Dataset Information

Nuclear Parkin Activates the ERR? Transcriptional Program and Drives Widespread Changes in Gene Expression Following Hypoxia.

ABSTRACT: Parkin is an E3 ubiquitin ligase well-known for facilitating clearance of damaged mitochondria by ubiquitinating proteins on the outer mitochondrial membrane. However, knowledge of Parkin's functions beyond mitophagy is still limited. Here, we demonstrate that Parkin has functions in the nucleus and that Parkinson's disease-associated Parkin mutants, ParkinR42P and ParkinG430D, are selectively excluded from the nucleus. Further, Parkin translocates to the nucleus in response to hypoxia which correlates with increased ubiquitination of nuclear proteins. The serine-threonine kinase PINK1 is responsible for recruiting Parkin to mitochondria, but translocation of Parkin to the nucleus occurs independently of PINK1. Transcriptomic analyses of HeLa cells overexpressing wild type or a nuclear-targeted Parkin revealed that during hypoxia, Parkin contributes to both increased and decreased transcription of genes involved in regulating multiple metabolic pathways. Furthermore, a proteomics screen comparing ubiquitinated proteins in hearts from Parkin^-/- and Parkin transgenic mice identified the transcription factor estrogen-related receptor ? (ERR?) as a potential Parkin target. Co-immunoprecipitation confirmed that nuclear-targeted Parkin interacts with and ubiquitinates ERR?. Further analysis uncovered that nuclear Parkin increases the transcriptional activity of ERR?. Overall, our study supports diverse roles for Parkin and demonstrates that nuclear Parkin regulates transcription of genes involved in multiple metabolic pathways.

SUBMITTER: Shires SE

PROVIDER: S-EPMC7244578 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Nuclear Parkin Activates the ERRα Transcriptional Program and Drives Widespread Changes in Gene Expression Following Hypoxia.

Shires Sarah E SE Quiles Justin M JM Najor Rita H RH Leon Leonardo J LJ Cortez Melissa Q MQ Lampert Mark A MA Mark Adam A Gustafsson Åsa B ÅB

Scientific reports 20200522 1

Parkin is an E3 ubiquitin ligase well-known for facilitating clearance of damaged mitochondria by ubiquitinating proteins on the outer mitochondrial membrane. However, knowledge of Parkin's functions beyond mitophagy is still limited. Here, we demonstrate that Parkin has functions in the nucleus and that Parkinson's disease-associated Parkin mutants, ParkinR42P and ParkinG430D, are selectively excluded from the nucleus. Further, Parkin translocates to the nucleus in response to hypoxia which cor ...[more]

PMID: 32444656

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Project description:Purpose: The E3 ubiquitin ligase Parkin is a well-characterized regulator of mitochondrial autophagy (mitophagy); however, it is becoming increasingly appreciated to perform additional roles in various compartments of the cell. Our laboratory confirmed the presence of Parkin in the nucleus of various tissues (biochemical fractionations) and cell types (immunofluorescent imaging). Hypoxia-induced nuclear translocation of Parkin occured independent of the mitophagy regulator PINK1, and Parkinson's disease-associated mutants were restricted from the nuclueus. Accordingly, we inserted a nuclear localization sequence (NLS) at the n-terminus of Parkin and overexpressed both NLS Parkin and the wild-type protein in HeLa cells cultured at normoxia and hypoxia. Next-generation RNA-sequencing (RNA-seq) was used to determine the effect of nuclear Parkin on cellular transcription. Methods: mCherry-tagged NLS and wild-type Parkin were overexpressed in HeLa cells. Differential expression analyses were performed on Parkin vs. mCherry control cells and NLS Parkin vs. mCherry control cells at normoxia and following 12hr of hypoxia (n=3/group/condition). Paired-end sequencing was performed using the HiSeq4000. FastQC v0.11.3 was used for quality control, Trimmomatic v0.36 was used to trim reads which were aligned to the human genome (GRCh37.p13) using the STAR aligner v2.5.3a. Read quantification was performed with RSEM v 1.3.0 and the Gencode release 19. The R BioConductor packages edgeR and limma were used to implement the limma-voom method for differential expression analysis. Results: During normoxia, Parkin had no effect on basal transcription; however, overexpression of NLS Parkin was associated with 168 differentially expressed genes (DEGs: fold-change </= 1.5, FDR < 0.05) relative to mCherry control cells. Following hypoxia, the transcriptome associated with the overexpression of wild-type Parkin more closely resembled that of NLS Parkin. Along these lines, Parkin overexpression during hypoxia coincided with a total of 158 DEGs, 37% of which were shared with NLS Parkin. Overlapping and shared DEGs among Parkin and NLS Parkin were implicated in cellular metabolism, HIF1 signaling and survival. Our follow up co-immunoprecipitation and real-time quantitative PCR studies demonstrated that Parkin interacts with the Estrogen Related Receptor Alpha (ERRa) to promote the induction of its downstream target genes. Conclusions: Nuclear translocation of Parkin is a novel means by which this cytoprotective protein contributes to cellular homeostasis and especially critical during hypoxia.

Project description:Oxidative stress underlies a number of pathological conditions, including cancer, neurodegeneration, and aging. Antioxidant-rich foods help maintain cellular redox homeostasis and mitigate oxidative stress, but the underlying mechanisms are not clear. For example, sulforaphane (SFN), an electrophilic compound that is enriched in cruciferous vegetables such as broccoli, is a potent inducer of cellular antioxidant responses. NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2), a transcriptional factor that controls the expression of multiple detoxifying enzymes through antioxidant response elements (AREs), is a proposed target of SFN. NFE2L2/NRF2 is a target gene of TFEB (transcription factor EB), a master regulator of autophagic and lysosomal functions, which we show here to be potently activated by SFN. SFN induces TFEB nuclear translocation via a Ca2+-dependent but MTOR (mechanistic target of rapamycin kinase)-independent mechanism through a moderate increase in reactive oxygen species (ROS). Activated TFEB then boosts the expression of genes required for autophagosome and lysosome biogenesis, which are known to facilitate the clearance of damaged mitochondria. Notably, TFEB activity is required for SFN-induced protection against both acute oxidant bursts and chronic oxidative stress. Hence, by simultaneously activating macroautophagy/autophagy and detoxifying pathways, natural compound SFN may trigger a self-defense cellular mechanism that can effectively mitigate oxidative stress commonly associated with many metabolic and age-related diseases.Abbreviations: ANOVA: analyzes of variance; AREs: antioxidant response elements; Baf-A1: bafilomycin A1; BHA: butylhydroxyanisole; CAT: catechin hydrate; CCCP: carbonyl cyanide m- chlorophenylhydrazone; CLEAR: coordinated lysosomal expression and regulation; DCFH-DA: 2',7'-dichlorofluorescin diacetate; FBS: fetal bovine serum; GFP: green fluorescent protein; HMOX1/HO-1: heme oxygenase 1; KD: knockdown; KEAP1: kelch like ECH associated protein 1; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MCOLN1/TRPML1: mucolipin 1; ML-SA1: mucolipin-specific synthetic agonist 1; ML-SI3: mucolipin-specific synthetic inhibitor 3; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; NAC: N-acetylcysteine; NFE2L2/NRF2: nuclear factor: erythroid 2 like 2; NPC: Niemann-Pick type C; PBS: phosphate-buffered saline; PPP2/PP2A: protein phosphatase 2; Q-PCR: real time polymerase chain reaction; ROS: reactive oxygen species; RPS6KB1/S6K1/p70S6K: ribosomal protein S6 kinase B1; SFN: sulforaphane; TFEB: transcription factor EB; WT, wild-type.

Dataset Information

Nuclear Parkin Activates the ERR? Transcriptional Program and Drives Widespread Changes in Gene Expression Following Hypoxia.

Publications

Nuclear Parkin Activates the ERRα Transcriptional Program and Drives Widespread Changes in Gene Expression Following Hypoxia.

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