Iwasaki Hajime, Suwanai Hirotsugu, Yakou Fumiyoshi, Sakai Hiroyuki, Ishii Keitaro, Hara Natsuko, Buckle Ashley M, Kanekura Kohsuke, Miyagi Tamami, Narumi Satoshi, Suzuki Ryo
Department of Diabetes, Metabolism, and Endocrinology, Tokyo Medical University, Tokyo, Japan.
Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.
Thyroid. 2024 May;34(5):659-667. doi: 10.1089/thy.2023.0514. Epub 2024 Apr 11.
Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) ( < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.
先天性甲状腺功能减退症（CH）是由半胱氨酸残基的突变引起的，包括在甲状腺过氧化物酶（TPO）中形成二硫键的Cys655和Cys825。这些二硫键很可能在TPO活性中发挥重要作用。然而，迄今为止，尚无研究全面分析在TPO中形成二硫键的半胱氨酸突变。在本研究中，我们诱导参与二硫键形成的半胱氨酸残基发生突变，并分析它们对亚细胞定位、降解和酶活性的影响，以评估二硫键在TPO活性中的重要性。已知发生在CH中的载体质粒TPO突变体C655F和C825R被转染到HEK293细胞中。通过Amplex red测定法和蛋白质印迹法测量TPO活性和蛋白质表达水平。在存在MG132蛋白酶体抑制剂的情况下进行相同的操作。使用免疫细胞化学和流式细胞术确定亚细胞定位。使用分析预测TPO内所有二硫键的位置。诱导了与二硫键相关的所有TPO突变。还使用Amplex red测定法和蛋白质印迹法在与二硫键相关的所有TPO突变体中测量TPO活性和蛋白质表达水平。与野生型（WT）相比，C655F和C825R的活性和蛋白质表达明显降低（<0.05）。在存在MG132蛋白酶体抑制剂的情况下，TPO的蛋白质表达水平增加到与WT相当的水平，但其活性没有增加。与WT TPO相比，突变体中TPO在细胞表面的亚细胞分布程度较低。24个半胱氨酸残基参与了TPO中12个二硫键的形成。每个半胱氨酸中发生氨基酸取代的所有TPO突变体均显示TPO活性和蛋白质表达水平显著降低。此外，TPO活性的差异取决于二硫键的位置。所有12个二硫键在TPO的活性中都起重要作用。此外，这些突变导致错误折叠、降解和膜插入。
