Dataset Information

Engineering a Cell-Based Orthogonal Ubiquitin Transfer Cascade for Profiling the Substrates of RBR E3 Parkin

ABSTRACT: The E3 ubiquitin (UB) ligase Parkin utilizes a Ring-Between-Ring (RBR) domain to mediate UB transfer to substrate proteins, and mutations affecting Parkin catalysis promote cancer and are associated with Parkinson’s disease. An essential role of Parkin is to initiate mitophagy by ubiquitinating mitochondrial proteins. Still, it is unclear how Parkin carries out other cellular functions, such as the regulation of the cell cycle, metabolism, and the neuronal synapse. Here, we used phage display to engineer the RBR domain of Parkin and assembled an orthogonal ubiquitin transfer (OUT) cascade to profile Parkin substrates in living cells. Guided by the substate profile from the OUT screen, we verified a panel of Rab GTPases and CDK5 as Parkin substrates. We also showed mitophagy stimulation enhanced Parkin-mediated ubiquitination of Rab proteins. Our work demonstrates that the Parkin OUT cascade can be an empowering tool for identifying Parkin substrates to elucidate its multifaceted cellular functions.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Duc Duong

LAB HEAD: Jun Yin

PROVIDER: PXD063080 | Pride | 2025-07-28

REPOSITORIES: Pride

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Engineering a cell-based orthogonal ubiquitin transfer cascade for profiling the substrates of RBR E3 Parkin.

Fang Shuai S Zhou Li L Chen Geng G Zhang Jing J Wang Xiaoyu X Jeong In Ho IH Jacobs Savannah E SE Kossmann Bradley R BR Wei Wei W Liu Shu S Jeong Geon H GH Xie Yayun Y Duong Duc D Seyfried Nicholas T NT Ivanov Ivaylo I Mabb Angela M AM Kiyokawa Hiroaki H Zhao Bo B Yin Jun J

iScience 20250617 7

The E3 ubiquitin (UB) ligase Parkin utilizes a Ring-Between-Ring (RBR) domain to mediate UB transfer to substrate proteins, and mutations affecting Parkin catalysis promote cancer and are associated with Parkinson's disease. An essential role of Parkin is to initiate mitophagy by ubiquitinating mitochondrial proteins. Still, it is unclear how Parkin carries out other cellular functions, such as the regulation of the cell cycle, metabolism, and the neuronal synapse. Here, we used phage display to ...[more]

PMID: 40687836

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Project description:Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.

Project description:Mutations in the E3 ubiquitin ligase Parkin cause autosomal recessive Parkinson’s disease. In concert with PINK1, Parkin regulates the clearance of dysfunctional mitochondria via lysosomes. In response, new mitochondria are generated through an interplay of nuclear- and mitochondrial-encoded proteins. Mouse and overexpression models suggests that Parkin also influences these processes, both in the nuclear cascade and at the level of the mitochondrial genome. Additionally, Parkin has been shown to prevent mitochondrial membrane permeation, impeding the escape of mitochondrial DNA (mtDNA). In line with this, serum from Parkin mutation carriers showed higher levels of circulating cell-free mtDNA (ccf-mtDNA) and inflammatory cytokines – a result of the innate immune response - which can be triggered by cytosolic mtDNA. However, Parkin’s relationship with the mitochondrial genome and the mitogenesis pathway has not been explored in patient-derived neurons. To investigate this aspect of Parkin’s cellular function endogenously, we generated induced pluripotent stem cell (iPSC)-derived midbrain neurons from Parkin mutation carriers and healthy controls. In Parkin-deficient cells, several factors in the mitochondrial biogenesis pathway were significantly reduced, resulting in impaired mtDNA homeostasis - a phenomenon that was exacerbated in dopaminergic neurons. Moreover, in response to a lack of freely accessible NAD+, the energy sensor Sirtuin 1, which simultaneously controls mtDNA maintenance processes and mitophagy, was downregulated in Parkin-deficient neurons. However, while impaired lysosomal degradation of mitochondria was only detectable in oxidative conditions, biogenesis defects were already apparent in untreated patient neurons. This may suggest that mitophagy disruption occurs in response to acute stress. By contrast, the biogenesis pathway may be continually impaired in Parkin-associated Parkinson’s disease. Next, using a mutagenic stress model in combination with Parkin knockdown, we detected an increase in ccf-mtDNA and the cytosolic DNA sensor cGAS. To explore if ccf-mtDNA can act as damage-associated molecular pattern in the brain, we used postmortem tissue from a Parkin mutation carrier and performed single-cell RNA sequencing. In the midbrain lacking Parkin, we found a higher percentage of microglia along with an upregulation of proinflammatory cytokines in these cells. Together, our findings suggest a role for Parkin in the control of mitochondrial biogenesis and mtDNA maintenance, which protects midbrain neurons from neuroinflammation-induced degeneration. Future research in iPSC-derived neuron-microglia co-culture systems could aim at developing PD treatment approaches that target the neuronal release or microglial uptake of ccf-mtDNA.

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