Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Ubiquiton – an inducible, linkage-specific polyubiquitylation tool

ABSTRACT: Here we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called ‘Ubiquiton’ (Ubo) system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. SILAC-based and label-free quantitative proteomics experiments were performed to verify the linkages of the polyubiquitin chains produced on a model substrate, GFP, in yeast and to identify potential off-target effects of a transgenic E3 system in human cells.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Culture

SUBMITTER: Jia-Xuan Chen

LAB HEAD: Helle Ulrich

PROVIDER: PXD046526 | Pride | 2023-12-15

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Ubiquiton – an inducible, linkage-specific polyubiquitylation tool

Project description:Here we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called ‘Ubiquiton’ (Ubo) system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. SILAC-based and label-free quantitative proteomics experiments were performed to verify the linkages of the polyubiquitin chains produced on a model substrate, GFP, in yeast and to identify potential off-target effects of a transgenic E3 system in human cells.

2023-12-15 | PXD045662 | Pride

Characterization of the acetylome associated with rapamycin-induced autophagy in HeLa cells

Project description:Autophagy is a lysosomal-mediated catabolic process that degrades cytoplasmic components to help maintain cellular homeostasis and cell survival during exposure to stress. Lysine acetylation is a reversible post-translational modification that plays an important role in the regulation of diverse cellular processes.

2019-01-02 | PXD005793 | Pride

Global, site-resolved analysis of ubiquitylation occupancy and turnover rate reveals systems properties

Project description:Ubiquitylation regulates most proteins and biological processes in a eukaryotic cell. However, the site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have not been quantified. Here we present an integrated picture of the global ubiquitylation site occupancy and half-life. Ubiquitylation site occupancy spans over four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and regulation of sites by proteasome inhibitors are strongly interrelated, and these attributes distinguish sites involved in proteasomal degradation and cellular signaling. Sites in structured protein regions exhibit longer half-lives and stronger upregulation by proteasome inhibitors than sites in unstructured regions. Importantly, we discovered a surveillance mechanism that rapidly and site-indiscriminately deubiquitylates all ubiquitin-specific E1 and E2 enzymes, protecting them against accumulation of bystander ubiquitylation. The work provides a systems-scale, quantitative view of ubiquitylation properties and reveals general principles of ubiquitylation-dependent governance.

2024-04-15 | PXD037009 | Pride

Phosphoproteome analysis reveals the importance of Greatwall-endosulfine pathway to regulate cell-cycle progression upon the rapamycin-induced autophagy

Project description:Protein phosphorylation plays an important role in the process of autophagy. Autophagy is associated with cell cycle, which is controlled by the balance between the cyclin-dependent kinases and phosphatases activities. However, up to now, the signal transduction pathways that underlied rapamycin-induced autophagy and cell cycle have remained elusive. Here, we used stable isotope labeling of amino acids in cell culture and high-throughput quantitative mass spectrometry to perform a global proteome and phosphoproteome analysis of rapamycin-induced autophagy in vitro. By monitoring proteome and phosphoproteome alterations with rapamycin treatment, we detected downregulation of mTOR signaling pathway and confirmed the occurrence of autophagy. Additionally, spliceosome and nuclear pore complexes were enriched to be regulated, which further validated autophagy as a stress response itself. More importantly, we discovered that mTORC1 could control cell cycle progression though Greatwall-endosulfine-PP2A pathway in HeLa cells. Firstly, we found the elevated profile of cell cycle-related substrates and determined the enrichment of CDK1, MAPK1 and MAPK3 kinases, which implied the activation of these kinases. Secondly, Pathway interrogation using kinase inhibitor treatment confirmed the effect of mTOR inhibition on CDK1 activity and cell cycle progression. Thirdly, we discovered that Greatwall-endosulfine complex was required to mediate mTOR-mediated cell-cycle progression. In conclusion, we presented a high-confidence map of phosphoproteomic of autophagy and unveiled the Greatwall-endosulfine pathway to regulate cell-cycle progression in the rapamycin-induced autophagy.

2021-04-06 | PXD014737 | Pride

Phase separation and stress granule association of SARS-CoV-2 Nucleocapsid protein

Project description:The current work identified the protein interactors of SARS-CoV-2 Nucleocapsid RNA binding protein (NCAP or N protein) in lung cancer A-549 cells, using a co-immunoprecipitation strategy, which is coupled with SILAC-based mass spec. A significant proportion of the identified NCAP interacting proteins included stress granule (SG) and immunoregulators. SG nucleator G3BP1 showed specific interaction wit NCAP in unstressed and oxidative- or heat-stressed cells. Following stress treatment, NCAP showed enhanced association with SGs. Recombinant NCAP exhibited in vitro liquid-liquid phase separation (LLPS)to form liquid droplets. RNA stimulated NCAP to develop droplets in vitro and SG assembly in cells, and RNase A treatment completely blocked both of these functions. An RNA intercalating mitoxantrone disrupted NCAP assembly in SGs invitro and in cells. This study provides insight into the biological processes and biophysical properties of the SARS-CoV-2 NCAP and identifies a candidate way to target this protein.

2021-11-25 | PXD023989 | Pride

Ligand-induced disorder-to-order transitions characterized by structural proteomics and molecular dynamics simulations

Project description:We have studied both the Spy and the FKBP protein-ligand systems by structural proteomics and molecular -dynamics simulations. We have used hydrogen/deuterium exchange, differential crosslinking, and surface modification to characterize the conformational changes that occur upon both peptide binding (Im7 with Spy) and small molecule binding (rapamycin with FKBP) to the protein. We have shown that, in both cases, the proteins are considerably disordered but their structures are different from the unfolded structure obtained with 8M urea in solution, and both proteins undergo a dramatic increase in secondary structure content upon ligand binding.

2021-09-08 | PXD015275 | Pride

The functional interactome of site-specifically ubiquitylated linker histone H1

Project description:Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. While this process is well studied for core histones and many of their PTMs, this is not the case for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the generation of site-specifically mono-ubiquitylated H1.2 via click chemistry and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that the H1 interactome is generally modulated by ubiquitylation and that site-specific ubiquitylation results in overlapping, but distinct interactomes. We further demonstrate that site-specific ubiquitylation of H1 affects the interaction with enzymes relevant for deubiquitylation and deacetylation. We finally show that site-specific ubiquitylation at position K64 impacts H1-dependent chromatosome assembly as well as H1-induced phase separation. In summary, we demonstrate that site-specific ubiquitylation is a general functional regulator for linker histone H1.

2021-07-20 | PXD020686 | Pride

Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals (single embryo)

Project description:Parental dietary conditions can influence the metabolic traits of offspring. In mice, paternal consumption of low protein diet alters cholesterol and lipid metabolism of progeny. Here, we examine RNA species expressed in male reproductive tissues of mice. Protein restriction leads to altered levels of multiple small RNAs in mature sperm, as well as throughout the male reproductive tract, with decreased levels of let-7 family members and increased levels of 5â?? fragments of tRNA-Gly isoacceptors. Intriguingly, tRNA fragments are scarce in the testis, but their levels increase in sperm during post-testicular maturation in the epididymis. We find that epididymosomes â?? extracellular vesicles which fuse with sperm during epididymal transit â?? exhibit RNA payloads closely matching those of mature sperm, and can deliver tRNA fragments to immature sperm in vitro both in mouse and in bull. Finally, we show that tRNA-Gly-GCC fragments play a role in repressing genes associated with the endogenous retroelement MERVL, both in ES cells and in preimplantation embryos. Our results shed light on small RNA biogenesis during post-testicular sperm maturation, and link tRNA fragments to regulation of endogenous retroelements active in the early embryo. Zygotes were generated by IVF from animals fed a control diet. These embryos were then microinjected with various combinations of small RNAs and control RNA (HIS3.3::GFP). Follwoing injections the zygotes were developed and allowed to develop until 2 cell (2C) or 4 cell (4C) stage when single embryo RNA seq was carried out to study gene expression changes

2015-12-31 | E-GEOD-75765 | biostudies-arrayexpress

Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα

Project description:Progenitor cells at the basal layer of skin epidermis play an essential role in maintaining tissue homeostasis and enhancing wound repair in skin. The proliferation, differentiation, and cell death of epidermal progenitor cells have to be delicately regulated, as deregulation of this process can lead to many skin diseases, including skin cancers. However, the underlying molecular mechanisms involved in skin homeostasis remain poorly defined. In this study, with quantitative proteomics approach, we identified an important interaction between KDF1 (Keratinocyte Differentiation Factor 1) and IKKα (IκB kinase α) in differentiating skin keratinocytes. Ablation of either KDF1 or IKKα in mice leads to similar but striking abnormalities in skin development, particularly in skin epidermal differentiation. With biochemical and mouse genetics approach, we further demonstrate that the interaction of IKKα and KDF1 is essential for epidermal differentiation. To probe deeper into the mechanisms, we find that KDF1 associates with a deubiquitinating protease, USP7 (Ubiquitin Specific Peptidase 7), and KDF1 can regulate skin differentiation through deubiquitination and stabilization of IKKα. Taken together, our study unravels an important molecular mechanism underlying skin tissue homeostasis and epidermal differentiation.

2020-05-13 | PXD015673 | Pride

p97/VCP targets Toxoplasma gondii vacuoles for parasite restriction in interferon-stimulated cells

Project description:Infection with the parasite Toxoplasma gondii leads to production of interferon gamma (IFN) that stimulates cells to upregulate defence proteins targeting the parasite for cell intrinsic elimination or growth restriction. Various host defence mechanisms operate at the parasitophorous vacuole (PV) in different human cell types leading to PV disruption, acidification, or membrane envelopment. Ubiquitin and p62 are players in all human host control mechanisms of Toxoplasma, but other unifying proteins have not been identified. Here, we show that p97/valosin-containing protein (VCP), as well as its associated proteins ANKRD13A and UBXD1 control Toxoplasma infection while recruited to the PV in IFN-stimulated endothelial cells. Convergent deposition of ANKRD13A, p97/VCP and UBXD1 onto the same vacuole is dependent on vacuolar ubiquitination and observed within 2h post-infection. ANKRD13A, p97/VCP and UBXD1 all drive the acidification mechanism of the vacuole, which is the IFN-dependent control pathway of Toxoplasma in endothelial cells. We assessed p97/VCP in Toxoplasma control in various human cells and demonstrate that p97/VCP is a universal IFN-dependent host restriction factor targeting the Toxoplasma PV in epithelial (HeLa) and endothelial cells (HUVEC), fibroblasts (HFF) and macrophages (THP1).

2023-11-15 | PXD042937 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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