Dataset Information

Proteomic analysis of U2OS cells treated with the dihydroorotate dehydrogenase (DHODH) inhibitor Brequinar

ABSTRACT: The mitochondrial respiratory chain assembles into higher order complexes termed supercomplexes (SCs) under certain physiological or metabolic stimuli. A small molecule screen developed by the lab identified DHODH inhibitors as potent activators of SC assembly in cancer cells. To investigate the proteomic regulation of SCs under nucleotide deficiency, we treated U2OS cells for 48 hours with Brequinar (500 nM). Proteomic analysis highlighted strong signatures of respiratory chain subunit abundance and peroxisomal-derived ether phospholipid synthesis enzymes. Bypassing DHODH inhibition through uridine supplementation prevented these alterations. These findings establish a coordinated cellular response to reduced nucleotide pools that stimulates ether phospholipid synthesis and respiratory chain supra-assembly.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Melanocyte, Cell Culture

DISEASE(S): Disease Free

SUBMITTER: mark Jedrychowski

LAB HEAD: Pere Puigserver

PROVIDER: PXD023882 | Pride | 2022-02-16

REPOSITORIES: Pride

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Publications

Peroxisomal-derived ether phospholipids link nucleotides to respirasome assembly.

Bennett Christopher F CF O'Malley Katherine E KE Perry Elizabeth A EA Balsa Eduardo E Latorre-Muro Pedro P Riley Christopher L CL Luo Chi C Jedrychowski Mark M Gygi Steven P SP Puigserver Pere P

Nature chemical biology 20210315 6

The protein complexes of the mitochondrial electron transport chain exist in isolation and in higher order assemblies termed supercomplexes (SCs) or respirasomes (SC I+III<sub>2</sub>+IV). The association of complexes I, III and IV into the respirasome is regulated by unknown mechanisms. Here, we designed a nanoluciferase complementation reporter for complex III and IV proximity to determine in vivo respirasome levels. In a chemical screen, we found that inhibitors of the de novo pyrimidine synt ...[more]

PMID: 33723432

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Project description:Establishment of an in vitro system to explore molecular mechanisms of mastitis susceptibility in cattle by comparative expression profiling of Escherichia coli and Staphylococcus aureus inoculated primary cells sampled from cows with different genetic predisposition for somatic cell score Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows that were selected for high and low mastitis susceptibility by applying a marker assisted selection strategy considering QTL and molecular marker information of a repetitively confirmed QTL for SCS in the telomeric region on BTA18 The cells were cultivated and subsequently inoculated with heat inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 hours the cells were harvested and comparatively analyzed using microarray expression chip technology to identify differences in mRNA expression profiles attributed to cultivation, inoculation or to genetic predisposition. Six heifers inheriting the favorable paternal QTL allele and five heifers inheriting the unfavorable QTL allele were selected by applying a marker assisted selection strategy. All heifers were kept under the same environmental conditions, had no clinical mastitis and did not show any indication of bacterial infection at slaughter. Primary bovine mammary gland epithelial cell cultures were established from cells sampled from the udder parenchyma of each cow. The cells were challenged with heat inactivated Escherichia coli and Staphylococcus aureus or with PBS for control. After 1, 6 and 24 hours the cells were harvested and mRNA expression was comparatively analyzed between time points for each treatment and each paternally inherited SCS-BTA18-QTL allele, respectively. In addition, the differences in gene expression at time points 1, 6 and 24h between inoculated and respective uninoculated control cells were investigated using the short time series expression miner STEM for co-expression profiling and GO cattegory enrichment analyses.

Project description:Staphylococcus Enterotoxin B (SEB) is an exotoxin produced by Staphylococcus aureus. It is a public health threat during nosocomial infections. It is also considered as a biological weapon. The Centers for Disease Control and Prevention (CDC) lists SEB as a category B agent of Biodefence. SEB is easily aerosolized and can enter the host through inhalation which could lead to Acute Lung Injury (ALI) and in severe cases, it may result in multiple organ failure, shock and death. It is considered to be a Super-antigen as it activates a large number (30%) of T cells and results in the production of cytokines SEB binds directly to the non polymorphic region of MHC Class II which is expressed on antigen presenting cells and this complex activates T cells expressing a specific variable region of the β chain (Vβ8) of the T cell receptor (TCR). Thus, inhalation of SEB leads to recruitment of immune cells into the lung and secretion of pro-inflammatory cytokines which results in severe damage to the lung by increasing the permeability, and induction of respiratory failure. MicroRNA (miRNAs) is considered one of the mechanisms by which the epigenetic regulation happens. MicroRNA are noncoding small RNAs that have recently been shown to play a role in gene expression. The action of miRNA is dependent on its binding to 3’ Untranslated Region (3’UTR) of mRNA resulting in transcriptional or translational repression. Specifically, the efficacy of gene silencing is determined by the interaction of miRNA seed sequence with the target mRNA. Endocannabinoids are lipid molecules produced endogenously by host cells and bind to cannabinoid receptors, CB1 and CB2. N-Arachidonoylethanolamide or anandamide (AEA) is an endocannabinoid that has been shown to have immunomodulatory effects by our lab and others, although the precise mechanism is not clear. So far through my research, I found that Endocannabinoid is involved in a very unique way in the anti-inflammatory response through upregulation of many anti-inflammatory genes such as FOXP3, ARG1, and IL-10 that targeted by miRNA23a-3p and miRNA 34a-5p as well as the anti-inflammatory phenotype population as it show in the RT-PCR and flow cytometry results.

Dataset Information

Proteomic analysis of U2OS cells treated with the dihydroorotate dehydrogenase (DHODH) inhibitor Brequinar

Publications

Peroxisomal-derived ether phospholipids link nucleotides to respirasome assembly.

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