Ubiquitin Ligase Parkin Research Articles

HaloTag-Targeted Sirtuin-Rearranging Ligand (SirReal) for the Development of Proteolysis-Targeting Chimeras (PROTACs) against the Lysine Deacetylase Sirtuin 2 (Sirt2)*.

We have discovered the sirtuin‐rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+‐dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so‐called proteolysis‐targeting chimera (PROTAC) enabled small‐molecule‐induced degradation of Sirt2. Herein, we report the structure‐based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo‐tagged E3 ubiquitin ligases. Using this orthogonal approach for Sirt2 degradation, we show that other E3 ligases than cereblon, such as the E3 ubiquitin ligase parkin, can also be harnessed for small‐molecule‐induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be used as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.

Estrogen receptor α controls metabolism in white and brown adipocytes by regulating Polg1 and mitochondrial remodeling.

Obesity is heightened during aging, and although the estrogen receptor α (ERα) has been implicated in the prevention of obesity, its molecular actions in adipocytes remain inadequately understood. Here, we show that adipose tissue ESR1/Esr1 expression inversely associated with adiposity and positively associated with genes involved in mitochondrial metabolism and markers of metabolic health in 700 Finnish men and 100 strains of inbred mice from the UCLA Hybrid Mouse Diversity Panel. To determine the anti-obesity actions of ERα in fat, we selectively deleted Esr1 from white and brown adipocytes in mice. In white adipose tissue, Esr1 controlled oxidative metabolism by restraining the targeted elimination of mitochondria via the E3 ubiquitin ligase parkin. mtDNA content was elevated, and adipose tissue mass was reduced in adipose-selective parkin knockout mice. In brown fat centrally involved in body temperature maintenance, Esr1 was requisite for both mitochondrial remodeling by dynamin-related protein 1 (Drp1) and uncoupled respiration thermogenesis by uncoupled protein 1 (Ucp1). In both white and brown fat of female mice and adipocytes in culture, mitochondrial dysfunction in the context of Esr1 deletion was paralleled by a reduction in the expression of the mtDNA polymerase γ subunit Polg1 We identified Polg1 as an ERα target gene by showing that ERα binds the Polg1 promoter to control its expression in 3T3L1 adipocytes. These findings support strategies leveraging ERα action on mitochondrial function in adipocytes to combat obesity and metabolic dysfunction.

Regulation of Iron Homeostasis through Parkin-Mediated Lactoferrin Ubiquitylation.

Somatic mutations that perturb Parkin ubiquitin ligase activity and the misregulation of iron homeostasis have both been linked to Parkinson's disease. Lactotransferrin (LTF) is a member of the family of transferrin iron binding proteins that regulate iron homeostasis, and increased levels of LTF and its receptor have been observed in neurodegenerative disorders like Parkinson's disease. Here, we report that Parkin binds to LTF and ubiquitylates LTF to influence iron homeostasis. Parkin-dependent ubiquitylation of LTF occurred most often on lysines (K) 182 and 649. Substitution of K182 or K649 with alanine (K182A or K649A, respectively) led to a decrease in the level of LTF ubiquitylation, and substitution at both sites led to a major decrease in the level of LTF ubiquitylation. Importantly, Parkin-mediated ubiquitylation of LTF was critical for regulating intracellular iron levels as overexpression of LTF ubiquitylation site point mutants (K649A or K182A/K649A) led to an increase in intracellular iron levels measured by ICP-MS/MS. Consistently, RNAi-mediated depletion of Parkin led to an increase in intracellular iron levels in contrast to overexpression of Parkin that led to a decrease in intracellular iron levels. Together, these results indicate that Parkin binds to and ubiquitylates LTF to regulate intracellular iron levels. These results expand our understanding of the cellular processes that are perturbed when Parkin activity is disrupted and more broadly the mechanisms that contribute to Parkinson's disease.

A Cell-Based High-Throughput Screening Identified Two Compounds that Enhance PINK1-Parkin Signaling.

SummaryEarly-onset Parkinson's disease-associated PINK1-Parkin signaling maintains mitochondrial health. Therapeutic approaches for enhancing PINK1-Parkin signaling present a potential strategy for treating various diseases caused by mitochondrial dysfunction. We report two chemical enhancers of PINK1-Parkin signaling, identified using a robust cell-based high-throughput screening system. These small molecules, T0466 and T0467, activate Parkin mitochondrial translocation in dopaminergic neurons and myoblasts at low doses that do not induce mitochondrial accumulation of PINK1. Moreover, both compounds reduce unfolded mitochondrial protein levels, presumably through enhanced PINK1-Parkin signaling. These molecules also mitigate the locomotion defect, reduced ATP production, and disturbed mitochondrial Ca2+ response in the muscles along with the mitochondrial aggregation in dopaminergic neurons through reduced PINK1 activity in Drosophila. Our results suggested that T0466 and T0467 may hold promise as therapeutic reagents in Parkinson's disease and related disorders.

Mdm2 enhances ligase activity of parkin and facilitates mitophagy

Loss-of-function mutations in the E3 ubiquitin ligase parkin have been implicated in the death of dopaminergic neurons in the substantia nigra, which is the root cause of dopamine deficit in the striatum in Parkinson's disease. Parkin ubiquitinates proteins on mitochondria that lost membrane potential, promoting the elimination of damaged mitochondria. Neuroprotective activity of parkin has been linked to its critical role in the mitochondria maintenance. Here we report a novel regulatory mechanism: another E3 ubiquitin ligase Mdm2 directly binds parkin and enhances its enzymatic activity in vitro and in intact cells. Mdm2 translocates to damaged mitochondria independently of parkin, enhances parkin-dependent ubiquitination of the outer mitochondria membrane protein mitofusin1. Mdm2 facilitates and its knockdown reduces parkin-dependent mitophagy. Thus, ubiquitously expressed Mdm2 might enhance cytoprotective parkin activity. The data suggest that parkin activation by Mdm2 could be targeted to increase its neuroprotective functions, which has implications for anti-parkinsonian therapy.

Gemcitabine induces Parkin-independent mitophagy through mitochondrial-resident E3 ligase MUL1-mediated stabilization of PINK1

Mitophagy plays an important role in the maintenance of mitochondrial homeostasis. PTEN-induced kinase (PINK1), a key regulator of mitophagy, is degraded constitutively under steady-state conditions. During mitophagy, it becomes stabilized in the outer mitochondrial membrane, particularly under mitochondrial stress conditions, such as in treatment with uncouplers, generation of excessive mitochondrial reactive oxygen species, and formation of protein aggregates in mitochondria. Stabilized PINK1 recruits and activates E3 ligases, such as Parkin and mitochondrial ubiquitin ligase (MUL1), to ubiquitinate mitochondrial proteins and induce ubiquitin-mediated mitophagy. Here, we found that the anticancer drug gemcitabine induces the stabilization of PINK1 and subsequent mitophagy, even in the absence of Parkin. We also found that gemcitabine-induced stabilization of PINK1 was not accompanied by mitochondrial depolarization. Interestingly, the stabilization of PINK1 was mediated by MUL1. These results suggest that gemcitabine induces mitophagy through MUL1-mediated stabilization of PINK1 on the mitochondrial membrane independently of mitochondrial depolarization.

PINK1 import regulation at a crossroad of mitochondrial fate: the molecular mechanisms of PINK1 import.

PTEN-induced kinase 1 (PINK1) is a mitochondrial kinase whose activity is tightly regulated by the mitochondrial health status. In response to mitochondrial damage, activated PINK1 can promote mitophagy, an autophagic elimination of damaged mitochondria, by cooperating with Parkin ubiquitin ligase. Loss-of-function of PINK1/Parkin-mediated mitophagy results in the accumulation of dysfunctional mitochondria, which could be one aetiology of Parkinson's disease (PD). Within step-by-step signalling cascades of PINK1/Parkin-mediated mitophagy, mitochondrial damage-dependent PINK1 kinase activation is a critical step to trigger the mitophagy signal. Recent investigation of this process reveals that this stress-dependent PINK1 kinase activation is achieved by its regulated import into different mitochondrial compartments. Thus, PINK1 import regulation stands at an important crossroad to determine the mitochondrial fate-'keep' or 'remove'? In this review, we will summarize how the PINK1 import is regulated in a mitochondrial health status-dependent manner and how this process could be pharmacologically modulated to activate the PINK1/Parkin pathway.

The PINK1 kinase-driven ubiquitin ligase Parkin promotes mitochondrial protein import through the presequence pathway in living cells

Most of over a thousand mitochondrial proteins are encoded by nuclear genes and must be imported from the cytosol. Little is known about the cytosolic events regulating mitochondrial protein import, partly due to the lack of appropriate tools for its assessment in living cells. We engineered an inducible biosensor for monitoring the main presequence-mediated import pathway with a quantitative, luminescence-based readout. This tool was used to explore the regulation of mitochondrial import by the PINK1 kinase-driven Parkin ubiquitin ligase, which is dysfunctional in autosomal recessive Parkinson’s disease. We show that mitochondrial import was stimulated by Parkin, but not by disease-causing Parkin variants. This effect was dependent on Parkin activation by PINK1 and accompanied by an increase in the abundance of K11 ubiquitin chains on mitochondria and by ubiquitylation of subunits of the translocase of outer mitochondrial membrane. Mitochondrial import efficiency was abnormally low in cells from patients with PINK1- and PARK2-linked Parkinson’s disease and was restored by phosphomimetic ubiquitin in cells with residual Parkin activity. Altogether, these findings uncover a role of ubiquitylation in mitochondrial import regulation and suggest that loss of this regulatory loop may underlie the pathophysiology of Parkinson’s disease, providing novel opportunities for therapeutic intervention.

Stress-induced phospho-ubiquitin formation causes parkin degradation

Mutations in the E3 ubiquitin ligase parkin are the most common known cause of autosomal recessive Parkinson’s disease (PD), and parkin depletion may play a role in sporadic PD. Here, we sought to elucidate the mechanisms by which stress decreases parkin protein levels using cultured neuronal cells and the PD-relevant stressor, L-DOPA. We find that L-DOPA causes parkin loss through both oxidative stress-independent and oxidative stress-dependent pathways. Characterization of the latter reveals that it requires both the kinase PINK1 and parkin’s interaction with phosphorylated ubiquitin (phospho-Ub) and is mediated by proteasomal degradation. Surprisingly, autoubiquitination and mitophagy do not appear to be required for such loss. In response to stress induced by hydrogen peroxide or CCCP, parkin degradation also requires its association with phospho-Ub, indicating that this mechanism is broadly generalizable. As oxidative stress, metabolic dysfunction and phospho-Ub levels are all elevated in PD, we suggest that these changes may contribute to a loss of parkin expression.

Parkin and AMPK team up against necroptosis.

Ubiquitin ligase parkin — a key mediator of mitophagy — is induced in necroptotic conditions through the activity of AMP-activated protein kinase (AMPK) to restrain necroptosis and limit associated pathologies.

Nilotinib: A Tyrosine Kinase Inhibitor Mediates Resistance to Intracellular Mycobacterium Via Regulating Autophagy.

Nilotinib, a tyrosine kinase inhibitor, has been studied extensively in various tumor models; however, no information exists about the pharmacological action of nilotinib in bacterial infections. Mycobacterium bovis (M. bovis) and Mycobacterium avium subspecies paratuberculosis (MAP) are the etiological agents of bovine tuberculosis and Johne’s disease, respectively. Although M. bovis and MAP cause distinct tissue tropism, both of them infect, reside, and replicate in mononuclear phagocytic cells of the infected host. Autophagy is an innate immune defense mechanism for the control of intracellular bacteria, regulated by diverse signaling pathways. Here we demonstrated that nilotinib significantly inhibited the intracellular survival and growth of M. bovis and MAP in macrophages by modulating host immune responses. We showed that nilotinib induced autophagic degradation of intracellular mycobacterium occurred via the inhibition of PI3k/Akt/mTOR axis mediated by abelson (c-ABL) tyrosine kinase. In addition, we observed that nilotinib promoted ubiquitin accumulation around M. bovis through activation of E3 ubiquitin ligase parkin. From in-vivo experiments, we found that nilotinib effectively controlled M. bovis growth and survival through enhanced parkin activity in infected mice. Altogether, our data showed that nilotinib regulates protective innate immune responses against intracellular mycobacterium, both in-vitro and in-vivo, and can be exploited as a novel therapeutic remedy for the control of M. bovis and MAP infections.

The E3 ubiquitin ligase parkin is dispensable for metabolic homeostasis in murine pancreatic β cells and adipocytes

The E3 ubiquitin ligase parkin is a critical regulator of mitophagy and has been identified as a susceptibility gene for type 2 diabetes (T2D). However, its role in metabolically active tissues that precipitate T2D development is unknown. Specifically, pancreatic β cells and adipocytes both rely heavily on mitochondrial function in the regulation of optimal glycemic control to prevent T2D, but parkin's role in preserving quality control of β cell or adipocyte mitochondria is unclear. Although parkin has been reported previously to control mitophagy, here we show that, surprisingly, parkin is dispensable for glucose homeostasis in both β cells and adipocytes during diet-induced insulin resistance in mice. We observed that insulin secretion, β cell formation, and islet architecture were preserved in parkin-deficient β cells and islets, suggesting that parkin is not necessary for control of β cell function and islet compensation for diet-induced obesity. Although transient parkin deficiency mildly impaired mitochondrial turnover in β cell lines, parkin deletion in primary β cells yielded no deficits in mitochondrial clearance. In adipocyte-specific deletion models, lipid uptake and β-oxidation were increased in cultured cells, whereas adipose tissue morphology, glucose homeostasis, and beige-to-white adipocyte transition were unaffected in vivo In key metabolic tissues where mitochondrial dysfunction has been implicated in T2D development, our experiments unexpectedly revealed that parkin is not an essential regulator of glucose tolerance, whole-body energy metabolism, or mitochondrial quality control. These findings highlight that parkin-independent processes maintain β cell and adipocyte mitochondrial quality control in diet-induced obesity.

Myoglobin induces mitochondrial fusion, thereby inhibiting breast cancer cell proliferation

Myoglobin is a monomeric heme protein expressed ubiquitously in skeletal and cardiac muscle and is traditionally considered to function as an oxygen reservoir for mitochondria during hypoxia. It is now well established that low concentrations of myoglobin are aberrantly expressed in a significant proportion of breast cancer tumors. Despite being expressed only at low levels in these tumors, myoglobin is associated with attenuated tumor growth and a better prognosis in breast cancer patients, but the mechanism of this myoglobin-mediated protection against further cancer growth remains unclear. Herein, we report a signaling pathway by which myoglobin regulates mitochondrial dynamics and thereby decreases cell proliferation. We demonstrate in vitro that expression of human myoglobin in MDA-MB-231, MDA-MB-468, and MCF7 breast cancer cells induces mitochondrial hyperfusion by up-regulating mitofusins 1 and 2, the predominant catalysts of mitochondrial fusion. This hyperfusion causes cell cycle arrest and subsequent inhibition of cell proliferation. Mechanistically, increased mitofusin expression was due to myoglobin-dependent free-radical production, leading to the oxidation and degradation of the E3 ubiquitin ligase parkin. We recapitulated this pathway in a murine model in which myoglobin-expressing xenografts exhibited decreased tumor volume with increased mitofusin, markers of cell cycle arrest, and decreased parkin expression. Furthermore, in human triple-negative breast tumor tissues, mitofusin and myoglobin levels were positively correlated. Collectively, these results elucidate a new function for myoglobin as a modulator of mitochondrial dynamics and reveal a novel pathway by which myoglobin decreases breast cancer cell proliferation and tumor growth by up-regulating mitofusin levels.

Midnolin promotes neurite outgrowth and expression of parkin ubiquitin ligase, and is associated with Parkinson's Disease.

Midnolin promotes neurite outgrowth and expression of parkin ubiquitin ligase, and is associated with Parkinson's Disease.

RAB7A phosphorylation by TBK1 promotes mitophagy via the PINK-PARKIN pathway.

Removal of damaged mitochondria is orchestrated by a pathway involving the PINK1 kinase and the PARKIN ubiquitin ligase. Ubiquitin chains assembled by PARKIN on the mitochondrial outer membrane recruit autophagy cargo receptors in complexes with TBK1 protein kinase. While TBK1 is known to phosphorylate cargo receptors to promote ubiquitin binding, it is unknown whether TBK1 phosphorylates other proteins to promote mitophagy. Using global quantitative proteomics, we identified S72 in RAB7A, a RAB previously linked with mitophagy, as a dynamic target of TBK1 upon mitochondrial depolarization. TBK1 directly phosphorylates RAB7AS72, but not several other RABs known to be phosphorylated on the homologous residue by LRRK2, in vitro, and this modification requires PARKIN activity in vivo. Interaction proteomics using nonphosphorylatable and phosphomimetic RAB7A mutants revealed loss of association of RAB7AS72E with RAB GDP dissociation inhibitor and increased association with the DENN domain-containing heterodimer FLCN-FNIP1. FLCN-FNIP1 is recruited to damaged mitochondria, and this process is inhibited in cells expressing RAB7AS72A. Moreover, nonphosphorylatable RAB7A failed to support efficient mitophagy, as well as recruitment of ATG9A-positive vesicles to damaged mitochondria. These data reveal a novel function for TBK1 in mitophagy, which parallels that of LRRK2-mediated phosphorylation of the homologous site in distinct RABs to control membrane trafficking.

Structural insights into ubiquitin phosphorylation by PINK1

Mutations of PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin (Ub) ligase parkin can cause familial parkinsonism. These two proteins are essential for ubiquitylation of damaged mitochondria and subsequent degradation. PINK1 phosphorylates Ser65 of Ub and the Ub-like (UBL) domain of parkin to allosterically relieve the autoinhibition of parkin. To understand the structural mechanism of the Ub/UBL-specific phosphorylation by PINK1, we determined the crystal structure of Tribolium castaneum PINK1 kinase domain (TcPINK1) in complex with a nonhydrolyzable ATP analogue at 2.5 Å resolution. TcPINK1 consists of the N- and C-terminal lobes with the PINK1-specific extension. The ATP analogue is bound in the cleft between the N- and C-terminal lobes. The adenine ring of the ATP analogue is bound to a hydrophobic pocket, whereas the triphosphate group of the ATP analogue and two coordinated Mg ions interact with the catalytic hydrophilic residues. Comparison with protein kinases A and C (PKA and PKC, respectively) unveils a putative Ub/UBL-binding groove, which is wider than the peptide-binding groove of PKA or PKC to accommodate the globular head of Ub or UBL. Further crosslinking analyses suggested a PINK1-interacting surface of Ub. Structure-guided mutational analyses support the findings from the present structural analysis of PINK1.

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.

Mechanism of parkin activation by PINK1.

Mutations in the E3 ubiquitin ligase parkin (PARK2, also known as PRKN) and the protein kinase PINK1 (also known as PARK6) are linked to autosomal-recessive juvenile parkinsonism (AR-JP)1,2; at the cellular level, these mutations cause defects in mitophagy, the process that organizes the destruction of damaged mitochondria3,4. Parkin is autoinhibited, and requires activation by PINK1, which phosphorylates Ser65 in ubiquitin and in the parkin ubiquitin-like (Ubl) domain. Parkin binds phospho-ubiquitin, which enables efficient parkin phosphorylation; however, the enzyme remains autoinhibited with an inaccessible active site5,6. It is unclear how phosphorylation of parkin activates the molecule. Here we follow the activation of full-length human parkin by hydrogen-deuterium exchange mass spectrometry, and reveal large-scale domain rearrangement in the activation process, during which the phospho-Ubl rebinds to the parkin core and releases the catalytic RING2 domain. A 1.8Å crystal structure of phosphorylated human parkin reveals the binding site of the phospho-Ubl on the unique parkin domain (UPD), involving a phosphate-binding pocket lined by AR-JP mutations. Notably, a conserved linker region between Ubl and the UPD acts as an activating element (ACT) that contributes to RING2 release by mimicking RING2 interactions on the UPD, explaining further AR-JP mutations. Our data show how autoinhibition in parkin is resolved, and suggest a mechanism for how parkin ubiquitinates its substrates via an untethered RING2 domain. These findings open new avenues for the design of parkin activators for clinical use.

OP-26 - Myoglobin induces mitochondrial fusion to inhibit cancer cell proliferation and tumor growth

Emerging data demonstrates that the monomeric heme protein myoglobin is aberrantly expressed in breast cancer tumors and is associated with slower tumor growth and better patient prognosis. However, the mechanism by which myoglobin slows tumor growth is unknown. In this study we hypothesized that myoglobin regulates mitochondrial structure/function to inhibit cell proliferation. Using a model of MDA-MB-231 breast cancer cells, we showed that stably expressing human myoglobin decreased cell proliferation and induced cell cycle arrest (characterized by increase p21 and decreased cyclin E expression). We demonstrate that mechanistically this cell cycle arrest is due to the myoglobin-dependent oxidation and degradation of the ubiquitin ligase parkin, which results in an increase in the expression of mitofusin-1. This protein is known to induce mitochondrial fusion and inhibit cell cycle progression. These data are recapitulated in vivo in a murine xenograft model. These data suggest a novel role for myoglobin as a modulator of mitochondrial dynamics, cell proliferation, and tumor growth.

Regulation of Mitophagy by the Parkin Ubiquitin Ligase and PINK1 Ubiquitin Kinase

Familial forms of Parkinson's disease arise from loss of the mitochondrial quality control pathway mediated by PINK1, a protein kinase, and Parkin, an E3 ubiquitin ligase. Together, they regulate the disposal of damaged mitochondria by autophagy (mitophagy). When damaged, mitochondria accumulate PINK1, which phosphorylates ubiquitin to recruit cytosolic Parkin. Normally quiescent due to autoinhibitory interactions, Parkin is activated by both phosphorylation by PINK1 and binding phosphorylated ubiquitin. The molecular mechanisms that activate activate Parkin have been the focus of much speculation. Here, I describe the crystal structure of the active conformation of Parkin that shows phosphorylation of parkin by PINK1 leads to a dramatic conformational change to derepress its E2‐binding and catalytic sites. Using a variety of experimental approaches, the activation pathways mediated by phospho‐ubiquitin binding and Parkin phosphorylation are compared. The study deepens our understanding of the mechanisms of Parkin activation and suggests new approaches for the treatment of Parkinson's disease by activating Parkin with small molecules.Support or Funding InformationCanadian Institutes of Health Research and the Michael J Fox FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.