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

Singh, Simran Jeet; Meyer-Jens, Moritz; Alizoti, Erda; Bacon, W. Clark; Davis, Gregory F.; Osińska, Hanna; Gulick, James; Reischmann-Düsener, Silke; Orthey, Ellen; McLendon, Patrick M.; Molkentin, Jeffery D.; Schlossarek, Saskia; Robbins, Jeffrey; Carrier, Lucie

doi:10.1080/15548627.2020.1856493

Cited by 23 publications

(12 citation statements)

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“…The isolation of proteins from 2D-cultured hiPSC-CMs is based on a recent publication, 57 with adjusted volumes as follow. For the isolation of protein from untreated 2D-cultured hiPSC-CMs, 50 µL water with protease inhibitor cocktail was added to obtain the cytosolic fraction and 75 µL SDS-buffer for the collection of the membrane-enriched fraction.…”

Section: Protein Isolationmentioning

confidence: 99%

“…Western blot analysis was performed according to previous publications, 57,58 with slight adaptations. For analysis of 30-day-old, 2D-cultured hiPSC-CMs, total crude protein lysates were evaluated by mixing 7.5 µg of protein with 6x Laemmli buffer and subsequent separations on 4-15% Mini-PROTEAN Precast Gels (Bio-Rad, Hercules, CA) or 10%/12% self-casted acrylamide/bisacrylamide (29:1) gels.…”

Section: Western Blot Analysismentioning

confidence: 99%

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ACTN2mutant causes proteopathy in human iPSC-derived cardiomyocytes

Zech

Prondzynski

Singh

et al. 2021

Preprint

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RationaleGenetic variants in ACTN2, encoding α-actinin 2 (ACTN2), are associated with several forms of (cardio)myopathy in the heterozygous state, and can cause progressive, severe cardiomyopathy in the homozygous state. We previously reported a heterozygous missense (c.740C>T) ACTN2 variant, associated with hypertrophic cardiomyopathy (HCM), which induced an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).ObjectiveTo evaluate the impact of heterozygosity and homozygosity of the c.740C>T ACTN2 variant on cardiomyocyte structure and function.Methods and ResultsNext to the previously established hiPSC line carrying the heterozygous ACTN2 variant (ACTN2het), we created a homozygous line (ACTN2hom) with CRISPR/Cas9. Differentiation into cardiomyocytes revealed myofibrillar disarray, increased cell area and volume, multinucleation, and ACTN2 aggregates in mutant hiPSC-CMs. Live cell imaging in hiPSC-CMs showed an inverse correlation between sarcomeric incorporation and aggregation of exogenously expressed mutant ACTN2. RNA-seq and proteomic analyses showed alteration of several canonical pathways involved in metabolism, oxidative and cellular stress, and proteostasis, including the ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) in mutant hiPSC-CMs. Detailed evaluation of the UPS and ALP, including a high-content screening with a mWasabi-mTagRFP-hLC3 tandem construct, revealed a global activation of these systems in mutant hiPSC-CMs. The ACTN2 missense variant caused allele dose-dependent and -independent effects on several cardiomyocyte features. ACTN2hom exhibited markedly lower levels of sarcomere-associated proteins, and hypocontractility in engineered heart tissues, resembling a dilated cardiomyopathy phenotype.ConclusionThis study showed allele dose-independent effects of the ACTN2 variant on multinucleation, ACTN2 aggregation, and UPS activation, and allele dose-dependent effects of the variant on cellular hypertrophy, myofibrillar disarray, and ALP activation. Activation of the proteolytic systems is likely to cope with ACTN2 aggregation. Our data indicates proteopathy as an additional cellular feature caused by the c.740C>T ACTN2 variant, which may contribute to human ACTN2-associated inherited cardiomyopathy.

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Section: Protein Isolationmentioning

confidence: 99%

Section: Western Blot Analysismentioning

confidence: 99%

ACTN2mutant causes proteopathy in human iPSC-derived cardiomyocytes

Zech

Prondzynski

Singh

et al. 2021

Preprint

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“…Ubiquitin, proteasome, and autophagy-related genes were selected based on the commonality of their classi cation in two datasets, the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology and that reported by Singh et al 16 (Table 1). Lysosome genes were obtained in the same way using the KEGG orthology and PathCards databases.…”

Section: Gene Selectionmentioning

confidence: 99%

A Bioinformatic Investigation of Proteasome And Autophagy Expression in The Central Nervous System

Watanabe

Takeda

Honda

et al. 2021

Preprint

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The ubiquitin proteasome system (UPS) and autophagy lysosome pathway (ALP) play major roles in protein quality control. However, data regarding the relative significance of UPS and ALP in the central nervous system (CNS) remain limited. In this study, we reckon the quantitative expression status of UPS- and ALP-related genes and their products in the CNS compared to that in other tissues. We collected human and mouse gene expression datasets from the reference expression dataset (RefEx) and Genevestigator (a tool for handling curated transcriptomic data from public repositories) and human proteomics data from the proteomics database (ProteomicsDB). The expression levels of genes and proteins in four categories—ubiquitin, proteasome, autophagy, and lysosome in cells and tissues were extracted. Perturbation of expression by drugs was also analyzed based on the four categories. Compared to that for the other three categories, proteasome gene expression was consistently low in the CNS of mice, and was more pronounced in humans. Neural stem cells and neurons showed low proteasome gene expressions when compared to non-neuronal stem cells. Proteomic analyses, however, did not show trends similar to those observed in the gene expression analyses. Perturbation analyses revealed that agents such as azithromycin and vitamin D3 upregulated the expression of both the UPS and ALP. Disproportional expression of the UPS and ALP might play a role in the pathophysiology of CNS disorders and this imbalance might be redressed by several therapeutic candidates.

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“…Traditionally, the UPS and ALP have been considered to act separately, but recent data suggest that they functionally cooperate with each other to maintain cellular homeostasis (Wang et al, 2008;Korolchuk et al, 2010;Lamark and Johansen, 2010;Singh et al, 2020). In the present study, we therefore aimed to investigate alterations of both the UPS and the ALP in a mouse model of eccentric left ventricular (LV) hypertrophy with preserved EF expressing both cyclin T1 and Gαq under the control of the cardiac-specific α-myosin heavy chain promoter.…”

Section: Introductionmentioning

confidence: 99%

A Transgenic Mouse Model of Eccentric Left Ventricular Hypertrophy With Preserved Ejection Fraction Exhibits Alterations in the Autophagy-Lysosomal Pathway

et al. 2021

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The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the main proteolytic systems involved in cellular homeostasis. Since cardiomyocytes, as terminally differentiated cells, lack the ability to share damaged proteins with their daughter cells, they are especially reliant on these protein degradation systems for their proper function. Alterations of the UPS and ALP have been reported in a wide range of cardiac diseases, including cardiomyopathies. In this study, we determined whether the UPS and ALP are altered in a mouse model of eccentric left ventricular (LV) hypertrophy expressing both cyclin T1 and Gαq under the control of the cardiac-specific α-myosin heavy chain promoter (double transgenic; DTG). Compared to wild-type (WT) littermates, DTG mice showed higher end-diastolic (ED) LV wall thicknesses and diameter with preserved ejection fraction (EF). The cardiomyopathic phenotype was further confirmed by an upregulation of the fetal gene program and genes associated with fibrosis as well as a downregulation of genes involved in Ca2+ handling. Likewise, higher NT-proBNP levels were detected in DTG mice. Investigation of the UPS showed elevated steady-state levels of (poly)ubiquitinated proteins without alterations of all proteasomal activities in DTG mice. Evaluation of ALP key marker revealed a mixed pattern with higher protein levels of microtubule-associated protein 1 light chain 3 beta (LC3)-I and lysosomal-associated membrane protein-2, lower protein levels of beclin-1 and FYVE and coiled-coil domain-containing protein 1 (FYCO1) and unchanged protein levels of p62/SQSTM1 in DTG mice when compared to WT. At transcriptional level, a > 1.2-fold expression was observed for Erbb2, Hdac6, Lamp2, Nrg1, and Sqstm1, while a < 0.8-fold expression was revealed for Fyco1 in DTG mice. The results related to the ALP suggested overall a repression of the ALP during the initiation process, but an induction of the ALP at the level of autophagosome-lysosome fusion and the delivery of ubiquitinated cargo to the ALP for degradation.

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

Abstract: Carrier (2020): A high-throughput screening identifies ZNF418 as a novel regulator of the ubiquitin-proteasome system and autophagy-lysosomal pathway, Autophagy,

Cited by 23 publications

References 68 publications

ACTN2mutant causes proteopathy in human iPSC-derived cardiomyocytes

ACTN2mutant causes proteopathy in human iPSC-derived cardiomyocytes

A Bioinformatic Investigation of Proteasome And Autophagy Expression in The Central Nervous System

A Transgenic Mouse Model of Eccentric Left Ventricular Hypertrophy With Preserved Ejection Fraction Exhibits Alterations in the Autophagy-Lysosomal Pathway

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