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A proteomic approach on the effects of TX527, a 1α,25-dihydroxyvitamin D3 analog, in human T lymphocytes.

The Journal of steroid biochemistry and molecular biology (2013-11-02)
G B Ferreira, F Baeke, A Verstuyf, P De Clercq, E Waelkens, C Mathieu, L Overbergh
ABSTRACT

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3), and its analogs (i.e. 14,20-bis-epi-19-nor-23-yne-1α,25(OH)2D3 - TX527) have been shown to prevent autoimmunity and prolong islet graft survival in the non-obese diabetic (NOD) mouse. Their effects are mediated by their action on various immune cell types, such as dendritic cells (DC) and T cells. We have previously reported important direct effects of TX527 on human T cells, on their cytokine/chemokine profiles, T regulatory cell markers, homing characteristics and chemotaxis. In order to fully understand the molecular mechanisms underlying the beneficial properties of TX527 on human T cells, we applied here 2-dimensional difference gel electrophoresis (2-D DIGE) to analyze the global protein alterations induced by TX527 on human synchronized T cells. We detected differential expression of 64 protein spots upon TX527 treatment, of which 65.6% could be successfully identified using tandem mass spectrometry (MALDI-TOF/TOF). The identified proteins function in various processes, such as metabolism and energy pathways, cytoskeleton and protein metabolism. When comparing the proteomics data to our previously performed microarray data on the same set of cells, we found an overlap of 17 different mRNAs/proteins. For some of these (e.g. PSME2, HSPA8), the direction of regulation was not similar, hereby reinforcing the important role of post-transcriptional/translational processes in the functionality of proteins. In addition, although 2-D DIGE offers the possibility of picking up post-translational processes, it lacks the ability to detect molecules with extreme molecular weight (MW) and isoelectrical point (pI) values, or very low abundant/hydrophobic proteins. This study highlights therefore the importance of combining different experimental approaches to obtain a complete picture of the underlying mechanisms and general processes being affected in T cells upon TX527 treatment. These processes lead altogether to the generation of T cells with interesting immunomodulatory features for clinical applications in the treatment of autoimmune diseases or in the prevention of graft rejection. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Cholecalciferol, ≥98% (HPLC)
Supelco
Cholecalciferol (Vitamin D3), Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Vitamin D3 solution, 1 mg/mL in ethanol, ampule of 1 mL, certified reference material, Cerilliant®
Supelco
Cholecalciferol (D3), analytical standard
USP
Cholecalciferol, United States Pharmacopeia (USP) Reference Standard
Cholecalciferol, European Pharmacopoeia (EP) Reference Standard
Cholecalciferol for system suitability, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Cholecalciferol, meets USP testing specifications
Sigma-Aldrich
Cholecalciferol, analytical standard