Characterization of S-homocysteinylation of Human Insulin and Its Implications in Diabetes

IF 1.4 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

The Protein Journal Pub Date : 2025-07-23 DOI:10.1007/s10930-025-10282-9

Amreen B. Sheikh, Swaraj M. Jathar, Vaishnavi Tammara, Atanu Das, Mahesh J. Kulkarni

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Abstract

Homocysteine thiolactone is a reactive thiol known for its interaction with various proteins. Nevertheless, there exists a paucity of information concerning the interaction between homocysteine thiolactone and human insulin, particularly regarding the mechanism by which homocysteine facilitates the reduction of disulfide bonds within insulin. In the present study, we have elucidated the binding sites of homocysteine to the cysteine residues (A6-B7 and A20-B19) that are implicated in the formation of intermolecular disulfide bonds in insulin through an in vitro reaction analyzed via LC-ESI MS/MS. This results in a reduction of disulfide bonds linking the A and B chains, which was corroborated by MALDI-TOF-MS and ESI-MS analysis. The secondary structure of insulin is affected by this modification, as evidenced by circular dichroism spectroscopy. In-silico studies also show that homocysteine affects the insulin structure. A glucose uptake assay conducted in Chinese hamster ovary (CHO) cells that stably express the insulin receptor revealed that HC-modified insulin is less effective in inducing glucose uptake compared to native insulin, suggesting that HC-induced structural modifications in insulin influence functional activity. This study provides insight into the HC-induced structural and functional changes in insulin and discusses the consequent implications for diabetes.

Graphical Abstract

Abstract Image

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人胰岛素的s -同型半胱氨酸化特征及其在糖尿病中的意义。

同型半胱氨酸硫内酯是一种活性硫醇，以其与各种蛋白质的相互作用而闻名。然而，关于同型半胱氨酸硫内酯与人胰岛素之间的相互作用，特别是同型半胱氨酸促进胰岛素内二硫键减少的机制，目前还缺乏相关信息。在本研究中，我们通过LC-ESI MS/MS分析了同型半胱氨酸与半胱氨酸残基（A6-B7和A20-B19）的结合位点，这些残基与胰岛素分子间二硫键的形成有关。这导致连接a链和B链的二硫键减少，MALDI-TOF-MS和ESI-MS分析证实了这一点。胰岛素的二级结构受到这种修饰的影响，圆二色光谱证明了这一点。计算机研究也表明同型半胱氨酸影响胰岛素结构。在稳定表达胰岛素受体的中国仓鼠卵巢（CHO）细胞中进行的葡萄糖摄取实验显示，与天然胰岛素相比，hc修饰的胰岛素诱导葡萄糖摄取的效果较差，这表明hc诱导的胰岛素结构修饰影响了功能活性。本研究提供了hc诱导的胰岛素结构和功能变化的见解，并讨论了其对糖尿病的影响。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

The Protein Journal 生物-生化与分子生物学

CiteScore

5.20

自引率

0.00%

发文量

审稿时长

12 months

期刊介绍： The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.