Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The V-ATPase/ATG16L1 axis is controlled by the V1H subunit

ABSTRACT: Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically de-acidified compartments targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to pH gradient disruption is unclear. Here, we reveal a direct role for the V1H subunit of the V-ATPase proton pump in recruiting ATG16L1. The interaction between V1H and ATG16L1 occurs within assembled V-ATPases, but not dissociated V1 complexes. This selectivity allows recruitment to be coupled to changes in V-ATPase assembly that follow pH dissipation. Cells lacking V1H undergo canonical macroautophagy but are unable to recruit ATG16L1 in response to influenza infection, STING activation or ionophore drugs. We identify a loop within V1H that mediates ATG16L1 binding, which is absent in a neuronal isoform of V1H. Thus, V1H controls ATG16L1 recruitment in response to proton gradient dissipation, suggesting that the V-ATPase acts autonomously as a cell-intrinsic damage sensor.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Mark Skehel

LAB HEAD: Rupert Beale

PROVIDER: PXD052945 | Pride | 2025-01-30

REPOSITORIES: pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	GF2475_CTL.raw	Raw
	GF2475_CTL_1.raw	Raw
	GF2475_CTL_2.raw	Raw
	GF2475_CTL_3.raw	Raw
	GF2475_CTL_Sid_plus.raw	Raw

Items per page:

1 - 5 of 51

Similar Datasets

Nucleocytoplasmic mRNA redistribution accompanies RNA binding protein mislocalisation in ALS motor neurons and is restored by VCP inhibition

Project description:Although the pathological hallmark of amyotrophic lateral sclerosis (ALS) is the nucleocytoplasmic mislocalisation of RNA binding proteins (RBPs), such as TDP-43 and FUS, the nucleocytoplasmic distribution of mRNA remains uncharacterised. Here, we used subcellular fractionation with RNA sequencing and proteomics to assess nucleocytoplasmic mRNA and protein localisation in human induced pluripotent stem cell-derived motor neurons (iPSNs) from ALS patients with TARDBP mutations, VCP mutations, and controls with VCP mutations knocked-in with CRISPR/Cas9. In each mutant group, we found substantial nucleocytoplasmic mRNA redistribution, particularly in transcripts involved in protein binding. Redistributed transcripts in ALS iPSNs were enriched in protein-coding biotypes, exhibited longer lengths, and had enhanced interactions with RBPs, including TDP-43 and FUS. Using mass spectrometry in TARDBP mutant, VCP mutant, and VCP mutant knock-in iPSNs, we reveal widespread protein mislocalisation, especially in RBPs. We find that mislocalised proteins exhibit substantially greater binding to redistributed transcripts than non-redistributed mRNAs, suggesting that RBP mislocalisation may be directly coupled to mRNA redistribution. Remarkably, treatment with the VCP D2 ATPase domain inhibitor ML240 restored both nucleocytoplasmic mRNA redistribution and protein mislocalisation not only in VCP mutants but also in TARDBP mutant iPSNs. Functional assays in VCP mutant iPSNs further confirmed that ML240 restores lysosomal localisation from the perinuclear region towards the neurites and reduces oxidative stress. Our findings demonstrate that ALS motor neurons exhibit concomitant nucleocytoplasmic mRNA redistribution and RBP

2023-10-13 | PXD037107 | Pride

Drosophila S2s Elongator identification

Project description:A collection of stable Drosophila S2 cell lines was established, each one expressing a (His)-PC SNAP tagged version of each of the six Elongator subunits. From these stable lines, the endogenous Elongator complex can be recovered by pulling on the exogenous subunit via the PC tag. This allowed us to test which subunit can be over-expressed and used to recover the full Elongator complex for further in vitro experiments.

2022-09-15 | PXD036556 | Pride

Structure of the native gamma-Tubulin Ring Complex capping spindle microtubules

Project description:Microtubules (MTs) are fundamental to cellular architecture, function and organismal development. They are nucleated from microtubule organizing centres by the evolutionary conserved ?-tubulin ring complex (?TuRC). However, the molecular mechanism of nucleation remains elusive. Here, we used cryo-electron tomography (cryo-ET) to determine the structure of the native ?TuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, ?TuRC presents a ring of ?-tubulin subunits to seed nucleation of exclusively 13-protofilament microtubules, and it adopts an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-ET reconstruction also revealed that a novel coiled-coil protein staples the first row of ?/?-tubulin molecules to alternating positions along the ?-tubulin ring. This positioning of ?/?-tubulin onto ?TuRC suggests a role for the coiled-coil protein in augmenting ?TuRC-mediated microtubule nucleation. Based on our results we describe a molecular model for budding yeast ?TuRC activation and MT nucleation.

2024-03-05 | PXD050372 | Pride

The deubiquitinase TRABID stabilises the K29/K48-specific E3 ubiquitin ligase HECTD1

Project description:The OTU deubiquitinase TRABID is highly tuned for the recognition and cleavage of K29 and K33-linked ubiquitin chains. Yet the cellular functions of these atypical ubiquitin signals remain unclear. Here, we compared the interactome of two different catalytically-dead TRABID constructs, one that lacks the OTU domain and the other a single point mutant that is catalytically inactive. Since both constructs also act as ubiquitin trapping mutants, this approach was used to differentiate between interactors and substrates. The E3 ubiquitin ligase HECTD1 was confirmed as a TRABID substrate, and using UbiCREST and Ubiquitin-AQUA proteomics, we determined that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that in contrast to UBE3C, HECTD1 requires the formation of branched K29/K48-linked chains for its full activity. Finally, we used transient knockdown and genetic knock out of TRABID in mammalian cells to show that TRABID stabilises HECTD1 protein levels. This study identifies TRABID-HECTD1 as a K29-specific DUB/E3 pair and provides novel insights on the assembly of branched ubiquitin signals.

2021-06-23 | PXD022703 | Pride

Mechanism-based traps enable protease and hydrolase substrate discovery

Project description:we report a strategy for creating mechanism-based, light activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid permits the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4. We demonstrate that RHBDL4 can shed luminal fragments of ER-resident type I transmembrane proteins to the extracellular space, as well as catalysing non-canonical secretion of endogenous soluble ER-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 is an aminopeptidase – with a preference for removing aromatic amino acids – in human cells. Our results exemplify a powerful paradigm for discovering the substrates and activities of hydrolase enzymes.

2022-02-10 | PXD030381 | Pride

Interactome analysis of C. elegans synapses by TurboID-based proximity labeling

Project description:Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. Using the highly active biotin ligase TurboID, we optimize a proximity labeling protocol for C. elegans. We use this protocol to characterise the proteomes of the worm’s gut, muscle, skin, and nervous system. We express TurboID exclusively in the pair of AFD neurons and show we can identify known and previously unknown proteins expressed selectively in AFD. We knock TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identify many known ELKS-1 interacting proteins as well as previously uncharacterised synaptic proteins. Versatile vectors, and the inherent advantage of C. elegans for biochemistry, make proximity labeling a valuable addition to the nematode’s armory.

2021-09-15 | PXD027068 | Pride

TAP-GluN1, PSD95-TAP, WT (control) BNP-LC-MS/MS proteomics

Project description:TAP-GluN1 (840 kDa and 1.5 MDa), PSD95-TAP (1.5 MDa) and WT (control; 1.5 MDa) native complexes from Grin1^TAP/TAP, Dlg4^TAP/TAP, and WT mouse forebrain (cortex and hippocampus), respectively. Purified samples were seperated by blue native PAGE. Bands were excised, digested with trypsin. Peptides were identified by LC-MS/MS.

2016-04-20 | PXD000011 | Pride

Shulin packages axonemal outer dynein arms for ciliary targeting

Project description:The main force generators in eukaryotic cilia and flagella are axonemal outer dynein arms (ODAs). During cilio-genesis, these ∼1.8 MDa complexes are assembled in the cytoplasm and targeted to cilia via an unknown mechanism. Here we use the ciliate Tetrahymena to identify two novel factors (Q22YU3 and Q22MS1) which bind ODAs in the cytoplasm and are required for their delivery to cilia. We show that Q22YU3, which we name Shulin, locks the ODA motor domains into a closed conformation and inhibits motor activity. Cryo-EM reveals how Shulin stabilizes this compact form of ODAs by binding to the dynein tails. Our findings provide a molecular explanation for how newly assembled dyneins are packaged for delivery to the cilia.

2021-09-09 | PXD022396 | Pride

Multi-signal regulation of the GSK-3β homolog Rim11 governs meiosis entry in yeast

Project description:Starvation in diploid budding yeast cells triggers a cell-fate program culminating in meiosis and spore formation. Transcription activation of early meiotic genes (EMGs) hinges on the transcription activator Ime1, its DNA-binding partner Ume6, and GSK-3 kinase Rim11. Phosphorylation of Ume6 by Rim11 is key for EMG activation. We report that Rim11 functions as the central signal integrator for controlling Ume6 phosphorylation and EMG transcription. In nutrient-rich conditions, PKA suppresses Rim11 levels, while TORC1 retains Rim11 in the cytoplasm. Inhibiting PKA and TORC1 induces Rim11 expression and nuclear localization. Remarkably, nuclear Rim11 is required, but not sufficient, for Rim11-dependent Ume6 phosphorylation. Additionally, Ime1 is an essential anchor protein for phosphorylating Ume6. Subsequently, Ume6-Ime1 coactivator complexes form, which drive EMG transcription. Our results demonstrate how varied signalling inputs (PKA/TORC1/Ime1) converge through Rim11 to regulate EMG expression and meiosis initiation. We posit that the signalling-regulatory network elucidated here generates robustness in cell-fate control.

2024-03-06 | PXD049212 | Pride

Wnt signalosome assembly is governed by conformational flexibility of Axin and AP2 clathrin adaptor

Project description:Wnt signal transduction relies on the direct inhibition of GSK3 by phosphorylated PPPSPxS motifs within the cytoplasmic tail of the LRP6 co-receptor. How GSK3 is recruited to LRP6 remains unclear. Here, we use nuclear magnetic resonance spectroscopy to identify the membrane-proximal PPPSPxS motif and its flanking sequences as the primary binding site for both Axin and GSK3, and an intrinsically disordered segment of Axin as its LRP6-interacting region (LIR). Co-immunoprecipitation and CRISPR-engineered mutations in endogenous Axin indicate that its docking at LRP6 is antagonized by a phospho-dependent foldback within LIR and by a PRTxR motif that allows Axin and GSK3 to form a multi-pronged interaction that favors their detachment from LRP6. Crucially, signaling by LRP6 also depends on its binding to AP2 clathrin adaptor. We propose that the Wnt-driven clustering of LRP6 within clathrin-coated locales allows the Axin-GSK complex to dock at adjacent LRP6 molecules, while also exposing it to co-targeted kinases that change its activity in Wnt signal transduction.

2025-04-30 | PXD061649 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data