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MS-analysis of pull downs to elucidate putative transcriptional regulators from phasin and depolymerase genes

ABSTRACT: LC-MS/MS analysis of promoter pull-down assays with crude protein extracts from R. eutropha Re2058/pCB113, to identify putative transcriptional regulators involved in the expression control of PHA metabolism, specifically targeting phasin phaP1 and depolymerase phaZ3 and phaZ5 genes.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Cupriavidus Necator (strain Atcc 17699 / H16 / Dsm 428 / Stanier 337) (ralstonia Eutropha)

SUBMITTER: Christian Forbrig

LAB HEAD: Juri Rappsilber

PROVIDER: PXD050193 | Pride | 2024-10-17

REPOSITORIES: Pride

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			Action	DRS
	B220113_19_HF_AS_CO_80_RH-P1_24h.raw	Raw
	B220113_20_HF_AS_CO_80_RH-P1_72h.raw	Raw
	B220113_23_HF_AS_CO_80_RH-Z3_24h.raw	Raw
	B220113_24_HF_AS_CO_80_RH-Z3_72h.raw	Raw
	B220113_27_HF_AS_CO_80_RH-Z5_24h.raw	Raw

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1 - 5 of 23

Publications

Elucidating regulation of polyhydroxyalkanoate metabolism in Ralstonia eutropha: Identification of transcriptional regulators from phasin and depolymerase genes.

Santolin Lara L Eichenroth Rosalie Sandra Josianne RSJ Cornehl Paul P Wortmann Henrike H Forbrig Christian C Schulze Anne A Haq Inam Ul IU Brantl Sabine S Rappsilber Juri J Riedel Sebastian Lothar SL Neubauer Peter P Gimpel Matthias M

The Journal of biological chemistry 20240704 8

Despite the ever-growing research interest in polyhydroxyalkanoates (PHAs) as green plastic alternatives, our understanding of the regulatory mechanisms governing PHA synthesis, storage, and degradation in the model organism Ralstonia eutropha remains limited. Given its importance for central carbon metabolism, PHA homeostasis is probably controlled by a complex network of transcriptional regulators. Understanding this fine-tuning is the key for developing improved PHA production strains thereby ...[more]

PMID: 38969063

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2017-07-07 | PXD006347 | Pride

Duplicate genes and robustness to transient gene knock-downs in Caenorhabditis elegans.

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Primate jumping genes elucidate strepsirrhine phylogeny.

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HaploReg v4: systematic mining of putative causal variants, cell types, regulators and target genes for human complex traits and disease.

Project description:More than 90% of common variants associated with complex traits do not affect proteins directly, but instead the circuits that control gene expression. This has increased the urgency of understanding the regulatory genome as a key component for translating genetic results into mechanistic insights and ultimately therapeutics. To address this challenge, we developed HaploReg (http://compbio.mit.edu/HaploReg) to aid the functional dissection of genome-wide association study (GWAS) results, the prediction of putative causal variants in haplotype blocks, the prediction of likely cell types of action, and the prediction of candidate target genes by systematic mining of comparative, epigenomic and regulatory annotations. Since first launching the website in 2011, we have greatly expanded HaploReg, increasing the number of chromatin state maps to 127 reference epigenomes from ENCODE 2012 and Roadmap Epigenomics, incorporating regulator binding data, expanding regulatory motif disruption annotations, and integrating expression quantitative trait locus (eQTL) variants and their tissue-specific target genes from GTEx, Geuvadis, and other recent studies. We present these updates as HaploReg v4, and illustrate a use case of HaploReg for attention deficit hyperactivity disorder (ADHD)-associated SNPs with putative brain regulatory mechanisms.

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The effect of multiple phaJ deletions Pseudomonas putida KT2440 on polyhydroxyalkanoates synthesis (part2)

Project description:Pseudomonas putida KT2440 is a well-known model organism for the medium chain length (mcl) PHA accumulation. (R)-Specific enoyl-coenzyme A hydratase (PhaJ) was considered to be the main supplier of monomers for PHA synthesis by converting the -oxidation intermediate, trans-2-enoyl-CoA to (R)-3-hydroxyacyl-CoA when fatty acids (FA) are used. Three PhaJ homologues, PhaJ1, PhaJ4 and MaoC are annotated in P. putida KT2440. To investigate the relationship of fatty acids - PHA metabolism and the role of each PhaJ in PHA biosynthesis in P. putida KT2440, a series of P. putida KT2440 knockouts was obtained. PHA content and monomer composition in WT and mutants under different growth conditions were analysed. However, when all three PhaJ homologues were deleted, the mutant still accumulated PHA up to 10.7 % of the cell dry weight (CDW). To identify other potential PHA monomer suppliers by analysing the proteome of the phaJ1maoCphaJ4. The deletion of (R)-3-hydroxydecanoyl-ACP:CoA transacylase (PhaG), which connects de novo FA and PHA synthesis pathways, while causing further 1.8-fold decrease in PHA content, did not abolish PHA accumulation. Further proteome analysis revealed quinoprotein alcohol dehydrogeanses PedE and PedH as potential monomer suppliers, but when these were deleted PHA level remained at 2.2 – 14.8 % CDW depending on the fatty acid used, and whether nitrogen limitation was applied. To identify the other non-specific dehydrogenases supply monomers for PHA synthesis, we analysed the proteome of the sextuple mutant under nitrogen limiting and non-limiting conditions.

2023-05-10 | PXD037937 | Pride

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Identification of phasin PhaF interaction partners and proteomic analysis of polyhydroxyalkanoate granules in Pseudomonas putida

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