Nature Chinese scientists reveal the molecular mechanism of autoimmune hyperthyroidism and hypothyroidism

In recent years, the incidence of thyroid-related diseases is increasing year by year, and the total prevalence of thyroid-related diseases in China is as high as 20%
.
Thyroid-related diseases include hyperthyroidism (hyperthyroidism), hypothyroidism (hypothyroidism), thyroid nodules, and thyroid cancer
.
The main physiological function of the thyroid gland is to secrete thyroxine to regulate the body's energy metabolism, and the realization of this function depends on the thyroid-stimulating hormone receptor (TSHR) on the surface of thyroid cells to sense the thyroid-stimulating hormone (TSH) signal secreted by pituitary cells
.
Clinical studies have found that diffuse toxic goiter (also known as Graves'disease, GD) is the main disease phenotype of hyperthyroidism, GD is an autoimmune disease, the total incidence in the population is about 1%, and the female The incidence is much higher than that of men.
The pathogenesis is that the immune system produces activating antibodies that can specifically recognize TSHR.
The TSHR on the surface of thyroid cells is continuously activated by the antibody, which causes the thyroid to secrete too much thyroxine to cause the disease
.
At the same time, the activating antibody also activates TSHR expressed on the membrane of orbital fibroblasts, which leads to excessive proliferation and differentiation of fibroblasts, resulting in swelling of eye muscles and causing Graves'orbitopathy (GO).
Nearly half of people with GD also suffer from Guerilla eye disease
.
In addition, there are also some patients with hypothyroidism, whose disease type is autoimmune Hashimoto's disease (also known as Hashimoto's disease)
.
In patients with Hashimoto's disease, the immune system produces inhibitory antibodies that target TSHR, in part
.
Therefore, in addition to mediating the normal physiological function of the thyroid, TSHR is also involved in the occurrence and development of thyroid-related diseases (Figure 1)
.

Figure 1 Physiological and pathological processes mediated by TSHR in vivo

TSH is a member of glycoprotein hormones, and together with chorionic gonadotropin CG, luteinizing hormone LH, and follicle-stimulating hormone FSH constitute the glycoprotein hormone family
.
In the human body, four glycoprotein hormones can act on three glycoprotein hormone receptors.
Except TSH can act on TSHR, CG and LH can act together on LHCGR, and FSH can act on FSHR
.
LHCGR and FSHR are mainly highly expressed in the gonads, and are involved in the regulation of human sex development, the maintenance of secondary sexual characteristics, and reproduction.
They are also the main targets of current research on infertility and in vitro assisted reproductive technology
.

　　The cooperative team has long been committed to the research on the structure and function of the glycoprotein hormone receptor family, and has made breakthroughs in the field of glycoprotein hormone research
.
In 2021, the team first reported four structures of the first full-length glycoprotein hormone receptor LHCGR in inactive and multiple activated states, revealing the detailed mode of interaction between CG and LHCGR for the first time, clarifying In order to determine the key amino acid residues that determine the selectivity of LHCGR and FSHR for CG, LH, and FSH, and to reveal the structural change of the receptor from the inactive state to the active state, the "push and pull" of the hormone CG-activated receptor is proposed.
” model, and further revealed the detailed mode of interaction between the small molecule compound Org43553 in phase 1 clinical trials and the receptor LHCGR, providing a structural basis for the clinical development of small molecule drugs targeting LHCGR 1
.
In 2022, the team published the first structure of full-length FSHR in the inactivated and hormone-activated state on bioRxiv, which reconfirmed that the activation of FSHR by hormone FSH still follows the "push and pull" model, and compared with LHCGR and TSHR.
, further explored in detail the key amino acid sites that determine the selective recognition of different small molecule allosteric agonists by FSHR, laying a structural foundation for the design of selective small molecule drugs targeting FSHR 2
.

　　TSH is an important member of glycoprotein hormones.
Due to the complexity and instability of its structure, no structural information of TSH has been reported yet
.
TSH is an important clinical drug for adjuvant treatment of thyroid cancer, and it is also an important target for many major rare diseases including McCune-Albright syndrome, pituitary TSH adenoma, and primary congenital hypothyroidism.
key molecule
.
However, the molecular mechanism of how TSH acts on TSHR to trigger thyroxine secretion has not been known; TSHR can trigger autoimmune hyperthyroidism or hypothyroidism, but how human activating antibodies and inhibitory antibodies act on TSHR and then trigger receptor activation The mechanism of TSHR or inhibition is still unknown, and the difference between activating antibodies and the way the hormone TSH activates TSHR remains to be further explored
.
In addition, the molecular mechanism of how TSHR-targeting small-molecule allosteric agonists activate the receptor has also been the focus of research
.

　　On August 8, 2022, Xu Huaqiang, a researcher from Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Jiang Yi , a researcher from Lingang Laboratory , and Zhang Shuyang, a professor from Peking Union Medical College Hospital , jointly published the latest research results "Hormone- and antibody-mediated activation of the thyrotropin receptor”, and analyzed for the first time the full-length TSHR in the activated state when the hormone TSH binds to the human activating antibody M22, and the inhibitory state structure when the human inhibitory antibody K1-70 binds
.
This work revealed for the first time the detailed mode of interaction between TSH and TSHR, elucidated the key amino acid residues that determine the specific recognition of TSH and TSHR, and revealed the molecular mechanism by which TSH activates TSHR, and revealed that antibodies activate or inhibit receptors to trigger The molecular mechanism of GD and Hashimoto's disease, and further revealed the detailed mode of interaction between the small-molecule allosteric agonist ML-109 and the receptor TSHR, providing a structural basis for the clinical development of antibodies or small-molecule drugs for the treatment of thyroid-related diseases
.

　

　　The research team used single-particle cryo-electron microscopy to restructure the Gs complex formed by TSH-activated TSHR, the Gs complex formed by the activation of TSHR by human activating antibody M22, and the TSHR bound by human inhibitory antibody.
Among them, TSH- The overall resolution of the TSHR-Gs complex structure is 2.
96 Å, the extracellular local resolution is 2.
67 Å, ​​the overall resolution of the M22-TSHR-Gs complex structure is 2.
78 Å, the extracellular local resolution is 2.
39 Å, and the K1- The resolution of the 70-TSHR complex is 5.
46 angstroms
.

　　Through structural analysis, the researchers identified key amino acid residues specifically recognized by the hormones TSH and TSHR for the first time
.
By comparing the inhibitory state of K1-70 binding with the activated state TSHR structure under TSH activation, the researchers found that K1-70 can stably bind to the surface of the receptor's extracellular domain (ECD) in an inactive state, while TSH and inactive When the receptor ECD binds, it will generate steric hindrance with the cell membrane, thereby pushing the receptor ECD upward deflection, that is, the effect of "pull"; at the same time, the researchers further confirmed that the loop extending from the receptor hinge region also exists with the hormone TSH Interaction, the loop will further pull the overall upward deflection of the receptors ECD and TSH, that is, the effect of "pull".
Combining the two effects, the researchers once again confirmed that the activation of TSHR by TSH still follows the "push and pull" activation model, that is, glycoproteins Hormone-activated receptors all follow a conservative activation mechanism of "push and pull"
.
At the same time, from the structural comparison, it was found that the inhibitory antibody mainly inhibited the activation of the receptor by competing with TSH to bind to TSHR
.
Compared with the inhibitory antibody K1-70, when the antibody M22 binds to the inactive receptor ECD, it will have steric hindrance with the cell membrane, thereby pushing the receptor ECD upward deflection and producing a "push" effect.
Further research It shows that there is no interaction between the antibody and the hinge region loop, that is, the antibody-activated receptor only has a mode of action of "push"
.
Finally, the researchers also analyzed the molecular details of the interaction between the small molecule agonist ML-109 and the receptor TSHR, and through further mutation experiments identified the key amino acid residues that determine the specificity of ML-109 to recognize and activate TSHR but not LHCGR base (Figure 2)
.

　

Figure 2 Comparison of the complex structures of TSH, M22 and K1-70 with TSHR and Gs proteins

　　In conclusion, this study systematically studied the structures of TSHR with endogenous hormone TSH and small molecule agonist ML-109, revealing the mechanism of receptor activation induced by hormone TSH and allosteric agonist ML-109 for the first time; The structures of the TSHR-type antibody M22 and the inhibitory-type antibody K1-70 and TSHR reveal the structural basis of how TSHR is activated or inhibited by autoimmune antibodies, thus providing antibody drugs and small molecules for autoimmune diseases caused by abnormal TSHR function Drug discovery provides clearer templates and ideas
.

　　This project was completed by researcher Xu Huaqiang of Shanghai Institute of Materia Medica , researcher Jiang Yi of Lingang Laboratory and Professor Zhang Shuyang of Peking Union Medical College Hospital .
Dr.
Duan Jia from Shanghai Institute of Materia Medica, Dr.
Xu Peiyu (now a postdoctoral fellow at MIT), and Dr.
Luan Xiaodong from Tsinghua University are the co-first authors of this article .
This work has been funded by the National Key R&D Program, the Shanghai Municipal Science and Technology Major Project, the Ministry of Science and Technology's Key R&D Program, the Chinese Academy of Sciences' Strategic Pilot Science and Technology Project, the National Natural Science Foundation of China, and the Innovation Project of the Chinese Academy of Medical Sciences .

1.
Duan, J.
et al .
Structures of full-length glycoprotein hormone receptor signalling complexes.
Nature , doi:10.
1038/s41586-021-03924-2 (2021).

2.
Duan, J.
et al.
Universal mechanism of hormone and allosteric agonist mediated activation of glycoprotein hormone receptors as revealed by structures of follicle stimulating hormone receptor.
bioRxiv (2022).