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RSK feedback modulates initiation of embryonic stem cell differentiation

ABSTRACT: In this study we use a SILAC based quantitative proteomics approach to examine the ERK-responsive phosphoproteome in mouse embryonic stem cells.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Embryonic Stem Cell

SUBMITTER: Isabelle Nett

LAB HEAD: Kathryn Lilley

PROVIDER: PXD003247 | Pride | 2018-06-18

REPOSITORIES: pride

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			Action	DRS
	MQ_N1_output_file.rar	Other
	MQ_S1_output_file.rar	Other
	SN_0808_SCX1.raw	Raw
	SN_0808_SCX10.raw	Raw
	SN_0808_SCX11.raw	Raw

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Publications

Negative feedback via RSK modulates Erk-dependent progression from naïve pluripotency.

Nett Isabelle Re IR Mulas Carla C Gatto Laurent L Lilley Kathryn S KS Smith Austin A

EMBO reports 20180612 8

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signalling is implicated in initiation of embryonic stem (ES) cell differentiation. The pathway is subject to complex feedback regulation. Here, we examined the ERK-responsive phosphoproteome in ES cells and identified the negative regulator RSK1 as a prominent target. We used CRISPR/Cas9 to create combinatorial mutations in <i>RSK</i> family genes. Genotypes that included homozygous null mutations in <i>Rps6ka1, ...[more]

PMID: 29895711

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Coisolation of peptide pairs for peptide identification and MS/MS-based quantification

Project description:SILAC-based metabolic labeling is a widely adopted proteomics approach that enables quantitative comparisons among a variety of experimental conditions. Despite its quantitative capacity, SILAC experiments analyzed with data dependent acquisition (DDA) do not fully leverage peptide pair information for identification and suffer from undersampling compared to label-free proteomic experiments. Herein, we developed a data dependent acquisition strategy that coisolates and fragments SILAC peptide pairs and uses y-ions for their relative quantification. To facilitate the analysis of this type of data, we adapted the Comet sequence database search engine to make use of SILAC peptide paired fragments and developed a tool to annotate and quantify MS/MS spectra of coisolated SILAC pairs. In an initial feasibility experiment, this peptide pair coisolation approach generally improved expectation scores compared to the traditional DDA approach. Fragment ion quantification performed similarly well to precursor quantification in the MS1 and achieved more quantifications. Lastly, our method enables reliable MS/MS quantification of SILAC proteome mixtures with overlapping isotopic distributions, which are difficult to deconvolute in MS1-based quantification. This study demonstrates the initial feasibility of the coisolation approach. Coupling this approach with intelligent acquisition strategies has the potential to improve SILAC peptide sampling and quantification.

2022-11-03 | PXD033016 | Pride

Pseudouridylation of tRNA-derived fragments steers translational control in stem cells

Project description:Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ ‘writer’ PUS7 modifies and activates a network of tRNA-derived fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translational regulation leading to increased protein biosynthesis and abnormal germ layer specification. Remarkably, dysregulation of PUS7 and tRFs in myeloid malignancies associates with altered translation rates, suggesting a role of Ψ in leukemogenesis. Our findings unveil a critical function of Ψ in directing translational control in stem cells with important implications for human disease.

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Project description:Identification of ncRNA differentially expressed in embryonic stem cells (ESCs) and the neuronal/glia cells differentiated

2018-01-01 | E-MTAB-6391 | biostudies-arrayexpress

Analysis of endogenous NOTCH1 from POFUT1 S162L patient fibroblasts reveals the importance of the O-fucose modification on EGF12 in human development

Project description:NOTCH1 (N1) is a transmembrane receptor interacting with membrane-tethered ligands on opposing cells that mediate the direct cell-cell interaction necessary for many cell fate decisions. Protein O-fucosyltransferase 1 (POFUT1) adds O-fucose to Epidermal Growth Factor (EGF)-like repeats in the NOTCH extracellular domain, which is required for signaling activation and trafficking. We previously showed that POFUT1 S162L caused a 90% loss of POFUT1 activity and global developmental defects in a patient; however, the mechanism by which POFUT1 contributes to these symptoms is still unclear. Compared to controls, POFUT1 S162L patient fibroblast cells had an equivalent amount of N1 on the cell surface but showed a 60% reduction of DLL1 ligand binding and a 70% reduction in JAG1 ligand binding. To determine if the reduction of O-fucose on N1 in POFUT1 S162L patient fibroblasts is the cause of these effects, we immunopurified endogenous N1 from control and patient fibroblasts and analyzed O-fucosylation using mass spectral glycoproteomics methods. N1 EGF8 to EGF12 comprise the ligand binding domain, and O-fucose on EGF8 and EGF12 physically interact with ligands to enhance affinity. Glycoproteomics of N1 from POFUT1 S162L patient fibroblasts showed WT fucosylation levels at all sites analyzed except for a large decrease at EGF9 and the complete absence of O-fucose at EGF12. Since the loss of O-fucose on EGF12 has significant effects on N1 activity, this may explain the symptoms observed in the POFUT1 S162L patient.

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Synapse integrity and function: dependence on protein synthesis and identification of potential failure points

Project description:Synaptic integrity and function depend on myriad proteins - labile molecules with finite lifetimes that need to be continually replaced with freshly synthesized copies. Here we describe experiments designed to expose synaptic (and neuronal) properties and functions that are particularly sensitive to disruptions in protein supply, identify proteins lost early upon such disruptions, and uncover potential, yet currently underappreciated failure points. We report here that acute suppressions of protein synthesis are followed within hours by reductions in spontaneous network activity levels, impaired oxidative phosphorylation and mitochondrial function, reduced tenacity of both excitatory and inhibitory synapses and loss of synapses of both classes. Conversely, gross impairments in presynaptic vesicle recycling are observed only after several days, as is overt cell death. Proteomic analysis identified groups of potentially essential ‘early-lost’ proteins including regulators of synapse stability, proteins related to bioenergetics, fatty acid and lipid metabolism, and, unexpectedly, numerous proteins involved in Alzheimer’s disease pathology and amyloid beta processing.

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