Structure of human PINK1 at a mitochondrial TOM/VDAC array
Ontology highlight
ABSTRACT:
INSTRUMENT(S): timsTOF Pro
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Laura Dagley
LAB HEAD: Prof. David Komander
PROVIDER: PXD058529 | Pride | 2025-05-05
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| P4467_01_1_1_5867.d.zip | Other | |||
| P4467_02_1_1_5868.d.zip | Other | |||
| P4467_03_1_1_5869.d.zip | Other | |||
| P4467_04_1_1_5870.d.zip | Other | |||
| P4467_05_1_1_5871.d.zip | Other |
Items per page: 5 1 - 5 of 15 |
Similar Datasets
Project description:Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive early onset Parkinson disease (PD). PINK1 is a Ser/Thr kinase that regulates mitochondrial quality control by triggering mitophagy mediated by the ubiquitin ligase Parkin. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane (OMM) forming a high molecular weight complex with the translocase of the outer membrane (TOM). PINK1 then phosphorylates ubiquitin, which enables recruitment and activation of Parkin followed by autophagic clearance of the damaged mitochondrion. Thus, Parkin-dependent mitophagy hinges on the stable accumulation of PINK1 on the TOM complex. Yet, the mechanism linking mitochondrial stressors to PINK1 accumulation and whether the translocases of the inner membrane (TIMs) are also involved, remain unclear. Herein, we demonstrate that mitochondrial stress induces the formation of a PINK1-TOM-TIM23 supercomplex in human cultured cell lines, dopamine neurons, and midbrain organoids. Moreover, we show that PINK1 is required to stably tether the TOM to TIM23 complexes in response to stress, such that the supercomplex fails to accumulate in cells lacking PINK1. This tethering is dependent on an interaction between the PINK1 NT-CTE module and the cytosolic domain of the Tom20 subunit of the TOM complex, the disruption of which, by either designer or PD-associated PINK1 mutations, inhibits downstream mitophagy. Together, the findings provide key insight into how PINK1 interfaces with the mitochondrial import machinery, with important implications for the mechanisms of mitochondrial quality control and PD pathogenesis.
2024-03-02 | PXD047765 | Pride
Project description:Mitochondrial protein import through the outer and inner membranes is key to mitochondrial biogenesis. Recent studies have explored how cells respond when import is impaired by a variety of different insults. Here, we developed a mammalian import blocking system using dihydrofolate reductase (DHFR) fused to the N-terminus of the inner membrane protein MIC60. While stabilization of the DHFR domain by methotrexate inhibited endogenous mitochondrial protein import, it neither activated the transcription factor ATF4, nor was affected by ATAD1 expression or by VCP/p97 inhibition. On the other hand, surprisingly, plugging the channel of translocase of the outer membrane (TOM) induced YME1L1, an ATP-dependent protease, to eliminate translocase of the inner membrane (TIM23) channel components TIMM17A and TIMM23. The data suggest that unoccupied TIM23 complexes expose a C-terminal degron on TIMM17A to YME1L1 for degradation. Import plugging caused a cell growth defect and loss of YME1L1 exacerbated the growth inhibition, showing the protective effect of YME1L1 activity. YME1L1 appears to play a crucial role in mitochondrial quality control to counteract precursor stalling in the TOM complex and unoccupied TIM23 channels.
2024-10-24 | MSV000096201 | MassIVE
Project description:The voltage-dependent anion-selective channels (VDACs), also known as eukaryotic porins, are pore-forming proteins which allow the passage of ions and small molecules across the outer mitochondrial membrane (OMM). They are involved in complex interactions regulating organelle and cellular metabolism. We have recently reported about the post-translational modifications (PTMs) of the three VDAC isoforms purified from rat liver mitochondria (rVDACs), showing for the first time the over-oxidation of the cysteine residues as an exclusive feature of VDACs. Noteworthy, this peculiar PTM is not detectable in other integral membrane mitochondrial proteins, as defined by their elution at low salt concentration by a hydroxyapatite column. In this study, the association of tryptic and chymotryptic proteolysis with UHPLC/High Resolution nESI-MS/MS, allowed us to extend the investigation to the human VDACs. Over-oxidation of the cysteine residues, essentially irreversible in cell conditions, was as certained also in VDAC isoforms from human cells. In human VDAC2 and 3 isoforms the permanently reduced state of a cluster of close cysteines indicates the possibility that disulfide bridges are formed in the proteins. Importantly, the detailed oxidative PTMs found in human VDACs confirm and sustain our previous findings in rat tissues, claiming for a predictable characterization that has to be conveyed in the functional role of VDAC proteins within the cell.
2020-03-09 | PXD017482 | Pride
Project description:The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinson’s Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation, that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are most prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. In this model, USP30 deubiquitylation of TOM complex components dampens the trigger that unleashes the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly PINK1 generation of phospho-Ser65 Ubiquitin proceeds more rapidly and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.
2020-07-03 | PXD019692 | Pride
Project description:The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinson’s Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation, that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are most prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. In this model, USP30 deubiquitylation of TOM complex components dampens the trigger that unleashes the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly PINK1 generation of phospho-Ser65 Ubiquitin proceeds more rapidly and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.
2020-07-03 | PXD018640 | Pride
Project description:Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organisation of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs. Here, we designed a precursor protein that is stalled during matrix transport in a TOM-TIM23-spanning manner and enables purification of the translocation intermediate. Combining chemical cross-linking with mass spectrometric analyses and structural modelling allowed us to map the molecular environment of the intermembrane space interface of TOM and TIM23 as well as the import motor interactions with amino acid resolution. Our analyses provide a framework for understanding presequence handover and translocation during matrix protein transport.
2021-12-13 | PXD028002 | Pride
Project description:This study aims to investigate the role and mechanism of DEK in asthmatic airway inflammation and in regulating PTEN-induced putative kinase 1 (PINK1)-Parkin mediated mitophagy, NLRP3 (NOD-like receptor family pyrin domain containing 3) inflammasome activation, and apoptosis. We found that recombinant DEK protein (rmDEK) promoted eosinophils recruitment, mitochondrial fragmentation, and outer membrane 20 (TOM20) and LC3 co-localization representing mitophagosomes in bronchoalveolar lavage fluid (BALF) in house dust mite (HDM) induced-asthma. rmDEK also reduced co-localization of mitochondrial fusion protein mitofusin1 (MFN1) and mitochondria, and the protein level of manganese superoxide dismutase (MnSOD), enhanced microtubule-associated protein1 light chain 3 (LC3) and voltage-dependent anion channels (VDAC) co-localization which also represent the mitophagosomes in airway epithelial cells, furthermore, increased dynamin-related protein 1 (DRP1) expression, PINK1-Parkin-mediated mitophagy, NLRP3 inflammasome activation, and apoptosis. In the DEK knockout mice, HDM induced asthmatic airway inflammation, MnSOD, PINK1-Parkin protein level, Parkin mediated mitophagy characterized by LC3 and Parkin co-localization in the airways, ROS generation, NLRP3 inflammation and apoptosis were fully reversed. Similar effects of rmDEK were also observed in the BEAS-2B cells, which were rescued by the autophagy inhibitor 3-MA. Moreover, DEK silencing diminished the Parkin, LC3, DRP1 translocation to mitochondria; as well as mitochondrial ROS; TOM20 and mitochondrial DNA mediated mitochondrial oxidative damage. ChIP-sequence analysis showed that DEK was enriched on the AAA domain-containing protein 3A (ATAD3A) promoter and could positively regulate ATAD3A expression. Additionally, ATAD3A was highly expressed in HDM-induced asthma models. Furthermore, ATAD3A interacted with DRP1, and knockdown of ATAD3A could down-regulate DRP1 and mitochondrial oxidative damage. Conclusively, DEK deficiency alleviates airway inflammation in asthma by down-regulating PINK1-Parkin mitophagy, NLRP3 inflammasome activation, and apoptosis. The mechanism may be through the DEK/ATAD3A/DRP1 signaling axis. Our findings may provide new potential therapeutic targets for asthma treatment.
2023-09-24 | GSE243532 | GEO
Project description:Parkinson's disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD. Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of M-NM-1-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. Thus, aging Pink1M-bM-^HM-^R/M-bM-^HM-^R mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death. Transcriptome microarray data of Pink1-/- mouse brains in absence of a stressor, even at old age, show remarkably sparse dysregulations. See Gispert-S et al 2009 PLOS ONE. Factorial design comparing Pink1 knock-out mice with wild type littermates in three different tissues (striatum, midbrain, cerebellum at four different timepoints (6, 12, 14 weeks, and 18 month)
2014-11-04 | E-GEOD-60413 | biostudies-arrayexpress
Project description:Parkinson's disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD. Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of α-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. Thus, aging Pink1−/− mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death. Transcriptome microarray data of Pink1-/- mouse brains in absence of a stressor, even at old age, show remarkably sparse dysregulations. See Gispert-S et al 2009 PLOS ONE.
2014-11-04 | GSE60413 | GEO
Project description:Proteomic analysis of N. crassa was performed to explore the role of VDAC
(voltage dependent anion-selective channels) in the regulation of cellular
processes. Differential label free quantitation was applied to both wild-type
and VDAC-knockout versions, involving both cytosolic and mitochondrial isolations.
Proteins involved in amino acid and protein synthesis were lowered by VDAC deletion,
while stress responses and pyruvate metabolism were over-expressed. The absence
of VDAC also impacted membrane lipids and hyphal morphology.
2021-12-14 | MSV000088567 | MassIVE