DESCRIPTION (provided by applicant) This collaborative study will be primarily performed in Hungary at the Institute of Experimental Medicine as an extension of NIH grant #RO1 DK-58538 to reveal novel molecular aspects of the selective proteolysis of the type 2 deiodinase (D2), the major enzyme activating thyroxine. It is proposed that as an extension of the parent grant these studies will identify D2 structures which are responsible for the metabolic instability of this key rate-limiting enzyme of thyroid hormone action serving as triggers for ubiquitination. In addition, D2 degradation will be investigated from a novel point of view. The hypothesis will be tested whether the intracellular localization of D2 plays a role in its degradation. An experimental model will be established in mammalian cells that enables the systematic testing of the functional role of different D2 domains in the half-life of type 2 deiodinase. Expression constructs will be created using standard recombinant DNA techniques suitable for fusion of D2 fragments to the carboxy terminus of Sec62. The stable and endoplasmic reticulum (ER) inserted Sec62 with both of its termini in the cytosol will drive D2 with no transmembrane domain into its native compartment, the ER. The amino terminus of Sec62 will be FLAG tagged and its carboxy terminus will be used for fusion with the amino terminus of different D2 and fragments, lacking the D2 transmembrane domain. Second, D2 fragments will be systematically tested to identify fragments involved in decreasing half-life of the D2/Sec62 fusion protein. As controls, D1/Sec62 controls will be used since D1 is a long-lived protein and is not expected to change Sec62 half-life. Constructs will be transiently transfected into HEK-293 cells and the half-life determined by pulse chase with 35S Met/Cys. FLAG-immunoprecipitation will be followed by SDS-PAGE and autoradiograpy. We will also investigate D2 degradation from a novel point of view studying whether the intracellular localization of D2 plays a role in the selected proteolysis of D2. By fusing D2 to a stable, plasma membrane located protein, such as the type 1 deiodinase, we will direct D2 into a different cell compartment and investigate, whether the D2 degradation is dependent on the ER based ERAD system or independent from its intracellular localization and is able to change the half-life of the D2/NIS fusion protein. Since the "low T3 syndrome" is associated with impaired T3 generation and in certain mesotheliomas overexpress D2, it is critical to understand the rate-limiting steps in D2 proteolysis.
|Effective start/end date||9/1/03 → 8/31/07|
- National Institutes of Health: $40,320.00
Proteasome Endopeptidase Complex
Euthyroid Sick Syndromes
Polyacrylamide Gel Electrophoresis