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Protein Modifications Driven by Nitric Oxide and Nitroxyl during Macrophage Proinflammatory Activation

ABSTRACT: Proinlfammatory stimulation of murine macrophages elicits profound metabolic changes. iNOS-derived Nitric oxide (NO) is both necessary and sufficient for the repression of OXPHOS and major mitochondrial rewiring of carbon fluxes during M1 polarization. Since inflammation results in broad proteomics changes with oxidative modifications of specific targets, we aim to understand how this is managed in absence of this important endogenous gas and its derived reductive nitrogen species like nitroxyl (HNO). We used proteomics to detail the mitochondrial proteome expression and status of cysteine modifications underlying proinflammatory stimulation and identified distinct targets of reversible and irreversible cysteine oxidations during this process in presence and absence of NOS2, RNS donors, and in exacerbating conditions of oxidative stress.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Daniel McVicar

PROVIDER: MSV000092336 | MassIVE | Mon Jul 03 12:02:00 BST 2023

REPOSITORIES: MassIVE

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Project description:This study describes a transcriptome-phenotype matching approach in which the starter L. lactis MG1363 was fermented under a variety of conditions that differed in the levels of oxygen and/or salt, as well as the fermentation pH and temperature. Samples derived from these fermentations in the exponential phase of bacterial growth were analyzed by full-genome transcriptomics and the assessment of heat and oxidative stress phenotypes. Variations in the fermentation conditions resulted in up to 1000-fold differences in survival during heat and oxidative stress. More specifically, aeration during fermentation induced protection against heat stress, whereas a relatively high fermentation temperature resulted in enhanced robustness towards oxidative stress. Concomitantly, oxygen levels and fermentation temperature induced differential expression of markedly more genes when compared with the other fermentation parameters. Correlation analysis of robustness phenotypes and gene expression levels revealed transcriptome signatures for oxidative and/or heat stress survival, including the metC-cysK operon involved in methionine and cysteine metabolism. To validate this transcriptome-phenotype association we grew L. lactis MG1363 in the absence of cysteine which led to enhanced robustness towards oxidative stress. Conclusions Overall, we demonstrated the importance of careful selection of fermentation parameters prior to industrial processing of starter cultures. Furthermore, established stress genes as well as novel genes were associated with robustness towards heat and/or oxidative stress. Assessment of the expression levels of this group of genes could function as an indicator for enhanced selection of fermentation parameters resulting in improved robustness during spray drying. The increased robustness after growth without cysteine appeared to confirm the role of expression of the metC-cysK operon as an indicator of robustness and suggests that sulfur amino acid metabolism plays a pivotal role in oxidative stress survival. two connected loops, both containing samples derived on a single day (sample 1-6, sample 7-13)

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Protein Modifications Driven by Nitric Oxide and Nitroxyl during Macrophage Proinflammatory Activation

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