scholarly journals Selective localization of Mfn2 near PINK1 enable its preferential ubiquitination by Parkin on mitochondria

2021 ◽  
Author(s):  
Marta Vranas ◽  
Yang Lu ◽  
Shafqat Rasool ◽  
Nathalie Croteau ◽  
Jonathan D. Krett ◽  
...  
Keyword(s):  
Intermediate Formation ◽  
Acyl Transfer ◽  
Substrate Selectivity ◽  
Outer Mitochondrial Membrane ◽  
Mitochondrial Depolarization ◽  
Proximity Ligation ◽  
Isolated Mitochondria ◽  
Selective Localization ◽  
Familial Parkinson’S Disease

Mutations in Parkin and PINK1 cause an early-onset familial Parkinson's disease. Parkin is a RING-In-Between-RING (RBR) E3 ligase that transfers ubiquitin from an E2 enzyme to a substrate in two steps: 1) thioester intermediate formation on Parkin, and 2) acyl transfer to a substrate lysine. The process is triggered by PINK1, which phosphorylates ubiquitin on damaged mitochondria, which in turn recruits and activates Parkin. This leads to the ubiquitination of outer mitochondrial membrane proteins and clearance of the organelle. While the targets of Parkin on mitochondria are known, the factors determining substrate selectivity remain unclear. To investigate this, we examined how Parkin catalyzes ubiquitin transfer to substrates. We found that His433 in the RING2 domain catalyzes acyl transfer. In cells, mutation of His433 impairs mitophagy. In vitro ubiquitination assays with isolated mitochondria show that Mfn2 is a kinetically preferred substrate. Using proximity-ligation assays, we show that Mfn2 specifically co-localizes with PINK1 and phospho-ubiquitin in U2OS cells upon mitochondrial depolarization. We propose a model whereby ubiquitination of Mfn2 is efficient by virtue of its localization near PINK1, which leads to the recruitment and activation of Parkin via phospho-ubiquitin at these sites.

scholarly journals Catalysis of ubiquitin transfer by the RBR ubiquitin ligase parkin

2014 ◽  
Vol 70 (a1) ◽  
pp. C845-C845
Author(s):  
Jean-François Trempe ◽  
Jonathan Krett ◽  
Véronique Sauvé ◽  
Marjan Seirafi ◽  
Karl Grenier ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Ubiquitin Ligase ◽  
Intermediate Formation ◽  
Acyl Transfer ◽  
Acceptor Site ◽  
E3 Ligases ◽  
Insight Into ◽  
In Cells

Mutations in the Parkin gene are responsible for an autosomal recessive form of Parkinson's disease. The parkin protein is a RING1-In-Between-RING2 (RBR) E3 ubiquitin ligase, which functions through a two-step mechanism involving a parkin~ubiquitin thioester intermediate [1]. However, compared to other ubiquitin ligases, parkin exhibits low basal activity and requires activation both in vitro and in cells. As parkin is neuroprotective in various models of Parkinson's disease, understanding how it catalyses ubiquitin transfer will be critical. We previously reported the crystal structure of full-length parkin [2]. The structure shows parkin in an auto-inhibited state and provides insight into how it is activated. The RING0 domain occludes the ubiquitin acceptor site Cys431 in RING2 whereas a novel Repressor Element of Parkin (REP) binds RING1 and blocks its E2-binding site. Remarkably, mutations that disrupt these inhibitory interactions activate parkin both in vitro and in cells. The structure also reveals that His433 and Glu444 form a catalytic dyad adjacent to Cys431. Here, we show that His433 catalyses the acyl transfer of ubiquitin carboxy terminus from Cys431 to a target lysine side-chain amino group. Mutation of His433 does not affect UbcH7~ubiquitin discharging or thioester intermediate formation, but prevents formation of polyubiquitin chains on parkin. However, mutation of His433 does not affect significantly parkin's mitochondrial recruitment and substrate ubiquitination, suggesting that other factors might be at play in vivo. We also investigate the catalytic role of other residues located around the Cys431, such as Trp462. The work provides insight into the mechanism of ubiquitination by RBR E3 ligases with important implications for Parkinson's disease.

scholarly journals Familial Parkinson's Disease Mutant E46K α-Synuclein Localizes to Membranous Structures, Forms Aggregates, and Induces Toxicity in Yeast Models

2011 ◽  
Vol 2011 ◽  
pp. 1-14 ◽  
Author(s):  
Michael Fiske ◽  
Michael White ◽  
Stephanie Valtierra ◽  
Sara Herrera ◽  
Keith Solvang ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Plasma Membrane ◽  
Mutant Form ◽  
Phospholipid Membrane ◽  
Endomembrane System ◽  
Wild Type ◽  
Significant Toxicity ◽  
Familial Parkinson’S Disease

In Parkinson’s disease (PD), midbrain dopaminergic neuronal death is linked to the accumulation of aggregated α-synuclein. The familial PD mutant form of α-synuclein, E46K, has not been thoroughly evaluated yet in an organismal model system. Here, we report that E46K resembled wild-type (WT) α-synuclein in Saccharomyces cerevisiae in that it predominantly localized to the plasma membrane, and it did not induce significant toxicity or accumulation. In contrast, in Schizosaccharomyces pombe, E46K did not associate with the plasma membrane. Instead, in one strain, it extensively aggregated in the cytoplasm and was as toxic as WT. Remarkably, in another strain, E46K extensively associated with the endomembrane system and was more toxic than WT. Our studies recapitulate and extend aggregation and phospholipid membrane association properties of E46K previously observed in vitro and cell culture. Furthermore, it supports the notion that E46K generates toxicity partly due to increased association with endomembrane systems within cells.

scholarly journals AHR:IKAROS Interaction Promotes Platelet Biogenesis in Response to SR1

Reports ◽  
2021 ◽  
Vol 4 (1) ◽  
pp. 7
Author(s):  
Lea Mallo ◽  
Valentin Do Sacramento ◽  
Christian Gachet ◽  
Susan Chan ◽  
Philippe Kastner ◽  
...  
Keyword(s):  
High Capacity ◽  
Proximity Ligation Assay ◽  
Physical Interaction ◽  
Short Report ◽  
Production Engineering ◽  
Platelet Production ◽  
Proximity Ligation ◽  
Aryl Hydrocarbon ◽  
Dependent Pathway

In vitro, the differentiation of megakaryocytes (MKs) is improved by aryl-hydrocarbon receptor (AHR) antagonists such as StemRegenin 1 (SR1), an effect physiologically recapitulated by the presence of stromal mesenchymal cells (MSC). This inhibition promotes the amplification of a CD34+CD41low population able to mature as MKs with a high capacity for platelet production. In this short report, we showed that the emergence of the thrombocytogenic precursors and the enhancement of platelet production triggered by SR1 involved IKAROS. The downregulation/inhibition of IKAROS (shRNA or lenalidomide) significantly reduced the emergence of SR1-induced thrombocytogenic population, suggesting a crosstalk between AHR and IKAROS. Interestingly, using a proximity ligation assay, we could demonstrate a physical interaction between AHR and IKAROS. This interaction was also observed in the megakaryocytic cells differentiated in the presence of MSCs. In conclusion, our study revealed a previously unknown AHR/ IKAROS -dependent pathway which prompted the expansion of the thrombocytogenic precursors. This AHR- IKAROS dependent checkpoint controlling MK maturation opens new perspectives to platelet production engineering.

A Relative Energy Failure Is Associated With Low-Mg2+ But Not With 4-Aminopyridine Induced Seizure-Like Events in Entorhinal Cortex

1999 ◽  
Vol 81 (1) ◽  
pp. 399-403 ◽  
Author(s):  
S. Schuchmann ◽  
K. Buchheim ◽  
H. Meierkord ◽  
U. Heinemann
Keyword(s):  
Entorhinal Cortex ◽  
Focal Epilepsy ◽  
Relative Energy ◽  
In Vitro Models ◽  
Mitochondrial Depolarization ◽  
Citrate Cycle ◽  
Cellular Energy ◽  
Intracellular Ca ◽  
Energy Failure

Schuchmann, S., K. Buchheim, H. Meierkord, and U. Heinemann. A relative energy failure is associated with low-Mg2+ but not with 4-aminopyridine induced seizure-like events in entorhinal cortex. J. Neurophysiol. 81: 399–403, 1999. During seizure-like events (SLEs), intracellular Ca2+ concentration ([Ca2+]i) increases causing depolarization of the mitochondrial membrane and subsequent intramitochondrial accumulation of Ca2+. Mitochondrial depolarization results in an interruption of oxidative phosphorylation and increase in reactive oxygen species. Calcium activates enzymes of the citrate cycle. A characteristic feature of the low-Mg2+–induced SLEs is that they are transformed to a late activity refractory to anticonvulsant drugs, which may be regarded as a model system of difficult to treat status epilepticus. In contrast, 4-aminopyridine (4-AP)–induced activity rarely evolves to such late activity. The autofluorescence of NAD(P)H was used to monitor changes in cellular energy metabolism in the entorhinal cortex in two in vitro models of focal epilepsy. During repetitive 4-AP–induced SLEs there was a short decrease followed by a long-lasting overshoot of the NAD(P)H signal. This sequence remained unaltered during recurring SLEs. In contrast, during recurrent low-Mg2+–induced SLEs, the brief initial NADH signal reduction was unchanged but the following overshoot of NADH displayed a continuous decrease. This indicates a relative energy failure, which may contribute to the transformation to late activity in the low-Mg2+ model.

The Phytotoxicity of Propanil

Weed Science ◽  
1968 ◽  
Vol 16 (1) ◽  
pp. 23-28 ◽  
Author(s):  
G. Hofstra ◽  
C. M. Switzer
Keyword(s):  
Oxygen Uptake ◽  
Beta Vulgaris ◽  
Field Experiments ◽  
Lycopersicum Esculentum ◽  
Chloroplast Membranes ◽  
Isolated Mitochondria ◽  
Treated Leaf ◽  
Leaf Tissues ◽  
Methods Of Application

Preliminary field experiments with 3,4-dichloropropionanilide (propanil) indicated that methods of application had little effect on the activity of this herbicide. Growth of tomato (Lycopersicum esculentum Mill.) plants was reduced for about 7 days. Susceptible plants were killed in 3 to 7 days. Propanil inhibited the growth of tomato radicles and the auxin-induced growth of Avena coleoptiles. Propanil destroyed the permeability of red beet (Beta vulgaris L.) membranes and also destroyed chloroplast membranes in vitro. Oxygen uptake by isolated mitochondria and treated leaf tissues, and phosphate esterification in mitochondria were severely inhibited by propanil. Photosynthesis was completely inhibited within 20 min after treatment but began to return to normal in resistant plants (tomato) after 6 hr.

scholarly journals Exercise-induced protection against reperfusion arrhythmia involves stabilization of mitochondrial energetics

2016 ◽  
Vol 310 (10) ◽  
pp. H1360-H1370 ◽  
Author(s):  
Rick J. Alleman ◽  
Alvin M. Tsang ◽  
Terence E. Ryan ◽  
Daniel J. Patteson ◽  
Joseph M. McClung ◽  
...  
Keyword(s):  
Simultaneous Measurement ◽  
Cardiac Electrophysiology ◽  
Ischemia Reperfusion ◽  
Antiarrhythmic Effect ◽  
Treadmill Running ◽  
Sprague Dawley ◽  
Isolated Mitochondria ◽  
Reperfusion Arrhythmia ◽  
Exercise Induced

Mitochondria influence cardiac electrophysiology through energy- and redox-sensitive ion channels in the sarcolemma, with the collapse of energetics believed to be centrally involved in arrhythmogenesis. This study was conducted to determine if preservation of mitochondrial membrane potential (ΔΨm) contributes to the antiarrhythmic effect of exercise. We utilized perfused hearts, isolated myocytes, and isolated mitochondria exposed to metabolic challenge to determine the effects of exercise on cardiac mitochondria. Hearts from sedentary (Sed) and exercised (Ex; 10 days of treadmill running) Sprague-Dawley rats were perfused on a two-photon microscope stage for simultaneous measurement of ΔΨm and ECG. After ischemia-reperfusion, the collapse of ΔΨm was commensurate with the onset of arrhythmia. Exercise preserved ΔΨm and decreased the incidence of fibrillation/tachycardia ( P < 0.05). Our findings in intact hearts were corroborated in isolated myocytes exposed to in vitro hypoxia-reoxygenation, with Ex rats demonstrating enhanced redox control and sustained ΔΨm during reoxygenation. Finally, we induced anoxia-reoxygenation in isolated mitochondria using high-resolution respirometry with simultaneous measurement of respiration and H2O2. Mitochondria from Ex rats sustained respiration with lower rates of H2O2 emission than Sed rats. Exercise helps sustain postischemic mitochondrial bioenergetics and redox homeostasis, which is associated with preserved ΔΨm and protection against reperfusion arrhythmia. The reduction of fatal ventricular arrhythmias through exercise-induced mitochondrial adaptations indicates that mitochondrial therapeutics may be an effective target for the treatment of heart disease.

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