Dataset Information

AMpylation is a hallmark of human neuronal differentiation

ABSTRACT: The AMPylation is novel protein post-translation modification. In consist of attachment of the adenosine monophosphate onto the serine, threonine or tyrosine amino acid residues of the protein. This process is catalysed by bacterial effectors or in human by protein FICD (also called HYPE) which contain conserved fic domain. In our study we have focused on identification of the unknown AMPylated proteins and their function in human cells (HeLa, A549, SH-SY5Y, iPSCs, NPCs and neurons) and cerebral organoids (COs). For this purpose we designed and synthesized the small molecule N6-propargyl phosphoramidate adenosine (PAK-76) which was used for in situ labelling of the AMPylation in cells and COs. Subsequnetly the the propargyl tag was employed for preparation of chemical-proteomic samples which were measured by LC-MS/MS. This approach confirmed the known AMPylation of heat shock protein HSPA5 and found diverse group of novel AMPylation targets. Several cathepsins found as AMPylation targets were further characterized by identification of AMP binding site. Based on our chemical proteomic data we found out that neurons and neuronal like cells contain distinct AMPylatino pattern from other studied cell types. This also suggested the importance of the AMPylation in these cell types which were then studied in cerebral organoids by overexpressing the FICD wt and E234G highly active mutant. This led to the accelerated differentiation of progenitors into the neurons. Overall our approach is suitable for identification of the AMPylated proteins in living cells and led to characterization of FICD function.

INSTRUMENT(S): Orbitrap Fusion ETD, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture, Hela Cell

SUBMITTER: Pavel Kielkowski

LAB HEAD: Stephan Axel Sieber

PROVIDER: PXD008394 | Pride | 2024-05-21

REPOSITORIES: Pride

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Publications

Single-Cell RNA Sequencing Reveals Cardiac Fibroblast-Specific Transcriptomic Changes in Dilated Cardiomyopathy.

Russell-Hallinan Adam A Cappa Oisín O Kerrigan Lauren L Tonry Claire C Edgar Kevin K Glezeva Nadezhda N Ledwidge Mark M McDonald Kenneth K Collier Patrick P Simpson David A DA Watson Chris J CJ

Cells 20240426 9

Dilated cardiomyopathy (DCM) is the most common cause of heart failure, with a complex aetiology involving multiple cell types. We aimed to detect cell-specific transcriptomic alterations in DCM through analysis that leveraged recent advancements in single-cell analytical tools. Single-cell RNA sequencing (scRNA-seq) data from human DCM cardiac tissue were subjected to an updated bioinformatic workflow in which unsupervised clustering was paired with reference label transfer to more comprehensiv ...[more]

PMID: 38727290

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Project description:Abstract: Chemogenomic fitness assays were combined with a transcriptome analysis to understand both the mode of action and the mechanisms of resistance to Chitosan oligosaccharides (COS). COS are deacetylated chitin compounds, with antimicrobial properties, that are presumed to act by disrupting the cell membrane. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified encode membrane proteins and members of the protein degradation/ proteosome pathway. The transcriptomes of wild-type and five suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and furthermore, treatment with environmental stressors does not provide resistance to COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the RAS signal transduction pathway. Down-regulated transcripts included those encoding protein folding components, and respiratory chain proteins. Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to amphotericin B, fluconazole and terbinafine as the wild-type (vector control). The gene targets of COS identified in this study suggest that COSM-bM-^@M-^Ys mechanism of action is different from other commonly studied fungicides, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens. We selected the 5 overexpressing strains based on the characteristics of the genes. ARL1, encoding a GTPase and is involved in membrane trafficking, was selected since it was observed in the HIP-HOP assay as a sensitive homozygous deletion strain and in the MSP assay as multicopy suppressor. The rest of selected overexpressing strains were BCK2 and MSG5, involved in cell integrity pathways, ERG24 in ergosterol synthesis, and RBA50 in transcription. BCK2 is a Ser-Thr rich protein with protein kinase C activity that acts in signal transduction. Overexpression of BCK2 can rescue defects in yeast cwh43M-NM-^T, which displays several cell wall defects [29]. BCK2 overexpression also can suppress a cell lysis defect of mpk1M-NM-^T and pck1M-NM-^T [30]. MSG5 is also involved in signal transduction. This gene encodes a protein phosphatase involved in cell cycle control through the dephosphorylation of MAPK and is indispensable for restricting the signaling by the cell integrity pathway in yeast [31]. The inhibition of MAPK signaling leads to inhibition of cell differentiation and cell division [32]. The functions of ARL1 and ERG24 and their potential roles in chitosan resistance will be described in more detail in the Discussion. To gain a further understanding on the mode of action and mechanisms of resistance to COS, we performed a transcriptome analysis of the above mentioned five overexpressing strains known to increase resistance to COS-5.44. Each overexpressing strain and the wild type (vector control) were treated with COS-5.44 and RNAs isolated from both treated and untreated cells. The RNAs were converted to labeled cDNA and hybridized to NimbleGen expression microarrays (see Methods). Three cDNA biological replicates either with or without a 60 minutes exposure to COS-5.44 for each of the five overexpressing strains as well as an untransformed wild type BY4743 cells (vector control; for a total of 36 samples) were hybridized to NimbleGen 4X72k microarrays (Roche NimbleGen, Inc. Design ID A6186-00-01, TI4932 60mer expr X4).

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