Novel molecular mechanism of de novo triiodothyronine (T3) formation induced by Fam20c in stimulated thyrocytes.

IGLESIAS, CINTHIA; GAUNA, ALICIA T.; ARVAN, PETER ; TARGOVNIK , HÉCTOR MANUEL; CITTERIO, CINTIA ELIANA

virtual

Congreso; Reunión Anual de Sociedades de Biociencia 2019. LXV Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica; 2020

Sociedad Argentina de Investigación Clínica

T4 derives from the N-terminus of thyroglobulin (Tg) de novo T3 involves coupling of mono- and di-iodotyrosine famously occurring at the opposite end of the Tg protein, whereas formation of Stimulation of thyroidal TSH receptors enhances T3 formation in secreted Tg, as occurs in autoimmune hyperthyroidism of Graves ́ disease, and promotes the expression of Fam20C, a Golgi-localized secretory (casein) kinase. Knockdown of Fam20C or mutation of mTg-S2718A (equivalent to hTg-S2721A, an established phosphorylation site) inhibits Fam20C-stimulated T3 formation, while enhancement is observed for the phosphomimetic mTg-S2718E mutant. We found that Fam20C-stimulated T3-formation in mTg still occurred when the classic T3-forming sites were eliminated: Y2519F, Y2552F and Y2744F (Tg-3xF). We hypothesize that Fam20C-stimulated T3-formation occurs at secondary T3 hormonogenic sites in Tg. To consider T3 formation involving Y5, Y130, Y239, Y973, or Y1290, we utilized Tg-3xF and further mutagenized selective additional Y residue to F. In parallel, we also created a single mutation of Tg-Y5F, Tg-Y130F, or Tg-Y239F. We co-expressed each Tg construct +/- Fam20C, performed enzymatic iodination of the secreted Tg, and monitored de novo T3-formation by immunoblotting with anti-T3 and anti-Tg Abs. Our data reveal that Fam20C could still increase de novo T3-formation in Tg even in the Tg-Y973F-3xF or Tg-Y1290F-3xF mutants. However, Fam20C-stimulated T3-formation was partially reduced when either Y130 or Y239 (iodotyrosyl donors to Y5) were disrupted. Moreover,when Fam20C was co- expressed with Tg-Y5F-3xF or Tg-Y5F, de novo T3 formation was inhibited nearly to control levels, suggesting that Fam20C shifts Tg to favor T3-formation at the classic T4-forming site at Y5. Our results show that TSH/Fam20C-stimulated de novo T3 formation in Tg entails a coupling reaction that includes Y5. Donor iodotyrosines, such as Y130 and Y239, may control the T4:T3 ratio of Tg in different thyroid diseases.