Thyroglobulin Interactome Profiling Defines Altered Proteostasis Topology Associated With Thyroid Dyshormonogenesis

Abstract

Thyroglobulin (Tg) is a secreted iodoglycoprotein serving as the precursor for triiodothyronine and thyroxine hormones. Many characterized Tg gene mutations produce secretion-defective variants resulting in congenital hypothyroidism. Tg processing and secretion is controlled by extensive interactions with chaperone, trafficking, and degradation factors comprising the secretory proteostasis network. While dependencies on individual proteostasis network components are known, the integration of proteostasis pathways mediating Tg protein quality control and the molecular basis of mutant Tg misprocessing remain poorly understood. We employ a multiplexed quantitative affinity purification-mass spectrometry approach to define the Tg proteostasis interactome and changes between WT and several congenital hypothyroidism variants. Mutant Tg processing is associated with common imbalances in proteostasis engagement including increased chaperoning, oxidative folding, and engagement by targeting factors for endoplasmic reticulum-associated degradation. Furthermore, we reveal mutation-specific changes in engagement with N-glycosylation components, suggesting distinct requirements for 1 Tg variant on dual engagement of both oligosaccharyltransferase complex isoforms for degradation. Modulating dysregulated proteostasis components and pathways may serve as a therapeutic strategy to restore Tg secretion and thyroid hormone biosynthesis.

Keywords: affinity purification - mass spectrometry; cell secretion; congenital hypothyroidism; protein folding; protein quality control; protein-protein interactions; tandem mass tags.

Graphical abstract

Fig. 1

Distinct Tg mutants present secretion defects.A, schematic of Tg domain organization consisting of cysteine rich repeats, a linker/N-terminal region, and hinge/flap region followed by a cholinesterase-like (ChEL) C-terminal domain. B, schematic detailing Tg processing and subsequent hormone production. Tg is synthesized in follicular cells and secreted into the follicular lumen where it undergoes iodination and is stored. Tg is later taken up and proteolyzed leading to the liberation of T3 and T4 hormones. C, immunoblot for Flag-tagged Tg expressed in transiently transfected HEK293T cells. All Tg variants are detected in the lysate while only WT is detected in cell culture media. D, Western blot for Tg probing EndoH sensitivity to remove high-mannose glycans of ER-localized Tg. Golgi-associated (resistant) and ER-associated (sensitive) bands are indicated by arrows. Only WT Tg develops EndoH resistance, indicating it is able to traverse the medial Golgi. All CH-associated Tg mutants are EndoH sensitive and unable to reach the medial Golgi. E, quantification of EndoH sensitivity in (D). All Tg mutants are 100% EndoH sensitive, showing they are retained within the ER, unable to reach the medial Golgi, and thus model a hypothyroidism phenotype. Error bars show SEM for four biological replicates. CH, congenital hypothyroidism; Tg, thyroglobulin; ER, endoplasmic reticulum.

Fig. 2

Defining the Tg interactome using multiplexed quantitative AP-MS.A, schematic detailing the multiplexed quantitative interactomics workflow utilizing in situ crosslinking, affinity purification—mass spectrometry (AP-MS), and tandem mass tags (TMT) for relative quantification of identified interactors to delineate interaction changes from WT to mutant variants. B, volcano plot showing TMT enrichment ratios (log₂ difference all Tg channels versus all mock channels) versus −log10 adjusted p values (Storey q values) for coimmunoprecipitated proteins (n = 13 biological replicates). Variable cutoffs were used to optimize confident interactors of Tg. Optimization described in supplemental Figure S1 and supplemental Table S3. C, proteins found to be confident interactors with Tg are enriched within the secretory pathway. D, Schematic detailing newly identified Tg interactors (red) compared with previously publishes interactors (gray). Tg interactors are organized by biological function and organellar localization. Tg, thyroglobulin.

Fig. 3

The secretion defect of Tg mutants is associated with both common and mutant specific changes in proteostasis interactions.A, dot plots displaying aggregate interactome changes of proteostasis pathways between the different mutant Tg variants (G2341R, L2284P, A2234D, C1264R) compared with WT. Proteostasis factors are grouped based on biological function as in Figure 3B, and dots represent interaction changes for individual high-confidence interactors of Tg belonging to each group. Source data found in supplemental Table S10. B, heatmap displaying altered interactions of mutant Tg variants with individual proteostasis components that were identified as high-confidence interactors. Interactors are grouped by biological function as in Figure 3A. Source data found in supplemental Table S10. Tg, thyroglobulin.

Fig. 4

Tg mutants are increasingly routed toward ER-associated degradation machineries but not degraded at faster rates.A, schematic detailing the ERAD pathway: degradation factors targeting proteins through glycan trimming, subsequent retrotranslocation and ubiquitination, followed by proteasome-mediated degradation in the cytosol. B–D, interaction changes of Tg mutants compared with WT with individual ERAD factors that were identified as high-confidence interactors of Tg. Error bars show SEM. Student’s parametric t test with adjusted p values (Storey q values) was used to determine significant changes. ∗q < 0.05. B, Mannosidases responsible for glycan trimming. C, an ERAD-specific lectin (OS-9) and another ERAD factor (FOXRED2), D, a subunit of the retrotranslocation complex. E, plot showing the percentage of Tg degradation measured in HEK293^DAX cells 4 h after treatment with 50 μg/ml of cycloheximide to block new protein translation. Error bars show SEM for three to six biological replicates. There is no significant difference in degradation for any of the Tg mutants compared with WT. Student’s parametric t test was used to determine significant changes (p < 0.05). G2341R: p = 0.849, L2284P: p = 0.942, A2234D: p = 0.464, C1264R: p = 0.813. Representative Western blots for the CHX chase experiments are shown in supplemental Figure S5B. F, interaction changes of Tg mutants compared with WT for cytosolic proteins involved in proteasome-mediated protein degradation. Error bars shows SEM. Student’s parametric t test with multiple testing correction (Storey q values) was used to determine significant changes. ∗q < 0.05. CHX, a cycloheximide; ER, endoplasmic reticulum; ERAD, ER-associated degradation; Tg, thyroglobulin.

Fig. 5

Perturbation of N-linked glycosylation distinctly impacts A2234D Tg.A, schematic detailing the N-glycosylation and lectin-mediated folding pathway. Glycosylation is carried out by two distinct OST complexes containing STT3A or STT3B as catalytic subunits. Glucosidases then trim terminal glucose residues, lectin chaperones (CANX, CALR), and PDIA3 assist in folding, followed by further glucose trimming. Subsequently, glucosyltransferases (UGGT1, UGGT2) serve as quality control sensors to re-glucosylate improperly folded proteins for iterative chaperoning cycles. B–E, interaction changes of Tg mutants compared with WT with individual N-glycosylation quality control factors that were identified as high-confidence interactors of Tg. Error bars show SEM. Student’s parametric t test with adjusted p values (Storey q values) was used to determine significant changes. ∗q < 0.05, ∗∗q < 0.005, ∗∗∗q < 0.0005. B, lectin chaperones and lectin-associated protein disulfide isomerase PDIA3. C, glucosyltransferases sensing misfolded proteins. D, glucosidases involved in glycan trimming. E, catalytic subunits of the OST complex STT3A responsible for cotranslational glycosylation and STT3B responsible for posttranslational glycosylation. F, comparison of WT Tg secretion in parental, STT3A, or STT3B KO HEK293T cells. WT Tg was transiently transfected into the respective cells, and newly synthesized proteins were metabolically labeled for 30 min with ³⁵S and then chased with unlabeled media. ³⁵S-labeled protein was quantified in the media, and lysate after 4 h % Tg secreted was calculated as $T{g}_{media,4h}/\left(T{g}_{lysate,0h}+T{g}_{media,0h}\right)$. Error bars represent SEM of three to four biological replicates. STT3A or STT3B KO do not significantly alter WT secretion. Student’s parametric t test was used to determine significant changes in Tg secretion. Representative autoradiograms are shown in supplemental Figure S5G. G, comparison of A2234D Tg degradation in parental, STT3A, or STT3B KO HEK293T cells. A2234D Tg was transiently transfected into the respective cells and subjected to the same ³⁵S-pulse labeling scheme as in F. % Tg degraded was calculated as $1-(T{g}_{lysate,4h}/T{g}_{lysate,0h})$. Error bars represent SEM of three to four biological replicates. Representative autoradiograms are shown in supplemental Figure S5H. Student’s parametric t test was used to determine significant (∗p < 0.05) changes in Tg degradation. OST, oligosaccharyl transferase complex; Tg, thyroglobulin; UGGT1, UDP-glucose glycoprotein glucosyltansferase 1;UGGT2, UDP-glucose glycoprotein glucosyltansferase 2.

Fig. 6

Model for common and mutant-specific proteostasis interactome changes mediating the secretion defect of CH-associated Tg variants.A, in the case of WT, Tg processing (top) the delicate balance of proper chaperoning, posttranslational modification, and trafficking is maintained to provide sufficient Tg processing, secretion, and subsequent hormone production (indicated by the arrow size and color code denoted in the key). B, in the case of secretion-defective, CH-associated Tg mutants (bottom), this balance between chaperoning is shifted in such a way that increased chaperoning and engagement with oxidative folding enzymes, possibly stemming from altered engagement with the OST complex dominates Tg processing (indicated by the arrow size and color code denoted in the key). Additionally, mutant Tg is increasingly marked for degradation, yet inefficient retrotranslocation or decreased engagement by the proteasome leads to degradation rates remaining consistent compared to WT. CH, congenital hypothyroidism; OST, oligosaccharyl transferase complex; Tg, thyroglobulin.