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LC-MSMS of cell secretome for MAP1LC3B interactors

ABSTRACT: Growing evidence implicates autophagy in cell secretion. Identifying the repertoire of proteins involved with autophagy dependent secretions is key for understanding the underlying mechanism. We use a proximity-dependent biotinylation proteomics strategy to label protein that engage the autophagy regulator MAP1LC3B (LC3/ATG8) in cells; the labeled proteins are then secreted, captured with neutravidin, tryptically digested, and identified by LC-MS/MS. Cells stably expressing BirA* alone serves as control for non-specific cytosolic labeling. SILAC is employed to quantify the degree of LC3B interaction over the background.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture, Fibroblast

DISEASE(S): Disease Free

SUBMITTER: Hector Huang

LAB HEAD: Arun Paul Wiita

PROVIDER: PXD006541 | Pride | 2022-03-01

REPOSITORIES: Pride

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The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles.

Leidal Andrew M AM Huang Hector H HH Marsh Timothy T Solvik Tina T Zhang Dachuan D Ye Jordan J Kai FuiBoon F Goldsmith Juliet J Liu Jennifer Y JY Huang Yu-Hsin YH Monkkonen Teresa T Vlahakis Ariadne A Huang Eric J EJ Goodarzi Hani H Yu Li L Wiita Arun P AP Debnath Jayanta J

Nature cell biology 20200113 2

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins ( ...[more]

PMID: 31932738

Similar Datasets

LC-MSMS of cell secretome for MAP1LC3B interactors

Project description:Description Growing evidence implicates autophagy in cell secretion. Identifying the repertoire of proteins involved with autophagy dependent secretions is key for understanding the underlying mechanism. We initially use a proximity-dependent biotinylation proteomics strategy to label protein that engage the autophagy regulator MAP1LC3B (LC3/ATG8) in cells; the labeled proteins are then secreted, captured with neutravidin, tryptically digested, and identified by LC-MS/MS. Cells stably expressing BirA* alone serves as control for non-specific cytosolic labeling. SILAC is employed to quantify the degree of LC3B interaction over the background. We follow up with proteomic comparisons of purified exosomes from HEK293T, and autophagy related, ATG7-/- and ATG12-/-, knockout HEK293T cell lines. We quantified differentially secreted proteins in the exosomes by isobaric TMT labels.

2020-01-09 | PXD015479 | Pride

Defining the NSD2 interactome: PARP1 PARylation reduces NSD2 histone methyltransferase activity and impedes chromatin binding

Project description:NSD2 is a histone methyltransferase that specifically dimethylates histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. Dramatic overexpression of NSD2 in t(4;14) multiple myeloma (MM) and an activating mutation of NSD2 discovered in acute lymphoblastic leukemia (ALL) are significantly associated with altered gene activation, transcription and DNA damage repair. The partner proteins through which NSD2 may influence critical cellular processes remain poorly defined. In this study, we utilized proximity-based labelling (BioID) combined with label-free quantitative mass spectrometry to identify high confidence NSD2 interacting partners in MM cells.

2019-06-27 | PXD013759 | Pride

Characterization of PARP-6 function in knock out mice and patients with developmental delay

Project description:PARP-6, a member of a family of enzymes (17 in humans) known as poly-ADP- ribose polymerases (PARPs), is a neuronally enriched PARP. While previous studies from our group show that PARP-6 is a regulator of dendrite morphogenesis in hippocampal neurons, its function in the nervous system in vivo is poorly understood. Here, we describe the generation of a PARP-6 loss-of-function mouse model for examining the function of PARP-6 during neurodevelopment in vivo. Using CRISPR-Cas9 mutagenesis, we generated a mouse line that expresses a PARP-6 truncated variant (PARP-6TR) in place of PARP-6WT. Unlike PARP-6WT, PARP-6TR is devoid of catalytic activity. Homozygous PARP-6TR do not exhibit obvious neuromorphological defects during development, but nevertheless die perinatally. This suggests that PARP-6 catalytic activity is important for postnatal survival. We also report PARP-6 mutations in six patients with several neurodevelopmental disorders, including microencephaly, intellectual disabilities, and epilepsy. The most severe mutation in PARP-6 (C563R) results in the loss of catalytic activity. Expression of the PARP-6C563R mutant in hippocampal neurons decreases dendrite morphogeneis. Taken together, these results suggest that PARP-6 is an essential gene in mice, and the loss of PARP-6 catalytic activity has detrimental effects on neuronal function in humans.

2021-07-21 | PXD025386 | Pride

Proximity-dependent biotinylation to elucidate the interactome of TNK2 non-receptor tyrosine kinase

Project description:Non-receptor tyrosine kinases represent an important class of signaling molecules which are involved in driving diverse cellular pathways. Although the large majority have been well-studied in terms of their protein binding partners, the interactomes of some important non-receptor tyrosine kinases such as TNK2 (also known as activated Cdc42-associated kinase 1 or ACK1) have not been systematically investigated. Aberrant expression and hyperphosphorylation of TNK2 have been implicated in a number of cancers, although the exact proteins and cellular events that mediate phenotypic changes downstream of TNK2 are unclear. Biological systems that employ proximity-dependent protein labeling methods, such as biotinylation identification (BioID), are being increasingly used to map protein-protein interactomes as they provide increased sensitivity in finding interaction partners. In the present study, we employ BioID coupled to a Biotinylation Site Identification Technology (BioSITe) method we recently developed to perform molecular mapping of intracellular protein interactors of TNK2. By performing a controlled comparative analysis between full-length TNK2 and its truncated counterpart, we were not only able to confidently identify site-level biotinylation of previously well-established TNK2 binders and substrates, but also several novel binders of TNK2 that may help explain its role in oncogenic signaling.

2021-08-25 | PXD010474 | Pride

Analysis of HeLa cells after transfection with miR-1, miR-124 and miR-181.

Project description:Analysis of HeLa cells at 24 hours after transfection with wild type miR-1, miR-124, miR-181 versus control transfected HeLa cells. Results were compared to protein down-regulation at 48 hours measured by SILAC-MS. Analysis of HeLa cells at 24 hours after transfection with wild type miR-1, miR-124, miR-181 versus control transfected HeLa cells. Results were compared to protein down-regulation at 48 hours measured by SILAC-MS.

2010-05-25 | E-GEOD-11968 | biostudies-arrayexpress

Identification of Stress Granule interactor proteins in yeast S. cerevisiae

Project description:We investigated the sub-cellular architecture of Stress Granules in the context of glucose starvation. In order to uncover transient and peripheral Stress Granule interactors, we optimized the proximity-dependent biotinylation (BioID) approach for use in yeast. This approach allowed us to visualize Stress Granule formation while simultaneously inducing the biotinylation of SG components and nearby proteins in live cells. We used two independent Stress Granule components Pub1 and Pab1.

2020-03-23 | PXD016988 | Pride

Proximity proteomics identifies PAK4 as a component of Afadin–Nectin junctions.

Project description:Human PAK4 is an ubiquitously expressed p21-activated kinase which acts downstream of Cdc42. Since PAK4 is demonstrably enriched in cell-cell junctions, we probed the local protein environment around the kinase with a view to understanding its location and substrates. In U2OS cells expressing PAK4-BirA-GFP the resultant SILAC proximity analyses revealed a subset of 28 proteins that are primarily cell-cell junction components. The protein with highest relative biotin labelling was Afadin/AF6, which associates with the nectin family of homophilic junctional proteins. Reciprocally >50% of the PAK4-proximal proteins were identified by Afadin BioID. Pull-down experiments failed to identify junctional proteins, emphasizing the advantage of the BioID method. Mechanistically PAK4 depended on Afadin for its junctional localization, which is similar to the situation in Drosophila. A highly ranked PAK4-proximal protein LZTS2 was immuno-localized with Afadin at cell-cell junctions. Though PAK4 and Cdc42 are junctional, BioID analysis did not yield conventional cadherins, indicating their spatial segregation. To identify cellular PAK4 substrates we then assessed changes in phospho-proteome after 12 min PF-3758309 treatment. Among the PAK4-proximal junctional proteins 17 PAK4 sites were identified. Thus we show PAK4 is selective for the mammalian Afadin/nectin sub-compartment, with a demonstrably distinct localization from tight and cadherin junctions.

2021-11-30 | PXD024464 | Pride

Cortical actin tension regulates secretory protein trafficking and the endoplasmic reticulum stress response

Project description:We used proximity-based biotinylation assay cells to isolate candidate plasma membrane-associated proteins whose localization are sensitive to cortical tension level. Using membrane-targeting ascorbate peroxidase (APEX2-CAAX) as a bait, we mapped the proteomic landscape at the cell cortex under high and low cortical tension conditions.

2022-06-10 | PXD018975 | Pride

HYPE mediated AMPylation profiling in human lysates

Project description:We describe here the first example of global chemoproteomic screening and substrate validation for HYPE mediated AMPylation in mammalian cell lysate. Through quantitative mass spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification we identified a total of 27 AMPylated proteins, including the previously validated substrate, ER chaperone BiP (HSPA5), along with novel substrates involved in pathways of gene expression, ATP biosynthesis and maintenance of cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation.

2015-12-02 | PXD002601 | Pride

Transcription factor trapping by RNA in gene regulatory elements (CLIP-Seq)

Project description:Transcription factors (TFs) bind specific sequences in promoter-proximal and distal DNA elements in order to regulate gene transcription. RNA is transcribed from both promoter-proximal and distal DNA elements, and some DNA-binding TFs have also been shown to bind RNA. These obsevations led us to postulate that RNA transcribed from regulatory elements contributes to stable TF occupancy at these regulatory elements. We show here that the ubiquitously expressed TF YY1 binds to both proximal and distal regulatory elements and to the RNA species associated with these elements near active genes in embryonic stem cells. Inhibition of transcription from these elements reduces YY1 occupancy. In contrast, tethering of RNA species near YY1 DNA binding sites enhances YY1 occupancy. We propose that RNA acts as trap to maintain certain TFs at active enhancer and promoter-proximal regulatory elements. Thus, transcriptional control generally involves a positive feedback loop, where YY1 and other TFs stimulate local transcription, and newly transcribed nascent RNA reinforces local TF occupancy. This model helps explain why TFs occupy only the small fraction of their consensus motifs in the mammalian genome where transcription is detected. CLIP-Seq for YY1 in mouse embryonic stem cells

2015-10-29 | E-GEOD-68196 | biostudies-arrayexpress

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