Proteomic analyses of thyroid hormone-sensitive tissues during frog tadpole metamorphosis




Domanski, Dominik

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Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. There is also increasing evidence that changes in the phosphoproteome influence TH action. In addition, the disruption of TH-action by endocrine-disrupting compounds (EDC) is an emerging field and the developmental process of metamorphosis is a target as well as a model system for this research. This work identifies components of the proteome and phosphoproteome in TH-sensitive tadpole tissues that are altered during the initiation stages of TH-induced metamorphosis prior to the overt remodeling of the tissues. Proteomic analyses included two-dimensional (2D) gel electrophoresis for the assessment of differential protein/phosphoprotein expression, combined with mass spectrometry (MS) protein analysis for protein identification. Initial proteomic approaches in Xenopus laevis identified a number of proteins that are differentially expressed in the tadpole tail within 48 h of exposure of premetamorphic tadpoles to 3,5,3’-triiodothyronine (T3). Additionally, a time-course analysis of brain tissue within this 48 h period revealed alterations in phosphoproteins. The importance of phosphoproteome modulation in the process of metamorphosis was further revealed in the TH-induced tail of Rana catesbeiana tadpoles, where the inhibition of cyclin-dependent kinase activity which prevents tail regression, altered the tail phosphoproteome profile. Failure to identify the phosphoproteins involved in these initial studies led me to develop and apply new proteomic approaches. To this end, subcellular and protein fractionation methods were developed and combined with 2D gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using MS. Here components of the proteome and phosphoproteome were identified in the tail fin that changed within 48 h of exposure of premetamorphic R. catesbeiana tadpoles to 10 nM T3. This approach allowed the identification of and led to the cloning of a novel Rana larval type I keratin, RLK I, which is a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript level was reduced during T3-induced and natural metamorphosis, consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, was changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T3 treatment. The success in identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. This study demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which limited genome information is currently available. The early appearance of caspase-cleaved RLK I in the TH-induced process led to its investigation as a contributor to apoptosis. Furthermore, the caspase-cleavage product of RLK I was used as a biomarker in the development of an assay for the detection of disruptors of TH-action based on ex-vivo multi-well culturing of R. catesbeiana tail fin biopsies. This assay was able to detect perturbations in TH-signalling within 48h of exposure demonstrating that it has utility as a novel system for screening of TH disrupting chemicals. The present study identified proteins whose levels and/or phosphorylation states are altered within 48 h of the induction of tadpole metamorphosis prior to overt tissue remodeling and provided important insight into the molecular mechanisms of this postembryonic development. In particular, I have identified a novel keratin that is a target for T3-mediated changes in the tail that can serve as an indicator of early response to this hormone and can be used for the detection of EDCs of TH-action in an ex vivo assay.



thyroid hormone, metamorphosis, proteomics, anuran, mass spectrometry, endocrine disruption