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RNAseq and quantitative proteomic analysis of Dictyostelium knock-out cells lacking the core autophagy proteins ATG9 and/or ATG16

ABSTRACT: RNAseq and quantitative proteomic analysis of Dictyostelium knock-out cells lacking the core autophagy proteins ATG9 and/or ATG16

PROVIDER: PRJNA680492 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13125300_1.fastq.gz	Fastqsanger.gz
	SRR13125300_2.fastq.gz	Fastqsanger.gz
	SRR13125301_1.fastq.gz	Fastqsanger.gz
	SRR13125301_2.fastq.gz	Fastqsanger.gz
	SRR13125302_1.fastq.gz	Fastqsanger.gz

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RNAseq and quantitative proteomic analysis of Dictyostelium knock-out cells lacking the core autophagy proteins ATG9 and/or ATG16

Project description:Purpose: Global survey of the transcriptome and comparison to the proteome in autophagy-deficient strains of D. discoideum. Results: Our RNAseq analysis of ATG9‾, ATG16‾ and ATG9‾/16‾ cells revealed major transcriptional changes in a large number of genes in comparison to AX2. Conclusions: Our study provides the first combined transcriptome and proteome analysis of ATG9‾, ATG16‾ and ATG9‾/16‾ cells and adds to our knowledge of functions of these proteins in Dictyostelium discoideum.

2021-05-19 | GSE162070 | GEO

RNAseq and quantitative proteomic analysis of Dictyostelium knock-out cells lacking the core autophagy proteins ATG9 and/or ATG16

Project description:Autophagy is an evolutionary ancient mechanism that sequesters substrates for degradation within autolysosomes. The process is driven by many autophagy-related (ATG) proteins, including the core members ATG9 and ATG16. Here, we applied RNAseq and tandem mass tag (TMT) proteomic approaches to identify differentially expressed genes (DEGs) and proteins (DEPs) in ATG9‾, ATG16‾ and ATG9‾/16‾ strains in comparison to AX2 wild-type cells. In total, we identified 332 (279 up and 53 down), 639 (487 up and 152 down) and 260 (114 up and 146 down) DEGs and 124 (83 up and 41 down), 431 (238 up and 193 down) and 677 (347 up and 330 down) DEPs in ATG9‾, ATG16‾ and ATG9‾/16‾ strains, respectively. Thus, in the single knock-out strains, the number of DEGs was higher than the number of DEPs while in the double knock-out strain the number of DEPs was higher. Comparison of RNAseq and proteomic data further revealed, that only a small proportion of the transcriptional changes were reflected on the protein level. This suggests that most changes in protein abundance were not caused by transcriptional changes but were rather due to changes in protein homeostasis. Gene ontology (GO) analysis revealed an enrichment of DEPs involved in lipid metabolism and oxidative phosphorylation. Furthermore, we found increased expression of the anti-oxidant enzymes glutathione reductase (gsr) and catalase A (catA) in ATG16‾ and ATG9‾/16‾ cells, respectively, indicating adaptation to excess reactive oxygen species (ROS). Our study provides the first combined transcriptome and proteome analysis of ATG9‾, ATG16‾ and ATG9‾/16‾ cells and adds to our knowledge of functions of these proteins.

2021-06-15 | PXD023730 | Pride

RNA<sub>seq</sub> and quantitative proteomic analysis of Dictyostelium knock-out cells lacking the core autophagy proteins ATG9 and/or ATG16.

Project description:BackgroundAutophagy is an evolutionary ancient mechanism that sequesters substrates for degradation within autolysosomes. The process is driven by many autophagy-related (ATG) proteins, including the core members ATG9 and ATG16. However, the functions of these two core ATG proteins still need further elucidation. Here, we applied RNAseq and tandem mass tag (TMT) proteomic approaches to identify differentially expressed genes (DEGs) and proteins (DEPs) in Dictyostelium discoideum ATG9‾, ATG16‾ and ATG9‾/16‾ strains in comparison to AX2 wild-type cells.ResultIn total, we identified 332 (279 up and 53 down), 639 (487 up and 152 down) and 260 (114 up and 146 down) DEGs and 124 (83 up and 41 down), 431 (238 up and 193 down) and 677 (347 up and 330 down) DEPs in ATG9‾, ATG16‾ and ATG9‾/16‾ strains, respectively. Thus, in the single knock-out strains, the number of DEGs was higher than the number of DEPs while in the double knock-out strain the number of DEPs was higher. Comparison of RNAseq and proteomic data further revealed, that only a small proportion of the transcriptional changes were reflected on the protein level. Gene ontology (GO) analysis revealed an enrichment of DEPs involved in lipid metabolism and oxidative phosphorylation. Furthermore, we found increased expression of the anti-oxidant enzymes glutathione reductase (gsr) and catalase A (catA) in ATG16‾ and ATG9‾/16‾ cells, respectively, indicating adaptation to excess reactive oxygen species (ROS).ConclusionsOur study provides the first combined transcriptome and proteome analysis of ATG9‾, ATG16‾ and ATG9‾/16‾ cells. Our results suggest, that most changes in protein abundance were not caused by transcriptional changes, but were rather due to changes in protein homeostasis. In particular, knock-out of atg9 and/or atg16 appears to cause dysregulation of lipid metabolism and oxidative phosphorylation.

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Functional Characterisation of the Core Autophagy Protein ATG5 in Dictyostelium discoideum

Project description:Functional Characterisation of the Core Autophagy Protein ATG5 in Dictyostelium discoideum

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Functional Characterisation of the Ubiquitin-like Core Autophagy Protein ATG12 in Dictyostelium discoideum

Project description:Functional Characterisation of the Ubiquitin-like Core Autophagy Protein ATG12 in Dictyostelium discoideum

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Functional Characterisation of the Core Autophagy Protein ATG5 in Dictyostelium discoideum

Project description:Purpose: Global survey of the transcriptome in autophagy-deficient strains of D. discoideum Results: Our RNAseq analysis of ATG5‾, ATG5‾/12‾ and ATG5‾/12‾/16‾ cells revealed major transcriptional changes in a large number of genes in comparison to AX2 Conclusions: Our study presents a first detailed analysis of the transcriptome in autophagy-deficient strains of D. discoideum. The data were generated by RNA-seq technology. The optimised data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell. We conclude that RNA-seq based transcriptome characterisation would expedite genetic network analyses and permit the dissection of complex biologic functions.

2022-12-13 | GSE142251 | GEO

Transcriptome profile of Dictyostelium cell during ER-stress

Project description:The Unfolded Protein Response (UPR) is an adaptive pathway that restores cellular homeostasis after endoplasmic reticulum (ER) stress caused by an impairment of its protein folding capacity. The ER-resident kinase/ribonuclease Ire1 is the only UPR sensor that has been conserved during evolution from yeast to mammals; in these organisms, Ire1 transmits information from the ER to the nucleus trough the non-conventional splicing of Hac1 (yeast)/Xbp1 (metazoans) mRNA. We described the Dictyostelium discoideum ER-stress response and characterized its single bonafide Ire1 orthologue, IreA. We found that tunicamycin (TN) triggers a gene-expression program that increases the protein folding capacity of the ER and that alleviates ER protein load. Further, IreA resulted essential not only for cell-survival after TN-induced ER-stress, but also to accomplish about nearly 40% of the transcriptional changes induced upon a TN treatment. In addition, we described that autophagy is activated in Dictyostelium cells after a TN treatment and that autophagy-defective mutants exhibited increased sensitivity to this drug. The response of Dictyostelium cells to ER-stress involves the combined activation of an IreA-dependent gene expression program and the autophagy pathway.

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Functional Characterisation of the Ubiquitin-like Core Autophagy Protein ATG12 in Dictyostelium discoideum

Project description:Purpose: First global survey of the transcriptome in autophagy-deficient strains of D. discoideum. Results: Our RNAseq analysis of ATG12‾, ATG16‾ and ATG12‾/16‾ cells revealed major transcriptional changes in a large number of genes in comparison to AX2. Conclusions: Our study represents the first detailed analysis of the transcriptome in autophagy-deficient strains of D. discoideum. The data were generated by RNA-seq technology. The optimised data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell. We conclude that RNA-seq based transcriptome characterisation would expedite genetic network analyses and permit the dissection of complex biologic functions.

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DcgA knock-out experiment in Dictyostelium discoideum

Project description:DcgA knock-out experiment in Dictyostelium discoideum

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