Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression changes in NFAT5/TonEBP under isotonic and hypertonic conditions

ABSTRACT: TonEBP is a transcription factor that promotes cellular accumulation of organic osmolytes in the hypertonic renal medulla by stimulating expression of its target genes. Genetically modified animals with deficient TonEBP activity in the kidney suffer from severe medullary atrophy in association with cell death, demonstrating that TonEBP is essential for the survival of the renal medullary cells. Using both TonEBP knockout cells and RNA interference of TonEBP, we found that TonEBP promoted cellular adaptation to hypertonic stress. Microarray analyses revealed that genetic response to hypertonicity was dominated by TonEBP in that expression of totally different sets of genes was increased by hypertonicity in those cells with TonEBP vs. those without TonEBP activity. Out of over 100 potentially new TonEBP regulated genes, we selected 7 for further analyses and found that their expression was all dependent on TonEBP. RNA interference experiments showed that some of these genes – asporin, insulin-like growth factor-binding protein 5 and 7, and an extracellular lysophosphlipase D – plus Hsp70, a known TonEBP target gene, contributed to the adaptation to hypertonicity without promoting organic osmolyte accumulation. We conclude that TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress in addition to organic osmolyte accumulation. Quadruplicate samples were collected for each condition and then pooled into a single sample for hybridization to microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: Todd Lamitina

PROVIDER: E-GEOD-25816 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Organisms across all kingdoms of life use small organic molecules called osmolytes to adapt to diverse environmental conditions and preserve viability. Osmolytes are thought to thermally stabilise proteins based on in vitro studies of a few purified proteins. The molecular mechanisms of osmolytes remain controversial and systematic global studies of osmolyte action within a complex cellular milieu are so far lacking. Here we present an in situ proteome-wide analysis of protein thermal stabilisation by osmolytes, covering all major classes of these molecules: trimethylamine N-oxide (TMAO), betaine, glycerol, proline, trehalose and glucose. Using a structural proteomics approach, we systematically probed the effects of osmolytes on protein thermal stability, native structures, and aggregation, thus revealing widespread mechanisms of osmolyte action, but also osmolyte- and protein- specific effects. All tested osmolytes stabilised the majority of the proteome. The stabilisation of most proteins could be explained by the preferential exclusion model, according to which osmolytes unfavourably interact with protein backbones thus promoting folded states. We also detected specific protein-osmolyte binding events which resulted in strong thermal stabilisation, although binding was not necessary for stabilisation. Aggregation of most proteins occurred at higher temperatures in the presence of osmolytes than in their absence. TMAO however promoted aggregation of small, unfolded proteins but also showed the strongest protein thermal stabilisation effects, which might reflect adaptation to the extreme living conditions of marine organisms that accumulate TMAO. Our global in situ analysis provides mechanistic insight into how osmolytes function within a complex biological matrix. Our data further provides a resource to guide the design or selection of stabilisers for protein-based drugs and the identification of conditions that increase thermotolerance of microorganisms in biotechnological applications.

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Gene expression changes in NFAT5/TonEBP under isotonic and hypertonic conditions

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