Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects.
ABSTRACT: The E3 ubiquitin ligase PARK2 and the mitochondrial protein kinase PINK1 are required for the initiation of mitochondrial damage-induced mitophagy. Together, PARK2 and PINK1 generate a phospho-ubiquitin signal on outer mitochondrial membrane proteins that triggers recruitment of the autophagy machinery. This paper describes the detection of a defined 500-kDa phospho-ubiquitin-rich PARK2 complex that accumulates on mitochondria upon treatment with the membrane uncoupler CCCP. Formation of this complex is dependent on the presence of PINK1 and is absent in mutant forms of PARK2, whereby mitophagy is also arrested. These results signify a functional signaling complex that is essential for the progression of mitophagy. The visualization of the PARK2 signaling complex represents a novel marker for this critical step in mitophagy and can be used to monitor mitophagy progression in PARK2 mutants and to uncover additional upstream factors required for PARK2-mediated mitophagy signaling.
Project description:The selective degradation of mitochondria by the process of autophagy, termed mitophagy, is one of the major mechanisms of mitochondrial quality control. The best-studied mitophagy pathway is the one mediated by PINK1 and PARK2/Parkin. From recent studies it has become clear that ubiquitin-ligation plays a pivotal role and most of the focus has been on the role of ubiquitination of mitochondrial proteins in mitophagy. Even though ubiquitination is a reversible process, very little is known about the role of deubiquitinating enzymes (DUBs) in mitophagy. Here, we report that 2 mitochondrial DUBs, USP30 and USP35, regulate PARK2-mediated mitophagy. We show that USP30 and USP35 can delay PARK2-mediated mitophagy using a quantitative mitophagy assay. Furthermore, we show that USP30 delays mitophagy by delaying PARK2 recruitment to the mitochondria during mitophagy. USP35 does not delay PARK2 recruitment, suggesting that it regulates mitophagy through an alternative mechanism. Interestingly, USP35 only associates with polarized mitochondria, and rapidly translocates to the cytosol during CCCP-induced mitophagy. It is clear that PARK2-mediated mitophagy is regulated at many steps in this important quality control pathway. Taken together, these findings demonstrate an important role of mitochondrial-associated DUBs in mitophagy. Because defects in mitochondria quality control are implicated in many neurodegenerative disorders, our study provides clear rationales for the design and development of drugs for the therapeutic treatment of neurodegenerative diseases such as Parkinson and Alzheimer diseases.
Project description:The PINK1 protein kinase activates the PARK2 ubiquitin ligase to promote mitochondrial ubiquitylation and recruitment of ubiquitin-binding mitophagy receptors typified by OPTN and TAX1BP1. Here, we combine proximity biotinylation of OPTN and TAX1BP1 with CRISPR-Cas9-based screens for mitophagic flux to develop a spatial proteogenetic map of PARK2-dependent mitophagy. Proximity labeling of OPTN allowed visualization of a "mitochondrial-autophagosome synapse" upon mitochondrial depolarization. Proximity proteomics of OPTN and TAX1BP1 revealed numerous proteins at the synapse, including both PARK2 substrates and autophagy components. Parallel mitophagic flux screens identified proteins with roles in autophagy, vesicle formation and fusion, as well as PARK2 targets, many of which were also identified via proximity proteomics. One protein identified in both approaches, HK2, promotes assembly of a high-molecular weight complex of PINK1 and phosphorylation of ubiquitin in response to mitochondrial damage. This work provides a resource for understanding the spatial and molecular landscape of PARK2-dependent mitophagy.
Project description:Mutations in PARK2 gene are the most frequent cause of familial forms of Parkinson's disease (PD). This gene encodes Parkin, an E3 ubiquitin ligase involved in several cellular mechanisms, including mitophagy. Parkin loss-of-function is responsible for the cellular accumulation of damaged mitochondria, which in turn determines an increment of reactive oxygen species (ROS) levels, lower ATP production, and apoptosis activation. Given the importance of mitochondrial dysfunction and mitophagy impairment in PD pathogenesis, the aim of the present study was to investigate both total and mitochondrial proteome alterations in human skin fibroblasts of PARK2-mutated patients. To this end, both total and mitochondria-enriched protein fractions from fibroblasts of five PARK2-mutated patients and five control subjects were analyzed by quantitative shotgun proteomics to identify proteins specifically altered by Parkin mutations (mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD015880). Both the network-based and gene set enrichment analyses pointed out pathways in which Rab GTPase proteins are involved. To have a more comprehensive view of the mitochondrial alterations due to PARK2 mutations, we investigated the impact of Parkin loss on mitochondrial function and network morphology. We unveiled that the mitochondrial membrane potential was reduced in PARK2-mutated patients, without inducing PINK1 accumulation, even when triggered with the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Lastly, the analysis of the mitochondrial network morphology did not reveal any significant alterations in PARK2-mutated patients compared to control subjects. Thus, our results suggested that the network morphology was not influenced by the mitochondrial depolarization and by the lack of Parkin, revealing a possible impairment of fission and, more in general, of mitochondrial dynamics. In conclusion, the present work highlighted new molecular factors and pathways altered by PARK2 mutations, which will unravel possible biochemical pathways altered in the sporadic form of PD.
Project description:Cigarette smoke (CS)-induced mitochondrial damage with increased reactive oxygen species (ROS) production has been implicated in COPD pathogenesis by accelerating senescence. Mitophagy may play a pivotal role for removal of CS-induced damaged mitochondria, and the PINK1 (PTEN-induced putative kinase 1)-PARK2 pathway has been proposed as a crucial mechanism for mitophagic degradation. Therefore, we sought to investigate to determine if PINK1-PARK2-mediated mitophagy is involved in the regulation of CS extract (CSE)-induced cell senescence and in COPD pathogenesis. Mitochondrial damage, ROS production, and cell senescence were evaluated in primary human bronchial epithelial cells (HBEC). Mitophagy was assessed in BEAS-2B cells stably expressing EGFP-LC3B, using confocal microscopy to measure colocalization between TOMM20-stained mitochondria and EGFP-LC3B dots as a representation of autophagosome formation. To elucidate the involvement of PINK1 and PARK2 in mitophagy, knockdown and overexpression experiments were performed. PINK1 and PARK2 protein levels in lungs from patients were evaluated by means of lung homogenate and immunohistochemistry. We demonstrated that CSE-induced mitochondrial damage was accompanied by increased ROS production and HBEC senescence. CSE-induced mitophagy was inhibited by PINK1 and PARK2 knockdown, resulting in enhanced mitochondrial ROS production and cellular senescence in HBEC. Evaluation of protein levels demonstrated decreased PARK2 in COPD lungs compared with non-COPD lungs. These results suggest that PINK1-PARK2 pathway-mediated mitophagy plays a key regulatory role in CSE-induced mitochondrial ROS production and cellular senescence in HBEC. Reduced PARK2 expression levels in COPD lung suggest that insufficient mitophagy is a part of the pathogenic sequence of COPD.
Project description:The autophagy protein BECN1/Beclin 1 is known to play a central role in autophagosome formation and maturation. The results presented here demonstrate that BECN1 interacts with the Parkinson disease-related protein PARK2. This interaction does not require PARK2 translocation to mitochondria and occurs mostly in cytosol. However, our results suggest that BECN1 is involved in PARK2 translocation to mitochondria because loss of BECN1 inhibits CCCP- or PINK1 overexpression-induced PARK2 translocation. Our results also demonstrate that the observed PARK2-BECN1 interaction is functionally important. Measurements of the level of MFN2 (mitofusin 2), a PARK2 substrate, demonstrate that depletion of BECN1 prevents PARK2 translocation-induced MFN2 ubiquitination and loss. BECN1 depletion also rescues the MFN2 loss-induced suppression of mitochondrial fusion. In sum, our results demonstrate that BECN1 interacts with PARK2 and regulates PARK2 translocation to mitochondria as well as PARK2-induced mitophagy prior to autophagosome formation.
Project description:Sepsis is the major cause of acute kidney injury (AKI) associated with high mortality rates. Mitochondrial dysfunction contributes to the pathophysiology of septic AKI. Mitophagy is an important mitochondrial quality control mechanism that selectively eliminates damaged mitochondria, but its role and regulation in septic AKI remain largely unknown. Here, we demonstrate the induction of mitophagy in mouse models of septic AKI induced by lipopolysaccharide (LPS) treatment or by cecal ligation and puncture. Mitophagy was also induced in cultured proximal tubular epithelial cells exposed to LPS. Induction of mitophagy under these experimental setting was suppressed by pink1 or park2 knockout, indicating the role of the PINK1/PARK2 pathway of mitophagy in septic AKI. In addition, sepsis induced more severe kidney injury and cell apoptosis in pink1 or park2 knockout mice than in wild-type mice, suggesting a beneficial role of mitophagy in septic AKI. Furthermore, in cultured renal tubular cells treated with LPS, knockdown of pink1 or park2 inhibited mitochondrial accumulation of the autophagy adaptor optineurin (OPTN) and silencing Optn inhibited LPS-induced mitophagy. Taken together, these findings suggest that the PINK1/PARK2 pathway of mitophagy plays an important role in mitochondrial quality control, tubular cell survival, and renal function in septic AKI.
Project description:Damaged or dysfunctional mitochondria are toxic to the cell by producing reactive oxygen species and releasing cell death factors. Therefore, timely removal of these organelles is critical to cellular homeostasis and viability. Mitophagy is the mechanism of selective degradation of mitochondria via autophagy. The significance of mitophagy in kidney diseases, including ischemic acute kidney injury (AKI), has yet to be established, and the involved pathway of mitophagy remains poorly understood. Here, we show that mitophagy is induced in renal proximal tubular cells in both in vitro and in vivo models of ischemic AKI. Mitophagy under these conditions is abrogated by Pink1 and Park2 deficiency, supporting a critical role of the PINK1-PARK2 pathway in tubular cell mitophagy. Moreover, ischemic AKI is aggravated in pink1 andpark2 single- as well as double-knockout mice. Mechanistically, Pink1 and Park2 deficiency enhances mitochondrial damage, reactive oxygen species production, and inflammatory response. Taken together, these results indicate that PINK1-PARK2-mediated mitophagy plays an important role in mitochondrial quality control, tubular cell survival, and renal function during AKI.
Project description:Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPR(mt)) to increase chaperone production, while the mitochondrial kinase PINK1 and the E3 ubiquitin ligase PARK2/Parkin, whose mutations cause familial Parkinson disease, remove depolarized mitochondria through mitophagy. It is unclear, however, if there is a connection between those different levels of quality control (QC). Here, we show that the expression of unfolded proteins in the matrix causes the accumulation of PINK1 on energetically healthy mitochondria, resulting in mitochondrial translocation of PARK2, mitophagy and subsequent reduction of unfolded protein load. Also, PINK1 accumulation is greatly enhanced by the knockdown of the LONP1 protease. We suggest that the accumulation of unfolded proteins in mitochondria is a physiological trigger of mitophagy.
Project description:Mitochondrial anomalies have been previously reported in patients' brain and peripheral tissue, suggesting their relevance in sporadic Alzheimer's disease (AD). The present work evaluates mitochondrial function and recycling in human fibroblasts and brain biopsies. Functional studies using patients' skin fibroblasts showed slower mitochondrial membrane potential recovery after a mitochondrial insult together with alterations in lysosomes and autophagy, accompanied by an increase of oxidized and ubiquitinated proteins. Impairment in mitophagy has been proven in these cells due to diminished PARK2 and insufficient vesicle induction, accumulating depolarized mitochondria and PINK1. Augmented ?1 PINK1 fragment levels suggest an inhibitory effect over PARK2 translocation to the mitochondria, causing the accumulation of activated PINK1. Moreover, the overexpression of PARK2 diminished ubiquitinated proteins accumulation, improves its targeting to mitochondria and potentiates autophagic vesicle synthesis. This allows the reversion of mitophagy failure reflected in the recovery of membrane potential and the decrease of PINK1 and mitochondria accumulation. Sporadic AD fibroblasts exhibited alterations similar to what it could be found in patients' hippocampal samples at early stages of the disease, where there was an accumulation of PINK1 and ?1 PINK1 together with abnormally increased mitochondrial content. Our findings indicate that mitophagy alterations can be considered a new hallmark of sporadic AD and validate the use of fibroblasts for modelling this pathology.
Project description:Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.