Twelve “Bridges” That Hold Thyroid Peroxidase Together: Disulfide Bonds and Congenital Hypothyroidism

Written by Hirotsugu Suwanai, MD, PhD

Journal Club of the Suwanai · Thyroid

Thyroid peroxidase (TPO) is the enzyme that makes thyroid hormone. Can losing just one of the “bridges” that hold its structure together stop it from working?

In 2024, the journal Thyroid published a comprehensive analysis of the disulfide bonds of TPO. The study came from our group at Tokyo Medical University, and I took part as a co-author (second author). Read the original paper ↗

Key point: why activity does not return when degradation is blocked ↓

It began with one patient’s mutation

Congenital hypothyroidism is the most common congenital endocrine disorder, and mutations in TPO are one of its causes.

The study grew out of a case we reported in 2020: a man born before newborn screening was introduced, diagnosed with congenital hypothyroidism at 10 months, in whom genetic testing at age 52 revealed a novel homozygous TPO mutation (p.Cys655Phe). Cysteine 655 forms a disulfide (S–S) bond with another cysteine — a bridge that maintains the protein’s shape. 2020 case report ↗

Are TPO’s other disulfide bonds equally important? No study had examined them systematically.

Disulfide bonds analyzed in this study. Source: Iwasaki et al., Thyroid (2024).

How the experiments worked

The team first expressed two mutations reported in congenital hypothyroidism, C655F and C825R, in cultured HEK293 cells, measuring enzyme activity with the Amplex Red assay and protein levels by Western blotting.

They also examined the effect of the proteasome inhibitor MG132 and how much TPO reached the cell surface, where it normally works. They then created mutants replacing each of the 24 cysteines forming the 12 disulfide bonds and repeated the analysis. Abstract

What they found

The two known mutations: less activity and less protein

C655F and C825R had significantly lower activity and protein levels than wild type, and less TPO reached the cell surface.

Activity fell with every one of the 24 mutations

Every cysteine mutant showed significantly reduced activity and protein expression: all 12 disulfide bonds are bridges TPO cannot do without. The size of the reduction depended on the bond’s position.

Blocking degradation does not restore activity

With MG132, mutant TPO protein recovered to wild-type levels — but activity did not.

This shows that mutant TPO is made but misfolds and is degraded by the cell’s quality-control machinery, and that stopping degradation cannot make a misshapen protein work. The authors conclude that broken disulfide bonds lead to misfolding, degradation and impaired membrane insertion.

Journal Club view: significance and limits

Clinically, the study helps interpret genetic tests. Variants of uncertain significance are common in congenital hypothyroidism; experimental evidence that replacing any of these 24 cysteines impairs function supports their assessment.

That activity is not rescued by blocking degradation is also instructive for therapy: stabilizing the protein is not enough — its correct structure must be restored.

The experiments used HEK293 overexpression rather than thyroid cells, and the Amplex Red assay measures peroxidase activity rather than the full process from iodide uptake to hormone synthesis. Real patients may carry one normal and one mutant allele, or two different mutations, and how position-dependent activity maps onto disease severity remains to be studied.

From one patient to the whole enzyme

Starting from a single mutation found in a single patient, the study tested every bridge in the enzyme — taking a clinical question into basic research and back to the clinic.

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