I- Transport Defect-Causing NIS Mutants Uncover a Critical Tryptophan-Acid Motif Required for Plasma Membrane Transport

MARTIN, M; GEYSELS, RC; TESTA, G; CHIESA, AE; NICOLA, JP; MODENUTTI, CP; BERNAL BARQUERO, CE; PAPENDIECK, P; MIRAS, M; PEYRET, V; SIGNORINO, M; MASINI-REPISO, AM; MARTI, MA

Buenos Aires

Congreso; XVII Latin American Thyroid Society Congress; 2019

Latin American Thyroid Society

I- transportdefect (ITD) is an autosomal recessive disorder characterized by impairedthyroidal I- accumulation due to loss-of-function mutations in the Na+/I-symporter (NIS)-coding SLC5A5 gene.To characterize novelhomozygous (p.G561E) and compound heterozygous (p.G543R and p.L562M) SLC5A5 mutants found in twoITD-suspected patients on the basis of non-detectable I-accumulation in a eutopic thyroid gland.SLC5A5 gene coding region was PCR-amplifiedand subjected to Sanger sequencing. Insilico and functional in vitro studiesof novel NIS variants were performed.Functional studiesrevealed that G561E and L562M markedly reduces I- uptake, when theproteins are heterologously expressed in non-thyroid epithelial cells, becausetheir targeting to the plasma membrane is severely impaired. G543R NIS was previouslyreported as non-functional. G561Q NIS, like G561E, is mainly retained in theendoplasmic reticulum (ER). Bioinformatics reveal a fully conservedtryptophan-acidic (WD) motif whose disruption leads to NIS retention in the ER.Computational and biochemical analysis indicate that G561E and L562M impair therecognition of the flanking WD motif by the kinesin light chain (KLC) 2, thusimpairing mutant NIS exit from the ER. Moreover, short-hairpin RNA-mediatedKLC2 knock-down in FRTL-5 cells reduces NIS expression at the plasma membrane,and consequently NIS-mediated I- accumulation.Newly identified NISvariants negatively impact on the three-dimensional structure of its flanking WDmotif severely reducing the interaction with the ER-to-Golgi transport adaptor KLC2,thus impairing NIS maturation beyond the ER and reducing I-accumulation in thyroid follicular cells.