Proteomics and transcriptomics analyses of ataxia telangiectasia cells treated with Dexamethasone

Abstract

Ataxia telangiectasia (A-T) is an incurable and rare hereditary syndrome. In recent times, treatment with glucocorticoid analogues has been shown to improve the neurological symptoms that characterize this condition, but the molecular mechanism of action of these analogues remains unknown. Hence, the aim of this study was to gain insight into the molecular mechanism of action of glucocorticoid analogues in the treatment of A-T by investigating the role of Dexamethasone (Dexa) in A-T lymphoblastoid cell lines. We used 2DE and tandem MS to identify proteins that were influenced by the drug in A-T cells but not in healthy cells. Thirty-four proteins were defined out of a total of 746±63. Transcriptome analysis was performed by microarray and showed the differential expression of 599 A-T and 362 wild type (WT) genes and a healthy un-matching between protein abundance and the corresponding gene expression variation. The proteomic and transcriptomic profiles allowed the network pathway analysis to pinpoint the biological and molecular functions affected by Dexamethasone in Dexa-treated cells. The present integrated study provides evidence of the molecular mechanism of action of Dexamethasone in an A-T cellular model but also the broader effects of the drug in other tested cell lines.

Fig 1. 2DE representative images.

A-T and WT samples treated or not treated with Dexa. Each gel image was elaborated with Melanie software. Three technical replicates were used.

Fig 2

(A-B) STRING (A) and Reactome FI (B) networks. The proteomic data were used to compute both networks. The functional interaction of the discovered proteins in the AT129RM sample was extremely elevated as reported in S1 and S2 Supplementary Files. The nodes colours (in B) represent the Reactome FI clustered genes and the numbers state the enrichment pathways of nodes in clusters as reported in S2 Supplementary File.

Fig 3. Western blot.

Representative images of western blots performed in all tested LCLs, subsequently quantified as reported in Fig 4.

Fig 4. Western blot analysis of all tested LCLs.

The protein abundance of selected targets in sample AT129RM is in agreement with the 2DE outcome (paired t-test p<0.05) except for 14.3.3 ζ/δ (see text). Only the AT50RM sample behaved in a similar manner to the AT129RM sample, despite the ATK13RM and ATK36RM cell lines. The W-N graphic reports the whole lane normalization data of the WB experiments.

Fig 5. FKBP5, TMEM2 and NFIL3 gene expression by qPCR.

The well-known genes altered by Dexa administration have been tested by qPCR in order to validate the microarray procedure.

Fig 6. Veen diagram.

The splicing and expression outputs were compared and plotted to show differences about spliced and altered expression genes between WT and AT. Only small amounts of gene symbols were shared in all tested comparisons.

Fig 7. HLC outcome obtained by microarray expression profile of A-T samples.

A total of 675 differentially expressed transcripts allowed us to classify AT129RM and AT50RM as similar to each other, while the other A-T samples behaved differently. The same behaviour pattern was inferred by western blot analysis.