Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Metabolic labeling of NMT substrates by photoactivatable/clickable myristate probes X3/X8/X10

ABSTRACT: Efficiency of labelling by photoactivatable/clickable myristate probes X3/X8/X10 is compared to the positive control probe YnMyr. Additionally, negative controls for each probe were obtained by treatment with NMT ihibitor IMP-366 (DDD85464). The resulting data indicates efficient labeling of NMT substrates by photoactivatable probes that is NMT-dependent.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Roman Fedoryshchak

LAB HEAD: Edward Tate

PROVIDER: PXD027239 | Pride | 2023-03-11

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	Perseus1553.rar	Other
	Results_-_MaxQuant_txt_folder.rar	Other
	X10-I-a.raw	Raw
	X10-I-b.raw	Raw
	X10-I-c.raw	Raw

Items per page:

1 - 5 of 28

Publications

Discovery of lipid-mediated protein-protein interactions in living cells using metabolic labeling with photoactivatable clickable probes.

Fedoryshchak Roman O RO Gorelik Andrii A Shen Mengjie M Shchepinova Maria M MM Pérez-Dorado Inmaculada I Tate Edward W EW

Chemical science 20230130 9

Protein-protein interactions (PPIs) are essential and pervasive regulatory elements in biology. Despite the development of a range of techniques to probe PPIs in living systems, there is a dearth of approaches to capture interactions driven by specific post-translational modifications (PTMs). Myristoylation is a lipid PTM added to more than 200 human proteins, where it may regulate membrane localization, stability or activity. Here we report the design and synthesis of a panel of novel photocros ...[more]

PMID: 36873846

Similar Datasets

Identification of protein-protein interaction mediated by N-myristoylated proteins using photocrosslinkable clickable myristoylation probes X3 and X10

Project description:HeLa cells were treated with myristoylation probes X3 and X10, which incorporate both alkyne and diazirine moieties. Cells were then treated with NMT ihibitor IMP-366 (DDD85464) or DMSO vehicle and UV-irradiated before lysis. The experiment identifies myristoylated proteins through comparison of vehicle and NMT inhibitor treated samples. A number of non-myristoylated proteins were enriched together with myristoylated proteins indicating possible photocrosslinked protein interactors.

2023-02-04 | PXD029944 | Pride

Validation and invalidation of chemical probes for the human N-myristoyltransferases.

Project description:On-target, cell-active chemical probes are of fundamental importance in both chemical and cell biology, whereas the application of poorly-characterised probes often leads to invalid conclusions. Human N-myristoyltransferase (NMT) has attracted increasing interest as a target in cancer and infectious diseases; here we report an in-depth comparison of five compounds widely applied as human NMT inhibitors, using a combination of quantitative whole-proteome N-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis and cell cycle assays. We find that N-myristoylation is unaffected by 2-hydroxymyristic acid (100 μM), D-NMAPPD (30 μM) or Tris-DBA palladium (10 μM), with the latter compounds causing cytotoxicity through mechanisms unrelated to NMT. In contrast, drug-like inhibitors IMP 366 and IMP 1088 delivered complete and specific inhibition of N-myristoylation in a range of cell lines at 1 μM and 100 nM, respectively. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies which used off-target compounds.

2019-11-14 | PXD012722 | Pride

Profiling N-myristoyltransferase activity and inhibition in breast cancer cells (MD-MB-231) using Sortase-A labelling

Project description:We report a novel application for Sortase A (SrtA), labeling N-terminal Gly proteins across the proteome at endogenous levels. We combined SrtA labeling of whole-cell lysates with a potent and selective NMT inhibitor, delivering a method capable of quantifying changes in cellular NMT activity in multiple substrates at the whole proteome level. This method shows good overlap in substrates with a previously reported YnMyr metabolic tagging strategy (PXD002687), and a similar capability to measure cellular NMT activity. The SrtA-based approach described here is complementary to YnMyr metabolic tagging, since the increase in signal for newly synthesized proteins exposing an N-terminal glycine mirrors the loss of signal seen with YnMyr. Importantly, this new method overcomes the limitations imposed by metabolic tagging. Since SrtA labeling is performed post-lysis this labeling strategy provides a readout of the cell status in the absence of any external perturbation and is applicable to virtually any type of biological sample, giving clear advantages over other in-cell labeling strategies. We anticipate that this method will enable the exploration of new avenues for NMT as a therapeutic target by providing a method for de novo identification of substrates most responsive to NMT inhibition, and a multifuctional target engagement biomarker assay suited to both animal models and patient biopsies. Besides shotgun proteomics, we coupled this robust NMT activity assay to a variety of other detection techniques, including gel-based analyses, and ELISA. This submission concerns myristoylated proteomes of human origin.

2018-10-25 | PXD010634 | Pride

Co-translational and Post-translational protein myristoylation in HeLa cells

Project description:Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. In E. Thinon, R. A. Serwa, et al., Nature Communications 2014, doi:10.1038/ncomms5919, we report the global N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells.

2018-07-31 | PXD002687 | Pride

Identification of myristoylated proteins in zebrafish (Danio rerio)

Project description:Multi-functional capture reagents reported here enable robust identification of metabolically-tagged myristoylated proteomes with unprecedented confidence resulting from the combination of chemical probe-based enrichment, and release and direct detection of lipid-modified peptides by MS. Whilst capture reagents containing enzymatically cleavable linkers have been previously reported they typically require an extra proteolytic step and their capacity to enable detection of lipidated peptides has not been demonstrated. Herein, we report the largest database (85 counts) of experimentally validated human proteins that are myristoylated at an endogenous level in living cells. Furthermore, we demonstrate the first profile of myristoylation in a living multicellular organism and the confident identification of over 50 novel targets. Importantly, this is also the first example of analysis of any protein lipidation event during development. Our methodology is novel in analytical/chemical approach, and provides quantitative and dynamic information. This submission concerns myristoylated proteomes of zebrafish origin.

2015-03-30 | PXD001876 | Pride

Identification of myristoylated proteins in Human cell lines (HEK293, HeLa, MCF7)

2015-03-30 | PXD001863 | Pride

Identification of protein-protein interactions in live cells using photoactivatable myristate probe X10

Project description:19 TST-tagged myristoylated proteins (baits) were transfected into cells and labeled with photoactivatable clickable myristate probe X10. mCitrine-TST was used as a negative control. X10-labeled proteins were photocrosslinked in live cells to their interacting partners with the aim of identifying new PTM-mediated protein-protein interactions. Proteins enriched with a certain TST-tagged bait were considered interactors when compared to the presence in all other baits, with statistical significance FDR<0.05.

2023-02-04 | PXD027394 | Pride

Global profiling of myristoylation in Toxoplasma gondii

Project description:N-myristoylation is a ubiquitous class of protein lipidation across eukaryotes and N-myristoyl transferase (NMT) has been proposed as an attractive drug target in several pathogens. Here we describe the first global chemoproteomic analysis of protein myristoylation in Toxoplasma gondii. Through quantitative mass spectrometry coupled with validated chemoproteomic tools (cleavable capture reagents (Broncel et al., 2015, Speers and Cravatt, 2005) and NMT inhibitor (Schlott et al., 2019)) that allow for experimental validation, we confidently identified 65 myristoylated proteins. This dataset represents a large fraction of the parasite’s myristoylated proteome and a prerequisite to investigate this modification in Toxoplasma.

2020-07-06 | PXD019677 | Pride

N-Myristoyltransferase inhibition in HeLa

Project description:N-Myristoyltransferase (NMT) covalently attaches a C14-fatty acid to the N-terminal glycine of proteins and has been proposed as a therapeutic target in cancer. We used quantitative proteomics to map protein expression changes for more than 2700 proteins in response to treatment with an NMT inhibitor in HeLa cells, and observed down-regulation of proteins involved in cell cycle regulation, and up-regulation of many proteins involved in the endoplasmic reticulum stress response. This study defines the cellular response to NMT inhibition at the proteome level, and provides a knowledgebase for targeting specific cancers with NMT inhibitors, potentially in combination with other targeted agents.

2016-06-13 | PXD003186 | Pride

Global profiling and inhibition of protein lipidation in vector and host stages of the sleeping sickness parasite Trypanosoma brucei via chemical proteomics

Project description:The enzyme N-myristoyltransferase (NMT) catalyses the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localisation of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labelling with an alkyne-tagged myristic acid analogue (“YnMyr”), enabling the capture of lipidated proteins in insect (PCF) and host (BSF) life stages of T. brucei. We further compare this with a longer chain palmitate analogue (“YnPal”) to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors (Cpds 1 and 2) and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites.

2016-05-17 | PXD004053 | 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