Insights Into the Mechanism of MCT8 Oligomerization.

Mutations in the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) result in MCT8 deficiency, characterized by severe intellectual and motor disability. The MCT8 protein is predicted to have 12 transmembrane domains (TMDs) and is expressed as monomers, homodimers, and homo-oligomers. This study aimed to delineate the mechanism of MCT8 oligomerization. Coimmunoprecipitation studies demonstrated that lithium dodecyl sulfate effectively disrupts MCT8 protein complexes, indicating the involvement of non-covalent interactions. Successive C-terminal truncations of the MCT8 protein altered the oligomerization pattern only if introduced in the N-terminal half of the protein (TMD1-6). The truncation at extracellular loop 1 (E206X) still allowed homodimerization, but completely abrogated homo-oligomerization, whereas both were preserved by the C231X mutant (at TMD2), suggesting that the minimally required oligomerization sites are located proximal of Cys231. However, mutant constructs lacking the intracellular N-terminus or TMD1 and 2 were still capable to form homo-oligomers. Therefore, other domains distal of Cys231 are also likely to be involved in the formation of extensive multidomain interactions. This hypothesis was supported by structural modeling. Despite multiple approaches, MCT8 oligomerization could not be fully abrogated unless a substantial part of the protein was removed, precluding detailed studies into its functional role. Together, our findings suggest that MCT8 oligomerization involves extensive noncovalent interactions between the N-terminal halves of MCT8 proteins. Most mutations identified in patients with MCT8 deficiency have only minor effects on MCT8 oligomerization and, thus, impaired oligomerization does not appear to be an important pathogenic mechanism.

In Vitro Characterization of Human, Mouse, and Zebrafish MCT8 Orthologues.

Background: Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause MCT8 deficiency, characterized by severe intellectual and motor disability and abnormal serum thyroid function tests. Various Mct8 knock-out mouse models as well as mct8 knock-out and knockdown zebrafish models are used as a disease model for MCT8 deficiency. Although important for model eligibility, little is known about the functional characteristics of the MCT8 orthologues in these species. Therefore, we here compared the functional characteristics of mouse (mm) MCT8 and zebrafish (dr) Mct8 to human (hs) MCT8. Methods: We performed extensive transport studies in COS-1 and JEG-3 cells transiently transfected with hsMCT8, drMct8, and mmMCT8. Protein expression levels and subcellular localization were assessed by immunoblotting, surface biotinylation, and immunocytochemistry. Sequence alignment and structural modeling were used to interpret functional differences between the orthologues. Results: hsMCT8, drMct8, and mmMCT8 all facilitated the uptake and efflux of 3,3'-diiodothyronine (3,3'-T2), rT3, triiodothyronine (T3), and thyroxine (T4), although the initial uptake rates of drMct8 were 1.5-4.0-fold higher than for hsMCT8 and mmMCT8. drMct8 exhibited 3-50-fold lower apparent IC50 values than hsMCT8 and mmMCT8 for all tested substrates, and substrate preference of drMct8 (3,3'-T2, T3 > T4 > rT3) differed from hsMCT8 and mmMCT8 (T3 > T4 > rT3, 3,3'-T2). Compared with hsMCT8 and mmMCT8, cis-inhibition studies showed that T3 uptake by drMct8 was inhibited at a lower concentration and by a broader spectrum of TH metabolites. Total and cell surface expression levels of drMct8 and hsMCT8 were equal and both significantly exceeded those of mmMCT8. Structural modeling located most non-conserved residues outside the substrate pore, except for H192 in hsMCT8, which is replaced by a glutamine in drMct8. However, a H192Q substituent of hsMCT8 did not alter its transporter characteristics. Conclusion: Our studies substantiate the eligibility of mice and zebrafish models for human MCT8 deficiency. However, differences in the intrinsic transporter properties of MCT8 orthologues may exist, which should be realized when comparing MCT8 deficiency in different in vivo models. Moreover, our findings may indicate that the protein domains outside the substrate channel may play a role in substrate selection and protein stability.

Effects of Chemical Chaperones on Thyroid Hormone Transport by MCT8 Mutants in Patient-Derived Fibroblasts.

Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) result in severe intellectual and motor disability. At present, no effective therapy is available to restore TH signaling in MCT8-dependent tissues. Recent in vitro studies in stable overexpression cell models suggested that the function of certain mutant MCT8 proteins, specifically those that affect protein stability and intracellular trafficking (e.g., p.F501del), could be partially recovered by chemical chaperones. However, the effects of chaperones have not been demonstrated in other commonly used models for MCT8 deficiency, including transient overexpression models and patient-derived fibroblasts. Here, we demonstrate that the chemical chaperone 4-phenylbutyric acid (PBA) similarly potentiates the T3 transport function of wild-type and p.F501del mutant MCT8 in transiently transfected COS-1 cells by increasing MCT8 messenger RNA, total protein, and cell surface expression levels. Although PBA also increased the cell surface expression levels of the p.R445L mutant, no functional improvement was observed, which is in line with the proposed important role of Arg445 in substrate translocation. In contrast, PBA showed only minimal effects in ex vivo studies using control or p.F501del patient-derived fibroblasts. Moreover, the MCT8-specific inhibitor silychristin did not change these minimal effects, suggesting that the underlying mechanism is unrelated to the rescue of functional MCT8. Together, these findings indicate that the potency of chaperones to rescue mutant MCT8 function strongly depends on the cellular model and stress the need for further preclinical studies before clinically available chaperones should be considered as a treatment option in patients with MCT8 deficiency.