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Detection of putative PKA target phosphorylation sites in Atg5flox:CamKII-Cre wildtype and knockout mice brain tissue

ABSTRACT: The experiment was performed to identify PKA phosphorylation substrates in wildtype and autophagy-deficient brains. Therefore, hippocampus and cortex of wildtype and conditional knockout mice were isolated and sliced. The brain slices were incubated with DMSO or Forskolin, a cAMP elevating agent, to induce PKA activity. Samples were measured by LC-MS/MS and were used to quantify proteomic and phosphoproteomic changes.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Frederik Tellkamp

LAB HEAD: Natalia L Kononenko

PROVIDER: PXD031106 | Pride | 2022-11-15

REPOSITORIES: Pride

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			Action	DRS
	MaxQuant_output.7z	Other
	RAW_files.7z	Other
	checksum.txt	Txt
	experimentalDesignTemplate.txt	Txt
	mqpar.xml	Xml

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Publications

Autophagy regulates neuronal excitability by controlling cAMP/protein kinase A signaling at the synapse.

Overhoff Melina M Tellkamp Frederik F Hess Simon S Tolve Marianna M Tutas Janine J Faerfers Marcel M Ickert Lotte L Mohammadi Milad M De Bruyckere Elodie E Kallergi Emmanouela E Delle Vedove Andrea A Nikoletopoulou Vassiliki V Wirth Brunhilde B Isensee Joerg J Hucho Tim T Puchkov Dmytro D Isbrandt Dirk D Krueger Marcus M Kloppenburg Peter P Kononenko Natalia L NL

The EMBO journal 20221011 22

Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, by combining mouse models of neuron-specific ATG5 deficiency in either excitatory or inhibitory neurons with quantitative proteomics, high-content microscopy, and live-imaging approaches, we show that autophagy protein ATG5 functions in neurons to regulate cAMP-dependent protein kinase A (PKA)-mediated p ...[more]

PMID: 36217825

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Proteome and phosphoproteome analysis of autophagy deficient ATG5flox:CamKII-Cre and ATG5flox:VGAT-Cre conditional knockout mice

Project description:The experiment was performed to find selective targets of autophagy in different subtypes of neurons. Therefore, brain tissue of conditional knockout mice lacking autophagy protein ATG5 in inhibitory or excitatory neurons was used. Cortices (ATG5flox:CamKII-Cre) / striata (ATG5flox:VGAT-Cre) of mice were mixed with lys6 labeled brain lysates as internal controls between the samples. Samples were measured by LC-MS/MS and resulting H/L ratios were used to quantify proteomic and phosphoproteomic changes.

2022-11-15 | PXD029435 | Pride

Global proteome of Atg5flox:Slc32a1-Cre:tdTomato FACS sorted WT and KO neurons

Project description:The experiment was performed to identify phosphorylation target proteins of cAMP-dependent kinase A (PKA) in mouse brain tissue. Therefore, coimmunoprecipitation analysis was performed using a PKA phosphorylation state-specific antibody (RRXS/T). Potential PKA target proteins were determined by LC-MS/MS.

2022-11-15 | PXD031553 | Pride

Global proteome of Atg5flox:Slc32a1-Cre:tdTomato FACS sorted WT and KO neurons

Project description:The experiment was performed to identify autophagy targets in wildtype and autophagy-deficient forebrain inhibitory neurons. Therefore, neurons were isolated from the cortex, hippocampus and striatum of 2-3 weeks old Atg5flox/flox:Slc32a1-Cretg/wt:tdTomato+ (KO) and Atg5wt/wt:Slc332a1-Cretg/wt:tdTomato+ (WT) mice. Neurons in suspension were FACS sorted and inhibitory forebrain neurons expressing tdTomato were forwarded to global proteome analysis assessed by LC-MS/MS.

2022-11-15 | PXD031535 | Pride

Global proteome of Atg5flox:CamKIIα-Cre:tdTomato FACS sorted WT and KO neurons

Project description:The experiment was performed to identify autophagy targets in wildtype and autophagy-deficient forebrain excitatory neurons. Therefore, neurons were isolated from the cortex, hippocampus and striatum of 2-3 weeks old Atg5flox/flox:CamKIIα-Cretg/wt:tdTomato+ (KO) and Atg5wt/wt:CamKIIα-Cretg/wt:tdTomato+ (WT) mice. Neurons in suspension were FACS sorted and excitatory forebrain neurons expressing tdTomato were forwarded to global proteome analysis assessed by LC-MS/MS.

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PKA C3 immunoprecipitation mass spectrometry

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2024-07-12 | PXD039896 | Pride

Spontaneous nonsense mutation in Tuft1 (tuftelin 1) gene is associated with abnormal hair appearance and amelioration of glucose and lipid metabolism in the rat

Project description:A new spontaneous mutation, abnormal hair appearance in the rat, has been identified as a non-functional Tuft1 (tuftelin 1) gene. The pleiotropic effects of this mutation regulate glucose and lipid metabolism. Analysis of the liver proteome revealed possible molecular mechanisms for the metabolic effects of the Tuft1 gene. Recently, we identified a recessive mutation, an abnormal coat appearance, in BXH6 strain, a member of the HXB/BXH set of recombinant inbred strains derived from the SHR (spontaneously hypertensive rat) and BN-Lx (Brown Norway rat) progenitors. Whole genome sequencing of the mutant rats identified the 195875980 G/A mutation in the Tuft1 (tuftelin 1) gene on chromosome 2, which resulted in a premature stop codon. Compared to wild-type BXH6 rats, BXH6-Tuft1 mutant rats exhibited lower body weight due to reduced visceral fat and ectopic fat accumulation in the liver and heart. Reduced adiposity was associated with decreased serum glucose and insulin, and increased insulin-stimulated glycogenesis in skeletal muscle. In addition, mutant rats had lower serum MCP-1 and leptin levels, indicative of reduced inflammation. Analysis of the liver proteome identified differentially expressed proteins from fatty acid metabolism and beta oxidation, peroxisomes, carbohydrate metabolism, inflammation, and proteasome pathways. These results provide evidence for an important role of Tuft1 gene in the regulation of lipid and glucose metabolism and suggest underlying molecular mechanisms.

2024-03-12 | PXD045880 | Pride

Bacteroides thetaiotaomicron OMV proteomics

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Comparison of OMV to membrane proteomes across different media (membrane experiments)

Project description:This project examines the impact of different growth media on the protein compositions of membranes of Bacteroides thetaiotaomicron.

2023-06-14 | PXD036272 | Pride

Hypolipidemic effects of beetroot juice in SHR-CRP and HHTg rat models of metabolic syndrome: Analysis of hepatic proteome.

Project description:Recently, red beetroot has attracted attention as a health promoting functional food. Studies showed that beetroot administration can reduce blood pressure and ameliorate parameters of glucose and lipid metabolism, however, mechanisms underlying these beneficial effects of beetroot are not fully understood. In the current study, we analyzed effects of beetroot on parameters of glucose and lipid metabolism in two models of metabolic syndrome, (i) transgenic spontaneously hypertensive rats expressing human C-reactive protein (SHR-CRP rats), and (ii) hereditary hypertriglyceridemic (HHTg) rats. Treatment with beetroot juice for 4 weeks was in both models associated with amelioration of oxidative stress, reduced circulating lipids, smaller visceral fat depots, and lower ectopic fat accumulation in the liver, compared to their respective untreated controls. On the other hand, beetroot treatment had no significant effects on sensitivity of muscle and adipose tissue to insulin action in both models. Analyses of hepatic proteome revealed significantly deregulated proteins involved in glycerophospholipid metabolism, mTOR signalling, inflammation, and cytoskeleton rearrangement.

2023-03-11 | PXD039605 | Pride

Transcriptomics and mutagenesis analysis uncover key mechanisms involved in polymyxin B resistance and heteroresistance of the neonatal sepsis pathogen Enterobacter bugandensis

Project description:Enterobacter bugandensis is one of species from the E. cloacae complex (ECC) that has been predominantly associated to neonatal sepsis. A major concern with E. bugandensis and ECC bacteria in general is the frequent appearance of multidrug resistant isolates including those resistant to last-resort antibiotics, such as polymyxins, for which these microbes are in the ESKAPE list of global threat pathogens. Here, we investigated polymyxin B (PmB) resistance and heteroresistance in E. bugandensis by transcriptomics and a gene deletion approach using two clinical isolates. Genes encoded in the CrrAB-regulated operon including crrC and kexD were highly upregulated in both strains in the presence of PmB. We show in one of these isolates that ∆crrC and ∆kexD mutants exhibited lower levels of PmB resistance and heteroresistance than the parental strain. Moreover, the heterologous expression of CrrC and KexD proteins increased PmB resistance in a sensitive E. ludwigii clinical isolate and in the Escherichia coli K12 strain W3110. We also showed that the efflux pump AcrAB and TolC contribute to PmB resistance and heteroresistance. Deletion of the regulatory genes phoPQ and crrAB cause reduced PmB resistance and heteroresistance, while deletion of pmrAB did not have any effect. Our results also reveal that the addition of L-Ara4N into the lipid A, mediated by the arnBCADTEF operon, is critical to determine PmB resistance, while the deletion of eptA, encoding a PEtN transferase had no effect. Finally, PmB resistance did not correlate with pathogenicity in the Galleria mellonella infection model.

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