A high-throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway - PubMed (original) (raw)

. 2021 Oct;17(10):3124-3139.

doi: 10.1080/15548627.2020.1856493. Epub 2020 Dec 27.

Sonia R Singh 1 2 3, Erda Alizoti 1, W Clark Bacon 3, Gregory Davis 3, Hanna Osinska 3, James Gulick 3, Silke Reischmann-Düsener 1 2, Ellen Orthey 1, Patrick M McLendon 3, Jeffery D Molkentin 3, Saskia Schlossarek 1 2, Jeffrey Robbins 3, Lucie Carrier 1 2

Affiliations

A high-throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway

Sonia R Singh et al. Autophagy. 2021 Oct.

Abstract

The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are two major protein degradation pathways in eukaryotic cells. Initially considered as two independent pathways, there is emerging evidence that they can work in concert. As alterations of UPS and ALP function can contribute to neurodegenerative disorders, cancer and cardiac disease, there is great interest in finding targets that modulate these catabolic processes. We undertook an unbiased, total genome high-throughput screen to identify novel effectors that regulate both the UPS and ALP. We generated a stable HEK293 cell line expressing a UPS reporter (UbG76V-mCherry) and an ALP reporter (GFP-LC3) and screened for genes for which knockdown increased both UbG76V-mCherry intensity and GFP-LC3 puncta. With stringent selection, we isolated 80 candidates, including the transcription factor ZNF418 (ZFP418 in rodents). After screen validation with Zfp418 overexpression in HEK293 cells, we evaluated Zfp418 knockdown and overexpression in neonatal rat ventricular myocytes (NRVMs). Endogenous and overexpressed ZFP418 were localized in the nucleus. Subsequent experiments showed that ZFP418 negatively regulates UPS and positively regulates ALP activity in NRVMs. RNA-seq from Zfp418 knockdown revealed altered gene expression of numerous ubiquitinating and deubiquitinating enzymes, decreased expression of autophagy activators and initiators and increased expression of autophagy inhibitors. We found that ZPF418 activated the promoters of Dapk2 and Fyco1, which are involved in autophagy. RNA-seq from Zfp418 knockdown revealed accumulation of several genes involved in cardiac development and/or hypertrophy. In conclusion, our study provides evidence that ZNF418 activates the ALP, inhibits the UPS and regulates genes associated with cardiomyocyte structure/function.Abbreviations: ACTN2, actinin alpha 2; ALP, autophagy-lysosomal pathway; COPB1, COPI coat complex subunit beta 1; DAPK2, death associated protein kinase 2; FYCO1, FYVE and coiled-coil domain autophagy adaptor 1; HEK293, human embryonic kidney cells 293; HTS, high-throughput screen; LC3, microtubule associated protein 1 light chain 3; NRVMs, neonatal rat ventricular myocytes; RNA-seq, RNA sequencing; RPS6, ribosomal protein S6; TNNI3, troponin I, cardiac 3; UPS, ubiquitin-proteasome system; shRNA, short hairpin RNA; SQSTM1/p62, sequestosome 1; VPS28, VPS28 subunit of ESCRT-I; ZNF418/ZFP418, zinc finger protein 418.

Keywords: ALP; UPS; ZFP418; ZNF418; autophagy; cardiomyocyte; proteasome; protein degradation; screen; ubiquitin.

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Conflict of interest statement

No potential conflict of interest was reported by the authors.

Figures

Figure 1.

Figure 1.

Generation of a stable HEK293 UPS and ALP reporter cell line (HEK293LU). (A) A schematic diagram of the cell line-generating protocol. (B) Comparison of the reporter cell line (HEK293LU) to normal HEK293 cells. Differential interference contrast (DIC) and fluorescence images of mCherry and GFP are depicted. (C) HEK293LU ubiquitinG76V-mCherry expression with or without (control; ctrl) proteasome inhibitor treatment (250 nM epoxomicin or 1 μM MG132 treatment for 24 h). (D) HEK293LU GFP-LC3 expression with or without autophagy modulators (100 nM of the autophagy inhibitor bafilomycin A1 or 50 nM of the autophagy activator torin 2 for 3 h)

Figure 2.

Figure 2.

Design of a high-throughput screen that identifies modulators of the UPS and ALP. (A) Example of candidate with increased UbG76V-mCherry intensity and increased number of GFP-LC3 positive puncta (arrows). (B) A flow diagram illustrating the goal of the screen and sequential steps in its implementation. (C) A flow diagram illustrating the HTS assay. (D) Assay plate set-up. The screen was performed in 384-well plates with 16 images taken per well. High and low controls (green) were placed at the bottom of each plate. Four wells on each plate were not transduced with lentivirus and treated with puromycin to validate puromycin efficacy (light blue). (E) Analyses masks. Nuclei (DAPI), mCherry and GFP puncta count. (F) Z’-factor distribution of positive controls MG132 and bafilomycin A1 for 10% of the plates

Figure 3.

Figure 3.

Zfp418 knockdown and overexpression in cardiomyocytes. (A) NRVMs were transfected with scramble siRNA (Scr) or siRNA targeting Zfp418 (si_Zfp418_; 100 nM) and Zfp418 mRNA levels were determined by RT-qPCR after 5–6 days. (B) NRVMs were transfected with scramble siRNA (Scr) or siRNA targeting Zfp418 (si_Zfp418_; 100 nM) and stained for ZFP418 (green), cardiomyocytes (TNNI3; red) and nuclei (DAPI; blue). (C) NRVMs were transduced with control adenovirus (AdV-Cre) or an adenovirus encoding Flag-Zfp418, and Zfp418 mRNA levels were determined by RT-qPCR after 5–6 days. (D) NRVMs were transduced with control adenovirus (Cre) or an adenovirus encoding Flag-Zfp418 and stained for ZFP418 (green), cardiomyocytes (TNNI3; orange) and nuclei (DAPI; blue). Data are presented as mean+SEM with **P< 0.01 and ****P< 0.0001, unpaired Student’s _t_-test (against Scr or AdV-Cre)

Figure 4.

Figure 4.

ZFP418 negatively regulates UPS activity. (A, B) NRVMs were transfected with scramble siRNA (Scr) or siRNA targeting Zfp418 (si_Zfp418_; 100 nM) and treated with epoxomicin (proteasome inhibitor; 250 nM for 24 h) or DMSO (control). (A) After 5–6 days, cells were harvested and (poly)ubiquitinated protein levels were determined by western blot and normalized to ACTN2. (B) After 5–6 days, cells were harvested and chymotrypsin-like activity of the proteasome was measured. (C, D) NRVMs were transduced with adenovirus encoding Cre (AdV-Cre; control) or Flag-Zfp418 (AdV-Flag-Zfp418) and treated with epoxomicin (proteasome inhibitor; 250 nM for 24 h) or DMSO (control). (C) After 5–6 days, cells were harvested and (poly)ubiquitinated protein levels were determined by western blot and normalized to RPS6. (D) After 5–6 days, cells were harvested and chymotrypsin-like activity of the proteasome was measured. (E, F, G, H, I, J) NRVMs were transfected with si_Zfp418_ or scramble siRNA and transduced with an adenovirus encoding GFPu (a reverse UPS reporter) or GFP (control). Where indicated, cells were treated with the proteasomal inhibitor epoxomicin (250 nM for 24 h) or DMSO (control) before harvest. (E) GFPu and GFP protein levels were determined by western blot. GAPDH was stained as loading control. (F) Immunofluorescence analysis of NRVMs stained for TNNI3 (red) and DAPI (blue) and GFPu or GFP (green). The ntensities of GFPu and GFP were quantified in NRVMs. (G) GFPu protein levels were determined by western blot. GAPDH was stained as loading control. (H) GFPu mRNA levels were determined by RT-qPCR and normalized to 18s. (I) NRVMs were scr-transfected and treated with 100 µM cycloheximide for indicated times and GFPu protein levels were determined by western blot. (J) NRVMs were treated with 100 µM cycloheximide for indicated times and GFPu protein levels were determined by western blot. Presented part of Ponceau was used as a loading control. (K) NRVMs were transduced with adenovirus encoding Cre recombinase (AdV-Cre; control) or Flag-Zfp418 and GFPu (a reverse UPS reporter). GFPu protein levels were determined by western blot. Presented part of Ponceau was used as a loading control. (L) NRVMs were transfected with scramble siRNA (Scr), siRNA targeting Zfp418 (si_Zfp418_; 100 nM) and siRNA targeting Psmd1 (si_Psmd1_; 20 nM; essential proteasomal subunit). Four hours after transfection, NRVMs were transduced with an adenovirus encoding GFP-CRYABR120G (mutant protein that forms aggregates). After 5–6 days, cells were fixed and stained for immunofluorescence microscopy. CRYABR120G aggregates are depicted in green, cardiomyocytes in red (TNNI3) and nuclei in blue (DAPI). Aggregate-to-myocyte area was quantified. (M) NRVMs were transfected with si_Psmd1_ or scramble siRNA, transduced with an adenovirus encoding GFPu and treated with the proteasomal inhibitor epoxomicin (250 nM for 15 h) or DMSO before harvest. GFPu protein levels were determined by western blot and normalized to ACTN2. Data are presented as mean+SEM with *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, two-way ANOVA plus Sidak multiple comparison test and #P < 0.05, unpaired Student’s _t_-test

Figure 5.

Figure 5.

ZFP418 positively regulates ALP activity. (A) A diagram depicting the effects of the activators torin 1 and torin 2 or the inhibitors leupeptin and bafilomycin A1 on autophagic activity. Torin 1 and torin 2 inhibit mTOR complex 1 and activate autophagy at an early step. Leupeptin inhibits lysosomal proteases and inhibits autophagy at a late step. Bafilomycin A1 is an inhibitor of the V-ATPase and inhibits lysosomal acidification. (B, C, D) NRVMs were transfected with scramble siRNA (Scr) or siRNA targeting Zfp418 (si_Zfp418_; 100 nM) and analyzed 6 days after transfection. (B) Electron microscope images after Scr or si_Zfp418_ transfection. ALP related vesicles are marked with blue arrows. Vesicles of uncertain identity were marked with black arrows. Nuclei are marked with asterisks. Scale bar indicates 800 nm. Extended images in Figure S6. (C) NRVMs were treated with autophagy inhibitor leupeptin (100 μM, 24 h) or water, fixed and stained for LC3B (green) to detect LC3-positive vesicles that are markers for autophagosomes (puncta), cardiomyocytes (TNNI3; red) and nuclei (DAPI; blue). (D) NRVMs were treated with the autophagy inhibitor leupeptin (100 μM, 24 h) or/and autophagy activator torin 1 (1 μM, 3 h) or DMSO. LC3 and SQSTM1/p62 protein levels were determined by western blot, ACTN2 was stained as a loading control. LC3-II:ACTN2 and SQSTM1/p62:ACTN2 were quantified to determine autophagic activity. (E) NRVMs were transduced with an adenovirus encoding Cre (AdV-Cre; control) or Flag-Zfp418 and treated with autophagy inhibitor bafilomycin A1 (30 nM for 3 h) or/and autophagy activator torin 2 (50 nM, 3 h) or DMSO. LC3 and SQSTM1/p62 protein levels were determined by western blot. RPS6 was stained as a loading control and LC3-II:RPS6 and SQSTM1/p62:RPS6 were quantified to determine autophagic activity. Data are presented as mean+SEM with *P< 0.05, **P< 0.01, ***P< 0.001 and ****P< 0.0001, two-way ANOVA plus Sidak multiple comparison test, and #P< 0.05, ##P< 0.01, ###P< 0.001 and ####P< 0.0001 unpaired Student’s _t_-test

Figure 6.

Figure 6.

Zfp418 knockdown changes expression of UPS and ALP genes. NRVMs were transfected with scramble siRNA (Scr) or siRNA targeting Zfp418 (si_Zfp418_; 100 nM), harvested 5 days post-transfection and RNA sequencing was performed. Significantly (_P_≤ 0.05) up- (red) or downregulated (blue) genes after Zfp418 knockdown were compared to databases of genes encoding proteins related to the proteasome and ubiquitin (A) or autophagy (B). Databases are available in Tables S4-S7

Figure 7.

Figure 7.

Validation of targets identified in RNA sequencing. (A) NRVMs were transfected with scramble siRNA (Scr) or siRNA targeting Zfp418 (si_Zfp418_; 100 nM) and indicated mRNA levels were determined by RT-qPCR after 5 days. (B) NRVMs were transfected with scramble siRNA (Scr) or siRNA targeting Zfp418 (si_Zfp418_; 100 nM) or transduced with control adenovirus (AdV-Cre) or an adenovirus encoding Flag-ZFP418. Fyco1 or Dapk2 mRNA levels were determined by RT-qPCR after 5 days. (C) ZFP418 and Gaussia Luciferase plasmids with Fyco1 or Dapk2 promoter (Fyco1p or Dapk2p) were transfected into HEK293 cells. Luciferase activities were determined after 1 day. Data are presented as mean+SEM with *P< 0.05, **P< 0.01 and ****P< 0.0001, unpaired Student’s _t_-test. Abbreviations: Dapk2, death associated protein kinase 2; Fyco1, FYVE and coiled-coil domain autophagy adaptor 1. Primer sequences are given in Table S14

Figure 8.

Figure 8.

Zfp418 knockdown increases expression of cardiac genes associated with cardiomyocyte structure and function. NRVMs were transfected with scramble siRNA (Scr) or siRNA targeting Zfp418 (si_Zfp418_; 100 nM), harvested 5 days post-transfection and RNA sequencing was performed. (A) Volcano plot. Significantly upregulated genes involved in cardiomyocyte maturation (actin-filament-based process) and hypertrophy are highlighted in red. Zfp418 down-regulation is shown in blue. Vertical dotted lines showed the 2-fold-change threshold, and the horizontal dotted line the limit of P < 0.05. (B) STRING protein network of highlighted genes of (A). Proteins with red nodes are involved in actin-filament-based processes. Proteins with blue nodes are involved in striated muscle development. Gene acronym and name are given in Table S15

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