Surface Proteomics of ADAM15 Knock-Down cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Fibroblast
SUBMITTER: 
Stephan Mueller
LAB HEAD: Simone Dario Scilabra
PROVIDER: PXD042402 | Pride | 2023-06-13
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| KD_1_F1.raw | Raw | |||
| KD_1_F2.raw | Raw | |||
| KD_2_F1.raw | Raw | |||
| KD_2_F2.raw | Raw | |||
| KD_3_F1.raw | Raw |
Items per page: 5 1 - 5 of 25 |
Similar Datasets
Project description:Here we aimed to improve native gel electrophoresis-based complexome profiling (CP) method for examination of mitochondrial (mt)DNA-/RNA-protein complexes. We compared the performance of Blue Native PAGE (BNE) and high resolution Clear Native PAGE (hrCNE). We found that hrCNE much better preserves the integrity of mtDNA-/RNA-protein complexes.To further improve the detection of mtDNA-binding proteins, we treated solubilized mitochondrial samples with DNase I prior to separation on hrCNE or BNE. To characterize the detected by hrCNE mtRNA-protein complexes, we performed CP with samples treated with RNase A. To validate the utility of the method, we performed CP using mitochondria isolated from cells treated with ethidium bromide as well as from cells recovering after this treatment. Our study not only helps to validate and unveil proteins involved in mitochondrial DNA- and RNA-related processes, but also offers a convenient and systematic way for analysing these interactions that can be used equally well for the investigation of the nucleus and other DNA-/RNA-containing cell compartments.
2023-08-11 | PXD040103 | Pride
Project description:The cell surface proteome is dynamic and has fundamental roles in cell signaling. Many surface membrane proteins are proteolytically released into a cell’s secretome, where they can have additional functions in cell-cell-communication. Yet, it remains challenging to determine the surface proteome and to compare it to the cell secretome, in particular under serum-containing cell culture conditions. Here, we set-up and evaluated the ‘surface-spanning protein enrichment with click sugars’ (SUSPECS) method for cell surface membrane glycoprotein biotinylation, enrichment and label-free quantitative mass spectrometry. SUSPECS is based on click chemistry-mediated labeling of glycoproteins, is fully compatible with labeling of living cells and can be combined with secretome analyses in the same experiment. Immunofluorescence-based confocal microscopy demonstrated that SUSPECS selectively labeled proteins at the cell surface, but not within cells. Nearly 700 transmembrane glycoproteins were quantified at the surface of primary murine neurons. To demonstrate the utility of SUSPECS, we tested how the protease BACE1, which is a key drug target in Alzheimer’s disease, affects the cell surface glycoproteome. Pharmacological inhibition of BACE1 selectively remodeled the neuronal surface proteome, resulting in up to seven-fold increased abundance of the BACE1 substrates APP, SEZ6, SEZ6L, CNTN2, CHL1 and L1, while other substrates were not or only mildly affected. Protein changes at the cell surface only partly correlated with changes in the secretome. Additionally, apparent non-substrates, such as TSPAN6, were also increased. Several altered proteins were validated by immunoblots in neurons and BACE1-deficient murine brains and indicate that BACE1-inhibition may lead to unexpected secondary effects.
2018-05-08 | PXD008619 | Pride
Project description:Proximity-dependent labelling methods are based on promiscuous labeling enzymes that produce reactive molecules that covalently bind neighbor proteins. Labeled proteins can be then purified and identified using affinity-purification coupled to mass spectrometry methods23. Proximity-dependent biotin identification (BioID)24 uses a promiscuously active Escherichia coli biotin ligase (BirA*) generated by a point mutation (R118G) to biotinylate lysines in nearby proteins within an estimated range of 10 nm25. By fusing BirA* to specific proteins, BioID efficiently identifies interactors at physiological levels in living cells26. It has been extensively used in the Ub field, for instance, to identify substrates of E3 ligases27,28. Recently, a more efficient version of BioID, termed TurboID, has been developed29, being this more suitable for transient protein-protein interaction (PPI) detection. Several studies have developed split-versions and applied “protein fragment complementation” to BioID and TurboID, where proximal biotinylation is dependent on the proximity of the fusion partners, opening new opportunities for spatial and temporal identification of complex-dependent interactomes30,31. To study how SUMOylation and SUMO-SIM interactions can lead to other roles and fates for particular substrates poses particular challenges. SUMOylation occurs transiently and often in a small percentage of a given substrate. Modified proteins can be readily deSUMOylated and SUMO can be recycled and passed to other substrates. SUMO-SIM interactions are also difficult to analyze due to their weak affinity (Kd 1-100 μM). To overcome those technical issues, we developed SUMO-ID, a new strategy based on Split-TurboID to identify interactors of specific substrates dependent on SUMO conjugation or interaction. Using PML as a model, we demonstrate that SUMO-ID can enrich for factors that depend on PML-SUMO interaction. Importantly, the identified proteins are represented among proximal interactors of PML identified using full-length TurboID. We also applied SUMO-ID to a less-characterized SUMO substrate, Spalt Like Transcription Factor 1 (SALL1), and identified both known and novel interactors that depend on intact SUMOylation sites in SALL1. SUMO-ID is thus a powerful tool to study transient and dynamic SUMO-dependent interaction events. The developed methodology is generic and therefore widely applicable in the Ub and UbL field to identify readers of these modifications for individual target proteins to improve our insight in non-covalent signal transduction by Ub and UbL.
2021-09-20 | PXD027759 | Pride
Project description:Plasmodium multigene families are thought to play important roles in the pathogenesis of malaria. Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium species. However, their expression pattern and localisation remain to be elucidated. Protein subcellular localisation is fundamental to be able to elucidate the functional importance and cell-cell interactions of the PIR proteins. Here, we use the rodent malaria parasite, Plasmodium chabaudi chabaudi, as a model to investigate the localisation pattern of this gene family. We found that most PIR proteins are co-expressed in clusters during acute and chronic infection; members of the S7 clade are predominantly expressed during the acute-phase, whereas members of the L1 clade dominate the chronic-phase of infection. Using peptide antisera specific for S7 or L1 PIRS, we show that S7 and L1 PIRs have different localisations within the infected red blood cells. S7 PIRs are exported into the infected red blood cells cytoplasm where they are co-localised with parasite-induced host cell modifications termed Maurer's clefts, whereas L1 PIRs are localised on or close to the parasitophorous vacuolar membrane. This localisation pattern changes following mosquito transmission and during progression from acute- to chronic-phase of infection. However, neither S7 nor L1 PIR proteins detected by the peptide antisera are localised on the surface of infected red blood cells, suggesting that they are unlikely to be targets of surface variant-specific antibodies or be involved directly in adhesion of infected red blood cells to host cells, as described for Plasmodium falciparum VAR proteins. Their presence on Maurer’s clefts, as seen for Plasmodium falciparum RIFIN and STEVOR proteins, might further suggest trafficking of the PIRs on the surface of the infected erythrocytes. The differences in subcellular localisation of the two major clades of Plasmodium chabaudi PIRs across the blood cycle, and the apparent lack of expression on the red cell surface strongly suggest that the function(s) of this gene family may differ from those of other multigene families of Plasmodium, such as the var genes of Plasmodium falciparum.
2022-06-14 | PXD031586 | Pride
Project description:Protein domains of transposable elements (TEs) and viruses increase the protein diversity of host genomes by recombining with other protein domains. By screening 10 million eukaryotic proteins, we identified several domains that define multi-copy gene families and frequently co-occur with TE/viral domains. Among these, a Tc1/Mariner transposase helix turn helix (HTH) domain was captured by F-box genes in the Caenorhabditis genus, creating a new class of F-box genes. For specific members of this class, like fbxa-215, we found that the HTH domain is required for diverse processes including germ granule localisation, fertility, and thermotolerance. Furthermore, we provide evidence that HSF-1 mediates the transcriptional integration of fbxa-215 into the heat-shock response by binding to Helitron TEs directly upstream of its locus. The interactome of HTH-bearing F-box factors suggests roles in post-translational regulation and proteostasis, consistent with established functions of F box proteins. Based on AlphaFold2 multimer proteome-wide screens, we propose that the HTH domain may diversify the repertoire of protein substrates that F-box factors regulate post-translationally. We further demonstrate that F-box genes repeatedly and independently captured TE domains throughout eukaryotic evolution, and describe an additional instance in zebrafish. In conclusion, we identify recurrent TE domain captures by F-box genes in eukaryotes and provide insights into how these novel proteins are integrated within host gene regulatory networks.
2024-12-16 | PXD048290 | Pride
Project description:C. elegans exhibits thermotaxis, where most of the animals that had been cultivated at a particular temperature ranging from 15°C to 25°C for a few hours with a food source and then placed on a thermal gradient for an hour migrate to the cultivation temperature. In addition, animals that were previously conditioned to migrate to a certain temperature are capable of migrating to a new cultivation temperature a few hours after the cultivation temperature was shifted to the new temperature To gain the detailed molecular insight into thermotactic behavior, the genome-wide microarray analysis during behavioral conditioning was performed. We compared the transcriptional profile of animals conditioned to migrate to the new temperature 17°C with that of animals conditioned to migrate to the previous temperature 23°C Synchronized adult wild-type animals cultivated at 23 degrees were selected for RNA extraction and hybridization on Affymetrix microarrays.
2011-06-23 | E-GEOD-28856 | biostudies-arrayexpress
Project description:Though the rhesus monkey is one of the most valuable non-human primate animal models for various human diseases because of its manageable size and genetic and proteomic similarities with humans, proteomic research using rhesus monkeys still remains challenging due to the lack of a complete protein sequence database and sufficient proteomic information. In this project, proteomic profiling of multiple organ tissues, 9 male and 11 female were performed in an automated, high-throughput manner employing annotated UniProtKB human database. Based on the success of this alternative interpretation of MS data, the list of proteins identified from total 12 organs of male and female subjects will benefit future rhesus monkey proteome research.
2015-05-19 | PXD001972 | Pride
Project description:Huh7.5 cells were transfected with HCV-JFH1 RNAand harvested after 48h of transfection. Total proteins were extracted in IP lysis buffer (Pierce, Thermo Scientific) containing 1× halt-protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA) with intermediate vortexing followed by mild sonication. The lysate was centrifuged at 14,000 × g for 30 min at 4 °C. The supernatant was quantified by the BCA method (Thermo Fisher Scientific, USA). An equivalent amount (4 mg) of proteins from each condition was incubated with 3g of anti-HuR antibody for overnight at 4 °C. The immunocomplex was captured by using protein G Sepharose 4 fast flow beads (17-0618-01/ GE Healthcare). The immunoprecipitated complex was washed two times with IP lysis buffer and one time with mili-Q. Bound protein complexes were eluted in 50 µl SDS-PAGE 2X Laemmli sample buffer. The samples were resolved on 12% SDS–PAGE and stained with Coomassie stain (0.1% w/v). The gel was subjected to further in-gel mass spectrometry analysis.
2023-09-16 | PXD035903 | Pride
Project description:The organic acids lactate and diacetate are commonly used in combination in ready-to-eat foods because they show synergistic, i.e. greater than additive, ability to inhibit the growth of Listeria monocytogenes. Full genome microarrays were used to investigate the synergistic transcriptomic response of two L. monocytogenes strains, h7858 (serotype 4b) and f6854 (serotype 1/2a), to organic acid, under conditions controlling for osmotic and cold stress. Strains were exposed to BHI broth at 7°C with 4.65% water phase (w.p.) NaCl at pH 6.1 treated with 2% w.p. potassium lactate, 0.14% w.p. sodium diacetate, the combination of both at the same levels, or no inhibitors as control. RNA was extracted 8h after exposure, during lag phase, to capture gene expression changes during adaptation to the organic acid stress. Treatment with organic acids induced massive global transcriptional changes, with 1041 and 640 genes differentially expressed in h7858 and f6854. Major effects of treatment with lactate and diacetate are (i) a total of 474 and 209 genes, for h7858 and f6854, that showed synergistic expression differences, (ii) differential expression of membrane ion transport genes including those encoding ABC transporters of metals and decreased multi-drug transporter expression many ABC, PTS, and drug transporter systems, including increased PTS sugar transport and decreased multi-drug transporter expression, and (iii) altered metabolism including induction of a nutrient limiting stress response, reduction of menaquinone biosynthesis, and a shift from fermentative production of lactate and acetate and lactate to energetically less favorable, neutral acetoin. These data suggest that additional synergies in L. monocytogenes growth inhibition could be achieved by treatments that interfere with cellular energy generation processes. The dye-swapped, single loop design hybridizes a single biological replicate of both 2% water phase lactate (PL) and 0.14% water phase acetate (SDA) treatments to both the control (CTRL) and combination (PLSDA) treatments (4 hybridizations), using opposite dye labels for each sample, and a second biological replicate is hybridized with dye assignments swapped (4 more hybridizations) to balance labeling effects. The design was repeated twice comprising 16 hybridizations over 4 biological replicates for each strain, and 32 total hybridizations over both h7858 and f6854.
2011-11-03 | E-GEOD-25195 | biostudies-arrayexpress
Project description:Human immunodeficiency virus 1 (HIV-1) infects blood monocytes that cross the blood-brain barrier to the central nervous system inducing neuronal damage. This damage is prompted by the secretion of viral and neurotoxic factors by HIV-infected macrophages and can result in HIV associated neurocognitive disorders (HAND). One of these neurotoxic factors secreted by HIV-infected macrophages is cathepsin B (CATB), a lysosomal cysteine protease that plays an important role in neurodegeneration. CATB interacts with Serum Amyloid P component (SAPC) contributing to HIV-induced neurotoxicity. However, the neuronal apoptosis pathways triggered by CATB and SAPC remain unknown. We aimed to elucidate these pathways in neurons exposed to HIV-infected macrophage conditioned media (MCM) before and after inhibition of CATB or SAPC using Tandem Mass Tag (TMT) proteomics labeling. Based on significant fold change (FC) ≥ ǀ2ǀ and p-value < 0.05 criteria, a total of 10, 48 and 13 proteins were deregulated after inhibiting CATB, SAPC antibodies and the cathepsin B inhibitor CA-074, respectively. We found that antibodies against CATB and SAPC, as well as the CATB inhibitor CA-074 downregulated apoptosis related proteins. CATB, SAPC or apoptotic related proteins could become potential targets against HIV-induced neuronal degeneration.
2022-01-20 | PXD024636 | Pride