdGFP-CryABR120G and then transfected with scrambled (Scr) or Ube2v1 (siv1) siRNA. Subsequently, cells were treated with the lysosomal inhibitor Bafilomycin A1 (BafA1) at 50 nmol/L for 3 hours. A, Western blot showing microtubule-associated light chain-I and -II (LC3–1, LC3-II) levels. GAPDH was used as a loading control. B, The Western blot was quantitated. C, Cells were fixed and immunostained with troponin I (TnI) and DAPI. Scale bar=100 μm. D, Aggregates were quantitated using NIS-elements software. At least 100 cells per group were quantitated. Two-way ANOVA with Tukey’s post-hoc multiple comparisons for B and D. Data are shown as mean±SEM. ns; non-significant (_P_>0.05). R120G; AdGFP-CryABR120G infected NRVMs.

Figure 7.

Figure 7.. Ube2v1-regulated K63 ubiquitination promotes CryABR120G-induced protein aggregation.

A, Venn diagram showing the total common gene numbers and the overlap as indicated. CTR; untreated, control NRVMs, RSIV1; AdGFP-CryABR120G infection followed by siUbe2v1 transfection, RADV1; coinfection with AdGFP-CryABR120G and AdUbe2v1-myc. B, NRVMs were infected with either AdGFP-CryABWT or AdGFP-CryABR120G, the cells lysed after 5 days and the protein lysates immunoprecipitated with K63-ubiquitinated antibody and subsequently immunoblotted with CryAB antibody. Normal IgG antibody was used as a control. C, Western blot showing insoluble K63-ubiquitinated proteins with α-actinin used as a loading control. Scr; scrambled siRNA control, siv1; siUbe2v1. D, Western blot showing insoluble K63-ubiquitinated protein levels in NRVMs coinfected with AdUbe2v1-myc and AdGFP-CryABR120G. α-actinin was used as a loading control. E, NRVMs were coinfected with AdGFP-CryABR120G and the different ubiquitin mutant adenoviruses. At 5 days post-infection, NRVMs were fixed and immunostained with troponin I (TnI; red) and DAPI (nuclear staining; blue). Scale bar=100 μm. F, Aggregates in NRVMs were quantitated using NIS-elements software. At least 100 cells per group were quantitated. G, Quantitation of aggregate area in NRVMs coinfected with AdGFP-CryABR120G and the different ubiquitin mutant adenoviruses as well as with scrambled (Scramble) or siUbe2v1. At least 100 cells per group were quantitated using NIS-elements software. H, Aggregates in NRVMs coinfected with AdGFP-CryABR120G and the different ubiquitin mutant adenoviruses as well as AdUbe2v1-myc were quantitated using NIS-elements software. At least 100 cells per group were quantitated. One-way ANOVA with Tukey’s post-hoc multiple comparisons for F. Two-way ANOVA with Tukey’s post-hoc multiple comparisons for G and H. Data are shown as mean±SEM. ns; non-significant (_P_>0.05). WT; AdGFP-CryABWT, R120G; AdGFP-CryABR120G.

Figure 8.

Figure 8.. Knockout of Ube2v1 alleviates protein aggregation and normalizes cardiac function in CryABR120G Tg hearts.

A, Touchdown PCR across the exon 2 locus in heart-derived samples. The top band at 913bp represents the expected position of full-length PCR amplicons and the lower, 500 bp band represents the expected position of PCR products with deletions caused by the indicated sgRNA combinations. B, Western blot showing Ube2v1 knockout efficiency. C, Quantitation of the Western blot data. D, CryAB transcript levels were determined by real-time PCR. E, Representative images showing aggregates in the heart. Scale bar=50 μm. F, Aggregates in NRVMs were quantitated using NIS-elements software. At least 10 fields per group were quantitated. n=3 per group (two males and one female for AAV-GFP, one male and two females for AAV-sgRNAv1). G and H, The transcript levels of both ANP and BNP were determined by real-time PCR. n=5 per group (two males and three females per group). I and J, Measurement of ejection fraction (EF) (panel I) and fractional shortening (FS) (panel J) by echocardiography in 6-month old mice. n=6–8 per group (two males and four females for Ntg, three males and four females for AAV-GFP, three males and five females for AAV-sgRNAv1). K, Kaplan-Meier survival curves of R120G; Myh6-Cas9 mice injected with control AAV9-GFP or AAV9-sgRNAv1. n=8 per group (three males and five females for AAV-GFP, four males and four females for AAV-sgRNAv1). Unpaired, two-tailed Student’s t-test for C and F. One-way ANOVA with Tukey’s post-hoc multiple comparisons for D, G, H, I and J. Log-rank test for K. Data are shown as mean±SEM. ns; non-significant (_P_>0.05). Ntg; non-transgenic, AAV-GFP; R120G; Myh6-Cas9 mice injected with AAV9-GFP, AAV-sgRNAv1; R120G; Myh6-Cas9 mice injected with AAV9-sgRNAv1.

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