Parkin Recruitment Research Articles

Bcl-2-associated athanogene 5 (BAG5) regulates Parkin-dependent mitophagy and cell death

As pathogenic Parkin mutations result in the defective clearance of damaged mitochondria, Parkin-dependent mitophagy is thought to be protective against the dopaminergic neurodegeneration observed in Parkinson’s disease. Recent studies, however, have demonstrated that Parkin can promote cell death in the context of severe mitochondrial damage by degrading the pro-survival Bcl-2 family member, Mcl-1. Therefore, Parkin may act as a ‘switch’ that can shift the balance between protective or pro-death pathways depending on the degree of mitochondrial damage. Here, we report that the Parkin interacting protein, Bcl-2-associated athanogene 5 (BAG5), impairs mitophagy by suppressing Parkin recruitment to damaged mitochondria and reducing the movement of damaged mitochondria into the lysosomes. BAG5 also enhanced Parkin-mediated Mcl-1 degradation and cell death following severe mitochondrial insult. These results suggest that BAG5 may regulate the bi-modal activity of Parkin, promoting cell death by suppressing Parkin-dependent mitophagy and enhancing Parkin-mediated Mcl-1 degradation.

Dissociation of mitochondrial HK-II elicits mitophagy and confers cardioprotection against ischemia

Preservation of mitochondrial integrity is critical for maintaining cellular homeostasis. Mitophagy is a mitochondria-specific type of autophagy which eliminates damaged mitochondria thereby contributing to mitochondrial quality control. Depolarization of the mitochondrial membrane potential is an established mechanism for inducing mitophagy, mediated through PINK1 stabilization and Parkin recruitment to mitochondria. Hexokinase-II (HK-II) which catalyzes the first step in glucose metabolism, also functions as a signaling molecule to regulate cell survival, and a significant fraction of cellular HK-II is associated with mitochondria (mitoHK-II). We demonstrate here that pharmacological interventions and adenoviral expression of a mitoHK-II dissociating peptide which reduce mitoHK-II levels lead to robust increases in mitochondrial Parkin and ubiquitination of mitochondrial proteins in cardiomyocytes and in a human glioblastoma cell line 1321N1, independent of mitochondrial membrane depolarization or PINK1 accumulation. MitoHK-II dissociation-induced mitophagy was demonstrated using Mito-Keima in cardiomyocytes and in 1321N1 cells. Subjecting cardiomyocytes or the in vivo heart to ischemia leads to modest dissociation of mitoHK-II. This response is potentiated by expression of the mitoHK-II dissociating peptide, which increases Parkin recruitment to mitochondria and, importantly, provides cardioprotection against ischemic stress. These results suggest that mitoHK-II dissociation is a physiologically relevant cellular event that is induced by ischemic stress, the enhancement of which protects against ischemic damage. The mechanism which underlies the effects of mitoHK-II dissociation can be attributed to the ability of Bcl2-associated athanogene 5 (BAG5), an inhibitor of Parkin, to localize to mitochondria and form a molecular complex with HK-II. Overexpression of BAG5 attenuates while knockdown of BAG5 sensitizes the effect of mitoHK-II dissociation on mitophagy. We suggest that HK-II, a glycolytic molecule, can function as a sensor for metabolic derangements at mitochondria to trigger mitophagy, and modulating the intracellular localization of HK-II could be a novel way of regulating mitophagy to prevent cell death induced by ischemic stress.

Melatonin Enhances Mitophagy by Upregulating Expression of Heat Shock 70 kDa Protein 1L in Human Mesenchymal Stem Cells under Oxidative Stress.

Human mesenchymal stem cells (hMSCs) are a potent source of cell-based regenerative therapeutics used to treat patients with ischemic disease. However, disease-induced oxidative stress disrupts mitochondrial homeostasis in transplanted hMSCs, resulting in hMSC apoptosis and reducing their efficacy post-transplantation. To address this issue, we evaluated the effects of melatonin on cellular defense mechanisms and mitophagy in hMSCs subjected to oxidative stress. H2O2-induced oxidative stress increases the levels of reactive oxygen species and reduces membrane potential in hMSCs, leading to mitochondrial dysfunction and cell death. Oxidative stress also decreases the expression of 70-kDa heat shock protein 1L (HSPA1L), a molecular chaperone that assists in the recruitment of parkin to the autophagosomal mitochondrial membrane. Decreased expression of HSPA1L destabilizes parkin, thereby impairing mitophagy. Our results indicate that treating hMSCs with melatonin significantly inhibited mitochondrial dysfunction induced by oxidative stress, which decreased hMSCs apoptosis. In damaged hMSCs, treatment with melatonin increased the levels of HSPA1L, which bound to parkin. The interaction between HSPA1L and parkin increased membrane potential and levels of oxidative phosphorylation, resulting in enhanced mitophagy. Our results indicate that melatonin increased the expression of HSPA1L, thereby upregulating mitophagy and prolonging cell survival under conditions of oxidative stress. In this study, we have shown that melatonin, a readily available compound, can be used to improve hMSC-based therapies for patients with pathologic conditions involving oxidative stress.

Temporal integration of mitochondrial stress signals by the PINK1:Parkin pathway

BackgroundThe PINK1:Parkin pathway regulates the autophagic removal of damaged and dysfunctional mitochondria. While the response of this pathway to complete loss of ΔΨm, as caused by high concentrations of mitochondrial ionophores, has been well characterized, it remains unclear how the pathway makes coherent decisions about whether to keep or purge mitochondria in situations where ΔΨm is only partially lost or exhibits fluctuations, as has been observed in response to certain types of cellular stress.ResultsTo investigate the responses of the PINK1:Parkin pathway to mitochondrial insults of different magnitude and duration, controlled titration of the mitochondrial protonophore, CCCP, was used to manipulate ΔΨm in live cells, and the dynamics of PINK1 and Parkin recruitment was measured by fluorescence microscopy. In contrast to the stable accumulation of PINK1 and Parkin seen at completely depolarized mitochondria, partial depolarization produced a transient pulse of PINK1 stabilization and rapid loss, which was driven by small fluctuations in ΔΨm. As the rate of Parkin dissociation from the mitochondria and phospho-polyubiquitin chain removal was comparatively slow, repetitive pulses of PINK1 were able to drive a slow step-wise accumulation of Parkin and phospho-polyubiquitin leading to deferred mitophagy.ConclusionThese data suggest that the PINK1:Parkin mitophagy pathway is able to exhibit distinct dynamic responses to complete and partial mitochondrial depolarization. In this way, the pathway is able to differentiate between irretrievably damaged mitochondria and those showing signs of dysfunction, promoting either rapid or delayed autophagy, respectively.

Parkin recruitment to impaired mitochondria for nonselective ubiquitylation is facilitated by MITOL

PINK1 (PARK6) and PARKIN (PARK2) are causal genes of recessive familial Parkinson's disease. Parkin is a ubiquitin ligase E3 that conjugates ubiquitin to impaired mitochondrial proteins for organelle degradation. PINK1, a Ser/Thr kinase that accumulates only on impaired mitochondria, phosphorylates two authentic substrates, the ubiquitin-like domain of Parkin and ubiquitin. Our group and others have revealed that both the subcellular localization and ligase activity of Parkin are regulated through interactions with phosphorylated ubiquitin. Once PINK1 localizes on impaired mitochondria, PINK1-catalyzed phosphoubiquitin recruits and activates Parkin. Parkin then supplies a ubiquitin chain to PINK1 for phosphorylation. The amplified ubiquitin functions as a signal for the sequestration and degradation of the damaged mitochondria. Although a bewildering variety of Parkin substrates have been reported, the basis for Parkin substrate specificity remains poorly understood. Moreover, the mechanism underlying initial activation and translocation of Parkin onto mitochondria remains unclear, because the presence of ubiquitin on impaired mitochondria is thought to be a prerequisite for the initial PINK1 phosphorylation process. Here, we show that artificial mitochondria-targeted proteins are ubiquitylated by Parkin, suggesting that substrate specificity of Parkin is not determined by its amino acid sequence. Moreover, recruitment and activation of Parkin are delayed following depletion of the mitochondrial E3, MITOL/March5. We propose a model in which the initial step in Parkin recruitment and activation requires protein ubiquitylation by MITOL/March5 with subsequent PINK1-mediated phosphorylation. Because PINK1 and Parkin amplify the ubiquitin signal via a positive feedback loop, the low substrate specificity of Parkin might facilitate this amplification process.

The plant triterpenoid celastrol blocks PINK1-dependent mitophagy by disrupting PINK1's association with the mitochondrial protein TOM20

A critical function of the PTEN-induced kinase 1 (PINK1)-Parkin pathway is to mediate the clearing of unhealthy or damaged mitochondria via mitophagy. Loss of either PINK1 or Parkin protein expression is associated with Parkinson's disease. Here, using a high-throughput screening approach along with recombinant protein expression and kinase, immunoblotting, and immunofluorescence live-cell imaging assays, we report that celastrol, a pentacyclic triterpenoid isolated from extracts of the medicinal plant Tripterygium wilfordii, blocks recruitment pof Parkin to mitochondria, preventing mitophagy in response to mitochondrial depolarization induced by carbonyl cyanide m-chlorophenylhydrazone or to gamitrinib-induced inhibition of mitochondrial heat shock protein 90 (HSP90). Celastrol's effect on mitophagy was independent of its known role in microtubule disruption. Instead, we show that celastrol suppresses Parkin recruitment by inactivating PINK1 and preventing it from phosphorylating Parkin and also ubiquitin. We also observed that PINK1 directly and strongly associates with TOM20, a component of the translocase of outer mitochondrial membrane (TOM) machinery and relatively weak binding to another TOM subunit, TOM70. Moreover, celastrol disrupted binding between PINK1 and TOM20 both in vitro and in vivo but did not affect binding between TOM20 and TOM70. Using native gel analysis, we also show that celastrol disrupts PINK1 complex formation upon mitochondrial depolarization and sequesters PINK1 to high-molecular-weight protein aggregates. These results reveal that celastrol regulates the mitochondrial quality control pathway by interfering with PINK1-TOM20 binding.

Mitochondrial Quality Control in Aging and Heart Failure: Influence of Ketone Bodies and Mitofusin-Stabilizing Peptides.

Aim: Aging and heart failure (HF) are each characterized by increased mitochondrial damage, which may contribute to further cardiac dysfunction. Mitophagy in response to mitochondrial damage can improve cardiovascular health. HF is also characterized by increased formation and consumption of ketone bodies (KBs), which may activate mitophagy and provide an endogenous mechanism to limit the adverse effects of mitochondrial damage. However, the role of KBs in activation of mitophagy in aging and HF has not been evaluated. Methods: We assessed mitophagy by measuring mitochondrial Parkin accumulation and LC3-mediated autophagosome formation in cardiomyocytes from young (2.5 months), aged (2.5 years), and aged rabbits with HF (2.5 years) induced by aortic insufficiency and stenosis. Levels of reactive oxygen species (ROS) generation and redox balance were monitored using genetically encoded sensors ORP1-roGFP2 and GRX1-roGFP2, targeted to mitochondrial or cytosolic compartments, respectively. Results: Young rabbits exhibited limited mitochondrial Parkin accumulation with small (~1 μm2) puncta. Those small Parkin puncta increased four-fold in aged rabbit hearts, accompanied by elevated LC3-mediated autophagosome formation. HF hearts exhibited fewer small puncta, but many very large Parkin-rich regions (4–5 μm2) with completely depolarized mitochondria. Parkin protein expression was barely detectable in young animals and was much higher in aged and maximal in HF hearts. Expression of mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1) was reduced by almost 50% in HF, consistent with improper fusion-fission, contributing to mitochondrial Parkin build-up. The KB β-hydroxybutyrate (β-OHB) enhanced mitophagy in young and aging myocytes, but not in HF where β-OHB further increased the number of cells with giant Parkin-rich regions. This β-OHB effect on Parkin-rich areas was prevented by cell-permeable TAT-MP1Gly peptide (thought to promote MFN2-dependent fusion). Basal levels of mitochondrial ROS were highest in HF, while cytosolic ROS was highest in aged compared to HF myocytes, suggesting that cytosolic ROS promotes Parkin recruitment to the mitochondria. Conclusion: We conclude that elevated KB levels were beneficial for mitochondrial repair in the aging heart. However, an impaired MFN2-DRP1-mediated fusion-fission process in HF reduced this benefit, as well as Parkin degradation and mitophagic signaling cascade.

Parkin the progression of sarcopenia.

Parkin the progression of sarcopenia.

Caloric Restriction Increases Parkin Recruitment in Hearts of Obese Insulin‐Resistant Rats

Mitochondria provide energy to the cells, making the task of mitochondrial recycling highly important. Impairments in mitochondrial number and function can lead to dysregulation of cellular metabolism and disrupted capacity to maintain homeostasis. The Parkin gene codes for the protein, Parkin, which is a component of the Parkin/PINK1 pathway that helps regulate mitophagy. An impairment in the Parkin gene may also results in nonfunctional mitochondria, potentially leading to many diseases such as type two diabetes mellitus and cardiovascular disease. Recent studies suggest that Parkin expression is downregulated during insulin resistance. To address the hypothesis that insulin resistance downregulates Parkin in the heart, a piece of the left ventricle of the hearts of 4 groups of rats were extracted: (1) Long Evans Tokushima Otsuka (LETO), (2) Otsuka Long‐Evans Tokushima Fatty (OLETF), (3) LETO with caloric restriction, and (4) OLETF with caloric restriction. Extracted Parkin from the hearts of each group was measured by Western blot. Because Parkin expression is downregulated with insulin resistance, we anticipated lower levels of express in the insulin resistant, OLETF rats compared to LETO, and that caloric restriction would restore Parkin expression in the OLETF rats. Parkin protein expression was 71% lower in OLETF than LETO. While the 16% increase in expression in caloric restricted LETO group was not statistically significant, the expression increased nearly 7.5‐fold with caloric restriction in OLETF group compared to the ad lib OLETF group. The data suggest that the benefits of CR on CV function and health go beyond the potential benefits of mass loss (usually adiposity) but include cellular energetics and metabolism in the heart of insulin resistant rats.Support or Funding InformationThe STRIDE Fellowship is supported by the American Physiological Society and a grant from the National Heart, Lung and Blood Institute (NHLBI; 1 R25 HL115473‐01).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Disease‐associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria

Accumulation of the protein tau characterises Alzheimer's disease and other tauopathies, including familial forms of frontotemporal dementia (FTD) that carry pathogenic tau mutations. Another hallmark feature of these diseases is the accumulation of dysfunctional mitochondria. Although disease‐associated tau is known to impair several aspects of mitochondrial function, it is still unclear whether it also directly impinges on mitochondrial quality control, specifically Parkin‐dependent mitophagy. Using the mito‐QC mitophagy reporter, we found that both human wild‐type (hTau) and FTD mutant tau (hP301L) inhibited mitophagy in neuroblastoma cells, by reducing mitochondrial translocation of Parkin. In the Caenorhabditis elegans nervous system, hTau expression reduced mitophagy, whereas hP301L expression completely inhibited it. These effects were not due to changes in the mitochondrial membrane potential or the cytoskeleton, as tau specifically impaired Parkin recruitment to defective mitochondria by sequestering it in the cytosol. This sequestration was mediated by aberrant interactions of Parkin with the projection domain of tau. As mitochondria are dysfunctional in neurodegenerative conditions, these data suggest a vicious cycle, with tau also inhibiting the degradation of damaged mitochondria.

Miro proteins prime mitochondria for Parkin translocation and mitophagy

The Parkinson's disease‐associated protein kinase PINK1 and ubiquitin ligase Parkin coordinate the ubiquitination of mitochondrial proteins, which marks mitochondria for degradation. Miro1, an atypical GTPase involved in mitochondrial trafficking, is one of the substrates tagged by Parkin after mitochondrial damage. Here, we demonstrate that a small pool of Parkin interacts with Miro1 before mitochondrial damage occurs. This interaction does not require PINK1, does not involve ubiquitination of Miro1 and also does not disturb Miro1 function. However, following mitochondrial damage and PINK1 accumulation, this initial pool of Parkin becomes activated, leading to the ubiquitination and degradation of Miro1. Knockdown of Miro proteins reduces Parkin translocation to mitochondria and suppresses mitophagic removal of mitochondria. Moreover, we demonstrate that Miro1 EF‐hand domains control Miro1's ubiquitination and Parkin recruitment to damaged mitochondria, and they protect neurons from glutamate‐induced mitophagy. Together, our results suggest that Miro1 functions as a calcium‐sensitive docking site for Parkin on mitochondria.

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early‐onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N‐terminal ubiquitin‐like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho‐ubiquitin‐loaded C‐terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re‐modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity.

PINK1 Protects Against Gentamicin-Induced Sensory Hair Cell Damage: Possible Relation to Induction of Autophagy and Inhibition of p53 Signal Pathway.

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) is a gatekeeper of mitochondrial quality control. The present study was aimed to examine whether PINK1 possesses a protective function against gentamicin (GM)-induced sensory hair cell (HC) damage in vitro. The formation of parkin particles (a marker revealing the activation of PINK1 pathway which is a substrate of PINK1 and could signal depolarized mitochondria for clearance) and autophagy were determined by immunofluorescence staining. The expressions of PINK1, LC3B, cleaved-caspase 3 and p53 were measured by Western blotting. The levels of reactive oxygen species (ROS) and apoptosis were respectively evaluated by DCFH-DA staining, Annexin V Apoptosis Detection Kit and TUNEL staining. Cell viability was tested by a CCK8 kit. We found that treatment of 400 μM GM elicited the formation of ROS, which, in turn, led to PINK1 degradation, parkin recruitment, autophagy formation, an increase of p53 and cleaved-caspase 3 in HEI-OC1 cells and murine HCs. In contrast, co-treatment with ROS scavenger N-acetyl-L-cysteine (NAC) inhibited parkin recruitment, alleviated autophagy and p53 pathway-related damaged-cell elimination. Moreover, PINK1 interference contributed to a decrease of autophagy but an increase of p53 level in HEI-OC1 cells in response to GM stimulus. Findings from this work indicate that PINK1 alleviates the GM-elicited ototoxicity via induction of autophagy and resistance the increase of p53 in HCs.

High-content analysis for mitophagy response to nanoparticles: A potential sensitive biomarker for nanosafety assessment

Mitophagy, a selective autophagy of mitochondria, clears up damaged mitochondria to maintain cell homeostasis. We performed high-content analysis (HCA) to detect the increase of PINK1, an essential protein controlling mitophagy, in hepatic cells treated with several nanoparticles (NPs). PINK1 immunofluorescence-based HCA was more sensitive than assays and detections for cell viability and mitochondrial functions. Of which, superparamagnetic iron oxide (SPIO)-NPs or graphene oxide-quantum dots (GO-QDs) was selected as representatives for positive or negative inducer of mitophagy. SPIO-NPs, but not GO-QDs, activated PINK1-dependent mitophagy as demonstrated by recruitment of PARKIN to mitochondria and degradation of injured mitochondria. SPIO-NPs caused the loss of mitochondrial membrane potential, decrease in ATP, and increase in mitochondrial reactive oxide species and Ca2+. Blocking mitophagy with PARKIN siRNA aggravated the cytotoxicity of SPIO-NPs. Taken together, PINK1 immunofluorescence-based HCA is considered to be an early, sensitive, and reliable approach to evaluate the bioimpacts of NPs.

Mechanism of parkin activation by phosphorylation.

Mutations in the ubiquitin ligase parkin are responsible for a familial form of Parkinson's disease. Parkin and the PINK1 kinase regulate a quality-control system for mitochondria. PINK1 phosphorylates ubiquitin on the outer membrane of damaged mitochondria, thus leading to recruitment and activation of parkin via phosphorylation of its ubiquitin-like (Ubl) domain. Here, we describe the mechanism of parkin activation by phosphorylation. The crystal structure of phosphorylated Bactrocera dorsalis (oriental fruit fly) parkin in complex with phosphorylated ubiquitin and an E2 ubiquitin-conjugating enzyme reveals that the key activating step is movement of the Ubl domain and release of the catalytic RING2 domain. Hydrogen/deuterium exchange and NMR experiments with the various intermediates in the activation pathway confirm and extend the interpretation of the crystal structure to mammalian parkin. Our results rationalize previously unexplained Parkinson's disease mutations and the presence of internal linkers that allow large domain movements in parkin.

AKT signalling selectively regulates PINK1 mitophagy in SHSY5Y cells and human iPSC-derived neurons

The discovery of mutations within genes associated with autosomal recessive Parkinson’s disease allowed for the identification of PINK1/Parkin regulated mitophagy as an important pathway for the removal of damaged mitochondria. While recent studies suggest that AKT-dependent signalling regulates Parkin recruitment to depolarised mitochondria, little is known as to whether this can also regulate PINK1 mitochondrial accumulation and downstream mitophagy. Here, we demonstrate that inhibition of AKT signalling decreases endogenous PINK1 accumulation in response to mitochondria depolarisation, subsequent Parkin recruitment, phosphorylation of ubiquitin, and ultimately mitophagy. Conversely, we show that upon stimulation of AKT signalling via insulin, the mitophagy pathway is increased in SHSY5Y cells. These data suggest that AKT signalling is an upstream regulator of PINK1 accumulation on damaged mitochondria. Importantly, we show that the AKT pathway also regulates endogenous PINK1-dependent mitophagy in human iPSC-derived neurons.

Sam50 Regulates PINK1-Parkin-Mediated Mitophagy by Controlling PINK1 Stability and Mitochondrial Morphology

PINK1 and Parkin mediate mitophagy, the cellular process that clears dysfunctional mitochondria. Mitophagy is regulated by mitochondrial dynamics, but the molecules linking these two processes remain poorly understood. Here, we show that Sam50, the core component of the sorting and assembly machinery (SAM), is a critical regulator of mitochondrial dynamics and PINK1-Parkin-mediated mitophagy. In response to Sam50 depletion, normal tubular mitochondria are first fragmented and subsequently merged into large spheres. Sam50 interacts with PINK1 to facilitate its processing and degradation. Depletion of Sam50 results in PINK1 accumulation, Parkin recruitment, and mitophagy. Interestingly, Sam50 deficiency induces a piecemeal mode of mitophagy that eliminates mitochondria "bit by bit" but spares mtDNA. In C.elegans, the Sam50 homolog gop-3 is required for the maintenance of mitochondrial morphology and mass. Our findings reveal that Sam50 directly links mitochondrial dynamics and mitophagy and that Sam50 depletion induces elimination of mitochondria without affecting mtDNA content.

Mitophagy controls beige adipocyte maintenance through a Parkin-dependent and UCP1-independent mechanism.

Beige adipocytes are an inducible form of mitochondria-enriched thermogenic adipocytes that emerge in response to external stimuli, such as chronic cold exposure. We have previously shown that after the withdrawal of external stimuli, beige adipocytes directly acquire a white fat-like phenotype through autophagy-mediated mitochondrial degradation. We investigated the upstream pathway that mediates mitochondrial clearance and report that Parkin-mediated mitophagy plays a key role in the beige-to-white adipocyte transition. Mice genetically deficient in Park2 showed reduced mitochondrial degradation and retained thermogenic beige adipocytes even after the withdrawal of external stimuli. Norepinephrine signaling through the PKA pathway inhibited the recruitment of Parkin protein to mitochondria in beige adipocytes. However, mitochondrial proton uncoupling by uncoupling protein 1 (UCP1) was dispensable for Parkin recruitment and beige adipocyte maintenance. These results suggest a physiological mechanism by which external cues control mitochondrial homeostasis in thermogenic fat cells through mitophagy.

Hepatitis B Virus X protein elevates Parkin-mediated mitophagy through Lon Peptidase in starvation

Hepatocellular Carcinoma (HCC) is the fifth most prevalent cancer worldwide. Specially, Hepatitis B viurs X protein (HBx) is a leading factor in the progression of Hepatitis B viurs-related HCC. Nutrient-deprived tumor microenvironment also contributes to tumor development. However, the role of HBx in nutrient-deprived HCC has received little investigation. Here, we show that HBx elevates PINK1-Parkin mediating mitophagy in starvation. HBx not only increases the PINK1/Parkin gene expression but also accelerates Parkin recruitment to partial mitochondria. Further analysis indicates that, HBx either promotes mitochondrial unfolded protein response, with remarkable mitochondrial LONP1 increases, or reduces LONP1 expression in cytosol inducing LONP1-Parkin pathway, both consequently enhancing mitophagy. Moreover, the enhanced mitophagy lowers mitochondrial apoptosis in starved hepatoma cells, and Bax is implied in the machinery. In addition, we define differential centrifuge, 3000 g or 12,000 g to pellet mitochondria, as an effective method to obtain distinct mitochondria. In collect, HBx regulates diverse aspects of LONP1 and Parkin, enhancing mitophagy in starvation. This study may shed new insights into the machinery development of hepatocellular carcinoma.

Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics

Flux through kinase and ubiquitin-driven signaling systems depends on the modification kinetics, stoichiometry, primary site specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons, and we model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site specificity of PARKIN-dependent target ubiquitylation, and we demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation isinhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry invivo is optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains.