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 (Ub^G76V-mCherry) and an ALP reporter (GFP-LC3) and screened for genes for which knockdown increased both Ub^G76V-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.

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